Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients

Investigation of mutations in the partial sequences... | F1000Research "use strict";function _typeof(t){return(_typeof="function"==typeof Symbol&&"symbol"==typeof Symbol.iterator?function(t){return typeof t}:function(t){return t&&"function"==typeof Symbol&&t.constructor===Symbol&&t!==Symbol.prototype?"symbol":typeof t})(t)}!function(){var t=function(){var t,e,o=[],n=window,r=n;for(;r;){try{if(r.frames.__tcfapiLocator){t=r;break}}catch(t){}if(r===n.top)break;r=r.parent}t||(!function t(){var e=n.document,o=!!n.frames.__tcfapiLocator;if(!o)if(e.body){var r=e.createElement("iframe");r.style.cssText="display:none",r.name="__tcfapiLocator",e.body.appendChild(r)}else setTimeout(t,5);return!o}(),n.__tcfapi=function(){for(var t=arguments.length,n=new Array(t),r=0;r 3&&2===parseInt(n[1],10)&&"boolean"==typeof n[3]&&(e=n[3],"function"==typeof n[2]&&n[2]("set",!0)):"ping"===n[0]?"function"==typeof n[2]&&n[2]({gdprApplies:e,cmpLoaded:!1,cmpStatus:"stub"}):o.push(n)},n.addEventListener("message",(function(t){var e="string"==typeof t.data,o={};if(e)try{o=JSON.parse(t.data)}catch(t){}else o=t.data;var n="object"===_typeof(o)&&null!==o?o.__tcfapiCall:null;n&&window.__tcfapi(n.command,n.version,(function(o,r){var a={__tcfapiReturn:{returnValue:o,success:r,callId:n.callId}};t&&t.source&&t.source.postMessage&&t.source.postMessage(e?JSON.stringify(a):a,"*")}),n.parameter)}),!1))};"undefined"!=typeof module?module.exports=t:t()}(); dataLayer = dataLayer || []; // Standard GTM initialization - Google Consent Mode handles consent automatically (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start': new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0], j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src= 'https://www.googletagmanager.com/gtm.js?id='+i+dl+ '>m_auth=hzk0Vc3qFsQYhCrIoHz68A>m_preview=env-1>m_cookies_win=x';f.parentNode.insertBefore(j,f); })(window,document,'script','dataLayer','GTM-MWFK8L5J'); ;window.NREUM||(NREUM={});NREUM.init={distributed_tracing:{enabled:true},privacy:{cookies_enabled:true},ajax:{deny_list:["bam.nr-data.net"]}}; ;NREUM.loader_config={accountID:"438030",trustKey:"438030",agentID:"772317073",licenseKey:"97f8f67f26",applicationID:"772317073"} ;NREUM.info={beacon:"bam.nr-data.net",errorBeacon:"bam.nr-data.net",licenseKey:"97f8f67f26",applicationID:"772317073",sa:1} ;/*! For license information please see nr-loader-spa-1.236.0.min.js.LICENSE.txt */ (()=>{"use strict";var e,t,r={5763:(e,t,r)=>{r.d(t,{P_:()=>l,Mt:()=>g,C5:()=>s,DL:()=>v,OP:()=>T,lF:()=>D,Yu:()=>y,Dg:()=>h,CX:()=>c,GE:()=>b,sU:()=>_});var n=r(8632),i=r(9567);const o={beacon:n.ce.beacon,errorBeacon:n.ce.errorBeacon,licenseKey:void 0,applicationID:void 0,sa:void 0,queueTime:void 0,applicationTime:void 0,ttGuid:void 0,user:void 0,account:void 0,product:void 0,extra:void 0,jsAttributes:{},userAttributes:void 0,atts:void 0,transactionName:void 0,tNamePlain:void 0},a={};function s(e){if(!e)throw new Error("All info objects require an agent identifier!");if(!a[e])throw new Error("Info for ".concat(e," was never set"));return a[e]}function c(e,t){if(!e)throw new Error("All info objects require an agent identifier!");a[e]=(0,i.D)(t,o),(0,n.Qy)(e,a[e],"info")}var u=r(7056);const d=()=>{const e={blockSelector:"[data-nr-block]",maskInputOptions:{password:!0}};return{allow_bfcache:!0,privacy:{cookies_enabled:!0},ajax:{deny_list:void 0,enabled:!0,harvestTimeSeconds:10},distributed_tracing:{enabled:void 0,exclude_newrelic_header:void 0,cors_use_newrelic_header:void 0,cors_use_tracecontext_headers:void 0,allowed_origins:void 0},session:{domain:void 0,expiresMs:u.oD,inactiveMs:u.Hb},ssl:void 0,obfuscate:void 0,jserrors:{enabled:!0,harvestTimeSeconds:10},metrics:{enabled:!0},page_action:{enabled:!0,harvestTimeSeconds:30},page_view_event:{enabled:!0},page_view_timing:{enabled:!0,harvestTimeSeconds:30,long_task:!1},session_trace:{enabled:!0,harvestTimeSeconds:10},harvest:{tooManyRequestsDelay:60},session_replay:{enabled:!1,harvestTimeSeconds:60,sampleRate:.1,errorSampleRate:.1,maskTextSelector:"*",maskAllInputs:!0,get blockClass(){return"nr-block"},get ignoreClass(){return"nr-ignore"},get maskTextClass(){return"nr-mask"},get blockSelector(){return e.blockSelector},set blockSelector(t){e.blockSelector+=",".concat(t)},get maskInputOptions(){return e.maskInputOptions},set maskInputOptions(t){e.maskInputOptions={...t,password:!0}}},spa:{enabled:!0,harvestTimeSeconds:10}}},f={};function l(e){if(!e)throw new Error("All configuration objects require an agent identifier!");if(!f[e])throw new Error("Configuration for ".concat(e," was never set"));return f[e]}function h(e,t){if(!e)throw new Error("All configuration objects require an agent identifier!");f[e]=(0,i.D)(t,d()),(0,n.Qy)(e,f[e],"config")}function g(e,t){if(!e)throw new Error("All configuration objects require an agent identifier!");var r=l(e);if(r){for(var n=t.split("."),i=0;i {r.d(t,{D:()=>i});var n=r(50);function i(e,t){try{if(!e||"object"!=typeof e)return(0,n.Z)("Setting a Configurable requires an object as input");if(!t||"object"!=typeof t)return(0,n.Z)("Setting a Configurable requires a model to set its initial properties");const r=Object.create(Object.getPrototypeOf(t),Object.getOwnPropertyDescriptors(t)),o=0===Object.keys(r).length?e:r;for(let a in o)if(void 0!==e[a])try{"object"==typeof e[a]&&"object"==typeof t[a]?r[a]=i(e[a],t[a]):r[a]=e[a]}catch(e){(0,n.Z)("An error occurred while setting a property of a Configurable",e)}return r}catch(e){(0,n.Z)("An error occured while setting a Configurable",e)}}},6818:(e,t,r)=>{r.d(t,{Re:()=>i,gF:()=>o,q4:()=>n});const n="1.236.0",i="PROD",o="CDN"},385:(e,t,r)=>{r.d(t,{FN:()=>a,IF:()=>u,Nk:()=>f,Tt:()=>s,_A:()=>o,il:()=>n,pL:()=>c,v6:()=>i,w1:()=>d});const n="undefined"!=typeof window&&!!window.document,i="undefined"!=typeof WorkerGlobalScope&&("undefined"!=typeof self&&self instanceof WorkerGlobalScope&&self.navigator instanceof WorkerNavigator||"undefined"!=typeof globalThis&&globalThis instanceof WorkerGlobalScope&&globalThis.navigator instanceof WorkerNavigator),o=n?window:"undefined"!=typeof WorkerGlobalScope&&("undefined"!=typeof self&&self instanceof WorkerGlobalScope&&self||"undefined"!=typeof globalThis&&globalThis instanceof WorkerGlobalScope&&globalThis),a=""+o?.location,s=/iPad|iPhone|iPod/.test(navigator.userAgent),c=s&&"undefined"==typeof SharedWorker,u=(()=>{const e=navigator.userAgent.match(/Firefox[/\s](\d+\.\d+)/);return Array.isArray(e)&&e.length>=2?+e[1]:0})(),d=Boolean(n&&window.document.documentMode),f=!!navigator.sendBeacon},1117:(e,t,r)=>{r.d(t,{w:()=>o});var n=r(50);const i={agentIdentifier:"",ee:void 0};class o{constructor(e){try{if("object"!=typeof e)return(0,n.Z)("shared context requires an object as input");this.sharedContext={},Object.assign(this.sharedContext,i),Object.entries(e).forEach((e=>{let[t,r]=e;Object.keys(i).includes(t)&&(this.sharedContext[t]=r)}))}catch(e){(0,n.Z)("An error occured while setting SharedContext",e)}}}},8e3:(e,t,r)=>{r.d(t,{L:()=>d,R:()=>c});var n=r(2177),i=r(1284),o=r(4322),a=r(3325);const s={};function c(e,t){const r={staged:!1,priority:a.p[t]||0};u(e),s[e].get(t)||s[e].set(t,r)}function u(e){e&&(s[e]||(s[e]=new Map))}function d(){let e=arguments.length>0&&void 0!==arguments[0]?arguments[0]:"",t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:"feature";if(u(e),!e||!s[e].get(t))return a(t);s[e].get(t).staged=!0;const r=[...s[e]];function a(t){const r=e?n.ee.get(e):n.ee,a=o.X.handlers;if(r.backlog&&a){var s=r.backlog[t],c=a[t];if(c){for(var u=0;s&&u {let[t,r]=e;return r.staged}))&&(r.sort(((e,t)=>e[1].priority-t[1].priority)),r.forEach((e=>{let[t]=e;a(t)})))}function f(e,t){var r=e[1];(0,i.D)(t[r],(function(t,r){var n=e[0];if(r[0]===n){var i=r[1],o=e[3],a=e[2];i.apply(o,a)}}))}},2177:(e,t,r)=>{r.d(t,{c:()=>f,ee:()=>u});var n=r(8632),i=r(2210),o=r(1284),a=r(5763),s="nr@context";let c=(0,n.fP)();var u;function d(){}function f(e){return(0,i.X)(e,s,l)}function l(){return new d}function h(){u.aborted=!0,u.backlog={}}c.ee?u=c.ee:(u=function e(t,r){var n={},c={},f={},g=!1;try{g=16===r.length&&(0,a.OP)(r).isolatedBacklog}catch(e){}var p={on:b,addEventListener:b,removeEventListener:y,emit:v,get:x,listeners:w,context:m,buffer:A,abort:h,aborted:!1,isBuffering:E,debugId:r,backlog:g?{}:t&&"object"==typeof t.backlog?t.backlog:{}};return p;function m(e){return e&&e instanceof d?e:e?(0,i.X)(e,s,l):l()}function v(e,r,n,i,o){if(!1!==o&&(o=!0),!u.aborted||i){t&&o&&t.emit(e,r,n);for(var a=m(n),s=w(e),d=s.length,f=0;fn,p:()=>i});var n=r(2177).ee.get("handle");function i(e,t,r,i,o){o?(o.buffer([e],i),o.emit(e,t,r)):(n.buffer([e],i),n.emit(e,t,r))}},4322:(e,t,r)=>{r.d(t,{X:()=>o});var n=r(5546);o.on=a;var i=o.handlers={};function o(e,t,r,o){a(o||n.E,i,e,t,r)}function a(e,t,r,i,o){o||(o="feature"),e||(e=n.E);var a=t[o]=t[o]||{};(a[r]=a[r]||[]).push([e,i])}},3239:(e,t,r)=>{r.d(t,{bP:()=>s,iz:()=>c,m$:()=>a});var n=r(385);let i=!1,o=!1;try{const e={get passive(){return i=!0,!1},get signal(){return o=!0,!1}};n._A.addEventListener("test",null,e),n._A.removeEventListener("test",null,e)}catch(e){}function a(e,t){return i||o?{capture:!!e,passive:i,signal:t}:!!e}function s(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2],n=arguments.length>3?arguments[3]:void 0;window.addEventListener(e,t,a(r,n))}function c(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2],n=arguments.length>3?arguments[3]:void 0;document.addEventListener(e,t,a(r,n))}},4402:(e,t,r)=>{r.d(t,{Ht:()=>u,M:()=>c,Rl:()=>a,ky:()=>s});var n=r(385);const i="xxxxxxxx-xxxx-4xxx-yxxx-xxxxxxxxxxxx";function o(e,t){return e?15&e[t]:16*Math.random()|0}function a(){const e=n._A?.crypto||n._A?.msCrypto;let t,r=0;return e&&e.getRandomValues&&(t=e.getRandomValues(new Uint8Array(31))),i.split("").map((e=>"x"===e?o(t,++r).toString(16):"y"===e?(3&o()|8).toString(16):e)).join("")}function s(e){const t=n._A?.crypto||n._A?.msCrypto;let r,i=0;t&&t.getRandomValues&&(r=t.getRandomValues(new Uint8Array(31)));const a=[];for(var s=0;s {r.d(t,{Bq:()=>n,Hb:()=>o,oD:()=>i});const n="NRBA",i=144e5,o=18e5},7894:(e,t,r)=>{function n(){return Math.round(performance.now())}r.d(t,{z:()=>n})},7243:(e,t,r)=>{r.d(t,{e:()=>o});var n=r(385),i={};function o(e){if(e in i)return i[e];if(0===(e||"").indexOf("data:"))return{protocol:"data"};let t;var r=n._A?.location,o={};if(n.il)t=document.createElement("a"),t.href=e;else try{t=new URL(e,r.href)}catch(e){return o}o.port=t.port;var a=t.href.split("://");!o.port&&a[1]&&(o.port=a[1].split("/")[0].split("@").pop().split(":")[1]),o.port&&"0"!==o.port||(o.port="https"===a[0]?"443":"80"),o.hostname=t.hostname||r.hostname,o.pathname=t.pathname,o.protocol=a[0],"/"!==o.pathname.charAt(0)&&(o.pathname="/"+o.pathname);var s=!t.protocol||":"===t.protocol||t.protocol===r.protocol,c=t.hostname===r.hostname&&t.port===r.port;return o.sameOrigin=s&&(!t.hostname||c),"/"===o.pathname&&(i[e]=o),o}},50:(e,t,r)=>{function n(e,t){"function"==typeof console.warn&&(console.warn("New Relic: ".concat(e)),t&&console.warn(t))}r.d(t,{Z:()=>n})},2587:(e,t,r)=>{r.d(t,{N:()=>c,T:()=>u});var n=r(2177),i=r(5546),o=r(8e3),a=r(3325);const s={stn:[a.D.sessionTrace],err:[a.D.jserrors,a.D.metrics],ins:[a.D.pageAction],spa:[a.D.spa],sr:[a.D.sessionReplay,a.D.sessionTrace]};function c(e,t){const r=n.ee.get(t);e&&"object"==typeof e&&(Object.entries(e).forEach((e=>{let[t,n]=e;void 0===u[t]&&(s[t]?s[t].forEach((e=>{n?(0,i.p)("feat-"+t,[],void 0,e,r):(0,i.p)("block-"+t,[],void 0,e,r),(0,i.p)("rumresp-"+t,[Boolean(n)],void 0,e,r)})):n&&(0,i.p)("feat-"+t,[],void 0,void 0,r),u[t]=Boolean(n))})),Object.keys(s).forEach((e=>{void 0===u[e]&&(s[e]?.forEach((t=>(0,i.p)("rumresp-"+e,[!1],void 0,t,r))),u[e]=!1)})),(0,o.L)(t,a.D.pageViewEvent))}const u={}},2210:(e,t,r)=>{r.d(t,{X:()=>i});var n=Object.prototype.hasOwnProperty;function i(e,t,r){if(n.call(e,t))return e[t];var i=r();if(Object.defineProperty&&Object.keys)try{return Object.defineProperty(e,t,{value:i,writable:!0,enumerable:!1}),i}catch(e){}return e[t]=i,i}},1284:(e,t,r)=>{r.d(t,{D:()=>n});const n=(e,t)=>Object.entries(e||{}).map((e=>{let[r,n]=e;return t(r,n)}))},4351:(e,t,r)=>{r.d(t,{P:()=>o});var n=r(2177);const i=()=>{const e=new WeakSet;return(t,r)=>{if("object"==typeof r&&null!==r){if(e.has(r))return;e.add(r)}return r}};function o(e){try{return JSON.stringify(e,i())}catch(e){try{n.ee.emit("internal-error",[e])}catch(e){}}}},3960:(e,t,r)=>{r.d(t,{K:()=>a,b:()=>o});var n=r(3239);function i(){return"undefined"==typeof document||"complete"===document.readyState}function o(e,t){if(i())return e();(0,n.bP)("load",e,t)}function a(e){if(i())return e();(0,n.iz)("DOMContentLoaded",e)}},8632:(e,t,r)=>{r.d(t,{EZ:()=>u,Qy:()=>c,ce:()=>o,fP:()=>a,gG:()=>d,mF:()=>s});var n=r(7894),i=r(385);const o={beacon:"bam.nr-data.net",errorBeacon:"bam.nr-data.net"};function a(){return i._A.NREUM||(i._A.NREUM={}),void 0===i._A.newrelic&&(i._A.newrelic=i._A.NREUM),i._A.NREUM}function s(){let e=a();return e.o||(e.o={ST:i._A.setTimeout,SI:i._A.setImmediate,CT:i._A.clearTimeout,XHR:i._A.XMLHttpRequest,REQ:i._A.Request,EV:i._A.Event,PR:i._A.Promise,MO:i._A.MutationObserver,FETCH:i._A.fetch}),e}function c(e,t,r){let i=a();const o=i.initializedAgents||{},s=o[e]||{};return Object.keys(s).length||(s.initializedAt={ms:(0,n.z)(),date:new Date}),i.initializedAgents={...o,[e]:{...s,[r]:t}},i}function u(e,t){a()[e]=t}function d(){return function(){let e=a();const t=e.info||{};e.info={beacon:o.beacon,errorBeacon:o.errorBeacon,...t}}(),function(){let e=a();const t=e.init||{};e.init={...t}}(),s(),function(){let e=a();const t=e.loader_config||{};e.loader_config={...t}}(),a()}},7956:(e,t,r)=>{r.d(t,{N:()=>i});var n=r(3239);function i(e){let t=arguments.length>1&&void 0!==arguments[1]&&arguments[1],r=arguments.length>2?arguments[2]:void 0,i=arguments.length>3?arguments[3]:void 0;return void(0,n.iz)("visibilitychange",(function(){if(t)return void("hidden"==document.visibilityState&&e());e(document.visibilityState)}),r,i)}},1214:(e,t,r)=>{r.d(t,{em:()=>v,u5:()=>N,QU:()=>S,_L:()=>I,Gm:()=>L,Lg:()=>M,gy:()=>U,BV:()=>Q,Kf:()=>ee});var n=r(2177);const i="nr@original";var o=Object.prototype.hasOwnProperty,a=!1;function s(e,t){return e||(e=n.ee),r.inPlace=function(e,t,n,i,o){n||(n="");var a,s,c,u="-"===n.charAt(0);for(c=0;c 2?n-2:0),o=2;o {r(A[T],e,w),r(E[T],e,w)})),r(l._A,"fetch",y),t.on(y+"end",(function(e,r){var n=this;if(r){var i=r.headers.get("content-length");null!==i&&(n.rxSize=i),t.emit(y+"done",[null,r],n)}else t.emit(y+"done",[e],n)})),t}const O={},j=["pushState","replaceState"];function S(e){const t=function(e){return(e||n.ee).get("history")}(e);return!l.il||O[t.debugId]++||(O[t.debugId]=1,s(t).inPlace(window.history,j,"-")),t}var P=r(3239);const C={},R=["appendChild","insertBefore","replaceChild"];function I(e){const t=function(e){return(e||n.ee).get("jsonp")}(e);if(!l.il||C[t.debugId])return t;C[t.debugId]=!0;var r=s(t),i=/[?&](?:callback|cb)=([^&#]+)/,o=/(.*)\.([^.]+)/,a=/^(\w+)(\.|$)(.*)$/;function c(e,t){var r=e.match(a),n=r[1],i=r[3];return i?c(i,t[n]):t[n]}return r.inPlace(Node.prototype,R,"dom-"),t.on("dom-start",(function(e){!function(e){if(!e||"string"!=typeof e.nodeName||"script"!==e.nodeName.toLowerCase())return;if("function"!=typeof e.addEventListener)return;var n=(a=e.src,s=a.match(i),s?s[1]:null);var a,s;if(!n)return;var u=function(e){var t=e.match(o);if(t&&t.length>=3)return{key:t[2],parent:c(t[1],window)};return{key:e,parent:window}}(n);if("function"!=typeof u.parent[u.key])return;var d={};function f(){t.emit("jsonp-end",[],d),e.removeEventListener("load",f,(0,P.m$)(!1)),e.removeEventListener("error",l,(0,P.m$)(!1))}function l(){t.emit("jsonp-error",[],d),t.emit("jsonp-end",[],d),e.removeEventListener("load",f,(0,P.m$)(!1)),e.removeEventListener("error",l,(0,P.m$)(!1))}r.inPlace(u.parent,[u.key],"cb-",d),e.addEventListener("load",f,(0,P.m$)(!1)),e.addEventListener("error",l,(0,P.m$)(!1)),t.emit("new-jsonp",[e.src],d)}(e[0])})),t}var k=r(5763);const H={};function L(e){const t=function(e){return(e||n.ee).get("mutation")}(e);if(!l.il||H[t.debugId])return t;H[t.debugId]=!0;var r=s(t),i=k.Yu.MO;return i&&(window.MutationObserver=function(e){return this instanceof i?new i(r(e,"fn-")):i.apply(this,arguments)},MutationObserver.prototype=i.prototype),t}const z={};function M(e){const t=function(e){return(e||n.ee).get("promise")}(e);if(z[t.debugId])return t;z[t.debugId]=!0;var r=n.c,o=s(t),a=k.Yu.PR;return a&&function(){function e(r){var n=t.context(),i=o(r,"executor-",n,null,!1);const s=Reflect.construct(a,[i],e);return t.context(s).getCtx=function(){return n},s}l._A.Promise=e,Object.defineProperty(e,"name",{value:"Promise"}),e.toString=function(){return a.toString()},Object.setPrototypeOf(e,a),["all","race"].forEach((function(r){const n=a[r];e[r]=function(e){let i=!1;[...e||[]].forEach((e=>{this.resolve(e).then(a("all"===r),a(!1))}));const o=n.apply(this,arguments);return o;function a(e){return function(){t.emit("propagate",[null,!i],o,!1,!1),i=i||!e}}}})),["resolve","reject"].forEach((function(r){const n=a[r];e[r]=function(e){const r=n.apply(this,arguments);return e!==r&&t.emit("propagate",[e,!0],r,!1,!1),r}})),e.prototype=a.prototype;const n=a.prototype.then;a.prototype.then=function(){var e=this,i=r(e);i.promise=e;for(var a=arguments.length,s=new Array(a),c=0;c e())),t};function m(e,t){i.inPlace(t,["onreadystatechange"],"fn-",E)}function b(){var e=this,t=r.context(e);e.readyState>3&&!t.resolved&&(t.resolved=!0,r.emit("xhr-resolved",[],e)),i.inPlace(e,f,"fn-",E)}if(function(e,t){for(var r in e)t[r]=e[r]}(o,p),p.prototype=o.prototype,i.inPlace(p.prototype,J,"-xhr-",E),r.on("send-xhr-start",(function(e,t){m(e,t),function(e){h.push(e),a&&(y?y.then(A):u?u(A):(w=-w,x.data=w))}(t)})),r.on("open-xhr-start",m),a){var y=c&&c.resolve();if(!u&&!c){var w=1,x=document.createTextNode(w);new a(A).observe(x,{characterData:!0})}}else t.on("fn-end",(function(e){e[0]&&e[0].type===d||A()}));function A(){for(var e=0;e {r.d(t,{t:()=>n});const n=r(3325).D.ajax},6660:(e,t,r)=>{r.d(t,{A:()=>i,t:()=>n});const n=r(3325).D.jserrors,i="nr@seenError"},3081:(e,t,r)=>{r.d(t,{gF:()=>o,mY:()=>i,t9:()=>n,vz:()=>s,xS:()=>a});const n=r(3325).D.metrics,i="sm",o="cm",a="storeSupportabilityMetrics",s="storeEventMetrics"},4649:(e,t,r)=>{r.d(t,{t:()=>n});const n=r(3325).D.pageAction},7633:(e,t,r)=>{r.d(t,{Dz:()=>i,OJ:()=>a,qw:()=>o,t9:()=>n});const n=r(3325).D.pageViewEvent,i="firstbyte",o="domcontent",a="windowload"},9251:(e,t,r)=>{r.d(t,{t:()=>n});const n=r(3325).D.pageViewTiming},3614:(e,t,r)=>{r.d(t,{BST_RESOURCE:()=>i,END:()=>s,FEATURE_NAME:()=>n,FN_END:()=>u,FN_START:()=>c,PUSH_STATE:()=>d,RESOURCE:()=>o,START:()=>a});const n=r(3325).D.sessionTrace,i="bstResource",o="resource",a="-start",s="-end",c="fn"+a,u="fn"+s,d="pushState"},7836:(e,t,r)=>{r.d(t,{BODY:()=>A,CB_END:()=>E,CB_START:()=>u,END:()=>x,FEATURE_NAME:()=>i,FETCH:()=>_,FETCH_BODY:()=>v,FETCH_DONE:()=>m,FETCH_START:()=>p,FN_END:()=>c,FN_START:()=>s,INTERACTION:()=>l,INTERACTION_API:()=>d,INTERACTION_EVENTS:()=>o,JSONP_END:()=>b,JSONP_NODE:()=>g,JS_TIME:()=>T,MAX_TIMER_BUDGET:()=>a,REMAINING:()=>f,SPA_NODE:()=>h,START:()=>w,originalSetTimeout:()=>y});var n=r(5763);const i=r(3325).D.spa,o=["click","submit","keypress","keydown","keyup","change"],a=999,s="fn-start",c="fn-end",u="cb-start",d="api-ixn-",f="remaining",l="interaction",h="spaNode",g="jsonpNode",p="fetch-start",m="fetch-done",v="fetch-body-",b="jsonp-end",y=n.Yu.ST,w="-start",x="-end",A="-body",E="cb"+x,T="jsTime",_="fetch"},5938:(e,t,r)=>{r.d(t,{W:()=>o});var n=r(5763),i=r(2177);class o{constructor(e,t,r){this.agentIdentifier=e,this.aggregator=t,this.ee=i.ee.get(e,(0,n.OP)(this.agentIdentifier).isolatedBacklog),this.featureName=r,this.blocked=!1}}},9144:(e,t,r)=>{r.d(t,{j:()=>m});var n=r(3325),i=r(5763),o=r(5546),a=r(2177),s=r(7894),c=r(8e3),u=r(3960),d=r(385),f=r(50),l=r(3081),h=r(8632);function g(){const e=(0,h.gG)();["setErrorHandler","finished","addToTrace","inlineHit","addRelease","addPageAction","setCurrentRouteName","setPageViewName","setCustomAttribute","interaction","noticeError","setUserId"].forEach((t=>{e[t]=function(){for(var r=arguments.length,n=new Array(r),i=0;i 1?r-1:0),i=1;i {e.exposed&&e.api[t]&&o.push(e.api[t](...n))})),o.length>1?o:o[0]}(t,...n)}}))}var p=r(2587);function m(e){let t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:{},m=arguments.length>2?arguments[2]:void 0,v=arguments.length>3?arguments[3]:void 0,{init:b,info:y,loader_config:w,runtime:x={loaderType:m},exposed:A=!0}=t;const E=(0,h.gG)();y||(b=E.init,y=E.info,w=E.loader_config),(0,i.Dg)(e,b||{}),(0,i.GE)(e,w||{}),(0,i.sU)(e,x),y.jsAttributes??={},d.v6&&(y.jsAttributes.isWorker=!0),(0,i.CX)(e,y),g();const T=function(e,t){t||(0,c.R)(e,"api");const h={};var g=a.ee.get(e),p=g.get("tracer"),m="api-",v=m+"ixn-";function b(t,r,n,o){const a=(0,i.C5)(e);return null===r?delete a.jsAttributes[t]:(0,i.CX)(e,{...a,jsAttributes:{...a.jsAttributes,[t]:r}}),x(m,n,!0,o||null===r?"session":void 0)(t,r)}function y(){}["setErrorHandler","finished","addToTrace","inlineHit","addRelease"].forEach((e=>h[e]=x(m,e,!0,"api"))),h.addPageAction=x(m,"addPageAction",!0,n.D.pageAction),h.setCurrentRouteName=x(m,"routeName",!0,n.D.spa),h.setPageViewName=function(t,r){if("string"==typeof t)return"/"!==t.charAt(0)&&(t="/"+t),(0,i.OP)(e).customTransaction=(r||"http://custom.transaction")+t,x(m,"setPageViewName",!0)()},h.setCustomAttribute=function(e,t){let r=arguments.length>2&&void 0!==arguments[2]&&arguments[2];if("string"==typeof e){if(["string","number"].includes(typeof t)||null===t)return b(e,t,"setCustomAttribute",r);(0,f.Z)("Failed to execute setCustomAttribute.\nNon-null value must be a string or number type, but a type of was provided."))}else(0,f.Z)("Failed to execute setCustomAttribute.\nName must be a string type, but a type of was provided."))},h.setUserId=function(e){if("string"==typeof e||null===e)return b("enduser.id",e,"setUserId",!0);(0,f.Z)("Failed to execute setUserId.\nNon-null value must be a string type, but a type of was provided."))},h.interaction=function(){return(new y).get()};var w=y.prototype={createTracer:function(e,t){var r={},i=this,a="function"==typeof t;return(0,o.p)(v+"tracer",[(0,s.z)(),e,r],i,n.D.spa,g),function(){if(p.emit((a?"":"no-")+"fn-start",[(0,s.z)(),i,a],r),a)try{return t.apply(this,arguments)}catch(e){throw p.emit("fn-err",[arguments,this,"string"==typeof e?new Error(e):e],r),e}finally{p.emit("fn-end",[(0,s.z)()],r)}}}};function x(e,t,r,i){return function(){return(0,o.p)(l.xS,["API/"+t+"/called"],void 0,n.D.metrics,g),i&&(0,o.p)(e+t,[(0,s.z)(),...arguments],r?null:this,i,g),r?void 0:this}}function A(){r.e(439).then(r.bind(r,7438)).then((t=>{let{setAPI:r}=t;r(e),(0,c.L)(e,"api")})).catch((()=>(0,f.Z)("Downloading runtime APIs failed...")))}return["actionText","setName","setAttribute","save","ignore","onEnd","getContext","end","get"].forEach((e=>{w[e]=x(v,e,void 0,n.D.spa)})),h.noticeError=function(e,t){"string"==typeof e&&(e=new Error(e)),(0,o.p)(l.xS,["API/noticeError/called"],void 0,n.D.metrics,g),(0,o.p)("err",[e,(0,s.z)(),!1,t],void 0,n.D.jserrors,g)},d.il?(0,u.b)((()=>A()),!0):A(),h}(e,v);return(0,h.Qy)(e,T,"api"),(0,h.Qy)(e,A,"exposed"),(0,h.EZ)("activatedFeatures",p.T),T}},3325:(e,t,r)=>{r.d(t,{D:()=>n,p:()=>i});const n={ajax:"ajax",jserrors:"jserrors",metrics:"metrics",pageAction:"page_action",pageViewEvent:"page_view_event",pageViewTiming:"page_view_timing",sessionReplay:"session_replay",sessionTrace:"session_trace",spa:"spa"},i={[n.pageViewEvent]:1,[n.pageViewTiming]:2,[n.metrics]:3,[n.jserrors]:4,[n.ajax]:5,[n.sessionTrace]:6,[n.pageAction]:7,[n.spa]:8,[n.sessionReplay]:9}}},n={};function i(e){var t=n[e];if(void 0!==t)return t.exports;var o=n[e]={exports:{}};return r[e](o,o.exports,i),o.exports}i.m=r,i.d=(e,t)=>{for(var r in t)i.o(t,r)&&!i.o(e,r)&&Object.defineProperty(e,r,{enumerable:!0,get:t[r]})},i.f={},i.e=e=>Promise.all(Object.keys(i.f).reduce(((t,r)=>(i.f[r](e,t),t)),[])),i.u=e=>(({78:"page_action-aggregate",147:"metrics-aggregate",242:"session-manager",317:"jserrors-aggregate",348:"page_view_timing-aggregate",412:"lazy-feature-loader",439:"async-api",538:"recorder",590:"session_replay-aggregate",675:"compressor",733:"session_trace-aggregate",786:"page_view_event-aggregate",873:"spa-aggregate",898:"ajax-aggregate"}[e]||e)+"."+{78:"ac76d497",147:"3dc53903",148:"1a20d5fe",242:"2a64278a",317:"49e41428",348:"bd6de33a",412:"2f55ce66",439:"30bd804e",538:"1b18459f",590:"cf0efb30",675:"ae9f91a8",733:"83105561",786:"06482edd",860:"03a8b7a5",873:"e6b09d52",898:"998ef92b"}[e]+"-1.236.0.min.js"),i.o=(e,t)=>Object.prototype.hasOwnProperty.call(e,t),e={},t="NRBA:",i.l=(r,n,o,a)=>{if(e[r])e[r].push(n);else{var s,c;if(void 0!==o)for(var u=document.getElementsByTagName("script"),d=0;d {s.onerror=s.onload=null,clearTimeout(h);var i=e[r];if(delete e[r],s.parentNode&&s.parentNode.removeChild(s),i&&i.forEach((e=>e(n))),t)return t(n)},h=setTimeout(l.bind(null,void 0,{type:"timeout",target:s}),12e4);s.onerror=l.bind(null,s.onerror),s.onload=l.bind(null,s.onload),c&&document.head.appendChild(s)}},i.r=e=>{"undefined"!=typeof Symbol&&Symbol.toStringTag&&Object.defineProperty(e,Symbol.toStringTag,{value:"Module"}),Object.defineProperty(e,"__esModule",{value:!0})},i.j=364,i.p="https://js-agent.newrelic.com/",(()=>{var e={364:0,953:0};i.f.j=(t,r)=>{var n=i.o(e,t)?e[t]:void 0;if(0!==n)if(n)r.push(n[2]);else{var o=new Promise(((r,i)=>n=e[t]=[r,i]));r.push(n[2]=o);var a=i.p+i.u(t),s=new Error;i.l(a,(r=>{if(i.o(e,t)&&(0!==(n=e[t])&&(e[t]=void 0),n)){var o=r&&("load"===r.type?"missing":r.type),a=r&&r.target&&r.target.src;s.message="Loading chunk "+t+" failed.\n("+o+": "+a+")",s.name="ChunkLoadError",s.type=o,s.request=a,n[1](s)}}),"chunk-"+t,t)}};var t=(t,r)=>{var n,o,[a,s,c]=r,u=0;if(a.some((t=>0!==e[t]))){for(n in s)i.o(s,n)&&(i.m[n]=s[n]);if(c)c(i)}for(t&&t(r);u {i.r(o);var e=i(3325),t=i(5763);const r=Object.values(e.D);function n(e){const n={};return r.forEach((r=>{n[r]=function(e,r){return!1!==(0,t.Mt)(r,"".concat(e,".enabled"))}(r,e)})),n}var a=i(9144);var s=i(5546),c=i(385),u=i(8e3),d=i(5938),f=i(3960),l=i(50);class h extends d.W{constructor(e,t,r){let n=!(arguments.length>3&&void 0!==arguments[3])||arguments[3];super(e,t,r),this.auto=n,this.abortHandler,this.featAggregate,this.onAggregateImported,n&&(0,u.R)(e,r)}importAggregator(){let e=arguments.length>0&&void 0!==arguments[0]?arguments[0]:{};if(this.featAggregate||!this.auto)return;const r=c.il&&!0===(0,t.Mt)(this.agentIdentifier,"privacy.cookies_enabled");let n;this.onAggregateImported=new Promise((e=>{n=e}));const o=async()=>{let t;try{if(r){const{setupAgentSession:e}=await Promise.all([i.e(860),i.e(242)]).then(i.bind(i,3228));t=e(this.agentIdentifier)}}catch(e){(0,l.Z)("A problem occurred when starting up session manager. This page will not start or extend any session.",e)}try{if(!this.shouldImportAgg(this.featureName,t))return void(0,u.L)(this.agentIdentifier,this.featureName);const{lazyFeatureLoader:r}=await i.e(412).then(i.bind(i,8582)),{Aggregate:o}=await r(this.featureName,"aggregate");this.featAggregate=new o(this.agentIdentifier,this.aggregator,e),n(!0)}catch(e){(0,l.Z)("Downloading and initializing ".concat(this.featureName," failed..."),e),this.abortHandler?.(),n(!1)}};c.il?(0,f.b)((()=>o()),!0):o()}shouldImportAgg(r,n){return r!==e.D.sessionReplay||!1!==(0,t.Mt)(this.agentIdentifier,"session_trace.enabled")&&(!!n?.isNew||!!n?.state.sessionReplay)}}var g=i(7633),p=i(7894);class m extends h{static featureName=g.t9;constructor(r,n){let i=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];if(super(r,n,g.t9,i),("undefined"==typeof PerformanceNavigationTiming||c.Tt)&&"undefined"!=typeof PerformanceTiming){const n=(0,t.OP)(r);n[g.Dz]=Math.max(Date.now()-n.offset,0),(0,f.K)((()=>n[g.qw]=Math.max((0,p.z)()-n[g.Dz],0))),(0,f.b)((()=>{const t=(0,p.z)();n[g.OJ]=Math.max(t-n[g.Dz],0),(0,s.p)("timing",["load",t],void 0,e.D.pageViewTiming,this.ee)}))}this.importAggregator()}}var v=i(1117),b=i(1284);class y extends v.w{constructor(e){super(e),this.aggregatedData={}}store(e,t,r,n,i){var o=this.getBucket(e,t,r,i);return o.metrics=function(e,t){t||(t={count:0});return t.count+=1,(0,b.D)(e,(function(e,r){t[e]=w(r,t[e])})),t}(n,o.metrics),o}merge(e,t,r,n,i){var o=this.getBucket(e,t,n,i);if(o.metrics){var a=o.metrics;a.count+=r.count,(0,b.D)(r,(function(e,t){if("count"!==e){var n=a[e],i=r[e];i&&!i.c?a[e]=w(i.t,n):a[e]=function(e,t){if(!t)return e;t.c||(t=x(t.t));return t.min=Math.min(e.min,t.min),t.max=Math.max(e.max,t.max),t.t+=e.t,t.sos+=e.sos,t.c+=e.c,t}(i,a[e])}}))}else o.metrics=r}storeMetric(e,t,r,n){var i=this.getBucket(e,t,r);return i.stats=w(n,i.stats),i}getBucket(e,t,r,n){this.aggregatedData[e]||(this.aggregatedData[e]={});var i=this.aggregatedData[e][t];return i||(i=this.aggregatedData[e][t]={params:r||{}},n&&(i.custom=n)),i}get(e,t){return t?this.aggregatedData[e]&&this.aggregatedData[e][t]:this.aggregatedData[e]}take(e){for(var t={},r="",n=!1,i=0;i t.max&&(t.max=e),e 2&&void 0!==arguments[2])||arguments[2];super(e,r,j.t,n),c.il&&((0,t.OP)(e).initHidden=Boolean("hidden"===document.visibilityState),(0,N.N)((()=>(0,s.p)("docHidden",[(0,p.z)()],void 0,j.t,this.ee)),!0),(0,O.bP)("pagehide",(()=>(0,s.p)("winPagehide",[(0,p.z)()],void 0,j.t,this.ee))),this.importAggregator())}}var P=i(3081);class C extends h{static featureName=P.t9;constructor(e,t){let r=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];super(e,t,P.t9,r),this.importAggregator()}}var R,I=i(2210),k=i(1214),H=i(2177),L={};try{R=localStorage.getItem("__nr_flags").split(","),console&&"function"==typeof console.log&&(L.console=!0,-1!==R.indexOf("dev")&&(L.dev=!0),-1!==R.indexOf("nr_dev")&&(L.nrDev=!0))}catch(e){}function z(e){try{L.console&&z(e)}catch(e){}}L.nrDev&&H.ee.on("internal-error",(function(e){z(e.stack)})),L.dev&&H.ee.on("fn-err",(function(e,t,r){z(r.stack)})),L.dev&&(z("NR AGENT IN DEVELOPMENT MODE"),z("flags: "+(0,b.D)(L,(function(e,t){return e})).join(", ")));var M=i(6660);class B extends h{static featureName=M.t;constructor(r,n){let i=!(arguments.length>2&&void 0!==arguments[2])||arguments[2];super(r,n,M.t,i),this.skipNext=0;try{this.removeOnAbort=new AbortController}catch(e){}const o=this;o.ee.on("fn-start",(function(e,t,r){o.abortHandler&&(o.skipNext+=1)})),o.ee.on("fn-err",(function(t,r,n){o.abortHandler&&!n[M.A]&&((0,I.X)(n,M.A,(function(){return!0})),this.thrown=!0,(0,s.p)("err",[n,(0,p.z)()],void 0,e.D.jserrors,o.ee))})),o.ee.on("fn-end",(function(){o.abortHandler&&!this.thrown&&o.skipNext>0&&(o.skipNext-=1)})),o.ee.on("internal-error",(function(t){(0,s.p)("ierr",[t,(0,p.z)(),!0],void 0,e.D.jserrors,o.ee)})),this.origOnerror=c._A.onerror,c._A.onerror=this.onerrorHandler.bind(this),c._A.addEventListener("unhandledrejection",(t=>{const r=function(e){let t="Unhandled Promise Rejection: ";if(e instanceof Error)try{return e.message=t+e.message,e}catch(t){return e}if(void 0===e)return new Error(t);try{return new Error(t+(0,D.P)(e))}catch(e){return new Error(t)}}(t.reason);(0,s.p)("err",[r,(0,p.z)(),!1,{unhandledPromiseRejection:1}],void 0,e.D.jserrors,this.ee)}),(0,O.m$)(!1,this.removeOnAbort?.signal)),(0,k.gy)(this.ee),(0,k.BV)(this.ee),(0,k.em)(this.ee),(0,t.OP)(r).xhrWrappable&&(0,k.Kf)(this.ee),this.abortHandler=this.#e,this.importAggregator()}#e(){this.removeOnAbort?.abort(),this.abortHandler=void 0}onerrorHandler(t,r,n,i,o){"function"==typeof this.origOnerror&&this.origOnerror(...arguments);try{this.skipNext?this.skipNext-=1:(0,s.p)("err",[o||new F(t,r,n),(0,p.z)()],void 0,e.D.jserrors,this.ee)}catch(t){try{(0,s.p)("ierr",[t,(0,p.z)(),!0],void 0,e.D.jserrors,this.ee)}catch(e){}}return!1}}function F(e,t,r){this.message=e||"Uncaught error with no additional information",this.sourceURL=t,this.line=r}let U=1;const q="nr@id";function G(e){const t=typeof e;return!e||"object"!==t&&"function"!==t?-1:e===c._A?0:(0,I.X)(e,q,(function(){return U++}))}function V(e){if("string"==typeof e&&e.length)return e.length;if("object"==typeof e){if("undefined"!=typeof ArrayBuffer&&e instanceof ArrayBuffer&&e.byteLength)return e.byteLength;if("undefined"!=typeof Blob&&e instanceof Blob&&e.size)return e.size;if(!("undefined"!=typeof FormData&&e instanceof FormData))try{return(0,D.P)(e).length}catch(e){return}}}var X=i(7243);class W{constructor(e){this.agentIdentifier=e,this.generateTracePayload=this.generateTracePayload.bind(this),this.shouldGenerateTrace=this.shouldGenerateTrace.bind(this)}generateTracePayload(e){if(!this.shouldGenerateTrace(e))return null;var r=(0,t.DL)(this.agentIdentifier);if(!r)return null;var n=(r.accountID||"").toString()||null,i=(r.agentID||"").toString()||null,o=(r.trustKey||"").toString()||null;if(!n||!i)return null;var a=(0,_.M)(),s=(0,_.Ht)(),c=Date.now(),u={spanId:a,traceId:s,timestamp:c};return(e.sameOrigin||this.isAllowedOrigin(e)&&this.useTraceContextHeadersForCors())&&(u.traceContextParentHeader=this.generateTraceContextParentHeader(a,s),u.traceContextStateHeader=this.generateTraceContextStateHeader(a,c,n,i,o)),(e.sameOrigin&&!this.excludeNewrelicHeader()||!e.sameOrigin&&this.isAllowedOrigin(e)&&this.useNewrelicHeaderForCors())&&(u.newrelicHeader=this.generateTraceHeader(a,s,c,n,i,o)),u}generateTraceContextParentHeader(e,t){return"00-"+t+"-"+e+"-01"}generateTraceContextStateHeader(e,t,r,n,i){return i+"@nr=0-1-"+r+"-"+n+"-"+e+"----"+t}generateTraceHeader(e,t,r,n,i,o){if(!("function"==typeof c._A?.btoa))return null;var a={v:[0,1],d:{ty:"Browser",ac:n,ap:i,id:e,tr:t,ti:r}};return o&&n!==o&&(a.d.tk=o),btoa((0,D.P)(a))}shouldGenerateTrace(e){return this.isDtEnabled()&&this.isAllowedOrigin(e)}isAllowedOrigin(e){var r=!1,n={};if((0,t.Mt)(this.agentIdentifier,"distributed_tracing")&&(n=(0,t.P_)(this.agentIdentifier).distributed_tracing),e.sameOrigin)r=!0;else if(n.allowed_origins instanceof Array)for(var i=0;i 2&&void 0!==arguments[2])||arguments[2];super(r,n,Z.t,i),(0,t.OP)(r).xhrWrappable&&(this.dt=new W(r),this.handler=(e,t,r,n)=>(0,s.p)(e,t,r,n,this.ee),(0,k.u5)(this.ee),(0,k.Kf)(this.ee),function(r,n,i,o){function a(e){var t=this;t.totalCbs=0,t.called=0,t.cbTime=0,t.end=E,t.ended=!1,t.xhrGuids={},t.lastSize=null,t.loadCaptureCalled=!1,t.params=this.params||{},t.metrics=this.metrics||{},e.addEventListener("load",(function(r){_(t,e)}),(0,O.m$)(!1)),c.IF||e.addEventListener("progress",(function(e){t.lastSize=e.loaded}),(0,O.m$)(!1))}function s(e){this.params={method:e[0]},T(this,e[1]),this.metrics={}}function u(e,n){var i=(0,t.DL)(r);i.xpid&&this.sameOrigin&&n.setRequestHeader("X-NewRelic-ID",i.xpid);var a=o.generateTracePayload(this.parsedOrigin);if(a){var s=!1;a.newrelicHeader&&(n.setRequestHeader("newrelic",a.newrelicHeader),s=!0),a.traceContextParentHeader&&(n.setRequestHeader("traceparent",a.traceContextParentHeader),a.traceContextStateHeader&&n.setRequestHeader("tracestate",a.traceContextStateHeader),s=!0),s&&(this.dt=a)}}function d(e,t){var r=this.metrics,i=e[0],o=this;if(r&&i){var a=V(i);a&&(r.txSize=a)}this.startTime=(0,p.z)(),this.listener=function(e){try{"abort"!==e.type||o.loadCaptureCalled||(o.params.aborted=!0),("load"!==e.type||o.called===o.totalCbs&&(o.onloadCalled||"function"!=typeof t.onload)&&"function"==typeof o.end)&&o.end(t)}catch(e){try{n.emit("internal-error",[e])}catch(e){}}};for(var s=0;s 1?e[1]=i:e.push(i)}else e[0]&&e[0].headers&&s(e[0].headers,n)&&(this.dt=n);function s(e,t){var r=!1;return t.newrelicHeader&&(e.set("newrelic",t.newrelicHeader),r=!0),t.traceContextParentHeader&&(e.set("traceparent",t.traceContextParentHeader),t.traceContextStateHeader&&e.set("tracestate",t.traceContextStateHeader),r=!0),r}}function x(e,t){this.params={},this.metrics={},this.startTime=(0,p.z)(),this.dt=t,e.length>=1&&(this.target=e[0]),e.length>=2&&(this.opts=e[1]);var r,n=this.opts||{},i=this.target;"string"==typeof i?r=i:"object"==typeof i&&i instanceof Y?r=i.url:c._A?.URL&&"object"==typeof i&&i instanceof URL&&(r=i.href),T(this,r);var o=(""+(i&&i instanceof Y&&i.method||n.method||"GET")).toUpperCase();this.params.method=o,this.txSize=V(n.body)||0}function A(t,r){var n;this.endTime=(0,p.z)(),this.params||(this.params={}),this.params.status=r?r.status:0,"string"==typeof this.rxSize&&this.rxSize.length>0&&(n=+this.rxSize);var o={txSize:this.txSize,rxSize:n,duration:(0,p.z)()-this.startTime};i("xhr",[this.params,o,this.startTime,this.endTime,"fetch"],this,e.D.ajax)}function E(t){var r=this.params,n=this.metrics;if(!this.ended){this.ended=!0;for(var o=0;o 2&&void 0!==arguments[2])||arguments[2];super(e,t,we.t,r),this.importAggregator()}}new class{constructor(e){let t=arguments.length>1&&void 0!==arguments[1]?arguments[1]:(0,_.ky)(16);c._A?(this.agentIdentifier=t,this.sharedAggregator=new y({agentIdentifier:this.agentIdentifier}),this.features={},this.desiredFeatures=new Set(e.features||[]),this.desiredFeatures.add(m),Object.assign(this,(0,a.j)(this.agentIdentifier,e,e.loaderType||"agent")),this.start()):(0,l.Z)("Failed to initial the agent. Could not determine the runtime environment.")}get config(){return{info:(0,t.C5)(this.agentIdentifier),init:(0,t.P_)(this.agentIdentifier),loader_config:(0,t.DL)(this.agentIdentifier),runtime:(0,t.OP)(this.agentIdentifier)}}start(){const t="features";try{const r=n(this.agentIdentifier),i=[...this.desiredFeatures];i.sort(((t,r)=>e.p[t.featureName]-e.p[r.featureName])),i.forEach((t=>{if(r[t.featureName]||t.featureName===e.D.pageViewEvent){const n=function(t){switch(t){case e.D.ajax:return[e.D.jserrors];case e.D.sessionTrace:return[e.D.ajax,e.D.pageViewEvent];case e.D.sessionReplay:return[e.D.sessionTrace];case e.D.pageViewTiming:return[e.D.pageViewEvent];default:return[]}}(t.featureName);n.every((e=>r[e]))||(0,l.Z)("".concat(t.featureName," is enabled but one or more dependent features has been disabled (").concat((0,D.P)(n),"). This may cause unintended consequences or missing data...")),this.features[t.featureName]=new t(this.agentIdentifier,this.sharedAggregator)}})),(0,T.Qy)(this.agentIdentifier,this.features,t)}catch(e){(0,l.Z)("Failed to initialize all enabled instrument classes (agent aborted) -",e);for(const e in this.features)this.features[e].abortHandler?.();const r=(0,T.fP)();return delete r.initializedAgents[this.agentIdentifier]?.api,delete r.initializedAgents[this.agentIdentifier]?.[t],delete this.sharedAggregator,r.ee?.abort(),delete r.ee?.get(this.agentIdentifier),!1}}}({features:[J,m,S,class extends h{static featureName=oe;constructor(t,r){if(super(t,r,oe,!(arguments.length>2&&void 0!==arguments[2])||arguments[2]),!c.il)return;const n=this.ee;let i;(0,k.QU)(n),this.eventsEE=(0,k.em)(n),this.eventsEE.on(se,(function(e,t){this.bstStart=(0,p.z)()})),this.eventsEE.on(ae,(function(t,r){(0,s.p)("bst",[t[0],r,this.bstStart,(0,p.z)()],void 0,e.D.sessionTrace,n)})),n.on(ce+ne,(function(e){this.time=(0,p.z)(),this.startPath=location.pathname+location.hash})),n.on(ce+ie,(function(t){(0,s.p)("bstHist",[location.pathname+location.hash,this.startPath,this.time],void 0,e.D.sessionTrace,n)}));try{i=new PerformanceObserver((t=>{const r=t.getEntries();(0,s.p)(te,[r],void 0,e.D.sessionTrace,n)})),i.observe({type:re,buffered:!0})}catch(e){}this.importAggregator({resourceObserver:i})}},C,xe,B,class extends h{static featureName=de;constructor(e,r){if(super(e,r,de,!(arguments.length>2&&void 0!==arguments[2])||arguments[2]),!c.il)return;if(!(0,t.OP)(e).xhrWrappable)return;try{this.removeOnAbort=new AbortController}catch(e){}let n,i=0;const o=this.ee.get("tracer"),a=(0,k._L)(this.ee),s=(0,k.Lg)(this.ee),u=(0,k.BV)(this.ee),d=(0,k.Kf)(this.ee),f=this.ee.get("events"),l=(0,k.u5)(this.ee),h=(0,k.QU)(this.ee),g=(0,k.Gm)(this.ee);function m(e,t){h.emit("newURL",[""+window.location,t])}function v(){i++,n=window.location.hash,this[ve]=(0,p.z)()}function b(){i--,window.location.hash!==n&&m(0,!0);var e=(0,p.z)();this[pe]=~~this[pe]+e-this[ve],this[ye]=e}function y(e,t){e.on(t,(function(){this[t]=(0,p.z)()}))}this.ee.on(ve,v),s.on(be,v),a.on(be,v),this.ee.on(ye,b),s.on(ge,b),a.on(ge,b),this.ee.buffer([ve,ye,"xhr-resolved"],this.featureName),f.buffer([ve],this.featureName),u.buffer(["setTimeout"+le,"clearTimeout"+fe,ve],this.featureName),d.buffer([ve,"new-xhr","send-xhr"+fe],this.featureName),l.buffer([me+fe,me+"-done",me+he+fe,me+he+le],this.featureName),h.buffer(["newURL"],this.featureName),g.buffer([ve],this.featureName),s.buffer(["propagate",be,ge,"executor-err","resolve"+fe],this.featureName),o.buffer([ve,"no-"+ve],this.featureName),a.buffer(["new-jsonp","cb-start","jsonp-error","jsonp-end"],this.featureName),y(l,me+fe),y(l,me+"-done"),y(a,"new-jsonp"),y(a,"jsonp-end"),y(a,"cb-start"),h.on("pushState-end",m),h.on("replaceState-end",m),window.addEventListener("hashchange",m,(0,O.m$)(!0,this.removeOnAbort?.signal)),window.addEventListener("load",m,(0,O.m$)(!0,this.removeOnAbort?.signal)),window.addEventListener("popstate",(function(){m(0,i>1)}),(0,O.m$)(!0,this.removeOnAbort?.signal)),this.abortHandler=this.#e,this.importAggregator()}#e(){this.removeOnAbort?.abort(),this.abortHandler=void 0}}],loaderType:"spa"})})(),window.NRBA=o})(); window.jQuery || document.write(' ') CKEDITOR_BASEPATH='https://f1000research.com/js/vendor/ckeditor/' window.reactTheme = 'research'; window.MathJax = { CommonHTML: { linebreaks: { automatic: true } }, 'HTML-CSS': { linebreaks: { automatic: true } }, SVG: { linebreaks: { automatic: true } }, AuthorInit: function() { MathJax.Hub.Register.MessageHook('End Process', function () { let timeout = false; // holder for timeout id const delay = 250; // delay after event is "complete" to run callback const reflowMath = function() { const dispFormulas = document.querySelectorAll('.disp-formula.panel'); if (!dispFormulas) { return; } for (const dispFormula of dispFormulas) { const child = dispFormula.querySelector('.MathJax_Preview').nextSibling.firstChild; const isMultiline = MathJax.Hub.getAllJax(dispFormula)[0].root.isMultiline; if (dispFormula.offsetWidth < child.offsetWidth || isMultiline) { MathJax.Hub.Queue(['Rerender', MathJax.Hub, dispFormula]); } } }; window.addEventListener('resize', function() { clearTimeout(timeout); // clear the timeout timeout = setTimeout(reflowMath, delay); // start timing for event "completion" }); }); }, }; if (window.location.hash == '#_=_'){ window.location = window.location.href.split('#')[0] } !function(f,b,e,v,n,t,s){if(f.fbq)return;n=f.fbq=function() {n.callMethod? n.callMethod.apply(n,arguments):n.queue.push(arguments)} ;if(!f._fbq)f._fbq=n; n.push=n;n.loaded=!0;n.version='2.0';n.queue=[];t=b.createElement(e);t.async=!0; t.src=v;s=b.getElementsByTagName(e)[0];s.parentNode.insertBefore(t,s)}(window, document,'script','https://connect.facebook.net/en_US/fbevents.js'); fbq('init', '1641728616063202'); fbq('track', "PixelInitialized", {}); (function(h,o,t,j,a,r){ h.hj=h.hj||function(){(h.hj.q=h.hj.q||[]).push(arguments)}; h._hjSettings={hjid:2318163,hjsv:6}; a=o.getElementsByTagName('head')[0]; r=o.createElement('script');r.async=1; r.src=t+h._hjSettings.hjid+j+h._hjSettings.hjsv; a.appendChild(r); })(window,document,'https://static.hotjar.com/c/hotjar-','.js?sv='); search file_upload Submit your research search menu close search Browse Gateways & Collections How to Publish Submit your Research My Submissions Article Guidelines Article Guidelines (New Versions) Open Data, Software and Code Guidelines Open Data and Accessible Source Materials Guidelines (HSS) Open Data, Software and Code Guidelines (PSE) Prepublication Checks Production Process Posters and Slides Guidelines Document Guidelines Article Processing Charges Peer Review Finding Article Reviewers About How it Works For Reviewers Our Advisors Policies Glossary FAQs For Developers Newsroom Contact My Research Submissions Content and Tracking Alerts My Details Sign In file_upload Submit your research { "@context": "https://schema.org", "@type": "ScholarlyArticle", "mainEntityOfPage": { "@type": "WebPage", "@id": "https://f1000research.com/articles/12-1232" }, "headline": "Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus...", "datePublished": "2023-09-27T13:24:30", "dateModified": "2025-08-21T14:05:35", "author": [ { "@type": "Person", "name": "Lorato Modise" }, { "@type": "Person", "name": "Nomathamsanqa Sithebe" }, { "@type": "Person", "name": "Hazel Mufhandu" } ], "publisher": { "@type": "Organization", "name": "F1000Research", "logo": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 480, "width": 60 } }, "image": { "@type": "ImageObject", "url": "https://f1000research.com/img/AMP/F1000Research_image.png", "height": 1200, "width": 150 }, "description": " Background Co-infection of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) has an impact on high HBV replication and progression to liver cancer. These may lead to cross-resistance of drugs due to natural mutations or therapeutic pressure. These require continuous monitoring of HBV variants for better diagnosis and treatment strategies. Methods Convenience sampling was used to collect fifty archival sera from Inkosi Albert Luthuli Central Hospital. Sera were subjected to HBsAg screening using ELISA, DNA extraction, PCR amplification, Sanger sequencing, genotype prediction and mutation analysis. Results Of the 50 samples, 86% (43/50) were HBsAg positive; 82% (41/50) PCR positive with 92% (38/41) sequenced and only 26 sequences were subjected to molecular characterization. The HBV sequences showed similarity to genotype A (73% [19/26]), genotypes G (5% [3/26]) and genotype C (15% [4/26]). Prevalence of the mutations in the surface region was (47% [18/38]); including diagnostic failure (K122R and T143S) and immune escape mutations (P127T, G145R, S207N, Y200T, E164D, Y206H and L209V). The mutations in the RT were at (36% [14/38]) with drug resistance mutations (DRM) at (50% [7/14]). Mutations showed resistance to lamivudine (LMV) at (35% [5/14]), telbivudine (LdT) at (29% [4/14]), (14% [2/14]) for entecavir (ETV) and (21% [3/14]) for adefovir (ADV). One sample had a combination of L180M, M204V, S202K, and M250I mutations. Conclusions Our findings highlight the prevalence of HBV genotype A in HIV-infected patients in South Africa. The study provides evidence of mutations linked to immune evasion and drug resistance; this infers that these mutations may have clinical implications for the diagnosis and treatment of HBV in HBV/HIV co-infected individuals. Further in vitro studies must be conducted to explore the impact of the identified mutation on the surface protein expression during diagnosis; phenotype impact of the mutant virus towards the antiviral drugs. " } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/12-1232/v4", "name": "Investigation of mutations in the partial sequences of the surface..." } } ] } Home Browse Investigation of mutations in the partial sequences of the surface... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Modise L, Sithebe N and Mufhandu H. Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.12688/f1000research.132498.4 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Research Article Revised Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] Previously titled: Investigation of hepatitis B virus mutations associated with immune escape and drug resistance in human immunodeficiency virus-infected patients Lorato Modise https://orcid.org/0000-0001-7008-157X 1 , Nomathamsanqa Sithebe 1 , Hazel Mufhandu https://orcid.org/0000-0002-1551-3314 1 Lorato Modise https://orcid.org/0000-0001-7008-157X 1 , Nomathamsanqa Sithebe 1 , Hazel Mufhandu https://orcid.org/0000-0002-1551-3314 1 PUBLISHED 21 Aug 2025 Author details Author details 1 Biological Sciences, North West University, Mahikeng, North West, 2735, South Africa Lorato Modise Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Validation, Writing – Original Draft Preparation, Writing – Review & Editing Nomathamsanqa Sithebe Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Hazel Mufhandu Roles: Data Curation, Formal Analysis, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Pathogens gateway. This article is included in the Genomics and Genetics gateway. This article is included in the Antimicrobial Resistance collection. Abstract Background Co-infection of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) has an impact on high HBV replication and progression to liver cancer. These may lead to cross-resistance of drugs due to natural mutations or therapeutic pressure. These require continuous monitoring of HBV variants for better diagnosis and treatment strategies. Methods Convenience sampling was used to collect fifty archival sera from Inkosi Albert Luthuli Central Hospital. Sera were subjected to HBsAg screening using ELISA, DNA extraction, PCR amplification, Sanger sequencing, genotype prediction and mutation analysis. Results Of the 50 samples, 86% (43/50) were HBsAg positive; 82% (41/50) PCR positive with 92% (38/41) sequenced and only 26 sequences were subjected to molecular characterization. The HBV sequences showed similarity to genotype A (73% [19/26]), genotypes G (5% [3/26]) and genotype C (15% [4/26]). Prevalence of the mutations in the surface region was (47% [18/38]); including diagnostic failure (K122R and T143S) and immune escape mutations (P127T, G145R, S207N, Y200T, E164D, Y206H and L209V). The mutations in the RT were at (36% [14/38]) with drug resistance mutations (DRM) at (50% [7/14]). Mutations showed resistance to lamivudine (LMV) at (35% [5/14]), telbivudine (LdT) at (29% [4/14]), (14% [2/14]) for entecavir (ETV) and (21% [3/14]) for adefovir (ADV). One sample had a combination of L180M, M204V, S202K, and M250I mutations. Conclusions Our findings highlight the prevalence of HBV genotype A in HIV-infected patients in South Africa. The study provides evidence of mutations linked to immune evasion and drug resistance; this infers that these mutations may have clinical implications for the diagnosis and treatment of HBV in HBV/HIV co-infected individuals. Further in vitro studies must be conducted to explore the impact of the identified mutation on the surface protein expression during diagnosis; phenotype impact of the mutant virus towards the antiviral drugs. READ ALL READ LESS Keywords Hepatitis B virus, HIV, Drug resistance, Vaccine escape, Mutation, Co-infection, Corresponding Author(s) Lorato Modise ( [email protected] ) Hazel Mufhandu ( [email protected] ) Close Corresponding authors: Lorato Modise, Hazel Mufhandu Competing interests: No competing interests were disclosed. Grant information: This work was supported by funds from the North-West University, National Research foundation Innovation bursary (Grant No: SFH150727131319), Health and Welfare Sector Education and Training Authority, South African Centre for Epidemiological Modelling and Analysis and Organisation for Women in Science for the Developing Worlds (Grant No: 3240287283). These funding agencies were not involved and responsible in the study design and writing of the thesis and manuscript. The Authors in this study are solely responsible for the content and not the funding agencies involved. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2025 Modise L et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Modise L, Sithebe N and Mufhandu H. Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.12688/f1000research.132498.4 ) First published: 27 Sep 2023, 12 :1232 ( https://doi.org/10.12688/f1000research.132498.1 ) Latest published: 21 Aug 2025, 12 :1232 ( https://doi.org/10.12688/f1000research.132498.4 ) Revised Amendments from Version 3 The title of the study has been revised to make it more descriptive and specific clearly stating that the investigation of the study is on the mutations in the overlapping partial sequences of the Surface (S) and Polymerase (pol) genes of hepatitis B virus which are associated with immune escape and drug resistance in HIV-Infected patients. Under the introduction, the HBV genome features are briefly characterized; context is provided on the surface and polymerase genes, the proteins they encode for including surface antigen (containing the major hydrophilic region) and polymerase. Description is briefly given on the roles of the encoded proteins and on the mutations in the genes and their association with causing viral immune escape and drug resistance. An update is done under table 1, since the N=50 mentioned and removed as see at (Table 1). I have changed N (study population) to n (sub-sample size of each demographic variable and not for total sample population (50). The same on the abstract. The title of the study has been revised to make it more descriptive and specific clearly stating that the investigation of the study is on the mutations in the overlapping partial sequences of the Surface (S) and Polymerase (pol) genes of hepatitis B virus which are associated with immune escape and drug resistance in HIV-Infected patients. Under the introduction, the HBV genome features are briefly characterized; context is provided on the surface and polymerase genes, the proteins they encode for including surface antigen (containing the major hydrophilic region) and polymerase. Description is briefly given on the roles of the encoded proteins and on the mutations in the genes and their association with causing viral immune escape and drug resistance. An update is done under table 1, since the N=50 mentioned and removed as see at (Table 1). I have changed N (study population) to n (sub-sample size of each demographic variable and not for total sample population (50). The same on the abstract. See the authors' detailed response to the review by Nishi Prabdial-Sing See the authors' detailed response to the review by Anna McNaughton READ REVIEWER RESPONSES Introduction The Orthohepadnavirus genus is a significant group of human viruses, and the HBV is a prototype of the Hepadnaviridae family ( Summers et al. , 1975 ). More than 300 million people worldwide are chronically infected with hepatitis B, which can lead to severe liver disease and hepatocellular carcinoma (HCC), which accounts for more than 1 million annual deaths ( Musyoki et al. , 2015 ). HBV/HIV co-infections in the region of South Africa (SA) are estimated to range from 5% to 23% ( Anastasiou et al ., 2017 ; Kaswa & de Villiers, 2023 ; King & Hagemeister, 2016 ). However, only limited evidence is available for co-infection in KwaZulu-Natal (KZN) province with a hepatitis B surface antigen (HBsAg) seroprevalence of 8.5% reported in HIV-infected people ( Msomi et al ., 2020 ). The management of Hepatitis B include prevention by vaccination and treatment using the antiviral drugs. The introduction of the hepatitis B vaccine into the South African Expanded Program of Immunization (EPI) in 1995 led to a reduction in liver diseases ( Spearman et al ., 2013 ). Treatment for co-infection with HBV/HIV uses drugs that are effective against both HIV and HBV including, a combination of tenofovir/(TDF) and lamivudine (LMV)/emtricitabine (FTC) /efavirenz (EFV) as outlined by Maponga et al . (2020) . However, HBV vaccination and treatment may be threatened by the appearance of mutations on the HBV genome that could cause unfavourable clinical effects, such as vaccine escape and drug resistance. The HBV genome features are characterized by overlapping genes, notably the surface (S) gene within the polymerase (pol) gene. The S gene encodes the surface antigen (HBsAg), contains the major hydrophilic region (MHR) along with ‘α’ determinant (aa124-aa147) region, which is crucial for immune recognition and diagnostic purposes ( Liu et al ., 2017 ). This overlap causes mutations in one gene to affect the other, impacting HBV evolution, replication, antiviral effectiveness, and immune escape. Mutations in the ‘α’ determinant hinder anti-HBs binding and can lead to the emergence of drug resistance mutations (DRM) in the YMDD motif of pol region ( Adesina et al ., 2021 ; Cooreman et al ., 2001 ). Moreover, changes near the YMDD motif contribute to drug resistance in treatments such as lamivudine, adefovir, and entecavir. Several studies have revealed M204V/I in the YMDD motif (amino acid 203±206) among HIV-infected drug-naive and drug-experienced people who received long term LMV therapy ( Mokaya et al ., 2018 ; Selabe et al ., 2007 ; Selabe et al ., 2009 ). Also, previous studies showed drug resistance resulting from a single or combination of the following mutations: V173L, L180M, M204V/I, and A181V/T to lamivudine, rtA181V/T and rtN236T to adefovir, or rtI169T, rtI184G, rtS202G/I and rtM250V to entecavir, in treatment experienced and treatment naïve people ( Colagrossi et al ., 2018 ; Lai et al ., 2003 ; Torresi, 2002 ; Zöllner et al ., 2001 ). Mutations in the pol gene also impact the surface gene with the previously identified DRM in pol leading to the emergence of immune escape mutations in the MHR ( Torresi, 2002 ). The prevalence of HBV has been documented in several studies among HIV positive people in KZN ( Millar et al ., 2023 ; Samsunder et al ., 2019 ). However, investigations on the HBV genotype, mutations associated with immune escape and drug resistance are still scarce in this region, and most studies have focused on reporting seroprevalence. The aim of the study was to describe the prevalence and molecular characterization of HBV mutations associated with immune escape and drug resistance in HIV-infected individuals in Durban, KZN, South Africa. Methods Study design and population We applied a convenience sampling method to collect fifty archival sera in this descriptive exploratory investigation. The sample size was not determined, and we used samples that were already available. Sera were from people who underwent HIV testing at the Inkosi Albert Luthuli Central Hospital (NHLS-IALCH-NHLS) in Durban, KwaZulu-Natal Province, South Africa. Based on the sample size of 50 sera, we used the confidence level of 95% and the margin of error for our study was 14%. The 50 participants included men and women who previously tested positive for HBsAg and HIV. Participants received a written informed consent form, the information on the consent form was given in a language the patient understands, which is english and their native language (Zulu). Demographic data (age, sex, and ethnicity) were also collected. The specimen’s codes were de-linked to keep patients anonymous; with only additional data on the age and gender of the study participants provided. Laboratory testing Hepatitis B surface antigen (HBsAg) assay The Monolisa HBsAg ultra-confirmatory kit was used in according to the manufacturer’s instructions to perform an enzyme-linked immunosorbent assay (ELISA) on samples previously positive for HBsAg to confirm the presence of HBsAg marker (BioRad, Raymond Poincare, Marnes-la-Coquette, France). To identify and measure the presence of HBsAg, excess antibodies (anti-HBs; anti-HBs diluent: neutralization reagent) were used to neutralize 200 μL of undiluted sera specimens. At 450 nm, the optical density (OD) index of the sample was determined and compared to the cut-off value (COV) mean of a negative control. Reactive specimens for HBsAg were defined as those with an index greater than or equal to the COV. DNA extraction of HBV The sera obtained from patients were used to extract HBV deoxyribonucleic acid (DNA) using the QIAamp DNA Mini kit (catalog number: 51304) from (Qiagen, Hilden, Germany) following the manufacturer’s instructions. This technique allows the isolation and purification of total DNA from contaminants, inhibitors, and nucleases in the serum. An aliquot of 200 μL of the serum was added into 1.5 μL Eppendorf tube, to which 20 μL of proteinase K and 200 μL buffer AL (binding buffer mixed with poly [A] carrier RNA) was added. The mixture was pulse-vortex for 15 seconds to allow lysis of the mixture and destroy RNA, followed by a 10-minute incubation at 56 °C. The mixture was then transferred to a QIAamp spin column to allow binding of the DNA and centrifuged for 1 minute at 8 000 rpm. The column was placed into a clean collection tube, then 500 μL of buffer AW1 was added, and it was centrifuged for 1 minute at 8 000 rpm. The solution was aspirated, 500 μL of buffer AW2 was added to purify the DNA, and it was followed by centrifugation for 3 minutes at 14 000 rpm. The QIAamp spin column was placed in a sterile 1.5 μL Eppendorf tube, and 50 μL of elution buffer (provided by the kit as buffer AE) was added directly into the column and incubated at room temperature for 5 minutes to precipitate the DNA. The DNA was eluted by centrifugation at 8 000 rpm for 1 minute and stored at -20 °C until further analyses were performed. The negative control, consisting of nuclease-free water, was included in the extraction procedure to identify contamination. Polymerase chain reaction First round and nested-PCR A nested polymerase chain reaction (PCR) amplification of the overlapping surface/polymerase gene covering nucleotides 256 to 796 from EcoRI site was done as described previously ( Mphahlele, 2008 ) with slight modification. Outer sense strand (forward primer) S1 (5′-CCT GCT GGT GGC TCC AGT TC-3′), and anti-sense strand (reverse primer) S2Na (5′-CCA CAA TTC KTTGAC ATA CTT TCC A-3′) were used. The master mix were prepared using Ampli Taq Gold DNA polymerase (ThermoFisher Scientific, Waltham, Massachusetts, United States ). For each sample the following reagent volumes and concentration of the master mix were prepared as follows: 18.5 μL nuclease-free water, 2.5 μL of 1x PCR buffer with MgCl 2 , 0.5 μL (0.2 mM dNTP mix), 0.5 μL (10 μM) forward primer S1; 0.5 μL (10 μM) reverse S2Na anti-sense primer, 0.125 Taq DNA polymerase. A total of 22.1 μL of master mix was aliquoted into a 0.5 mL thin-walled PCR tube and 3 μL of DNA template was added. The PCR reaction mixtures (25.1 μL) was subjected to amplification of HBV DNA, carried out in an automated touch down thermal cycler CFX96 (Bio-Rad, Raymond Poincare, Marnes-la-Coquette, France). The HBV DNA amplification conditions were initial denaturation at 95 °C for 4 minutes, followed by 40× cycles involving denaturation at 95 °C for 4 minutes, annealing at 58 °C for 30 seconds, elongation at 72 °C for 1 minute, and final extension at 72 °C for 10 minutes. Nested PCR First round PCR product was used as a template for nested PCR. An aliquot of 3 μL of the first round PCR reaction was subjected to a nested PCR, the master mix volume and concentration were prepared as same for the first round PCR. The nested PCR conditions used were the same as first round PCR protocol except the annealing temperature at 55 °C for 45 seconds. Forward primers S6E (5′-GAGAAT TCCGAGGACTGG GGA CCC TG-3′) and reverse primer S7B (5′-CGG GAT CCT TAG GGT TTA AAT GTATAC C-3′) were used during nested PCR. The negative control consisting of nuclease-free water and a positive control were included in the PCR amplification assays. PCR products verification PCR amplification products were verified using 1% agarose gel (ThermoFischer, Waltham, Massachusetts) stained with 0.15 U/μL ethidium bromide (Biorad, California, USA). Aliquot of 10 μL PCR amplicon product was mixed with 2 μL 10x loading buffer (ThermoFischer, Waltham, Massachusetts). The mixtures were run on 1% gel along with a 1 Kb Invitrogen molecular weight maker (ThermoFischer, Waltham, Massachusetts) as a band size reference. The agarose gel was run at 100 V for 45 minutes. The gel was placed inside the ultraviolet (UV) transilluminator (Bio-rad, Hercules, California, United States ) to visualise and image capturing. Sequencing reaction The PCR products and the nested PCR primers S6E and S7B were sent for bi-directional Sanger sequencing at the Inqaba (Inqaba Biotechnological Industry, Pretoria, South Africa). The amplicons were prepared for direct sequencing using the BigDye terminator v3.0 cycle sequencing ready reaction kit (catalog number: 4458687) from (ThermoFischer, Waltham, Massachusetts). Briefly, an aliquot of 50 μL of the 1:1 ratio of sodium acetate: ethanol (NaAc:EtOH) was added to the amplicons solution and centrifuged at 2000 g for 30 minutes. The well plates were inverted and centrifuged at 150 g for 1 minute. Pre-chilled 70% ethanol was added into the wells and then centrifuged at 2000 g for 5 minutes. The pellets were dried at 65 °C for 5 minutes followed by an addition of 10 μL Hi-Di formamide for 5 minutes and loaded into the sequencing machine ABI 3130XL genetic analyser (Applied Biosystems, Foster City, CA). Sequences analysis Nucleotide sequences of HBV chromatograms were viewed and edited by removing unwanted and mixed nucleotides character from the sequences by ChromasPro, version.1. The contiguous sequences were formed by joining overlapping DNA sequences of a gene using BioEdit. The consensus sequences were compared with the GenBank complementary genotype sequences using the basic local alignment search tool (BLAST). Representative sequences belonging to different genotypes were redeemed from GenBank to make comparisons with the study sequences. Multiple sequences alignment was performed with ClustalW within the MEGA software package version 7.0 (TomHall, North Carolina State University). Genotyping and identification of the sequence was done on Geno2pheno ( Geno2pheno, 2023 ). Sequences were uploaded into the website where they were aligned at the sequence position 1 to 3182 (~3.18 kbp) relative to the Hepatitis B virus (taxon:10407) and a boostscan analysis of ≥ 80.0 was considered positive. Mutations analysis The aligned sequences were uploaded into the BioEdit and analysed for nucleotide base and amino acids changes. The sequences were further uploaded into the Geno2pheno to identify DRMs in the reverse transcriptase (RT) of polymerase and mutations in the overlapping S region. Statistical analyses Microsoft Excel and the data science statistical program STATA were used for data analyses (version 15). Excel (.csv.) file was imported into STATA and was used to calculate the frequency of age as numeric values and the frequency of HBsAg and mutations as categorical and numeric variables. The proportions of data reported as medians with interquartile ranges (IQR) for continuous variables (age) as frequencies and percentages for categorical variables (sex, ethnicity and mutations). Ethical approval The study ethics certificate was provided by the North-West University research ethics regulatory committee (ref. NWU-00068-15-A9). Results Baseline demographic data The baseline demographics of the study showed that all 50 HIV positive samples included were female at 64% (n=32/50) and 36% (n=18/50) male as shown in Table 1 . The median age was 33 years [IQR: 18-55] and there was no statistical difference on age between the female and male black Africans at p=0.8. Table 1. Demographics data of the patients by age, sex, HBV and HIV antigens markers. Age in years (median, IQR) Sex HIV p24 antigen HBsAg antigen [%; n/50] 33[9.25,2736.25] Female Male HIV (+) Female HIV (+) Male HBsAg (+) Female HBsAg (+) Male 18-35 59% (n=19) 66% (n=12) 40% (n=20) 14% (n=7) 34% (n=17) 12% (n=6) 36-55 41% (n=13) 33% (n=6) 12% (n=12) 22% (n=11) 24% (n=12) 16% (n=8) Total 64% (n=32) 36% (n=18) 64% (n=32) 36% (n=18) 58% (n=29) 28% (n=14) p-value 0.8 0.1 0.6 Laboratory testing HBsAg assay The confirmatory screening of HBsAg reported a prevalence of 86% (n=43/50) with 12% (n=6/50) being negative and 2% (n=1/50) missing data ( Table 1 ). Majority of the female’s participant were HBsAg positive at 58% (n=29/50) when compared to the males at 28% (n=14/50) as shown in Table 1 . PCR amplification and genotyping The PCR amplification of HBV DNA amplicons was successful in 82% (n=41/50) of the samples and consisting of overlapping surface/polymerase region. PCR amplification could not be obtained for 18% (n=09/50) samples. Only 92% (n=38/41) sequence products could be obtained by Sanger sequencing with only 26 sequences used to perform genotyping and 12 sequences were excluded due to the poor quality of the sequence reads. The genotypes of the sequences were confirmed by depositing nucleotides sequences into the Genotype2pheno database which showed that most of the nucleotide sequences had homology similarity to genotype A at 73% (n=19/26) and 12% (n=3/26) as genotype G and 15% (n=4/26) being genotype C ( Table 2 ). The study sequences with the highest similarity identity to genotype A were: (Q4P7 and Q7P7 to KX520697.1|South Africa; Q9P7 to U87728.1|South Africa; Q27P7 to AY233274.1|South Africa. Reference sequence of AY233277.1|South Africa had between 97%-99% similarity to fifteen sequences (Q5P7, Q8P7, Q10P7, Q13P7, Q18P7, Q19P7, Q20P7, Q21P7, Q22P7, Q23P7, Q24P7, Q26P7, Q27P7, Q29P7 and Q39P7). The sequences Q6P7, Q17P7, and Q42P7 had similarity to genotype G (EU694179.1|South Africa and sequences Q11P7, Q43P7, Q44P7, Q45P7 showed similarity to genotype C AB562444.1|Vietnam ( Table 2 ). Sub-genotypes of the sequences were confirmed by depositing nucleotides sequences into the Genotype2pheno database. The results retrieved from the Geno2Pheno database had a 96.85%-99.0% percentage of similarity to sub-genotype A1 for the genotype A sequences only. Table 2. Genotype identity of the study sequences by comparing with standard representative sequences obtained from GenBank. Sample code Reference accession no 1 Genotype identity Homology similarity (%) Q4P7 KX520697.1|South Africa A 99.46 Q5P7 AY233277.1|South Africa A 98.96 Q6P7 EU694179.1|South Africa G 99.83 Q7P7 KX520697.1|South Africa A 98.76 Q8P7 AY233277.1|South Africa A 98.42 Q9P7 U87728.1|South Africa A 98.98 Q10P7 AY233277.1|South Africa A 97.62 Q11P7 AB562444.1|Vietnam C 99.39 Q13P7 AY233277.1|South Africa A 97.95 Q17P7 EU694179.1|South Africa G 99.32 Q18P7 AY233277.1|South Africa A 98.56 Q19P7 AY233277.1|South Africa A 99.18 Q20P7 AY233277.1|South Africa A 99.78 Q21P7 AY233277.1|South Africa A 99.96 Q22P7 AY233277.1|South Africa A 98.49 Q23P7 AY233277.1|South Africa A 99.28 Q24P7 AY233277.1|South Africa A 99.94 Q26P7 AY233277.1|South Africa A 97.96 Q27P7 AY233274.1|South Africa A 99.08 Q29P7 AY233277.1|South Africa A 99.20 Q39P7 AY233277.1|South Africa A 97.50 Q42P7 EU694179.1|South Africa G 98.82 Q43P7 AB562444.1|Vietnam C 99.28 Q44P7 AB562444.1|Vietnam C 97.18 Q45P7 AB562444.1|Vietnam C 99.42 1 Reference sequences obtained from GenBank (designated by accession numbers). The study sequences are represented by the letters Q, P and are followed by numeric values. Mutations within the surface region The prevalence of mutations in the surface gene was 47% (n=18/38) and mutations were found in the “α”, “β”, “T” and outside the “α” epitope as shown in Table 3 . The most common mutations on the surface region and their frequencies were S207N at 71% (27/38), followed by L216V and A194V at 23%, and the least prevalent being S204R, S117N, T143S, G145R, Y206H, P127T, Y200T, F129T and K122R all at 3% as shown in Table 3 . Table 3. Amino acids substitution in surface, polymerase and their associated functions. Surface region Reverse transcriptase Epitope Mutation Frequency Function Mutation Drugs of resistance “α” epitope K122R 3 Diagnostic failure M129L Compensatory/epistasis to LMV F134L 5 Diagnostic failure M204V LMV, LdT S117N 3 Diagnostic failure L180M LMV, LdT T143S 3 Diagnostic failure V163I LMV, LdT “β” epitope S207N 71 Vaccine escape V173L LMV Y200T 3 Vaccine escape A236T ADV G145R 3 Vaccine escape Diagnostic failure Q125E Compensatory/epistasis to LMV T-helper epitope P127T 3 Vaccine escape Diagnostic failure S202K LMV, LdT, ETV Outside “α” E164D 5 Vaccine escape L217R Compensatory/epistasis to LMV L209V 18 Vaccine escape/ cause RT mutations (M204V/M204I/V173L) V214A Compensatory/epistasis to LMV Y206H 3 Vaccine escape V204I, S169T LMV, ETV L216V 23 Vaccine escape I253Y/M205I ADV A194V 23 Vaccine escape L181T/L80V LMV, P70H 21 Unknown L80V LMV P127L 8 Vaccine escape L180M+M204V+S202K+ M250I LMV+LdT+ADV and EFV T189I 5 Vaccine escape S204R 3 Vaccine escape F129T 3 Unknown Mutations within the polymerase region The prevalence of mutations in the RT of polymerase was reported at 36% (n=14/38). Mutations showed resistance to lamivudine (LMV) at (35% [5/14]), telbivudine (LdT) at (29% [4/14]), (14% [2/14]) for entecavir (ETV) and (21% [3/14]) for adefovir (ADV). Mutations causing resistance to LMV and LdT were M204V, L180M, V163I, and S202K. The S202K mutation causes resistance to ETV in addition to V204I and S169T. The ADV resistance mutations were I253Y, A236T and M250I in addition to complementary resistant mutation (L80I) not shown in the table. Multiple DRMs within a single sample were identified in one sample containing L180M, M204V, S202K and M250I mutations ( Table 3 ). Discussion HBV continues to be endemic in South Africa ( Maepa et al ., 2022 ). However, there are still areas in our country with limited published data on circulating genotypes. Therefore, this study considered to determine the HBV genotype and mutations circulating in HIV-infected people from KZN. The partially overlapping surface/polymerase gene region was successfully amplified in 78% (41/50), confirming the active replication of HBV in HIV co-infected people. Geno2pheno characterized majority of the sequences as HBV genotype A, followed by fewer sequences showing similar homology to genotype C and G. The predominance of HBV genotype A over other genotypes is consistent with previous studies in South Africa ( Bowyer et al ., 1997 ; Kimbi et al ., 2004 ; Kramvis, 2014 ; Kramvis et al ., 2005 ; Kramvis & Kew, 2007 ; Selabe et al ., 2009 ). Genotype A was found to be the most prevalent among the antiretroviral therapy (ART) naive HIV-infected people in South Africa ( Audsley et al ., 2010 ). This suggest that the mutant HBV is common in both naive and experience HIV positive people. The genotype A mutant virus may have a high transmission rate and low response to ART by an individual. The uncommon genotype G showed similarity to a reference sequence from South Africa with HIV co-infection ( Lukhwareni et al ., 2020 ). The occurrence of genotype C may be due to migration into South Africa. The predominance of HBV genotype A in our study indicates a less occurrence of genetic diversity and suggests that these strains are circulating in this geographic location due to the substandard immigration of new strains into the study region ( Kramvis et al ., 2008 ; Kramvis & Kew, 2007 ). A total of 47% variants were observed from all sequences at different locations within the surface region (upstream and downstream of MHR). We identified K122R and T143S which are characterized to be associated with diagnostic failure. Also, we found G145R, P127T, S207N, Y200T, E164D, Y206H and L209V which are immune escape mutations, and the findings correlate to previous studies ( El-Mokhtar et al ., 2020 ; Ireland et al ., 2000 ; Mokaya et al ., 2018 ). Contrary some of the mutations have been previously reported to have dual function as diagnostic failure and immune escape such as the G145R and P127T ( Archampong et al ., 2019 ; Colagrossi et al ., 2018 ; Kuzin et al ., 2012 ; Mokaya et al ., 2018 ; Yan et al ., 2017 ; Yousif et al ., 2014 ). We identified some mutations on the upstream position (aa147 (to aa216) including: T189I, A194V, S207N, Y200T, Y206H, L209V, L216V and S204R. These mutations were previously linked with vaccination escape diagnostic failure ( Colagrossi et al ., 2018 ). The G145R and E164D mutations either occurring alone or in combination have shown to result from a substitution change in the overlapping polymerase region caused by the mutation M204V, M204I, and V173L which cause lamivudine resistance ( Colagrossi et al ., 2018 ; Torresi, 2002 ). The frequency of DRMs in the RT region was 50% (n=7/14). M204V, L180M, V163I, and S202K mutations are linked to LMV and LdT resistance; S202K in addition confer resistance to EFV together with V204I. ADV resistance mutations included the I253Y, A236T, and complementary M250I. A single sample sequence contained a combination of L180M, M204V, S202K, and M250I mutations that showed to be associated with high resistance to LMV, LdT, EFV and ADV drugs. This is not opposing to previous studies on combination of DRMs ( Anastasiou et al ., 2017 ; Mokaya et al ., 2018 ). Contrary, the presence of multiple mutations combination may also induce cross-resistance to other drugs. Both L180M and M204V increased cross-resistance to other drugs and decreased sensitivity to ETV ( Sheldon et al ., 2005 ). On the contrary, these combined L180M + M204V + S202K + M250I and L180M + M204V + V173L + S202K + M250I mutations are reported to increase susceptibility to ADV due to the presence of the secondary mutation V173L which is missing from our combination of DRMs. However, the in vitro functional studies on the combined mutations L180M + M204V + S202K + M250I have not been established but we suggest they could have implication on the treatment of HBV in HIV-coinfected people. The following compensatory mutations S202K, Q125E, L217R, V124A, V204I, and T128A were identified, and other identified DRMs were T189I, Y206H, L216V, and S209N. These mutations have effects on treatment resistance; further research is required to fully comprehend how they affect antiviral drugs. The identification of DRMs in HBV treatment-naive patient increases the possibility that the treatment-naive patient may be infected with a mutant HBV virus that was either naturally occurring resistance mutations or derived from a patient who developed resistance after antiviral treatment ( Belyhun et al ., 2017 ; Tan et al ., 2012 ; Vutien et al ., 2014 ; Zhao et al ., 2016 ). We conclude that additional research is required to identify the mechanism producing the DRMs in HIV-positive patients who have never received HBV therapy. Although they can’t extrapolate on past treatment histories, the DRMs identified in this study provide information on the present status of treatment resistance. Second, the DRMs identified in this study suggest that those with mutations resistance to ARTs may have fewer options for therapy. The study does not support whether dual HBV/HIV therapy was effective on HBV or the effect of HIV treatment on ART-naïve HBV. This does not change how the DRMs sequences results are interpreted, which highlights the need of HBV testing by genotyping in addition to HBV and HCV testing prior to starting ART in HIV patients, as recommended by WHO et al . (2017) . Limitations The findings of the study should be considered, bearing in mind limitations, including the design of a cross-sectional study with a small sample size. Genotyping of the HBV was not based on full genome sequences which could have identified clearly the mixed base clustering of the sequences to the genotypes. The results encourage a larger sample size to provide a true representative of dominant mutations in the study site and other areas, which improves the generalization of the results. There is no availability of the presumptuous treatment history, and it effect on the understanding the impact of HIV treatment on the HBV mutations profile and treatment. Conclusion This study reports on the prevalence of HBV genotype A among HIV-infected patients. Furthermore, it provides evidence on the presence of HBV mutations related to immune escape and drug resistance in people co-infected with HIV in KwaZulu-Natal, South Africa. A thorough-synchronizes diagnosis and antiviral therapy for HBV patients with HIV co-infection should include proper HBV diagnosis by resistance testing to minimize the emergence and spread of antiviral drug resistance. We recommend that further in vitro studies be conducted to explore the impact and functions of the S and pol mutation on the protein expression and DRMs towards the antiviral resistance. Data availability Underlying data Figshare: data set for patients’ demographics, HIV and HBV test 2017-2019.xlsx, https://doi.org/10.6084/m9.figshare.23946621.v1 ( Modise, 2023a ). Figshare: PCR amplicon gel electrophoresis of HBV overlapping surface region, https://doi.org/10.6084/m9.figshare.23815278.v1 ( Modise, 2023b ). Figshare: PCR amplicon gel image of HBV overlapping surface region.pdf, https://doi.org/10.6084/m9.figshare.23946639.v1 ( Modise, 2023c ). Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Acknowledgements The authors would like to thank the NHLS-IALCH-NHLS in KZN for donating samples for this study. We thank the State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences for providing training on HBV genotyping and training on cell culture techniques. References Adesina OA, Akanbi OA, Opaleye OO, et al. : Detection of Q129H Immune Escape Mutation in Apparently Healthy Hepatitis B Virus Carriers in Southwestern Nigeria. Viruses. 2021; 13 (7). PubMed Abstract | Publisher Full Text | Free Full Text Anastasiou OE, Almpani F, Herrmann A, et al. : HBV reactivation in allogeneic stem cell transplant recipients: Risk factors, outcome, and role of hepatitis B virus mutations. Hepatol. Commun. 2017; 1 (10): 1014–1023. PubMed Abstract | Publisher Full Text | Free Full Text Archampong T, Ojewale O, Bears K, et al. : Brief Report: Relationship Between ABCC4 SNPs and Hepatitis B Virus Suppression During Tenofovir-Containing Antiretroviral Therapy in Patients With HIV/HBV Coinfection. J. Acquir. Immune. Defic. Syndr. 2019; 82 (4): 421–425. PubMed Abstract | Publisher Full Text | Free Full Text Audsley J, Littlejohn M, Yuen L, et al. : HBV mutations in untreated HIV-HBV co-infection using genomic length sequencing. Virology. 2010; 405 (2): 539–547. PubMed Abstract | Publisher Full Text | Free Full Text Belyhun Y, Maier M, Liebert UG: HIV therapy with unknown HBV status is responsible for higher rate of HBV genome variability in Ethiopia. Antivir. Ther. 2017; 22 (2): 97–111. PubMed Abstract | Publisher Full Text Bi X, Tong S: Impact of immune escape mutations and N-linked glycosylation on the secretion of hepatitis B virus virions and subviral particles: Role of the small envelope protein. Virology. 2018; 518 : 358–368. PubMed Abstract | Publisher Full Text | Free Full Text Bowyer SM, van Staden L , Kew MC, et al. : A unique segment of the hepatitis B virus group A genotype identified in isolates from South Africa. J. Gen. Virol. 1997; 78 (Pt 7): 1719–1729. Publisher Full Text Colagrossi L, Hermans LE, Salpini R, et al. : Immune-escape mutations and stop-codons in HBsAg develop in a large proportion of patients with chronic HBV infection exposed to anti-HBV drugs in Europe. BMC Infect. Dis. 2018; 18 (1): 251. PubMed Abstract | Publisher Full Text | Free Full Text Cooreman MP, Leroux-Roels G, Paulij WP: Vaccine- and hepatitis B immune globulin-induced escape mutations of hepatitis B virus surface antigen. J. Biomed. Sci. 2001; 8 (3): 237–247. PubMed Abstract | Publisher Full Text El-Mokhtar MA, Othman ER, Khashbah MY, et al. : Evidence of the Extrahepatic Replication of Hepatitis E Virus in Human Endometrial Stromal Cells. Pathogens. 2020; 9 (4). PubMed Abstract | Publisher Full Text | Free Full Text Geno2pheno: HBV genotyping. 2023. Reference Source Ireland JH, O’Donnell B, Basuni AA, et al. : Reactivity of 13 in vitro expressed hepatitis B surface antigen variants in 7 commercial diagnostic assays. Hepatology. 2000; 31 (5): 1176–1182. PubMed Abstract | Publisher Full Text Kaswa R, de Villiers M : Prevalence of hepatitis-B virus co-infection among people living with HIV in Mthatha region of South Africa. Afr. Health Sci. 2023; 23 (1): 149–156. PubMed Abstract | Publisher Full Text | Free Full Text Kimbi GC, Kramvis A, Kew MC: Distinctive sequence characteristics of subgenotype A1 isolates of hepatitis B virus from South Africa. J. Gen. Virol. 2004; 85 (Pt 5): 1211–1220. PubMed Abstract | Publisher Full Text King J, Hagemeister DT: Hepatitis B co-infection in HIV-infected patients receiving antiretroviral therapy at the TC Newman Anti Retroviral Treatment Clinic in Paarl, Western Cape. South Afr. J. HIV Med. 2016; 17 (1): 336. PubMed Abstract | Publisher Full Text | Free Full Text Kramvis A: Genotypes and genetic variability of hepatitis B virus. Intervirology. 2014; 57 (3-4): 141–150. Publisher Full Text Kramvis A, Arakawa K, Yu MC, et al. : Relationship of serological subtype, basic core promoter and precore mutations to genotypes/subgenotypes of hepatitis B virus. J. Med. Virol. 2008; 80 (1): 27–46. PubMed Abstract | Publisher Full Text Kramvis A, Kew M, François G: Hepatitis B virus genotypes. Vaccine. 2005; 23 (19): 2409–2423. Publisher Full Text Kramvis A, Kew MC: Epidemiology of hepatitis B virus in Africa, its genotypes and clinical associations of genotypes. Hepatol. Res. 2007; 37 (s1): S9–S19. Publisher Full Text Kuzin SN, Zabotina EE, Zabelin NN, et al. : Heterogeneity of hepatitis B virus and diagnostic potential of modern test systems for the detection of HBsAg. Zh. Mikrobiol. Epidemiol. Immunobiol. 2012; 1 : 68–75. Lai CL, Dienstag J, Schiff E, et al. : Prevalence and clinical correlates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B. Clin. Infect. Dis. 2003; 36 (6): 687–696. PubMed Abstract | Publisher Full Text Liu J, Lv J, Yan B, et al. : Comparison between two population-based hepatitis B serosurveys with an 8-year interval in Shandong Province, China. Int. J. Infect. Dis. 2017; 61 : 13–19. Publisher Full Text Lukhwareni A, Gededzha MP, Amponsah-Dacosta E, et al. : Impact of Lamivudine-Based Antiretroviral Treatment on Hepatitis B Viremia in HIV-Coinfected South Africans. Viruses. 2020 Jun 11; 12 (6):634. PubMed Abstract | Publisher Full Text | Free Full Text Maepa MB, Ely A, Kramvis A, et al. : Hepatitis B Virus Research in South Africa. Viruses. 2022; 14 (9). PubMed Abstract | Publisher Full Text | Free Full Text Maponga TG, McNaughton AL, van Schalkwyk M , et al. : Treatment advantage in HBV/HIV coinfection compared to HBV monoinfection in a South African cohort. J. Infect. 2020; 81 (1): 121–130. PubMed Abstract | Publisher Full Text | Free Full Text Millar J, Cromhout GZL, Mchunu N, et al. : Hepatitis B Virus Prevalence and Mother-to-Child Transmission Risk in an HIV Early Intervention Cohort in KwaZulu-Natal, South Africa. Open Forum Infect. Dis. 2023; 10 (8). PubMed Abstract | Publisher Full Text | Free Full Text Modise L: data set for patients’ demographics, HIV and HBV test 2017-2019.xlsx. Dataset. figshare. 2023a. Publisher Full Text Modise L: PCR amplicon gel electrophoresis of HBV overlapping surface region. figshare. Figure. 2023b. Publisher Full Text Modise L: PCR amplicon gel image of HBV overlapping surface region.pdf. figshare. Figure. 2023c. Publisher Full Text Mokaya J, McNaughton AL, Hadley MJ, et al. : A systematic review of hepatitis B virus (HBV) drug and vaccine escape mutations in Africa: A call for urgent action. PLoS Negl. Trop. Dis. 2018; 12 (8): e0006629. PubMed Abstract | Publisher Full Text | Free Full Text Mphahlele MJ: Impact of HIV co-infection on hepatitis B prevention and control: a view from sub-Saharan Africa. South Afr. J. Epidemiol. Infect. 2008; 23 (1): 14–18. Publisher Full Text Msomi N, Naidoo K, Yende-Zuma N, et al. : High incidence and persistence of hepatitis B virus infection in individuals receiving HIV care in KwaZulu-Natal, South Africa. BMC Infect. Dis. 2020; 20 (1): 847. Publisher Full Text | Free Full Text Musyoki AM, Msibi TL, Motswaledi MH, et al. : Active co-infection with HBV and/or HCV in South African HIV positive patients due for cancer therapy. J. Med. Virol. 2015; 87 (2): 213–221. Publisher Full Text Samsunder N, Ngcapu S, Lewis L, et al. : Seroprevalence of hepatitis B virus: Findings from a population-based household survey in KwaZulu-Natal, South Africa. Int. J. Infect. Dis. 2019; 85 : 150–157. PubMed Abstract | Publisher Full Text | Free Full Text Selabe SG, Lukhwareni A, Song E, et al. : Mutations associated with lamivudine-resistance in therapy-naïve hepatitis B virus (HBV) infected patients with and without HIV co-infection: implications for antiretroviral therapy in HBV and HIV co-infected South African patients. J. Med. Virol. 2007; 79 (11): 1650–1654. PubMed Abstract | Publisher Full Text Selabe SG, Song E, Burnett RJ, et al. : Frequent detection of hepatitis B virus variants associated with lamivudine resistance in treated South African patients infected chronically with different HBV genotypes. J. Med. Virol. 2009; 81 (6): 996–1001. PubMed Abstract | Publisher Full Text Sheldon J, Camino N, Rodés B, et al. : Selection of hepatitis B virus polymerase mutations in HIV-coinfected patients treated with tenofovir. Antivir. Ther. 2005; 10 (6): 727–734. PubMed Abstract | Publisher Full Text Spearman CW, Sonderup MW, Botha JF, et al. : South African guideline for the management of chronic hepatitis B: 2013. S. Afr. Med. J. 2013; 103 (5 Pt 2): 337–349. Summers J, O’Connell A, Millman I: Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles. Proc. Natl. Acad. Sci. U. S. A. 1975; 72 (11): 4597–4601. PubMed Abstract | Publisher Full Text | Free Full Text Tan Y, Ding K, Su J, et al. : The naturally occurring YMDD mutation among patients chronically infected HBV and untreated with lamivudine: a systematic review and meta-analysis. PLoS One. 2012; 7 (3): e32789. Publisher Full Text Torresi J: The virological and clinical significance of mutations in the overlapping envelope and polymerase genes of hepatitis B virus. J. Clin. Virol. 2002; 25 (2): 97–106. Publisher Full Text Vutien P, Trinh HN, Garcia RT, et al. : Mutations in HBV DNA polymerase associated with nucleos(t) ide resistance are rare in treatment-naive patients. Clin. Gastroenterol. Hepatol. 2014; 12 (8): 1363–1370. Publisher Full Text World Health Organization: Guidelines on Hepatiti B and C Testing. 2017. Reference Source Yan B, Lv J, Feng Y, et al. : Temporal trend of hepatitis B surface mutations in the post-immunization period: 9 years of surveillance (2005-2013) in eastern China. Sci. Rep. 2017; 7 (1): 6669. PubMed Abstract | Publisher Full Text | Free Full Text Yousif M, Mudawi H, Hussein W, et al. : Genotyping and virological characteristics of hepatitis B virus in HIV-infected individuals in Sudan. Int. J. Infect. Dis. 2014; 29 : 125–132. Publisher Full Text Zhang TY, Yuan Q, Zhao JH, et al. : Prolonged suppression of HBV in mice by a novel antibody that targets a unique epitope on hepatitis B surface antigen. Gut. 2016; 65 (4), 658–671. PubMed Abstract | Publisher Full Text Zhao Y, Wu J, Sun L, et al. : Prevalence of mutations in HBV DNA polymerase gene associated with nucleos(t) ide resistance in treatment-naive patients with Chronic Hepatitis B in Central China. Braz. J. Infect. Dis. 2016; 20 (2), 173–178. PubMed Abstract | Publisher Full Text | Free Full Text Zöllner B, Petersen J, Schröter M, et al. : 20-fold increase in risk of lamivudine resistance in hepatitis B virus subtype adw. Lancet. 2001; 357 (9260): 934–935. PubMed Abstract | Publisher Full Text Comments on this article Comments (0) Version 4 VERSION 4 PUBLISHED 27 Sep 2023 ADD YOUR COMMENT Comment Author details Author details 1 Biological Sciences, North West University, Mahikeng, North West, 2735, South Africa Lorato Modise Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Validation, Writing – Original Draft Preparation, Writing – Review & Editing Nomathamsanqa Sithebe Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing Hazel Mufhandu Roles: Data Curation, Formal Analysis, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information This work was supported by funds from the North-West University, National Research foundation Innovation bursary (Grant No: SFH150727131319), Health and Welfare Sector Education and Training Authority, South African Centre for Epidemiological Modelling and Analysis and Organisation for Women in Science for the Developing Worlds (Grant No: 3240287283). These funding agencies were not involved and responsible in the study design and writing of the thesis and manuscript. The Authors in this study are solely responsible for the content and not the funding agencies involved. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (4) version 4 Revised Published: 21 Aug 2025, 12:1232 https://doi.org/10.12688/f1000research.132498.4 version 3 Revised Published: 23 Apr 2025, 12:1232 https://doi.org/10.12688/f1000research.132498.3 version 2 Revised Published: 30 May 2024, 12:1232 https://doi.org/10.12688/f1000research.132498.2 version 1 Published: 27 Sep 2023, 12:1232 https://doi.org/10.12688/f1000research.132498.1 Copyright © 2025 Modise L et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Modise L, Sithebe N and Mufhandu H. Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.12688/f1000research.132498.4 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 4 VERSION 4 PUBLISHED 21 Aug 2025 Revised Views 0 Cite How to cite this report: Gelanew T. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.186606.r407676 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v4#referee-response-407676 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 11 Sep 2025 Tesfaye Gelanew , Armauer Hansen Research Institute, Addis Ababa, Addis Ababa, Ethiopia Approved VIEWS 0 https://doi.org/10.5256/f1000research.186606.r407676 I have no further comment. ... Continue reading READ ALL I have no further comment. I endorsed the manuscript for indexing. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Gelanew T. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.186606.r407676 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v4#referee-response-407676 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 3 VERSION 3 PUBLISHED 23 Apr 2025 Revised Views 0 Cite How to cite this report: Prabdial-Sing N. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.179250.r380330 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v3#referee-response-380330 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 05 Aug 2025 Nishi Prabdial-Sing , Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.179250.r380330 Thank you to the authors for addressing some of the reviewer’s comments. There are still grammatical errors in the abstract. Abstract: Background ‘”cross-resistance of drugs”” what does this mean exactly, that sites on the HIV ... Continue reading READ ALL Thank you to the authors for addressing some of the reviewer’s comments. There are still grammatical errors in the abstract. Abstract: Background ‘”cross-resistance of drugs”” what does this mean exactly, that sites on the HIV and HBV genome have shown cross-resistance of HIV antiviral drugs and to HBV antiviral drugs? I have not seen this and should be better written Begin the next sentence “Continuous monitoring of HBV variants is required for better…” Results: Also as you found all genotype A to be subgenotype A1, then indicate A1 in your findings and conclusions Genotype c, should be C Introduction: The abbreviation “DRM”, needs to be explained when used for the first time Method English should be English used in according should be used according Results: Mutations showed resistance to lamivudine (LMV) at (35% [5/14]), telbivudine (LdT) at (29% [4/14]), (14% [2/14]) for entecavir (ETV) and (21% [3/14]) for adefovir (ADV). Mutations causing resistance to LMV and LdT were M204V, L180M, V163I, and S202K” “the statement implies that the patients were treated for HBV and these DRMs were found. However, it was mentioned that these patients were Treatment naïve? Please clarify whether the patients were treatment naïve for HIV or HBV or both? Discussion: This suggest that the mutant HBV is common in both naive and experience HIV positive people””. This sentence is not referenced. There is only one reference for naïve, but not HIV treated persons. Conclusion thorough-synchronizes diagnosis” what does this mean? What do the authors mean by substandard immigration of new strains? Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Prabdial-Sing N. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.179250.r380330 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v3#referee-response-380330 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Gelanew T. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.179250.r393935 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v3#referee-response-393935 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 28 Jul 2025 Tesfaye Gelanew , Armauer Hansen Research Institute, Addis Ababa, Addis Ababa, Ethiopia Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.179250.r393935 The manuscript by Modise et al entitled "Investigation of Hepatitis B Virus Mutations Associated with Immune Escape and Drug Resistance in HIV-Infected Patients" is highly informative, and I concur with the authors’ observation that most studies conducted in Africa focus ... Continue reading READ ALL The manuscript by Modise et al entitled "Investigation of Hepatitis B Virus Mutations Associated with Immune Escape and Drug Resistance in HIV-Infected Patients" is highly informative, and I concur with the authors’ observation that most studies conducted in Africa focus primarily on prevalence, while only a limited number address genotyping and drug resistance profiling. The paper is well written, with sound methodological approaches. The conclusions are appropriately toned down, clearly derived from the data, and the limitations are well articulated. I recommend acceptance in its current form. However, I have the following few minor comments. 1. - The title lacks specificity. It should be revised to: Investigation of Mutations in the Partial Sequences of the S and Polymerase Genes of Hepatitis B Virus Associated with Immune Escape and Drug Resistance in HIV-Infected Patients 2. - The introduction would benefit from expanding the brief overview of the specific genes targeted in this study—namely those encoding the surface antigen and polymerase—and their roles in immune escape and antiviral drug resistance. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: My expertise is clinical microbiology and Immunology/molecular biology. My ongoing research areas are dengue virus, HBV, RSV, and pathogens causing STIs. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Gelanew T. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.179250.r393935 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v3#referee-response-393935 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Ike AC. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.179250.r386531 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v3#referee-response-386531 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 14 Jun 2025 Anthony C. Ike , University of Nigeria, Nsukka, Enugu, Nigeria Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.179250.r386531 The article is well designed and carried out and was also well written or corrected to bring it to its present level. I have made few observations as listed below: In Page 4 under DNA extraction of HBV in ... Continue reading READ ALL The article is well designed and carried out and was also well written or corrected to bring it to its present level. I have made few observations as listed below: In Page 4 under DNA extraction of HBV in Lines 4 and 10, the Eppendorf tube is 1.5 mL and not 1.5 μL. In the same page under Polymerase chain reaction First round and nested-PCR, in Line 8 of that section, the authors need to state whether the 0.125 of Taq DNA polymerase is the concentration (units) or volume (μL) of the enzyme. Under Nested PCR, the last line of the section, Line 6, the authors should state what was used as positive control and state the source if possible. In page 6 under laboratory testing HBsAg assay Line 2 of the section, it should be females and not female's. Also the HBsAg positive for the females should be 91% (N=29/32) and not 58% (N=29/50), since there were only 32 females and not 50. similarly that of the males should be 78% (N=14/18) and not 28% (N=14/50), males ere 18 and not 50. This should be corrected in Table 1 and be extended to the results of HIV (+) Female and HIV (+) Male. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Medical Microbiology with Virology bias. I have researched and published works on Noroviruses, Rotaviruses, influenza virus, hepatitis B and C viruses, Dengue virus among others. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Ike AC. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.179250.r386531 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v3#referee-response-386531 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 2 VERSION 2 PUBLISHED 30 May 2024 Revised Views 0 Cite How to cite this report: McNaughton A. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.163315.r284916 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v2#referee-response-284916 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 06 Jun 2024 Anna McNaughton , Nuffield Department of Medicine, University of Oxford Medawar Building, Oxford, England, UK Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.163315.r284916 The authors have clearly revised the manuscript, taking into account some of the feedback provided previously and their metadata now looks to be more accurate. However, there are still several issues with the revised document, and particularly the analysis and ... Continue reading READ ALL The authors have clearly revised the manuscript, taking into account some of the feedback provided previously and their metadata now looks to be more accurate. However, there are still several issues with the revised document, and particularly the analysis and presentation of the results which need correcting before the manuscript can be approved. HBsAg assay – COV needs defining at first use. Baseline demographic data – the median age is presented and the range is but I can’t see the standard deviation, despite the authors stating it in the paragraph. Table 1 should be revised to be more informative (i.e. present proportions) – if all individuals were black africans and HIV positive, this is not particularly useful information to present? I would encourage the authors to revise the data in table 1 to make it more informative, such as is there any data on their HIV treatment? Phylogenetic analysis – the authors state that sequences were obtained for 38/41 samples that were PCR positive, but only analysed n=26 sequences. Why were 12 sequences excluded from the analysis? The sequences analysed are relatively short, and as the authors state, this can affect the clustering but the phylogenetic analysis presented remains very problematic for genotyping. The reference sequences do not cluster by genotype, making it difficult to use the tree to confidently genotype the sequences presented. Whilst the authors checked a number of gtA the sequences with geno2pheno, I am puzzled by the interpretation of the other sequences (assumed not to be gtA). A number of sequences do not clearly cluster with any of the reference sequences, and the authors describe these as “24% (N=7/26) as genotypes B, C, D, E, F and G”, which is difficult to interpret. It is also a little unclear why sequences at the bottom of the tree ‘Q6P7, Q17P7, and Q42P7’ were decided to be closer to genotype H, D, B, and E than to genotype A without presenting bootstrapping data? This analysis needs revising, and perhaps the authors should consider a maximum-likelihood analysis as it may be more discriminatory for genotyping than the approach currently used. Alternatively, they may consider using geno2pheno to genotype their sequences, and present this data in a table rather than a tree? Association of mutations on the surface and polymerase regions – authors state the mean and standard deviation of the SHB mutations, but only provide a single number? Are the numbers presented also the number of mutations per sequence, as this is not clear? The discussion is too long and should be more concise – the discussion of the wider literature on drug resistant mutations could be reduced. Some context may also be useful to discuss – these samples were taken from HIV-positive individuals, so were any of them on treatment for their HIV infections and were these treatments active against HBV also? The authors mention that some patients are ART treatment naïve late in the discussion – but a little more detail on this would add to the interpretation of this study. Competing Interests: No competing interests were disclosed. Reviewer Expertise: Virology, genomics, epidemiology, viral hepatitis I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT McNaughton A. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.163315.r284916 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v2#referee-response-284916 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 17 Sep 2025 Lorato Mosetsanagape Modise , Biological Sciences, North West University, Mahikeng, 2735, South Africa 17 Sep 2025 Author Response Dear reviewer We truly appreciate knowledge, and insightful comments on our paper. We appreciate your reviews and recommendations, which have greatly enhanced the manuscript. We have carefully considered all your comments ... Continue reading Dear reviewer We truly appreciate knowledge, and insightful comments on our paper. We appreciate your reviews and recommendations, which have greatly enhanced the manuscript. We have carefully considered all your comments and revised them accordingly. Specific reviewer comment HBsAg assay – COV needs defining at first use. Response : Cut off value (COV) has been defined at first use. Reviewer comments Baseline demographic data – the median age is presented, and the range is, but I can’t see the standard deviation, despite the authors stating it in the paragraph. Table 1 should be revised to be more informative (i.e. present proportions) – if all individuals were black Africans and HIV positive, this is not particularly useful information to present. I would encourage the authors to revise the data in table 1 to make it more informative, such as is there any data on their HIV treatment? Response : Since all the samples were from the same group, the information on the ethnicity of Black African has been deleted from the description in Table 1. The description of the statistical analysis has been revised in the text and Table 1 to be specific to the standard deviation. The median age is presented with the interquartile range (IQR) the range and did not present the standard deviation because the data in not normally distributed and there is poor statistical power. Table 1 presents the demographic data collected from the participants which includes the age, sex and test done before. The data is already presented in proportions expressed by either a count by the total number and the proportion as a percentage. The age is already presented in proportions as indicated in the text (n=19/50). The results are on demographic data which is the primary data on the participants HIV status. We can only report on the data that was collected and available for this study, The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. This is highlighted in the limitations section. Reviewer comments Phylogenetic analysis – the authors state that sequences were obtained for 38/41 samples that were PCR positive but only analysed n=26 sequences. Why were 12 sequences excluded from the analysis? The sequences analysed are relatively short, and as the authors state, this can affect the clustering, but the phylogenetic analysis presented remains very problematic for genotyping. The reference sequences do not cluster by genotype, making it difficult to use the tree to confidently genotype the sequences presented. Whilst the authors checked a few gtA the sequences with geno2pheno, I am puzzled by the interpretation of the other sequences (assumed not to be gtA). A few sequences do not clearly cluster with any of the reference sequences, and the authors describe these as “24% (N=7/26) as genotypes B, C, D, E, F and G”, which is difficult to interpret. It is also a little unclear why sequences at the bottom of the tree ‘Q6P7, Q17P7, and Q42P7’ were decided to be closer to genotype H, D, B, and E than to genotype A without presenting bootstrapping data? This analysis needs revising, and perhaps the authors should consider a maximum-likelihood analysis as it may be more discriminatory for genotyping than the approach currently used. Alternatively, they may consider using geno2pheno to genotype their sequences, Response : The 12 sequences excluded from the analysis had poor sequence reads on the chromatogram, and they could not form the proper consensus sequences thus they were not considered for the analysis. The genotyping of the sequences is revised with the use of geno2pheno software as advised and the data is presented in a table instead of a phylogenetic tree (Table 2). The sequences were uploaded onto the geno2pheno software and compared against standard (wildtype) genome with the highest nucleotide similarity to the study sequences. The sequences that identify as genotype A have been clarified through revision. Additionally, the 24% (N=7/26) that represent a combination of other genotypes (C and G) have been broken down to demonstrate the individual and specific genotypes C and G; 12% (N=3/26) represent genotypes G, and 15% (N=4/26) represent genotype C. Association of the mutations Data on the association of mutations on the surface and polymerase regions has been removed due to the poor statistical power and statistical association. We focused on reporting the data on the mutations associated with drug resistance with a focus on their frequency as depicted in Table 3. Reviewer comment The discussion is too long and should be more concise – the discussion of the wider literature on drug resistant mutations could be reduced. Some contexts may also be useful to discuss – these samples were taken from HIV-positive individuals, so were any of them on treatment for their HIV infections and were these treatments active against HBV also? Response : The discussion has been summarised. The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. Secondly, we don’t know if HIV treatments is active against HBV because we do not have the information currently available to support that. However, we know the treatment history of HBV and that the participants are naïve to HBV ARTs. We have also added information on the results interpretation of the mutations in HBV patients that are HBV treatment naïve (last paragraph under the discussion section). Results Table 1: The median age was 33 years [IQR: 18-55] and there was no statistical difference on age between the genders with p=0.8. 92% (N=38/41) sequence products could be obtained by Sanger sequencing with only 26/38 sequences used to perform genotyping, that is, 12 sequences were excluded due to the poor quality of their sequence reads. The genotype of the sequences was confirmed by depositing nucleotide sequences into the Genotype2pheno database, which showed that most of the nucleotide sequences had homology with genotype A at 73% (N=19/26), genotype G at 12% (N=3/26), and genotype C at 15% (N=4/26) (Table 2). The study sequences with the highest similarity to Genotype A were: (Q4P7 and Q7P7 to KX520697.1|South Africa; Q9P7 to U87728.1|South Africa; Q27P7 to AY233274.1|South Africa. Reference sequence of AY233277.1|South Africa had between 97%-99% similarity to fifteen sequences (Q5P7, Q8P7, Q10P7, Q13P7, Q18P7, Q19P7, Q20P7, Q21P7, Q22P7, Q23P7, Q24P7, Q26P7, Q27P7, Q29P7 and Q39P7). The sequences Q6P7, Q17P7 and Q42P7 had similarity to genotype G (EU694179.1|South Africa; sequences Q11P7, Q43P7, Q44P7 and Q45P7 showed similarity to genotype C AB562444.1|Vietnam (Table 2). Sub-genotypes of the sequences were confirmed by depositing nucleotides sequences into the Genotype2pheno database. The results retrieved from the Geno2Pheno database had a 96.85%-99.0% percentage of similarity to sub-genotype A1 for the genotype A sequences only. Discussion Discussion on the interpretation of mutations in treatment-naïve has been revised. This includes clarity on the impact of the mutations on the treatment’s status of the participants. We also discuss how the study cannot answer on whether the treatment was active against HBV (see the last paragraph under the discussion section). However, we made recommendation that further in vitro studies must investigate the mechanism of action by DRMs on ARTs in vitro assays (last sentence under conclusion). Additional correction and response Reference removed from the list 1. Kimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 1980; 16(2): 111–120. PubMed Abstract|Publisher Full Text. 2. Kumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief. Bioinform. 2004; 5(2): 150–163. PubMed Abstract|Publisher Full Text. 3. Malik A, Singhal DK, Albanyan A, et al.: Hepatitis B virus gene mutations in liver diseases: a report from New Delhi. PLoS One. 2012; 7(6): e39028. PubMed Abstract|Publisher Full Text|Free Full Text. 4. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987; 4(4): 406–425. . Updated references and citation 1. Belyhun, Yeshambel, Melanie Maier, and Uwe Gerd Liebert. "HIV therapy with unknown HBV status is responsible for higher rate of HBV genome variability in Ethiopia." Antiviral therapy 22.2 (2017): 97-111. 2. Geno2pheno. HBV genotyping 2023 [Available from: https://hbv.geno2pheno.org/. 3. World Health Organization. Guidelines on Hepatitis B and C Testing 2017 update (available at https://apps.who.int/iris/bitstream/handle/10665/254621/9789241549981- eng.pdf.). Dear reviewer We truly appreciate knowledge, and insightful comments on our paper. We appreciate your reviews and recommendations, which have greatly enhanced the manuscript. We have carefully considered all your comments and revised them accordingly. Specific reviewer comment HBsAg assay – COV needs defining at first use. Response : Cut off value (COV) has been defined at first use. Reviewer comments Baseline demographic data – the median age is presented, and the range is, but I can’t see the standard deviation, despite the authors stating it in the paragraph. Table 1 should be revised to be more informative (i.e. present proportions) – if all individuals were black Africans and HIV positive, this is not particularly useful information to present. I would encourage the authors to revise the data in table 1 to make it more informative, such as is there any data on their HIV treatment? Response : Since all the samples were from the same group, the information on the ethnicity of Black African has been deleted from the description in Table 1. The description of the statistical analysis has been revised in the text and Table 1 to be specific to the standard deviation. The median age is presented with the interquartile range (IQR) the range and did not present the standard deviation because the data in not normally distributed and there is poor statistical power. Table 1 presents the demographic data collected from the participants which includes the age, sex and test done before. The data is already presented in proportions expressed by either a count by the total number and the proportion as a percentage. The age is already presented in proportions as indicated in the text (n=19/50). The results are on demographic data which is the primary data on the participants HIV status. We can only report on the data that was collected and available for this study, The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. This is highlighted in the limitations section. Reviewer comments Phylogenetic analysis – the authors state that sequences were obtained for 38/41 samples that were PCR positive but only analysed n=26 sequences. Why were 12 sequences excluded from the analysis? The sequences analysed are relatively short, and as the authors state, this can affect the clustering, but the phylogenetic analysis presented remains very problematic for genotyping. The reference sequences do not cluster by genotype, making it difficult to use the tree to confidently genotype the sequences presented. Whilst the authors checked a few gtA the sequences with geno2pheno, I am puzzled by the interpretation of the other sequences (assumed not to be gtA). A few sequences do not clearly cluster with any of the reference sequences, and the authors describe these as “24% (N=7/26) as genotypes B, C, D, E, F and G”, which is difficult to interpret. It is also a little unclear why sequences at the bottom of the tree ‘Q6P7, Q17P7, and Q42P7’ were decided to be closer to genotype H, D, B, and E than to genotype A without presenting bootstrapping data? This analysis needs revising, and perhaps the authors should consider a maximum-likelihood analysis as it may be more discriminatory for genotyping than the approach currently used. Alternatively, they may consider using geno2pheno to genotype their sequences, Response : The 12 sequences excluded from the analysis had poor sequence reads on the chromatogram, and they could not form the proper consensus sequences thus they were not considered for the analysis. The genotyping of the sequences is revised with the use of geno2pheno software as advised and the data is presented in a table instead of a phylogenetic tree (Table 2). The sequences were uploaded onto the geno2pheno software and compared against standard (wildtype) genome with the highest nucleotide similarity to the study sequences. The sequences that identify as genotype A have been clarified through revision. Additionally, the 24% (N=7/26) that represent a combination of other genotypes (C and G) have been broken down to demonstrate the individual and specific genotypes C and G; 12% (N=3/26) represent genotypes G, and 15% (N=4/26) represent genotype C. Association of the mutations Data on the association of mutations on the surface and polymerase regions has been removed due to the poor statistical power and statistical association. We focused on reporting the data on the mutations associated with drug resistance with a focus on their frequency as depicted in Table 3. Reviewer comment The discussion is too long and should be more concise – the discussion of the wider literature on drug resistant mutations could be reduced. Some contexts may also be useful to discuss – these samples were taken from HIV-positive individuals, so were any of them on treatment for their HIV infections and were these treatments active against HBV also? Response : The discussion has been summarised. The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. Secondly, we don’t know if HIV treatments is active against HBV because we do not have the information currently available to support that. However, we know the treatment history of HBV and that the participants are naïve to HBV ARTs. We have also added information on the results interpretation of the mutations in HBV patients that are HBV treatment naïve (last paragraph under the discussion section). Results Table 1: The median age was 33 years [IQR: 18-55] and there was no statistical difference on age between the genders with p=0.8. 92% (N=38/41) sequence products could be obtained by Sanger sequencing with only 26/38 sequences used to perform genotyping, that is, 12 sequences were excluded due to the poor quality of their sequence reads. The genotype of the sequences was confirmed by depositing nucleotide sequences into the Genotype2pheno database, which showed that most of the nucleotide sequences had homology with genotype A at 73% (N=19/26), genotype G at 12% (N=3/26), and genotype C at 15% (N=4/26) (Table 2). The study sequences with the highest similarity to Genotype A were: (Q4P7 and Q7P7 to KX520697.1|South Africa; Q9P7 to U87728.1|South Africa; Q27P7 to AY233274.1|South Africa. Reference sequence of AY233277.1|South Africa had between 97%-99% similarity to fifteen sequences (Q5P7, Q8P7, Q10P7, Q13P7, Q18P7, Q19P7, Q20P7, Q21P7, Q22P7, Q23P7, Q24P7, Q26P7, Q27P7, Q29P7 and Q39P7). The sequences Q6P7, Q17P7 and Q42P7 had similarity to genotype G (EU694179.1|South Africa; sequences Q11P7, Q43P7, Q44P7 and Q45P7 showed similarity to genotype C AB562444.1|Vietnam (Table 2). Sub-genotypes of the sequences were confirmed by depositing nucleotides sequences into the Genotype2pheno database. The results retrieved from the Geno2Pheno database had a 96.85%-99.0% percentage of similarity to sub-genotype A1 for the genotype A sequences only. Discussion Discussion on the interpretation of mutations in treatment-naïve has been revised. This includes clarity on the impact of the mutations on the treatment’s status of the participants. We also discuss how the study cannot answer on whether the treatment was active against HBV (see the last paragraph under the discussion section). However, we made recommendation that further in vitro studies must investigate the mechanism of action by DRMs on ARTs in vitro assays (last sentence under conclusion). Additional correction and response Reference removed from the list 1. Kimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 1980; 16(2): 111–120. PubMed Abstract|Publisher Full Text. 2. Kumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief. Bioinform. 2004; 5(2): 150–163. PubMed Abstract|Publisher Full Text. 3. Malik A, Singhal DK, Albanyan A, et al.: Hepatitis B virus gene mutations in liver diseases: a report from New Delhi. PLoS One. 2012; 7(6): e39028. PubMed Abstract|Publisher Full Text|Free Full Text. 4. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987; 4(4): 406–425. . Updated references and citation 1. Belyhun, Yeshambel, Melanie Maier, and Uwe Gerd Liebert. "HIV therapy with unknown HBV status is responsible for higher rate of HBV genome variability in Ethiopia." Antiviral therapy 22.2 (2017): 97-111. 2. Geno2pheno. HBV genotyping 2023 [Available from: https://hbv.geno2pheno.org/. 3. World Health Organization. Guidelines on Hepatitis B and C Testing 2017 update (available at https://apps.who.int/iris/bitstream/handle/10665/254621/9789241549981- eng.pdf.). Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 17 Sep 2025 Lorato Mosetsanagape Modise , Biological Sciences, North West University, Mahikeng, 2735, South Africa 17 Sep 2025 Author Response Dear reviewer We truly appreciate knowledge, and insightful comments on our paper. We appreciate your reviews and recommendations, which have greatly enhanced the manuscript. We have carefully considered all your comments ... Continue reading Dear reviewer We truly appreciate knowledge, and insightful comments on our paper. We appreciate your reviews and recommendations, which have greatly enhanced the manuscript. We have carefully considered all your comments and revised them accordingly. Specific reviewer comment HBsAg assay – COV needs defining at first use. Response : Cut off value (COV) has been defined at first use. Reviewer comments Baseline demographic data – the median age is presented, and the range is, but I can’t see the standard deviation, despite the authors stating it in the paragraph. Table 1 should be revised to be more informative (i.e. present proportions) – if all individuals were black Africans and HIV positive, this is not particularly useful information to present. I would encourage the authors to revise the data in table 1 to make it more informative, such as is there any data on their HIV treatment? Response : Since all the samples were from the same group, the information on the ethnicity of Black African has been deleted from the description in Table 1. The description of the statistical analysis has been revised in the text and Table 1 to be specific to the standard deviation. The median age is presented with the interquartile range (IQR) the range and did not present the standard deviation because the data in not normally distributed and there is poor statistical power. Table 1 presents the demographic data collected from the participants which includes the age, sex and test done before. The data is already presented in proportions expressed by either a count by the total number and the proportion as a percentage. The age is already presented in proportions as indicated in the text (n=19/50). The results are on demographic data which is the primary data on the participants HIV status. We can only report on the data that was collected and available for this study, The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. This is highlighted in the limitations section. Reviewer comments Phylogenetic analysis – the authors state that sequences were obtained for 38/41 samples that were PCR positive but only analysed n=26 sequences. Why were 12 sequences excluded from the analysis? The sequences analysed are relatively short, and as the authors state, this can affect the clustering, but the phylogenetic analysis presented remains very problematic for genotyping. The reference sequences do not cluster by genotype, making it difficult to use the tree to confidently genotype the sequences presented. Whilst the authors checked a few gtA the sequences with geno2pheno, I am puzzled by the interpretation of the other sequences (assumed not to be gtA). A few sequences do not clearly cluster with any of the reference sequences, and the authors describe these as “24% (N=7/26) as genotypes B, C, D, E, F and G”, which is difficult to interpret. It is also a little unclear why sequences at the bottom of the tree ‘Q6P7, Q17P7, and Q42P7’ were decided to be closer to genotype H, D, B, and E than to genotype A without presenting bootstrapping data? This analysis needs revising, and perhaps the authors should consider a maximum-likelihood analysis as it may be more discriminatory for genotyping than the approach currently used. Alternatively, they may consider using geno2pheno to genotype their sequences, Response : The 12 sequences excluded from the analysis had poor sequence reads on the chromatogram, and they could not form the proper consensus sequences thus they were not considered for the analysis. The genotyping of the sequences is revised with the use of geno2pheno software as advised and the data is presented in a table instead of a phylogenetic tree (Table 2). The sequences were uploaded onto the geno2pheno software and compared against standard (wildtype) genome with the highest nucleotide similarity to the study sequences. The sequences that identify as genotype A have been clarified through revision. Additionally, the 24% (N=7/26) that represent a combination of other genotypes (C and G) have been broken down to demonstrate the individual and specific genotypes C and G; 12% (N=3/26) represent genotypes G, and 15% (N=4/26) represent genotype C. Association of the mutations Data on the association of mutations on the surface and polymerase regions has been removed due to the poor statistical power and statistical association. We focused on reporting the data on the mutations associated with drug resistance with a focus on their frequency as depicted in Table 3. Reviewer comment The discussion is too long and should be more concise – the discussion of the wider literature on drug resistant mutations could be reduced. Some contexts may also be useful to discuss – these samples were taken from HIV-positive individuals, so were any of them on treatment for their HIV infections and were these treatments active against HBV also? Response : The discussion has been summarised. The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. Secondly, we don’t know if HIV treatments is active against HBV because we do not have the information currently available to support that. However, we know the treatment history of HBV and that the participants are naïve to HBV ARTs. We have also added information on the results interpretation of the mutations in HBV patients that are HBV treatment naïve (last paragraph under the discussion section). Results Table 1: The median age was 33 years [IQR: 18-55] and there was no statistical difference on age between the genders with p=0.8. 92% (N=38/41) sequence products could be obtained by Sanger sequencing with only 26/38 sequences used to perform genotyping, that is, 12 sequences were excluded due to the poor quality of their sequence reads. The genotype of the sequences was confirmed by depositing nucleotide sequences into the Genotype2pheno database, which showed that most of the nucleotide sequences had homology with genotype A at 73% (N=19/26), genotype G at 12% (N=3/26), and genotype C at 15% (N=4/26) (Table 2). The study sequences with the highest similarity to Genotype A were: (Q4P7 and Q7P7 to KX520697.1|South Africa; Q9P7 to U87728.1|South Africa; Q27P7 to AY233274.1|South Africa. Reference sequence of AY233277.1|South Africa had between 97%-99% similarity to fifteen sequences (Q5P7, Q8P7, Q10P7, Q13P7, Q18P7, Q19P7, Q20P7, Q21P7, Q22P7, Q23P7, Q24P7, Q26P7, Q27P7, Q29P7 and Q39P7). The sequences Q6P7, Q17P7 and Q42P7 had similarity to genotype G (EU694179.1|South Africa; sequences Q11P7, Q43P7, Q44P7 and Q45P7 showed similarity to genotype C AB562444.1|Vietnam (Table 2). Sub-genotypes of the sequences were confirmed by depositing nucleotides sequences into the Genotype2pheno database. The results retrieved from the Geno2Pheno database had a 96.85%-99.0% percentage of similarity to sub-genotype A1 for the genotype A sequences only. Discussion Discussion on the interpretation of mutations in treatment-naïve has been revised. This includes clarity on the impact of the mutations on the treatment’s status of the participants. We also discuss how the study cannot answer on whether the treatment was active against HBV (see the last paragraph under the discussion section). However, we made recommendation that further in vitro studies must investigate the mechanism of action by DRMs on ARTs in vitro assays (last sentence under conclusion). Additional correction and response Reference removed from the list 1. Kimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 1980; 16(2): 111–120. PubMed Abstract|Publisher Full Text. 2. Kumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief. Bioinform. 2004; 5(2): 150–163. PubMed Abstract|Publisher Full Text. 3. Malik A, Singhal DK, Albanyan A, et al.: Hepatitis B virus gene mutations in liver diseases: a report from New Delhi. PLoS One. 2012; 7(6): e39028. PubMed Abstract|Publisher Full Text|Free Full Text. 4. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987; 4(4): 406–425. . Updated references and citation 1. Belyhun, Yeshambel, Melanie Maier, and Uwe Gerd Liebert. "HIV therapy with unknown HBV status is responsible for higher rate of HBV genome variability in Ethiopia." Antiviral therapy 22.2 (2017): 97-111. 2. Geno2pheno. HBV genotyping 2023 [Available from: https://hbv.geno2pheno.org/. 3. World Health Organization. Guidelines on Hepatitis B and C Testing 2017 update (available at https://apps.who.int/iris/bitstream/handle/10665/254621/9789241549981- eng.pdf.). Dear reviewer We truly appreciate knowledge, and insightful comments on our paper. We appreciate your reviews and recommendations, which have greatly enhanced the manuscript. We have carefully considered all your comments and revised them accordingly. Specific reviewer comment HBsAg assay – COV needs defining at first use. Response : Cut off value (COV) has been defined at first use. Reviewer comments Baseline demographic data – the median age is presented, and the range is, but I can’t see the standard deviation, despite the authors stating it in the paragraph. Table 1 should be revised to be more informative (i.e. present proportions) – if all individuals were black Africans and HIV positive, this is not particularly useful information to present. I would encourage the authors to revise the data in table 1 to make it more informative, such as is there any data on their HIV treatment? Response : Since all the samples were from the same group, the information on the ethnicity of Black African has been deleted from the description in Table 1. The description of the statistical analysis has been revised in the text and Table 1 to be specific to the standard deviation. The median age is presented with the interquartile range (IQR) the range and did not present the standard deviation because the data in not normally distributed and there is poor statistical power. Table 1 presents the demographic data collected from the participants which includes the age, sex and test done before. The data is already presented in proportions expressed by either a count by the total number and the proportion as a percentage. The age is already presented in proportions as indicated in the text (n=19/50). The results are on demographic data which is the primary data on the participants HIV status. We can only report on the data that was collected and available for this study, The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. This is highlighted in the limitations section. Reviewer comments Phylogenetic analysis – the authors state that sequences were obtained for 38/41 samples that were PCR positive but only analysed n=26 sequences. Why were 12 sequences excluded from the analysis? The sequences analysed are relatively short, and as the authors state, this can affect the clustering, but the phylogenetic analysis presented remains very problematic for genotyping. The reference sequences do not cluster by genotype, making it difficult to use the tree to confidently genotype the sequences presented. Whilst the authors checked a few gtA the sequences with geno2pheno, I am puzzled by the interpretation of the other sequences (assumed not to be gtA). A few sequences do not clearly cluster with any of the reference sequences, and the authors describe these as “24% (N=7/26) as genotypes B, C, D, E, F and G”, which is difficult to interpret. It is also a little unclear why sequences at the bottom of the tree ‘Q6P7, Q17P7, and Q42P7’ were decided to be closer to genotype H, D, B, and E than to genotype A without presenting bootstrapping data? This analysis needs revising, and perhaps the authors should consider a maximum-likelihood analysis as it may be more discriminatory for genotyping than the approach currently used. Alternatively, they may consider using geno2pheno to genotype their sequences, Response : The 12 sequences excluded from the analysis had poor sequence reads on the chromatogram, and they could not form the proper consensus sequences thus they were not considered for the analysis. The genotyping of the sequences is revised with the use of geno2pheno software as advised and the data is presented in a table instead of a phylogenetic tree (Table 2). The sequences were uploaded onto the geno2pheno software and compared against standard (wildtype) genome with the highest nucleotide similarity to the study sequences. The sequences that identify as genotype A have been clarified through revision. Additionally, the 24% (N=7/26) that represent a combination of other genotypes (C and G) have been broken down to demonstrate the individual and specific genotypes C and G; 12% (N=3/26) represent genotypes G, and 15% (N=4/26) represent genotype C. Association of the mutations Data on the association of mutations on the surface and polymerase regions has been removed due to the poor statistical power and statistical association. We focused on reporting the data on the mutations associated with drug resistance with a focus on their frequency as depicted in Table 3. Reviewer comment The discussion is too long and should be more concise – the discussion of the wider literature on drug resistant mutations could be reduced. Some contexts may also be useful to discuss – these samples were taken from HIV-positive individuals, so were any of them on treatment for their HIV infections and were these treatments active against HBV also? Response : The discussion has been summarised. The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. Secondly, we don’t know if HIV treatments is active against HBV because we do not have the information currently available to support that. However, we know the treatment history of HBV and that the participants are naïve to HBV ARTs. We have also added information on the results interpretation of the mutations in HBV patients that are HBV treatment naïve (last paragraph under the discussion section). Results Table 1: The median age was 33 years [IQR: 18-55] and there was no statistical difference on age between the genders with p=0.8. 92% (N=38/41) sequence products could be obtained by Sanger sequencing with only 26/38 sequences used to perform genotyping, that is, 12 sequences were excluded due to the poor quality of their sequence reads. The genotype of the sequences was confirmed by depositing nucleotide sequences into the Genotype2pheno database, which showed that most of the nucleotide sequences had homology with genotype A at 73% (N=19/26), genotype G at 12% (N=3/26), and genotype C at 15% (N=4/26) (Table 2). The study sequences with the highest similarity to Genotype A were: (Q4P7 and Q7P7 to KX520697.1|South Africa; Q9P7 to U87728.1|South Africa; Q27P7 to AY233274.1|South Africa. Reference sequence of AY233277.1|South Africa had between 97%-99% similarity to fifteen sequences (Q5P7, Q8P7, Q10P7, Q13P7, Q18P7, Q19P7, Q20P7, Q21P7, Q22P7, Q23P7, Q24P7, Q26P7, Q27P7, Q29P7 and Q39P7). The sequences Q6P7, Q17P7 and Q42P7 had similarity to genotype G (EU694179.1|South Africa; sequences Q11P7, Q43P7, Q44P7 and Q45P7 showed similarity to genotype C AB562444.1|Vietnam (Table 2). Sub-genotypes of the sequences were confirmed by depositing nucleotides sequences into the Genotype2pheno database. The results retrieved from the Geno2Pheno database had a 96.85%-99.0% percentage of similarity to sub-genotype A1 for the genotype A sequences only. Discussion Discussion on the interpretation of mutations in treatment-naïve has been revised. This includes clarity on the impact of the mutations on the treatment’s status of the participants. We also discuss how the study cannot answer on whether the treatment was active against HBV (see the last paragraph under the discussion section). However, we made recommendation that further in vitro studies must investigate the mechanism of action by DRMs on ARTs in vitro assays (last sentence under conclusion). Additional correction and response Reference removed from the list 1. Kimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 1980; 16(2): 111–120. PubMed Abstract|Publisher Full Text. 2. Kumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief. Bioinform. 2004; 5(2): 150–163. PubMed Abstract|Publisher Full Text. 3. Malik A, Singhal DK, Albanyan A, et al.: Hepatitis B virus gene mutations in liver diseases: a report from New Delhi. PLoS One. 2012; 7(6): e39028. PubMed Abstract|Publisher Full Text|Free Full Text. 4. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987; 4(4): 406–425. . Updated references and citation 1. Belyhun, Yeshambel, Melanie Maier, and Uwe Gerd Liebert. "HIV therapy with unknown HBV status is responsible for higher rate of HBV genome variability in Ethiopia." Antiviral therapy 22.2 (2017): 97-111. 2. Geno2pheno. HBV genotyping 2023 [Available from: https://hbv.geno2pheno.org/. 3. World Health Organization. Guidelines on Hepatitis B and C Testing 2017 update (available at https://apps.who.int/iris/bitstream/handle/10665/254621/9789241549981- eng.pdf.). Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Prabdial-Sing N. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.163315.r284915 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v2#referee-response-284915 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 03 Jun 2024 Nishi Prabdial-Sing , Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.163315.r284915 Thank you to the authors for addressing some of the reviewer’s comments. There are still grammatical errors in the abstract. Abstract: Background: “…cross-resistance to drugs…”, not "of drugs". ... Continue reading READ ALL Thank you to the authors for addressing some of the reviewer’s comments. There are still grammatical errors in the abstract. Abstract: Background: “…cross-resistance to drugs…”, not "of drugs". Begin the next sentence “Continuous monitoring of HBV variants is required for better…” Methods: “Sera” is in the middle of the sentence and should be “sera”. Results: “Surface” is in the middle of the sentence and should be “surface”. Also as you found all genotype A to be subgenotype A1, then indicate A1 in your findings and conclusions. Introduction: The abbreviation “MHR”, needs to be explained when used for the first time. Results: The abbreviation “SHB”, needs to be explained when used for the first time. Discussion: “Therefore, this study should be considered…” should be written as: “This study determined the HBV genotype…” What do the authors mean by substandard immigration of new strains? in vitro should be in vitro (in italics) Competing Interests: No competing interests were disclosed. Reviewer Expertise: virology, diagnostics, molecular biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Prabdial-Sing N. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.163315.r284915 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v2#referee-response-284915 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 27 Sep 2023 Views 0 Cite How to cite this report: McNaughton A. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.145424.r224163 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v1#referee-response-224163 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 22 Nov 2023 Anna McNaughton , Nuffield Department of Medicine, University of Oxford Medawar Building, Oxford, England, UK Not Approved VIEWS 0 https://doi.org/10.5256/f1000research.145424.r224163 In this study, the authors sequenced the HBsAg region from a number of samples from individuals co-infected with HIV and HBV. Whilst this data can be informative, there are a number of issues with the analysis, results are not consistent ... Continue reading READ ALL In this study, the authors sequenced the HBsAg region from a number of samples from individuals co-infected with HIV and HBV. Whilst this data can be informative, there are a number of issues with the analysis, results are not consistent with their metadata and the conclusions need re-evaluating before this manuscript can be reconsidered. The manuscript is also too long in several parts, and should be more focused on relevant information. Abstract - Sampling site should be mentioned in the abstract – important to understand how/where these patients were identified, and has a link to the genotype distribution. Introduction - Very lengthy, with a large amount of information on HBV biology that is not relevant to this study. The authors should make this section more concise, and focused on HBV/HIV coinfection in the region these patients were sampled from, and the implications of treatment. Sampling approach – it is unexpected that 100% of n=50 people tested for HIV would randomly also test positive for HBV via convenience sampling. Were samples screened in advance as part of the clinical testing? The available metadata online (Figshare) also seems to state that 43/50 samples were HBV HBsAg positive, with 7 samples either negative or missing so the authors need to clarify this result. This sampling approach also means that discussions about prevalence and comparing with other studies (in results/particularly in discussion) are not appropriate and should be removed from the study. Methods are too detailed and could be more concise. Figure 1 is not required in the paper, particularly if sequences were generated. PCR Amplification – the authors state that 41/50 amplicons were obtained but that they were not obtained for 11/50 samples – this is 52/50? Sequence analyses of overlapping surface/polymerase gene region - The authors state that ‘Phylogenetic tree analysis identified nucleotide sequences from this study as genotype A as depicted in (Figure 2) but the current analysis has issues and cannot be used to inform genotyping assumptions as a result (see below). Phylogenetic analysis/Figure 2 - Authors should say what type of phylogenetic analysis this was, and how many study sequences were included in it. What length were these sequences? List of reference sequences listed in Figshare does not tally with the sequences in the tree where there appears to be more sequences? Unclear why this is. Methods says bootstrap analysis was done but the data is not presented on the tree anywhere. There appears to be a few issues with the clustering of the reference sequences in the tree – the clade at the bottom shows genotypes D/B/E/C clustering, but then other genotype B/C sequences can be seen to cluster with genotypes F/H at the top of the tree? Sequences of the same genotype should cluster together and they do not appear to be in this analysis. This analysis needs revising. For the mutation analysis, it is unclear what reference sequence the authors were comparing their sequences to? I am not keen of the approach of simply comparing sequences to a reference sequence to determine ‘mutations’ as these are likely to mostly just be reflective of expected sequence diversity. I think looking at a more focused list of sequences with known phenotypic associations would be more informative (as has been done with the resistance-associated mutations). Some rephrasing might help also – for example, saying 47% of sequences had mutations could imply that 53% of sequences were identical in the region? Be clear that these are amino acid differences. Table 3 - add the specific drug the mutation provides resistance against. Table 4 needs revising to say n= and % for the age groups. Typo of Fisher’s exact test also. I think figure 3 can also be removed – this is just showing that sequences more distant from a reference sequence in surface, and also more distant in RT. This is not surprising as the two regions overlap. Are the numbers listed amino acid positions in the sequences – it would be useful to know where in the genome this is? The conclusions need re-evaluating after addressing the concerns elsewhere in the paper. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? No Competing Interests: No competing interests were disclosed. Reviewer Expertise: Virology, genomics, epidemiology, viral hepatitis I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT McNaughton A. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.145424.r224163 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v1#referee-response-224163 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 31 May 2024 Lorato Mosetsanagape Modise , Biological Sciences, North West University, Mahikeng, 2735, South Africa 31 May 2024 Author Response Dear Reviewer We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The ... Continue reading Dear Reviewer We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below. In this study, the authors sequenced the HBsAg region from a number of samples from individuals co-infected with HIV and HBV. Whilst this data can be informative, there are several issues with the analysis, results are not consistent with their metadata and the conclusions need re-evaluating. before this manuscript can be reconsidered. The manuscript is also too long in several parts, and should be more focused on relevant information. Reviewer’s comments : Abstract - Sampling site should be mentioned in the abstract – important to understand how/where these patients were identified and has a link to the genotype distribution. Response: Yes, We have added information in the abstract on where the samples were collected (Inkosi Albert Luthuli Central Hospital) and how they were collected ( Convenience sampling). Introduction - Very lengthy, with a large amount of information on HBV biology that is not relevant to this study. The authors should make this section more concise and focused on HBV/HIV coinfection in the region these patients were sampled from, and the implications of treatment. Response : The introduction has been revised and shortened by removing information on the HBV biology and history. We have included information on the prevalence of HBV/HIV coinfection in the study region and implication of treatment. Sampling approach – it is unexpected that 100% of n=50 people tested for HIV would randomly also test positive for HBV via convenience sampling. Were samples screened in advance as part of the clinical testing? The available metadata online (Figshare) also seems to state that 43/50 samples were HBV HBsAg positive, with 7 samples either negative or missing so the authors need to clarify this result. This sampling approach also means that discussions about prevalence and comparing with other studies (in results/particularly in discussion) are not appropriate and should be removed from the study. Response : The sampling method and sample size is now clarified in the methodology section in page 3 under the title “Study design and population”. Convenience sampling was used to collect 43/50 samples that were previously tested for HIV and HBsAg. The sample size has been revised by reporting the correct size which correspond to the metadata value. The confirmatory screening of HBsAg reported a prevalence of 86% (N=43/50) with 12% (N=6/50) being negative and2% (N=1/50) missing data (Table 1). Reviewer comment: Methods are too detailed and could be more concise. Response : The methods have been revised and summarized. Figure 1 is not required in the paper, particularly if sequences were generated. Response : Figure has been removed from the paper and included as supplementary data on the repository site. PCR Amplification – the authors state that 41/50 amplicons were obtained but that they were not obtained for 11/50 samples – this is 52/50? Response : The result of failed PCR amplification has been corrected to 08/50 instead of 11/50. Sequence analyses of overlapping surface/polymerase gene region ‘Phylogenetic tree analysis identified nucleotide sequences from this study as genotype A as depicted in (Figure 2) but the current analysis has issues and cannot be used to inform genotyping assumptions as a result (see below). Phylogenetic analysis/Figure 2 - Authors should say what type of phylogenetic analysis this was, and how many study sequences were included in it. What length were these sequences? List of reference sequences listed in Figshare does not tally with the sequences in the tree? there appears to be more sequences. Unclear why this is. Methods says bootstrap analysis was done but the data is not presented on the tree anywhere. There appears to be a few issues with the clustering of the reference sequences in the tree – the clade at the bottom show's genotypes D/B/E/C clustering, but then other genotype B/C sequences can be seen to cluster with genotypes F/H at the top of the tree? Sequences of the same genotype should cluster together, and they do not appear to be in this analysis. This analysis needs revising. Response: The prevalence of mutations associated with drug resistance has been revised, to focus only on reporting the mutations associated with specific drug (shown in section titled: Mutations within the polymerase region and table 3) The phylogenetic tree results have been revised. The length these sequences is reported under the PCR amplification results as 547 bp in size and included 24 sequences and 39 reference sequences. The reference sequences listed in the metadata included some of the sequences and not all the reference sequences, but the list has been revised to correspond to the phylogenetic tree reference sequences. The neighbor joining method was used to construct a tree. The clustering of the sequences was revised, and the clustering of the sequences was mixed due to the short PCR product of 547 bp, unlike the full genome which would have given a homogenous clustering pattern. The sequence clustering results have been revised. For the mutation analysis, it is unclear what reference sequence the authors were comparing their sequences to? I am not keen of the approach of simply comparing sequences to a reference sequence to determine ‘mutations’ as these are likely to mostly just be reflective of expected sequence diversity. I think looking at a more focused list of sequences with known phenotypic associations would be more informative (as has been done with the resistance-associated mutations). Some rephrasing might help also – for example, saying 47% of sequences had mutations could imply that 53% of sequences were identical in the region? Be clear that these are amino acid differences. Response: The sequences were uploaded into geno2pheno and compared against standard genome with reported mutations using bioinformatics and statistical packages to check for sequence diversity and mutations. We did not investigate the phenotypic associations these mutants have on the virus. The focus was on identifying the mutations in the overlapping pol and S and their clinical implications. Table 3 - add the specific drug the mutation provides resistance against. Response : Table 3 has been revised, the initial table has been removed and replaced with the showing specific drug associated with resistance Table 4 needs revising to say n= and % for the age groups. Typo of Fisher’s exact test also. I think figure 3 can also be removed – this is just showing that sequences more distant from a reference sequence in surface, and also more distant in RT. This is not surprising as the two regions overlap. Are the numbers listed amino acid positions in the sequences – it would be useful to know where in the genome this is? Response: Table 4 has been removed. The conclusions need re-evaluating after addressing the concerns elsewhere in the paper. Response: The conclusion has been revised. The sequences of the studies Q cluster with each other and this is what is shown with my study sequences. There is a clustering between B/C and F/H reference sequences. The sequences were compared with the reference sequences on Geno2Pheno, which is an online server tool. Sequence alignment to the deposited HBV consensus sequence of the respective genotype. We looked at genetic mutations, not phenotypic mutations. that will acquire in vitro studies which is not the focus of the study. Under results section the subheadings 1. mutations within the surface region and 2. mutations within the polymerase region have been combined and reported under the heading: Mutations analysis. Dear Reviewer We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below. In this study, the authors sequenced the HBsAg region from a number of samples from individuals co-infected with HIV and HBV. Whilst this data can be informative, there are several issues with the analysis, results are not consistent with their metadata and the conclusions need re-evaluating. before this manuscript can be reconsidered. The manuscript is also too long in several parts, and should be more focused on relevant information. Reviewer’s comments : Abstract - Sampling site should be mentioned in the abstract – important to understand how/where these patients were identified and has a link to the genotype distribution. Response: Yes, We have added information in the abstract on where the samples were collected (Inkosi Albert Luthuli Central Hospital) and how they were collected ( Convenience sampling). Introduction - Very lengthy, with a large amount of information on HBV biology that is not relevant to this study. The authors should make this section more concise and focused on HBV/HIV coinfection in the region these patients were sampled from, and the implications of treatment. Response : The introduction has been revised and shortened by removing information on the HBV biology and history. We have included information on the prevalence of HBV/HIV coinfection in the study region and implication of treatment. Sampling approach – it is unexpected that 100% of n=50 people tested for HIV would randomly also test positive for HBV via convenience sampling. Were samples screened in advance as part of the clinical testing? The available metadata online (Figshare) also seems to state that 43/50 samples were HBV HBsAg positive, with 7 samples either negative or missing so the authors need to clarify this result. This sampling approach also means that discussions about prevalence and comparing with other studies (in results/particularly in discussion) are not appropriate and should be removed from the study. Response : The sampling method and sample size is now clarified in the methodology section in page 3 under the title “Study design and population”. Convenience sampling was used to collect 43/50 samples that were previously tested for HIV and HBsAg. The sample size has been revised by reporting the correct size which correspond to the metadata value. The confirmatory screening of HBsAg reported a prevalence of 86% (N=43/50) with 12% (N=6/50) being negative and2% (N=1/50) missing data (Table 1). Reviewer comment: Methods are too detailed and could be more concise. Response : The methods have been revised and summarized. Figure 1 is not required in the paper, particularly if sequences were generated. Response : Figure has been removed from the paper and included as supplementary data on the repository site. PCR Amplification – the authors state that 41/50 amplicons were obtained but that they were not obtained for 11/50 samples – this is 52/50? Response : The result of failed PCR amplification has been corrected to 08/50 instead of 11/50. Sequence analyses of overlapping surface/polymerase gene region ‘Phylogenetic tree analysis identified nucleotide sequences from this study as genotype A as depicted in (Figure 2) but the current analysis has issues and cannot be used to inform genotyping assumptions as a result (see below). Phylogenetic analysis/Figure 2 - Authors should say what type of phylogenetic analysis this was, and how many study sequences were included in it. What length were these sequences? List of reference sequences listed in Figshare does not tally with the sequences in the tree? there appears to be more sequences. Unclear why this is. Methods says bootstrap analysis was done but the data is not presented on the tree anywhere. There appears to be a few issues with the clustering of the reference sequences in the tree – the clade at the bottom show's genotypes D/B/E/C clustering, but then other genotype B/C sequences can be seen to cluster with genotypes F/H at the top of the tree? Sequences of the same genotype should cluster together, and they do not appear to be in this analysis. This analysis needs revising. Response: The prevalence of mutations associated with drug resistance has been revised, to focus only on reporting the mutations associated with specific drug (shown in section titled: Mutations within the polymerase region and table 3) The phylogenetic tree results have been revised. The length these sequences is reported under the PCR amplification results as 547 bp in size and included 24 sequences and 39 reference sequences. The reference sequences listed in the metadata included some of the sequences and not all the reference sequences, but the list has been revised to correspond to the phylogenetic tree reference sequences. The neighbor joining method was used to construct a tree. The clustering of the sequences was revised, and the clustering of the sequences was mixed due to the short PCR product of 547 bp, unlike the full genome which would have given a homogenous clustering pattern. The sequence clustering results have been revised. For the mutation analysis, it is unclear what reference sequence the authors were comparing their sequences to? I am not keen of the approach of simply comparing sequences to a reference sequence to determine ‘mutations’ as these are likely to mostly just be reflective of expected sequence diversity. I think looking at a more focused list of sequences with known phenotypic associations would be more informative (as has been done with the resistance-associated mutations). Some rephrasing might help also – for example, saying 47% of sequences had mutations could imply that 53% of sequences were identical in the region? Be clear that these are amino acid differences. Response: The sequences were uploaded into geno2pheno and compared against standard genome with reported mutations using bioinformatics and statistical packages to check for sequence diversity and mutations. We did not investigate the phenotypic associations these mutants have on the virus. The focus was on identifying the mutations in the overlapping pol and S and their clinical implications. Table 3 - add the specific drug the mutation provides resistance against. Response : Table 3 has been revised, the initial table has been removed and replaced with the showing specific drug associated with resistance Table 4 needs revising to say n= and % for the age groups. Typo of Fisher’s exact test also. I think figure 3 can also be removed – this is just showing that sequences more distant from a reference sequence in surface, and also more distant in RT. This is not surprising as the two regions overlap. Are the numbers listed amino acid positions in the sequences – it would be useful to know where in the genome this is? Response: Table 4 has been removed. The conclusions need re-evaluating after addressing the concerns elsewhere in the paper. Response: The conclusion has been revised. The sequences of the studies Q cluster with each other and this is what is shown with my study sequences. There is a clustering between B/C and F/H reference sequences. The sequences were compared with the reference sequences on Geno2Pheno, which is an online server tool. Sequence alignment to the deposited HBV consensus sequence of the respective genotype. We looked at genetic mutations, not phenotypic mutations. that will acquire in vitro studies which is not the focus of the study. Under results section the subheadings 1. mutations within the surface region and 2. mutations within the polymerase region have been combined and reported under the heading: Mutations analysis. Competing Interests: No competing interests. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 31 May 2024 Lorato Mosetsanagape Modise , Biological Sciences, North West University, Mahikeng, 2735, South Africa 31 May 2024 Author Response Dear Reviewer We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The ... Continue reading Dear Reviewer We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below. In this study, the authors sequenced the HBsAg region from a number of samples from individuals co-infected with HIV and HBV. Whilst this data can be informative, there are several issues with the analysis, results are not consistent with their metadata and the conclusions need re-evaluating. before this manuscript can be reconsidered. The manuscript is also too long in several parts, and should be more focused on relevant information. Reviewer’s comments : Abstract - Sampling site should be mentioned in the abstract – important to understand how/where these patients were identified and has a link to the genotype distribution. Response: Yes, We have added information in the abstract on where the samples were collected (Inkosi Albert Luthuli Central Hospital) and how they were collected ( Convenience sampling). Introduction - Very lengthy, with a large amount of information on HBV biology that is not relevant to this study. The authors should make this section more concise and focused on HBV/HIV coinfection in the region these patients were sampled from, and the implications of treatment. Response : The introduction has been revised and shortened by removing information on the HBV biology and history. We have included information on the prevalence of HBV/HIV coinfection in the study region and implication of treatment. Sampling approach – it is unexpected that 100% of n=50 people tested for HIV would randomly also test positive for HBV via convenience sampling. Were samples screened in advance as part of the clinical testing? The available metadata online (Figshare) also seems to state that 43/50 samples were HBV HBsAg positive, with 7 samples either negative or missing so the authors need to clarify this result. This sampling approach also means that discussions about prevalence and comparing with other studies (in results/particularly in discussion) are not appropriate and should be removed from the study. Response : The sampling method and sample size is now clarified in the methodology section in page 3 under the title “Study design and population”. Convenience sampling was used to collect 43/50 samples that were previously tested for HIV and HBsAg. The sample size has been revised by reporting the correct size which correspond to the metadata value. The confirmatory screening of HBsAg reported a prevalence of 86% (N=43/50) with 12% (N=6/50) being negative and2% (N=1/50) missing data (Table 1). Reviewer comment: Methods are too detailed and could be more concise. Response : The methods have been revised and summarized. Figure 1 is not required in the paper, particularly if sequences were generated. Response : Figure has been removed from the paper and included as supplementary data on the repository site. PCR Amplification – the authors state that 41/50 amplicons were obtained but that they were not obtained for 11/50 samples – this is 52/50? Response : The result of failed PCR amplification has been corrected to 08/50 instead of 11/50. Sequence analyses of overlapping surface/polymerase gene region ‘Phylogenetic tree analysis identified nucleotide sequences from this study as genotype A as depicted in (Figure 2) but the current analysis has issues and cannot be used to inform genotyping assumptions as a result (see below). Phylogenetic analysis/Figure 2 - Authors should say what type of phylogenetic analysis this was, and how many study sequences were included in it. What length were these sequences? List of reference sequences listed in Figshare does not tally with the sequences in the tree? there appears to be more sequences. Unclear why this is. Methods says bootstrap analysis was done but the data is not presented on the tree anywhere. There appears to be a few issues with the clustering of the reference sequences in the tree – the clade at the bottom show's genotypes D/B/E/C clustering, but then other genotype B/C sequences can be seen to cluster with genotypes F/H at the top of the tree? Sequences of the same genotype should cluster together, and they do not appear to be in this analysis. This analysis needs revising. Response: The prevalence of mutations associated with drug resistance has been revised, to focus only on reporting the mutations associated with specific drug (shown in section titled: Mutations within the polymerase region and table 3) The phylogenetic tree results have been revised. The length these sequences is reported under the PCR amplification results as 547 bp in size and included 24 sequences and 39 reference sequences. The reference sequences listed in the metadata included some of the sequences and not all the reference sequences, but the list has been revised to correspond to the phylogenetic tree reference sequences. The neighbor joining method was used to construct a tree. The clustering of the sequences was revised, and the clustering of the sequences was mixed due to the short PCR product of 547 bp, unlike the full genome which would have given a homogenous clustering pattern. The sequence clustering results have been revised. For the mutation analysis, it is unclear what reference sequence the authors were comparing their sequences to? I am not keen of the approach of simply comparing sequences to a reference sequence to determine ‘mutations’ as these are likely to mostly just be reflective of expected sequence diversity. I think looking at a more focused list of sequences with known phenotypic associations would be more informative (as has been done with the resistance-associated mutations). Some rephrasing might help also – for example, saying 47% of sequences had mutations could imply that 53% of sequences were identical in the region? Be clear that these are amino acid differences. Response: The sequences were uploaded into geno2pheno and compared against standard genome with reported mutations using bioinformatics and statistical packages to check for sequence diversity and mutations. We did not investigate the phenotypic associations these mutants have on the virus. The focus was on identifying the mutations in the overlapping pol and S and their clinical implications. Table 3 - add the specific drug the mutation provides resistance against. Response : Table 3 has been revised, the initial table has been removed and replaced with the showing specific drug associated with resistance Table 4 needs revising to say n= and % for the age groups. Typo of Fisher’s exact test also. I think figure 3 can also be removed – this is just showing that sequences more distant from a reference sequence in surface, and also more distant in RT. This is not surprising as the two regions overlap. Are the numbers listed amino acid positions in the sequences – it would be useful to know where in the genome this is? Response: Table 4 has been removed. The conclusions need re-evaluating after addressing the concerns elsewhere in the paper. Response: The conclusion has been revised. The sequences of the studies Q cluster with each other and this is what is shown with my study sequences. There is a clustering between B/C and F/H reference sequences. The sequences were compared with the reference sequences on Geno2Pheno, which is an online server tool. Sequence alignment to the deposited HBV consensus sequence of the respective genotype. We looked at genetic mutations, not phenotypic mutations. that will acquire in vitro studies which is not the focus of the study. Under results section the subheadings 1. mutations within the surface region and 2. mutations within the polymerase region have been combined and reported under the heading: Mutations analysis. Dear Reviewer We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below. In this study, the authors sequenced the HBsAg region from a number of samples from individuals co-infected with HIV and HBV. Whilst this data can be informative, there are several issues with the analysis, results are not consistent with their metadata and the conclusions need re-evaluating. before this manuscript can be reconsidered. The manuscript is also too long in several parts, and should be more focused on relevant information. Reviewer’s comments : Abstract - Sampling site should be mentioned in the abstract – important to understand how/where these patients were identified and has a link to the genotype distribution. Response: Yes, We have added information in the abstract on where the samples were collected (Inkosi Albert Luthuli Central Hospital) and how they were collected ( Convenience sampling). Introduction - Very lengthy, with a large amount of information on HBV biology that is not relevant to this study. The authors should make this section more concise and focused on HBV/HIV coinfection in the region these patients were sampled from, and the implications of treatment. Response : The introduction has been revised and shortened by removing information on the HBV biology and history. We have included information on the prevalence of HBV/HIV coinfection in the study region and implication of treatment. Sampling approach – it is unexpected that 100% of n=50 people tested for HIV would randomly also test positive for HBV via convenience sampling. Were samples screened in advance as part of the clinical testing? The available metadata online (Figshare) also seems to state that 43/50 samples were HBV HBsAg positive, with 7 samples either negative or missing so the authors need to clarify this result. This sampling approach also means that discussions about prevalence and comparing with other studies (in results/particularly in discussion) are not appropriate and should be removed from the study. Response : The sampling method and sample size is now clarified in the methodology section in page 3 under the title “Study design and population”. Convenience sampling was used to collect 43/50 samples that were previously tested for HIV and HBsAg. The sample size has been revised by reporting the correct size which correspond to the metadata value. The confirmatory screening of HBsAg reported a prevalence of 86% (N=43/50) with 12% (N=6/50) being negative and2% (N=1/50) missing data (Table 1). Reviewer comment: Methods are too detailed and could be more concise. Response : The methods have been revised and summarized. Figure 1 is not required in the paper, particularly if sequences were generated. Response : Figure has been removed from the paper and included as supplementary data on the repository site. PCR Amplification – the authors state that 41/50 amplicons were obtained but that they were not obtained for 11/50 samples – this is 52/50? Response : The result of failed PCR amplification has been corrected to 08/50 instead of 11/50. Sequence analyses of overlapping surface/polymerase gene region ‘Phylogenetic tree analysis identified nucleotide sequences from this study as genotype A as depicted in (Figure 2) but the current analysis has issues and cannot be used to inform genotyping assumptions as a result (see below). Phylogenetic analysis/Figure 2 - Authors should say what type of phylogenetic analysis this was, and how many study sequences were included in it. What length were these sequences? List of reference sequences listed in Figshare does not tally with the sequences in the tree? there appears to be more sequences. Unclear why this is. Methods says bootstrap analysis was done but the data is not presented on the tree anywhere. There appears to be a few issues with the clustering of the reference sequences in the tree – the clade at the bottom show's genotypes D/B/E/C clustering, but then other genotype B/C sequences can be seen to cluster with genotypes F/H at the top of the tree? Sequences of the same genotype should cluster together, and they do not appear to be in this analysis. This analysis needs revising. Response: The prevalence of mutations associated with drug resistance has been revised, to focus only on reporting the mutations associated with specific drug (shown in section titled: Mutations within the polymerase region and table 3) The phylogenetic tree results have been revised. The length these sequences is reported under the PCR amplification results as 547 bp in size and included 24 sequences and 39 reference sequences. The reference sequences listed in the metadata included some of the sequences and not all the reference sequences, but the list has been revised to correspond to the phylogenetic tree reference sequences. The neighbor joining method was used to construct a tree. The clustering of the sequences was revised, and the clustering of the sequences was mixed due to the short PCR product of 547 bp, unlike the full genome which would have given a homogenous clustering pattern. The sequence clustering results have been revised. For the mutation analysis, it is unclear what reference sequence the authors were comparing their sequences to? I am not keen of the approach of simply comparing sequences to a reference sequence to determine ‘mutations’ as these are likely to mostly just be reflective of expected sequence diversity. I think looking at a more focused list of sequences with known phenotypic associations would be more informative (as has been done with the resistance-associated mutations). Some rephrasing might help also – for example, saying 47% of sequences had mutations could imply that 53% of sequences were identical in the region? Be clear that these are amino acid differences. Response: The sequences were uploaded into geno2pheno and compared against standard genome with reported mutations using bioinformatics and statistical packages to check for sequence diversity and mutations. We did not investigate the phenotypic associations these mutants have on the virus. The focus was on identifying the mutations in the overlapping pol and S and their clinical implications. Table 3 - add the specific drug the mutation provides resistance against. Response : Table 3 has been revised, the initial table has been removed and replaced with the showing specific drug associated with resistance Table 4 needs revising to say n= and % for the age groups. Typo of Fisher’s exact test also. I think figure 3 can also be removed – this is just showing that sequences more distant from a reference sequence in surface, and also more distant in RT. This is not surprising as the two regions overlap. Are the numbers listed amino acid positions in the sequences – it would be useful to know where in the genome this is? Response: Table 4 has been removed. The conclusions need re-evaluating after addressing the concerns elsewhere in the paper. Response: The conclusion has been revised. The sequences of the studies Q cluster with each other and this is what is shown with my study sequences. There is a clustering between B/C and F/H reference sequences. The sequences were compared with the reference sequences on Geno2Pheno, which is an online server tool. Sequence alignment to the deposited HBV consensus sequence of the respective genotype. We looked at genetic mutations, not phenotypic mutations. that will acquire in vitro studies which is not the focus of the study. Under results section the subheadings 1. mutations within the surface region and 2. mutations within the polymerase region have been combined and reported under the heading: Mutations analysis. Competing Interests: No competing interests. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Prabdial-Sing N. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.145424.r211197 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v1#referee-response-211197 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 27 Oct 2023 Nishi Prabdial-Sing , Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.145424.r211197 The authors undertook a small study to characterize HBV among HIV infected individuals. The evidence and findings that the authors have revealed in the study indicates the necessity to engage in larger studies and population size to understand the extent ... Continue reading READ ALL The authors undertook a small study to characterize HBV among HIV infected individuals. The evidence and findings that the authors have revealed in the study indicates the necessity to engage in larger studies and population size to understand the extent of HBV sequence variation and diversity seen in South African patients coinfected with HIV. The study provides remarkable technical aspects for many others to undertake studies such as these and the authors are commended on their scientific output. There were a few grammatical errors and writing of the manuscript requires improvement. It is imperative that the writing of sequence variation indicates that it is the virus that was sequenced and not host or patient genes, because this can cause confusion to the reader. This has been commented on in the manuscript but the authors need to identify more of these to correct throughout the paper. The discussion is too long and too much information is provided that can be summarized and placed more appropriately in the introduction, especially on the vaccine escape mutations. The discussion should focus on the findings from the study with comparison on other similar studies in other countries or within the same country. On the results and discussion, the scatter plot needs to be explained as the X- and Y-axes are not well labeled and the interpretation and relevance of the plot does not seem to be evident in the discussion. The statistical significance is commented on for the RT gene but not shown for the HBsAg gene, but the latter was commented on. The sample size is small to conclude from these statistical significant data. It is recommended that a statistician look at the data and recommend a direction. Overall, the manuscript requires major improvement to improve on the aim and discussion and to draw on strengths from the authors' findings. The edits and comments are available as a PDF attachment which can be found here . Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: virology, diagnostics, molecular biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Prabdial-Sing N. Reviewer Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.145424.r211197 ) The direct URL for this report is: https://f1000research.com/articles/12-1232/v1#referee-response-211197 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 30 May 2024 Lorato Mosetsanagape Modise , Biological Sciences, North West University, Mahikeng, 2735, South Africa 30 May 2024 Author Response Dear Reviewer, We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The ... Continue reading Dear Reviewer, We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below. Reviewer’s general comments : The authors undertook a small study to characterize HBV among HIV infected individuals. The evidence and findings that the authors have revealed in the study indicates the necessity to engage in larger studies and population size to understand the extent of HBV sequence variation and diversity seen in South African patients coinfected with HIV. The study provides remarkable technical aspects for many others to undertake studies such as these and the authors are commended on their scientific output. Reviewer’s comments : There were a few grammatical errors and writing of the manuscript requires improvement. It is imperative that the writing of sequence variation indicates that it is the virus that was sequenced and not host or patient genes, because this can cause confusion to the reader. This has been commented on in the manuscript, but the authors need to identify more of these to correct throughout the paper. Response 1 : Yes, we have made the necessary improvement on the grammar and writing of the paper. Response 2: We have made clarification on the use and writing of virus sequence and virus nucleotide sequences and HBV sequence instead of host or patients’ genes. Reviewer’s comments : The discussion is too long and too much information is provided that can be summarized and placed more appropriately in the introduction, especially on the vaccine escape mutations. The discussion should focus on the findings from the study with comparison on other similar studies in other countries or within the same country. Response : Thank you for noting this. We have excluded the opening of the discussion about the seroprevalence. The discussion section has been revised, summarised, and focusing more on the sequence analysis of the viruses (genotyping and mutations analysis). The information on the vaccine escape mutations has been moved to the introduction. Reviewer’s specific comments per section Abstract Reviewer’s comments : Add a line on where and how samples were collected? Response : The type of samples and the location where the samples were collected is now clarified in the abstract and methodology section in page 3 under the title “Study design and population” Reviewer’s comments : So, here you indicate statistical significance of RT mutations. Were the mutations in the HBsAg statistically significant? as compared to RT. Response : We have provided the statistical significance of association between the RT and HBsAg mutations as outlined in the abstract and results section on page 10 “Association of mutations on the surface and polymerase” Reviewer’s comments : The aim of the study be revised. Response: We have revised the aim of the study Methods Reviewers’ comments : Add a line on where and how samples were collected? Response: We added information of how and where the samples were collected. We also describe the sampling method and how the sample size was calculated. Results Reviewers’ comments : Is the prevalence of HBsAg correctly reported and how were samples collected? Response : The seroprevalence of HBV has been revised by reporting the correct percentage values of HBsAg. Reviewers’ comments : It is not clear what these numbers are? is the first number, the number of mutations found in the age group and the second number is a %? Figure 3 is not well explained as how to read this plot because the X-and Y- axes are not well labelled . Response : Table 4 and Figure 3 are removed. The probability of association between the RT and SHB which was represented on Table 4 and Figure 3 is reported as depicted on page 10 under section “Association of mutations on the surface and polymerase” to report that there was no statistical significance association between the SHB and RT mutations at P > 0.005. Phylogenetic analysis was also revised. Discussion Reviewer’s comments: This is not a valid finding as it may reflect how samples were chosen for the study (previous point above). Also, the sample size is very small and not representative. I would avoid starting the discussion with this. Rather begin at the sequence analyses as this is the strength of the study. Response 1 : Thank you for noting this. We have excluded the statement on sample size being statistically significant and included it as a study limitation (last paragraph under discussion) on page 11. Response 2 : We begin the discussion on the sequence analyses and focus on the genotype identification and diversity. Dear Reviewer, We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below. Reviewer’s general comments : The authors undertook a small study to characterize HBV among HIV infected individuals. The evidence and findings that the authors have revealed in the study indicates the necessity to engage in larger studies and population size to understand the extent of HBV sequence variation and diversity seen in South African patients coinfected with HIV. The study provides remarkable technical aspects for many others to undertake studies such as these and the authors are commended on their scientific output. Reviewer’s comments : There were a few grammatical errors and writing of the manuscript requires improvement. It is imperative that the writing of sequence variation indicates that it is the virus that was sequenced and not host or patient genes, because this can cause confusion to the reader. This has been commented on in the manuscript, but the authors need to identify more of these to correct throughout the paper. Response 1 : Yes, we have made the necessary improvement on the grammar and writing of the paper. Response 2: We have made clarification on the use and writing of virus sequence and virus nucleotide sequences and HBV sequence instead of host or patients’ genes. Reviewer’s comments : The discussion is too long and too much information is provided that can be summarized and placed more appropriately in the introduction, especially on the vaccine escape mutations. The discussion should focus on the findings from the study with comparison on other similar studies in other countries or within the same country. Response : Thank you for noting this. We have excluded the opening of the discussion about the seroprevalence. The discussion section has been revised, summarised, and focusing more on the sequence analysis of the viruses (genotyping and mutations analysis). The information on the vaccine escape mutations has been moved to the introduction. Reviewer’s specific comments per section Abstract Reviewer’s comments : Add a line on where and how samples were collected? Response : The type of samples and the location where the samples were collected is now clarified in the abstract and methodology section in page 3 under the title “Study design and population” Reviewer’s comments : So, here you indicate statistical significance of RT mutations. Were the mutations in the HBsAg statistically significant? as compared to RT. Response : We have provided the statistical significance of association between the RT and HBsAg mutations as outlined in the abstract and results section on page 10 “Association of mutations on the surface and polymerase” Reviewer’s comments : The aim of the study be revised. Response: We have revised the aim of the study Methods Reviewers’ comments : Add a line on where and how samples were collected? Response: We added information of how and where the samples were collected. We also describe the sampling method and how the sample size was calculated. Results Reviewers’ comments : Is the prevalence of HBsAg correctly reported and how were samples collected? Response : The seroprevalence of HBV has been revised by reporting the correct percentage values of HBsAg. Reviewers’ comments : It is not clear what these numbers are? is the first number, the number of mutations found in the age group and the second number is a %? Figure 3 is not well explained as how to read this plot because the X-and Y- axes are not well labelled . Response : Table 4 and Figure 3 are removed. The probability of association between the RT and SHB which was represented on Table 4 and Figure 3 is reported as depicted on page 10 under section “Association of mutations on the surface and polymerase” to report that there was no statistical significance association between the SHB and RT mutations at P > 0.005. Phylogenetic analysis was also revised. Discussion Reviewer’s comments: This is not a valid finding as it may reflect how samples were chosen for the study (previous point above). Also, the sample size is very small and not representative. I would avoid starting the discussion with this. Rather begin at the sequence analyses as this is the strength of the study. Response 1 : Thank you for noting this. We have excluded the statement on sample size being statistically significant and included it as a study limitation (last paragraph under discussion) on page 11. Response 2 : We begin the discussion on the sequence analyses and focus on the genotype identification and diversity. Competing Interests: No competing interest. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 30 May 2024 Lorato Mosetsanagape Modise , Biological Sciences, North West University, Mahikeng, 2735, South Africa 30 May 2024 Author Response Dear Reviewer, We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The ... Continue reading Dear Reviewer, We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below. Reviewer’s general comments : The authors undertook a small study to characterize HBV among HIV infected individuals. The evidence and findings that the authors have revealed in the study indicates the necessity to engage in larger studies and population size to understand the extent of HBV sequence variation and diversity seen in South African patients coinfected with HIV. The study provides remarkable technical aspects for many others to undertake studies such as these and the authors are commended on their scientific output. Reviewer’s comments : There were a few grammatical errors and writing of the manuscript requires improvement. It is imperative that the writing of sequence variation indicates that it is the virus that was sequenced and not host or patient genes, because this can cause confusion to the reader. This has been commented on in the manuscript, but the authors need to identify more of these to correct throughout the paper. Response 1 : Yes, we have made the necessary improvement on the grammar and writing of the paper. Response 2: We have made clarification on the use and writing of virus sequence and virus nucleotide sequences and HBV sequence instead of host or patients’ genes. Reviewer’s comments : The discussion is too long and too much information is provided that can be summarized and placed more appropriately in the introduction, especially on the vaccine escape mutations. The discussion should focus on the findings from the study with comparison on other similar studies in other countries or within the same country. Response : Thank you for noting this. We have excluded the opening of the discussion about the seroprevalence. The discussion section has been revised, summarised, and focusing more on the sequence analysis of the viruses (genotyping and mutations analysis). The information on the vaccine escape mutations has been moved to the introduction. Reviewer’s specific comments per section Abstract Reviewer’s comments : Add a line on where and how samples were collected? Response : The type of samples and the location where the samples were collected is now clarified in the abstract and methodology section in page 3 under the title “Study design and population” Reviewer’s comments : So, here you indicate statistical significance of RT mutations. Were the mutations in the HBsAg statistically significant? as compared to RT. Response : We have provided the statistical significance of association between the RT and HBsAg mutations as outlined in the abstract and results section on page 10 “Association of mutations on the surface and polymerase” Reviewer’s comments : The aim of the study be revised. Response: We have revised the aim of the study Methods Reviewers’ comments : Add a line on where and how samples were collected? Response: We added information of how and where the samples were collected. We also describe the sampling method and how the sample size was calculated. Results Reviewers’ comments : Is the prevalence of HBsAg correctly reported and how were samples collected? Response : The seroprevalence of HBV has been revised by reporting the correct percentage values of HBsAg. Reviewers’ comments : It is not clear what these numbers are? is the first number, the number of mutations found in the age group and the second number is a %? Figure 3 is not well explained as how to read this plot because the X-and Y- axes are not well labelled . Response : Table 4 and Figure 3 are removed. The probability of association between the RT and SHB which was represented on Table 4 and Figure 3 is reported as depicted on page 10 under section “Association of mutations on the surface and polymerase” to report that there was no statistical significance association between the SHB and RT mutations at P > 0.005. Phylogenetic analysis was also revised. Discussion Reviewer’s comments: This is not a valid finding as it may reflect how samples were chosen for the study (previous point above). Also, the sample size is very small and not representative. I would avoid starting the discussion with this. Rather begin at the sequence analyses as this is the strength of the study. Response 1 : Thank you for noting this. We have excluded the statement on sample size being statistically significant and included it as a study limitation (last paragraph under discussion) on page 11. Response 2 : We begin the discussion on the sequence analyses and focus on the genotype identification and diversity. Dear Reviewer, We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below. Reviewer’s general comments : The authors undertook a small study to characterize HBV among HIV infected individuals. The evidence and findings that the authors have revealed in the study indicates the necessity to engage in larger studies and population size to understand the extent of HBV sequence variation and diversity seen in South African patients coinfected with HIV. The study provides remarkable technical aspects for many others to undertake studies such as these and the authors are commended on their scientific output. Reviewer’s comments : There were a few grammatical errors and writing of the manuscript requires improvement. It is imperative that the writing of sequence variation indicates that it is the virus that was sequenced and not host or patient genes, because this can cause confusion to the reader. This has been commented on in the manuscript, but the authors need to identify more of these to correct throughout the paper. Response 1 : Yes, we have made the necessary improvement on the grammar and writing of the paper. Response 2: We have made clarification on the use and writing of virus sequence and virus nucleotide sequences and HBV sequence instead of host or patients’ genes. Reviewer’s comments : The discussion is too long and too much information is provided that can be summarized and placed more appropriately in the introduction, especially on the vaccine escape mutations. The discussion should focus on the findings from the study with comparison on other similar studies in other countries or within the same country. Response : Thank you for noting this. We have excluded the opening of the discussion about the seroprevalence. The discussion section has been revised, summarised, and focusing more on the sequence analysis of the viruses (genotyping and mutations analysis). The information on the vaccine escape mutations has been moved to the introduction. Reviewer’s specific comments per section Abstract Reviewer’s comments : Add a line on where and how samples were collected? Response : The type of samples and the location where the samples were collected is now clarified in the abstract and methodology section in page 3 under the title “Study design and population” Reviewer’s comments : So, here you indicate statistical significance of RT mutations. Were the mutations in the HBsAg statistically significant? as compared to RT. Response : We have provided the statistical significance of association between the RT and HBsAg mutations as outlined in the abstract and results section on page 10 “Association of mutations on the surface and polymerase” Reviewer’s comments : The aim of the study be revised. Response: We have revised the aim of the study Methods Reviewers’ comments : Add a line on where and how samples were collected? Response: We added information of how and where the samples were collected. We also describe the sampling method and how the sample size was calculated. Results Reviewers’ comments : Is the prevalence of HBsAg correctly reported and how were samples collected? Response : The seroprevalence of HBV has been revised by reporting the correct percentage values of HBsAg. Reviewers’ comments : It is not clear what these numbers are? is the first number, the number of mutations found in the age group and the second number is a %? Figure 3 is not well explained as how to read this plot because the X-and Y- axes are not well labelled . Response : Table 4 and Figure 3 are removed. The probability of association between the RT and SHB which was represented on Table 4 and Figure 3 is reported as depicted on page 10 under section “Association of mutations on the surface and polymerase” to report that there was no statistical significance association between the SHB and RT mutations at P > 0.005. Phylogenetic analysis was also revised. Discussion Reviewer’s comments: This is not a valid finding as it may reflect how samples were chosen for the study (previous point above). Also, the sample size is very small and not representative. I would avoid starting the discussion with this. Rather begin at the sequence analyses as this is the strength of the study. Response 1 : Thank you for noting this. We have excluded the statement on sample size being statistically significant and included it as a study limitation (last paragraph under discussion) on page 11. Response 2 : We begin the discussion on the sequence analyses and focus on the genotype identification and diversity. Competing Interests: No competing interest. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 4 VERSION 4 PUBLISHED 27 Sep 2023 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 4 Version 4 (revision) 21 Aug 25 read Version 3 (revision) 23 Apr 25 read read read Version 2 (revision) 30 May 24 read read Version 1 27 Sep 23 read read Nishi Prabdial-Sing , National Institute for Communicable Diseases, Johannesburg, South Africa Anna McNaughton , University of Oxford Medawar Building, Oxford, UK Anthony C. Ike , University of Nigeria, Nsukka, Nigeria Tesfaye Gelanew , Armauer Hansen Research Institute, Addis Ababa, Ethiopia Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Gelanew T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 11 Sep 2025 | for Version 4 Tesfaye Gelanew , Armauer Hansen Research Institute, Addis Ababa, Addis Ababa, Ethiopia 0 Views copyright © 2025 Gelanew T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions I have no further comment. I endorsed the manuscript for indexing. Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Gelanew T. Peer Review Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.186606.r407676) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-1232/v4#referee-response-407676 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Prabdial-Sing N. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 05 Aug 2025 | for Version 3 Nishi Prabdial-Sing , Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa 0 Views copyright © 2025 Prabdial-Sing N. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Thank you to the authors for addressing some of the reviewer’s comments. There are still grammatical errors in the abstract. Abstract: Background ‘”cross-resistance of drugs”” what does this mean exactly, that sites on the HIV and HBV genome have shown cross-resistance of HIV antiviral drugs and to HBV antiviral drugs? I have not seen this and should be better written Begin the next sentence “Continuous monitoring of HBV variants is required for better…” Results: Also as you found all genotype A to be subgenotype A1, then indicate A1 in your findings and conclusions Genotype c, should be C Introduction: The abbreviation “DRM”, needs to be explained when used for the first time Method English should be English used in according should be used according Results: Mutations showed resistance to lamivudine (LMV) at (35% [5/14]), telbivudine (LdT) at (29% [4/14]), (14% [2/14]) for entecavir (ETV) and (21% [3/14]) for adefovir (ADV). Mutations causing resistance to LMV and LdT were M204V, L180M, V163I, and S202K” “the statement implies that the patients were treated for HBV and these DRMs were found. However, it was mentioned that these patients were Treatment naïve? Please clarify whether the patients were treatment naïve for HIV or HBV or both? Discussion: This suggest that the mutant HBV is common in both naive and experience HIV positive people””. This sentence is not referenced. There is only one reference for naïve, but not HIV treated persons. Conclusion thorough-synchronizes diagnosis” what does this mean? What do the authors mean by substandard immigration of new strains? Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Prabdial-Sing N. Peer Review Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.179250.r380330) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-1232/v3#referee-response-380330 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Gelanew T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 28 Jul 2025 | for Version 3 Tesfaye Gelanew , Armauer Hansen Research Institute, Addis Ababa, Addis Ababa, Ethiopia 0 Views copyright © 2025 Gelanew T. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript by Modise et al entitled "Investigation of Hepatitis B Virus Mutations Associated with Immune Escape and Drug Resistance in HIV-Infected Patients" is highly informative, and I concur with the authors’ observation that most studies conducted in Africa focus primarily on prevalence, while only a limited number address genotyping and drug resistance profiling. The paper is well written, with sound methodological approaches. The conclusions are appropriately toned down, clearly derived from the data, and the limitations are well articulated. I recommend acceptance in its current form. However, I have the following few minor comments. 1. - The title lacks specificity. It should be revised to: Investigation of Mutations in the Partial Sequences of the S and Polymerase Genes of Hepatitis B Virus Associated with Immune Escape and Drug Resistance in HIV-Infected Patients 2. - The introduction would benefit from expanding the brief overview of the specific genes targeted in this study—namely those encoding the surface antigen and polymerase—and their roles in immune escape and antiviral drug resistance. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise My expertise is clinical microbiology and Immunology/molecular biology. My ongoing research areas are dengue virus, HBV, RSV, and pathogens causing STIs. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Gelanew T. Peer Review Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.179250.r393935) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-1232/v3#referee-response-393935 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Ike A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 14 Jun 2025 | for Version 3 Anthony C. Ike , University of Nigeria, Nsukka, Enugu, Nigeria 0 Views copyright © 2025 Ike A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The article is well designed and carried out and was also well written or corrected to bring it to its present level. I have made few observations as listed below: In Page 4 under DNA extraction of HBV in Lines 4 and 10, the Eppendorf tube is 1.5 mL and not 1.5 μL. In the same page under Polymerase chain reaction First round and nested-PCR, in Line 8 of that section, the authors need to state whether the 0.125 of Taq DNA polymerase is the concentration (units) or volume (μL) of the enzyme. Under Nested PCR, the last line of the section, Line 6, the authors should state what was used as positive control and state the source if possible. In page 6 under laboratory testing HBsAg assay Line 2 of the section, it should be females and not female's. Also the HBsAg positive for the females should be 91% (N=29/32) and not 58% (N=29/50), since there were only 32 females and not 50. similarly that of the males should be 78% (N=14/18) and not 28% (N=14/50), males ere 18 and not 50. This should be corrected in Table 1 and be extended to the results of HIV (+) Female and HIV (+) Male. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Medical Microbiology with Virology bias. I have researched and published works on Noroviruses, Rotaviruses, influenza virus, hepatitis B and C viruses, Dengue virus among others. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Ike AC. Peer Review Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.179250.r386531) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-1232/v3#referee-response-386531 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 McNaughton A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 06 Jun 2024 | for Version 2 Anna McNaughton , Nuffield Department of Medicine, University of Oxford Medawar Building, Oxford, England, UK 0 Views copyright © 2024 McNaughton A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors have clearly revised the manuscript, taking into account some of the feedback provided previously and their metadata now looks to be more accurate. However, there are still several issues with the revised document, and particularly the analysis and presentation of the results which need correcting before the manuscript can be approved. HBsAg assay – COV needs defining at first use. Baseline demographic data – the median age is presented and the range is but I can’t see the standard deviation, despite the authors stating it in the paragraph. Table 1 should be revised to be more informative (i.e. present proportions) – if all individuals were black africans and HIV positive, this is not particularly useful information to present? I would encourage the authors to revise the data in table 1 to make it more informative, such as is there any data on their HIV treatment? Phylogenetic analysis – the authors state that sequences were obtained for 38/41 samples that were PCR positive, but only analysed n=26 sequences. Why were 12 sequences excluded from the analysis? The sequences analysed are relatively short, and as the authors state, this can affect the clustering but the phylogenetic analysis presented remains very problematic for genotyping. The reference sequences do not cluster by genotype, making it difficult to use the tree to confidently genotype the sequences presented. Whilst the authors checked a number of gtA the sequences with geno2pheno, I am puzzled by the interpretation of the other sequences (assumed not to be gtA). A number of sequences do not clearly cluster with any of the reference sequences, and the authors describe these as “24% (N=7/26) as genotypes B, C, D, E, F and G”, which is difficult to interpret. It is also a little unclear why sequences at the bottom of the tree ‘Q6P7, Q17P7, and Q42P7’ were decided to be closer to genotype H, D, B, and E than to genotype A without presenting bootstrapping data? This analysis needs revising, and perhaps the authors should consider a maximum-likelihood analysis as it may be more discriminatory for genotyping than the approach currently used. Alternatively, they may consider using geno2pheno to genotype their sequences, and present this data in a table rather than a tree? Association of mutations on the surface and polymerase regions – authors state the mean and standard deviation of the SHB mutations, but only provide a single number? Are the numbers presented also the number of mutations per sequence, as this is not clear? The discussion is too long and should be more concise – the discussion of the wider literature on drug resistant mutations could be reduced. Some context may also be useful to discuss – these samples were taken from HIV-positive individuals, so were any of them on treatment for their HIV infections and were these treatments active against HBV also? The authors mention that some patients are ART treatment naïve late in the discussion – but a little more detail on this would add to the interpretation of this study. Competing Interests No competing interests were disclosed. Reviewer Expertise Virology, genomics, epidemiology, viral hepatitis I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (1) Author Response 17 Sep 2025 Lorato Mosetsanagape Modise, Biological Sciences, North West University, Mahikeng, 2735, South Africa Dear reviewer We truly appreciate knowledge, and insightful comments on our paper. We appreciate your reviews and recommendations, which have greatly enhanced the manuscript. We have carefully considered all your comments and revised them accordingly. Specific reviewer comment HBsAg assay – COV needs defining at first use. Response : Cut off value (COV) has been defined at first use. Reviewer comments Baseline demographic data – the median age is presented, and the range is, but I can’t see the standard deviation, despite the authors stating it in the paragraph. Table 1 should be revised to be more informative (i.e. present proportions) – if all individuals were black Africans and HIV positive, this is not particularly useful information to present. I would encourage the authors to revise the data in table 1 to make it more informative, such as is there any data on their HIV treatment? Response : Since all the samples were from the same group, the information on the ethnicity of Black African has been deleted from the description in Table 1. The description of the statistical analysis has been revised in the text and Table 1 to be specific to the standard deviation. The median age is presented with the interquartile range (IQR) the range and did not present the standard deviation because the data in not normally distributed and there is poor statistical power. Table 1 presents the demographic data collected from the participants which includes the age, sex and test done before. The data is already presented in proportions expressed by either a count by the total number and the proportion as a percentage. The age is already presented in proportions as indicated in the text (n=19/50). The results are on demographic data which is the primary data on the participants HIV status. We can only report on the data that was collected and available for this study, The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. This is highlighted in the limitations section. Reviewer comments Phylogenetic analysis – the authors state that sequences were obtained for 38/41 samples that were PCR positive but only analysed n=26 sequences. Why were 12 sequences excluded from the analysis? The sequences analysed are relatively short, and as the authors state, this can affect the clustering, but the phylogenetic analysis presented remains very problematic for genotyping. The reference sequences do not cluster by genotype, making it difficult to use the tree to confidently genotype the sequences presented. Whilst the authors checked a few gtA the sequences with geno2pheno, I am puzzled by the interpretation of the other sequences (assumed not to be gtA). A few sequences do not clearly cluster with any of the reference sequences, and the authors describe these as “24% (N=7/26) as genotypes B, C, D, E, F and G”, which is difficult to interpret. It is also a little unclear why sequences at the bottom of the tree ‘Q6P7, Q17P7, and Q42P7’ were decided to be closer to genotype H, D, B, and E than to genotype A without presenting bootstrapping data? This analysis needs revising, and perhaps the authors should consider a maximum-likelihood analysis as it may be more discriminatory for genotyping than the approach currently used. Alternatively, they may consider using geno2pheno to genotype their sequences, Response : The 12 sequences excluded from the analysis had poor sequence reads on the chromatogram, and they could not form the proper consensus sequences thus they were not considered for the analysis. The genotyping of the sequences is revised with the use of geno2pheno software as advised and the data is presented in a table instead of a phylogenetic tree (Table 2). The sequences were uploaded onto the geno2pheno software and compared against standard (wildtype) genome with the highest nucleotide similarity to the study sequences. The sequences that identify as genotype A have been clarified through revision. Additionally, the 24% (N=7/26) that represent a combination of other genotypes (C and G) have been broken down to demonstrate the individual and specific genotypes C and G; 12% (N=3/26) represent genotypes G, and 15% (N=4/26) represent genotype C. Association of the mutations Data on the association of mutations on the surface and polymerase regions has been removed due to the poor statistical power and statistical association. We focused on reporting the data on the mutations associated with drug resistance with a focus on their frequency as depicted in Table 3. Reviewer comment The discussion is too long and should be more concise – the discussion of the wider literature on drug resistant mutations could be reduced. Some contexts may also be useful to discuss – these samples were taken from HIV-positive individuals, so were any of them on treatment for their HIV infections and were these treatments active against HBV also? Response : The discussion has been summarised. The data on the treatment history of the participants specifically HIV treatment is not available hence, it is not included. Secondly, we don’t know if HIV treatments is active against HBV because we do not have the information currently available to support that. However, we know the treatment history of HBV and that the participants are naïve to HBV ARTs. We have also added information on the results interpretation of the mutations in HBV patients that are HBV treatment naïve (last paragraph under the discussion section). Results Table 1: The median age was 33 years [IQR: 18-55] and there was no statistical difference on age between the genders with p=0.8. 92% (N=38/41) sequence products could be obtained by Sanger sequencing with only 26/38 sequences used to perform genotyping, that is, 12 sequences were excluded due to the poor quality of their sequence reads. The genotype of the sequences was confirmed by depositing nucleotide sequences into the Genotype2pheno database, which showed that most of the nucleotide sequences had homology with genotype A at 73% (N=19/26), genotype G at 12% (N=3/26), and genotype C at 15% (N=4/26) (Table 2). The study sequences with the highest similarity to Genotype A were: (Q4P7 and Q7P7 to KX520697.1|South Africa; Q9P7 to U87728.1|South Africa; Q27P7 to AY233274.1|South Africa. Reference sequence of AY233277.1|South Africa had between 97%-99% similarity to fifteen sequences (Q5P7, Q8P7, Q10P7, Q13P7, Q18P7, Q19P7, Q20P7, Q21P7, Q22P7, Q23P7, Q24P7, Q26P7, Q27P7, Q29P7 and Q39P7). The sequences Q6P7, Q17P7 and Q42P7 had similarity to genotype G (EU694179.1|South Africa; sequences Q11P7, Q43P7, Q44P7 and Q45P7 showed similarity to genotype C AB562444.1|Vietnam (Table 2). Sub-genotypes of the sequences were confirmed by depositing nucleotides sequences into the Genotype2pheno database. The results retrieved from the Geno2Pheno database had a 96.85%-99.0% percentage of similarity to sub-genotype A1 for the genotype A sequences only. Discussion Discussion on the interpretation of mutations in treatment-naïve has been revised. This includes clarity on the impact of the mutations on the treatment’s status of the participants. We also discuss how the study cannot answer on whether the treatment was active against HBV (see the last paragraph under the discussion section). However, we made recommendation that further in vitro studies must investigate the mechanism of action by DRMs on ARTs in vitro assays (last sentence under conclusion). Additional correction and response Reference removed from the list 1. Kimura M: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 1980; 16(2): 111–120. PubMed Abstract|Publisher Full Text. 2. Kumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief. Bioinform. 2004; 5(2): 150–163. PubMed Abstract|Publisher Full Text. 3. Malik A, Singhal DK, Albanyan A, et al.: Hepatitis B virus gene mutations in liver diseases: a report from New Delhi. PLoS One. 2012; 7(6): e39028. PubMed Abstract|Publisher Full Text|Free Full Text. 4. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987; 4(4): 406–425. . Updated references and citation 1. Belyhun, Yeshambel, Melanie Maier, and Uwe Gerd Liebert. "HIV therapy with unknown HBV status is responsible for higher rate of HBV genome variability in Ethiopia." Antiviral therapy 22.2 (2017): 97-111. 2. Geno2pheno. HBV genotyping 2023 [Available from: https://hbv.geno2pheno.org/. 3. World Health Organization. Guidelines on Hepatitis B and C Testing 2017 update (available at https://apps.who.int/iris/bitstream/handle/10665/254621/9789241549981- eng.pdf.). View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern McNaughton A. Peer Review Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.163315.r284916) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-1232/v2#referee-response-284916 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2024 Prabdial-Sing N. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 03 Jun 2024 | for Version 2 Nishi Prabdial-Sing , Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa 0 Views copyright © 2024 Prabdial-Sing N. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Thank you to the authors for addressing some of the reviewer’s comments. There are still grammatical errors in the abstract. Abstract: Background: “…cross-resistance to drugs…”, not "of drugs". Begin the next sentence “Continuous monitoring of HBV variants is required for better…” Methods: “Sera” is in the middle of the sentence and should be “sera”. Results: “Surface” is in the middle of the sentence and should be “surface”. Also as you found all genotype A to be subgenotype A1, then indicate A1 in your findings and conclusions. Introduction: The abbreviation “MHR”, needs to be explained when used for the first time. Results: The abbreviation “SHB”, needs to be explained when used for the first time. Discussion: “Therefore, this study should be considered…” should be written as: “This study determined the HBV genotype…” What do the authors mean by substandard immigration of new strains? in vitro should be in vitro (in italics) Competing Interests No competing interests were disclosed. Reviewer Expertise virology, diagnostics, molecular biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (0) Prabdial-Sing N. Peer Review Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.163315.r284915) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-1232/v2#referee-response-284915 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2023 McNaughton A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 22 Nov 2023 | for Version 1 Anna McNaughton , Nuffield Department of Medicine, University of Oxford Medawar Building, Oxford, England, UK 0 Views copyright © 2023 McNaughton A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Not Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions In this study, the authors sequenced the HBsAg region from a number of samples from individuals co-infected with HIV and HBV. Whilst this data can be informative, there are a number of issues with the analysis, results are not consistent with their metadata and the conclusions need re-evaluating before this manuscript can be reconsidered. The manuscript is also too long in several parts, and should be more focused on relevant information. Abstract - Sampling site should be mentioned in the abstract – important to understand how/where these patients were identified, and has a link to the genotype distribution. Introduction - Very lengthy, with a large amount of information on HBV biology that is not relevant to this study. The authors should make this section more concise, and focused on HBV/HIV coinfection in the region these patients were sampled from, and the implications of treatment. Sampling approach – it is unexpected that 100% of n=50 people tested for HIV would randomly also test positive for HBV via convenience sampling. Were samples screened in advance as part of the clinical testing? The available metadata online (Figshare) also seems to state that 43/50 samples were HBV HBsAg positive, with 7 samples either negative or missing so the authors need to clarify this result. This sampling approach also means that discussions about prevalence and comparing with other studies (in results/particularly in discussion) are not appropriate and should be removed from the study. Methods are too detailed and could be more concise. Figure 1 is not required in the paper, particularly if sequences were generated. PCR Amplification – the authors state that 41/50 amplicons were obtained but that they were not obtained for 11/50 samples – this is 52/50? Sequence analyses of overlapping surface/polymerase gene region - The authors state that ‘Phylogenetic tree analysis identified nucleotide sequences from this study as genotype A as depicted in (Figure 2) but the current analysis has issues and cannot be used to inform genotyping assumptions as a result (see below). Phylogenetic analysis/Figure 2 - Authors should say what type of phylogenetic analysis this was, and how many study sequences were included in it. What length were these sequences? List of reference sequences listed in Figshare does not tally with the sequences in the tree where there appears to be more sequences? Unclear why this is. Methods says bootstrap analysis was done but the data is not presented on the tree anywhere. There appears to be a few issues with the clustering of the reference sequences in the tree – the clade at the bottom shows genotypes D/B/E/C clustering, but then other genotype B/C sequences can be seen to cluster with genotypes F/H at the top of the tree? Sequences of the same genotype should cluster together and they do not appear to be in this analysis. This analysis needs revising. For the mutation analysis, it is unclear what reference sequence the authors were comparing their sequences to? I am not keen of the approach of simply comparing sequences to a reference sequence to determine ‘mutations’ as these are likely to mostly just be reflective of expected sequence diversity. I think looking at a more focused list of sequences with known phenotypic associations would be more informative (as has been done with the resistance-associated mutations). Some rephrasing might help also – for example, saying 47% of sequences had mutations could imply that 53% of sequences were identical in the region? Be clear that these are amino acid differences. Table 3 - add the specific drug the mutation provides resistance against. Table 4 needs revising to say n= and % for the age groups. Typo of Fisher’s exact test also. I think figure 3 can also be removed – this is just showing that sequences more distant from a reference sequence in surface, and also more distant in RT. This is not surprising as the two regions overlap. Are the numbers listed amino acid positions in the sequences – it would be useful to know where in the genome this is? The conclusions need re-evaluating after addressing the concerns elsewhere in the paper. Is the work clearly and accurately presented and does it cite the current literature? Partly Is the study design appropriate and is the work technically sound? No Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? No Competing Interests No competing interests were disclosed. Reviewer Expertise Virology, genomics, epidemiology, viral hepatitis I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. reply Respond to this report Responses (1) Author Response 31 May 2024 Lorato Mosetsanagape Modise, Biological Sciences, North West University, Mahikeng, 2735, South Africa Dear Reviewer We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below. In this study, the authors sequenced the HBsAg region from a number of samples from individuals co-infected with HIV and HBV. Whilst this data can be informative, there are several issues with the analysis, results are not consistent with their metadata and the conclusions need re-evaluating. before this manuscript can be reconsidered. The manuscript is also too long in several parts, and should be more focused on relevant information. Reviewer’s comments : Abstract - Sampling site should be mentioned in the abstract – important to understand how/where these patients were identified and has a link to the genotype distribution. Response: Yes, We have added information in the abstract on where the samples were collected (Inkosi Albert Luthuli Central Hospital) and how they were collected ( Convenience sampling). Introduction - Very lengthy, with a large amount of information on HBV biology that is not relevant to this study. The authors should make this section more concise and focused on HBV/HIV coinfection in the region these patients were sampled from, and the implications of treatment. Response : The introduction has been revised and shortened by removing information on the HBV biology and history. We have included information on the prevalence of HBV/HIV coinfection in the study region and implication of treatment. Sampling approach – it is unexpected that 100% of n=50 people tested for HIV would randomly also test positive for HBV via convenience sampling. Were samples screened in advance as part of the clinical testing? The available metadata online (Figshare) also seems to state that 43/50 samples were HBV HBsAg positive, with 7 samples either negative or missing so the authors need to clarify this result. This sampling approach also means that discussions about prevalence and comparing with other studies (in results/particularly in discussion) are not appropriate and should be removed from the study. Response : The sampling method and sample size is now clarified in the methodology section in page 3 under the title “Study design and population”. Convenience sampling was used to collect 43/50 samples that were previously tested for HIV and HBsAg. The sample size has been revised by reporting the correct size which correspond to the metadata value. The confirmatory screening of HBsAg reported a prevalence of 86% (N=43/50) with 12% (N=6/50) being negative and2% (N=1/50) missing data (Table 1). Reviewer comment: Methods are too detailed and could be more concise. Response : The methods have been revised and summarized. Figure 1 is not required in the paper, particularly if sequences were generated. Response : Figure has been removed from the paper and included as supplementary data on the repository site. PCR Amplification – the authors state that 41/50 amplicons were obtained but that they were not obtained for 11/50 samples – this is 52/50? Response : The result of failed PCR amplification has been corrected to 08/50 instead of 11/50. Sequence analyses of overlapping surface/polymerase gene region ‘Phylogenetic tree analysis identified nucleotide sequences from this study as genotype A as depicted in (Figure 2) but the current analysis has issues and cannot be used to inform genotyping assumptions as a result (see below). Phylogenetic analysis/Figure 2 - Authors should say what type of phylogenetic analysis this was, and how many study sequences were included in it. What length were these sequences? List of reference sequences listed in Figshare does not tally with the sequences in the tree? there appears to be more sequences. Unclear why this is. Methods says bootstrap analysis was done but the data is not presented on the tree anywhere. There appears to be a few issues with the clustering of the reference sequences in the tree – the clade at the bottom show's genotypes D/B/E/C clustering, but then other genotype B/C sequences can be seen to cluster with genotypes F/H at the top of the tree? Sequences of the same genotype should cluster together, and they do not appear to be in this analysis. This analysis needs revising. Response: The prevalence of mutations associated with drug resistance has been revised, to focus only on reporting the mutations associated with specific drug (shown in section titled: Mutations within the polymerase region and table 3) The phylogenetic tree results have been revised. The length these sequences is reported under the PCR amplification results as 547 bp in size and included 24 sequences and 39 reference sequences. The reference sequences listed in the metadata included some of the sequences and not all the reference sequences, but the list has been revised to correspond to the phylogenetic tree reference sequences. The neighbor joining method was used to construct a tree. The clustering of the sequences was revised, and the clustering of the sequences was mixed due to the short PCR product of 547 bp, unlike the full genome which would have given a homogenous clustering pattern. The sequence clustering results have been revised. For the mutation analysis, it is unclear what reference sequence the authors were comparing their sequences to? I am not keen of the approach of simply comparing sequences to a reference sequence to determine ‘mutations’ as these are likely to mostly just be reflective of expected sequence diversity. I think looking at a more focused list of sequences with known phenotypic associations would be more informative (as has been done with the resistance-associated mutations). Some rephrasing might help also – for example, saying 47% of sequences had mutations could imply that 53% of sequences were identical in the region? Be clear that these are amino acid differences. Response: The sequences were uploaded into geno2pheno and compared against standard genome with reported mutations using bioinformatics and statistical packages to check for sequence diversity and mutations. We did not investigate the phenotypic associations these mutants have on the virus. The focus was on identifying the mutations in the overlapping pol and S and their clinical implications. Table 3 - add the specific drug the mutation provides resistance against. Response : Table 3 has been revised, the initial table has been removed and replaced with the showing specific drug associated with resistance Table 4 needs revising to say n= and % for the age groups. Typo of Fisher’s exact test also. I think figure 3 can also be removed – this is just showing that sequences more distant from a reference sequence in surface, and also more distant in RT. This is not surprising as the two regions overlap. Are the numbers listed amino acid positions in the sequences – it would be useful to know where in the genome this is? Response: Table 4 has been removed. The conclusions need re-evaluating after addressing the concerns elsewhere in the paper. Response: The conclusion has been revised. The sequences of the studies Q cluster with each other and this is what is shown with my study sequences. There is a clustering between B/C and F/H reference sequences. The sequences were compared with the reference sequences on Geno2Pheno, which is an online server tool. Sequence alignment to the deposited HBV consensus sequence of the respective genotype. We looked at genetic mutations, not phenotypic mutations. that will acquire in vitro studies which is not the focus of the study. Under results section the subheadings 1. mutations within the surface region and 2. mutations within the polymerase region have been combined and reported under the heading: Mutations analysis. View more View less Competing Interests No competing interests. reply Respond Report a concern McNaughton A. Peer Review Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.145424.r224163) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-1232/v1#referee-response-224163 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2023 Prabdial-Sing N. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 27 Oct 2023 | for Version 1 Nishi Prabdial-Sing , Centre for Vaccines and Immunology, Division of the National Health Laboratory Service, National Institute for Communicable Diseases, Johannesburg, South Africa 0 Views copyright © 2023 Prabdial-Sing N. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors undertook a small study to characterize HBV among HIV infected individuals. The evidence and findings that the authors have revealed in the study indicates the necessity to engage in larger studies and population size to understand the extent of HBV sequence variation and diversity seen in South African patients coinfected with HIV. The study provides remarkable technical aspects for many others to undertake studies such as these and the authors are commended on their scientific output. There were a few grammatical errors and writing of the manuscript requires improvement. It is imperative that the writing of sequence variation indicates that it is the virus that was sequenced and not host or patient genes, because this can cause confusion to the reader. This has been commented on in the manuscript but the authors need to identify more of these to correct throughout the paper. The discussion is too long and too much information is provided that can be summarized and placed more appropriately in the introduction, especially on the vaccine escape mutations. The discussion should focus on the findings from the study with comparison on other similar studies in other countries or within the same country. On the results and discussion, the scatter plot needs to be explained as the X- and Y-axes are not well labeled and the interpretation and relevance of the plot does not seem to be evident in the discussion. The statistical significance is commented on for the RT gene but not shown for the HBsAg gene, but the latter was commented on. The sample size is small to conclude from these statistical significant data. It is recommended that a statistician look at the data and recommend a direction. Overall, the manuscript requires major improvement to improve on the aim and discussion and to draw on strengths from the authors' findings. The edits and comments are available as a PDF attachment which can be found here . Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise virology, diagnostics, molecular biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 30 May 2024 Lorato Mosetsanagape Modise, Biological Sciences, North West University, Mahikeng, 2735, South Africa Dear Reviewer, We appreciate the efforts, comments, and concerns in making the manuscript better. We have gone through the comments and edited the manuscript accordingly in the revised manuscript. The inputs made in response to your comments are listed below. Reviewer’s general comments : The authors undertook a small study to characterize HBV among HIV infected individuals. The evidence and findings that the authors have revealed in the study indicates the necessity to engage in larger studies and population size to understand the extent of HBV sequence variation and diversity seen in South African patients coinfected with HIV. The study provides remarkable technical aspects for many others to undertake studies such as these and the authors are commended on their scientific output. Reviewer’s comments : There were a few grammatical errors and writing of the manuscript requires improvement. It is imperative that the writing of sequence variation indicates that it is the virus that was sequenced and not host or patient genes, because this can cause confusion to the reader. This has been commented on in the manuscript, but the authors need to identify more of these to correct throughout the paper. Response 1 : Yes, we have made the necessary improvement on the grammar and writing of the paper. Response 2: We have made clarification on the use and writing of virus sequence and virus nucleotide sequences and HBV sequence instead of host or patients’ genes. Reviewer’s comments : The discussion is too long and too much information is provided that can be summarized and placed more appropriately in the introduction, especially on the vaccine escape mutations. The discussion should focus on the findings from the study with comparison on other similar studies in other countries or within the same country. Response : Thank you for noting this. We have excluded the opening of the discussion about the seroprevalence. The discussion section has been revised, summarised, and focusing more on the sequence analysis of the viruses (genotyping and mutations analysis). The information on the vaccine escape mutations has been moved to the introduction. Reviewer’s specific comments per section Abstract Reviewer’s comments : Add a line on where and how samples were collected? Response : The type of samples and the location where the samples were collected is now clarified in the abstract and methodology section in page 3 under the title “Study design and population” Reviewer’s comments : So, here you indicate statistical significance of RT mutations. Were the mutations in the HBsAg statistically significant? as compared to RT. Response : We have provided the statistical significance of association between the RT and HBsAg mutations as outlined in the abstract and results section on page 10 “Association of mutations on the surface and polymerase” Reviewer’s comments : The aim of the study be revised. Response: We have revised the aim of the study Methods Reviewers’ comments : Add a line on where and how samples were collected? Response: We added information of how and where the samples were collected. We also describe the sampling method and how the sample size was calculated. Results Reviewers’ comments : Is the prevalence of HBsAg correctly reported and how were samples collected? Response : The seroprevalence of HBV has been revised by reporting the correct percentage values of HBsAg. Reviewers’ comments : It is not clear what these numbers are? is the first number, the number of mutations found in the age group and the second number is a %? Figure 3 is not well explained as how to read this plot because the X-and Y- axes are not well labelled . Response : Table 4 and Figure 3 are removed. The probability of association between the RT and SHB which was represented on Table 4 and Figure 3 is reported as depicted on page 10 under section “Association of mutations on the surface and polymerase” to report that there was no statistical significance association between the SHB and RT mutations at P > 0.005. Phylogenetic analysis was also revised. Discussion Reviewer’s comments: This is not a valid finding as it may reflect how samples were chosen for the study (previous point above). Also, the sample size is very small and not representative. I would avoid starting the discussion with this. Rather begin at the sequence analyses as this is the strength of the study. Response 1 : Thank you for noting this. We have excluded the statement on sample size being statistically significant and included it as a study limitation (last paragraph under discussion) on page 11. Response 2 : We begin the discussion on the sequence analyses and focus on the genotype identification and diversity. View more View less Competing Interests No competing interest. reply Respond Report a concern Prabdial-Sing N. Peer Review Report For: Investigation of mutations in the partial sequences of the surface and polymerase genes of hepatitis B virus associated with immune escape and drug resistance in HIV-infected patients [version 4; peer review: 1 approved, 2 approved with reservations, 1 not approved] . F1000Research 2025, 12 :1232 ( https://doi.org/10.5256/f1000research.145424.r211197) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/12-1232/v1#referee-response-211197 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Adjust parameters to alter display View on desktop for interactive features Includes Interactive Elements View on desktop for interactive features Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list: Examples of 'Non-Financial Competing Interests' Within the past 4 years, you have held joint grants, published or collaborated with any of the authors of the selected paper. You have a close personal relationship (e.g. parent, spouse, sibling, or domestic partner) with any of the authors. You are a close professional associate of any of the authors (e.g. scientific mentor, recent student). You work at the same institute as any of the authors. You hope/expect to benefit (e.g. favour or employment) as a result of your submission. You are an Editor for the journal in which the article is published. Examples of 'Financial Competing Interests' You expect to receive, or in the past 4 years have received, any of the following from any commercial organisation that may gain financially from your submission: a salary, fees, funding, reimbursements. You expect to receive, or in the past 4 years have received, shared grant support or other funding with any of the authors. You hold, or are currently applying for, any patents or significant stocks/shares relating to the subject matter of the paper you are commenting on. Stay Updated Sign up for content alerts and receive a weekly or monthly email with all newly published articles Register with F1000Research Already registered? Sign in Not now, thanks close PLEASE NOTE If you are an AUTHOR of this article, please check that you signed in with the account associated with this article otherwise we cannot automatically identify your role as an author and your comment will be labelled as a “User Comment”. If you are a REVIEWER of this article, please check that you have signed in with the account associated with this article and then go to your account to submit your report, please do not post your review here. If you do not have access to your original account, please contact us . All commenters must hold a formal affiliation as per our Policies . The information that you give us will be displayed next to your comment. User comments must be in English, comprehensible and relevant to the article under discussion. We reserve the right to remove any comments that we consider to be inappropriate, offensive or otherwise in breach of the User Comment Terms and Conditions . Commenters must not use a comment for personal attacks. When criticisms of the article are based on unpublished data, the data should be made available. I accept the User Comment Terms and Conditions Please confirm that you accept the User Comment Terms and Conditions. Affiliation ✕ refresh Please enter your institution. Note: To add your institution or organisation, start typing the name and then select the correct name from the list. Where applicable, the name will appear in both the original language and in English. Do not paste in the name. If the name does not appear in the drop-down list, we will display the information you have entered. ✕ refresh Country/Region * USA UK Canada China France Germany Afghanistan Aland Islands Albania Algeria American Samoa Andorra Angola Anguilla Antarctica Antigua and Barbuda Argentina Armenia Aruba Australia Austria Azerbaijan Bahamas Bahrain Bangladesh Barbados Belarus Belgium Belize Benin Bermuda Bhutan Bolivia Bosnia and Herzegovina Botswana Bouvet Island Brazil British Indian Ocean Territory British Virgin Islands Brunei Bulgaria Burkina Faso Burundi Cambodia Cameroon Canada Cape Verde Cayman Islands Central African Republic Chad Chile China Christmas Island Cocos (Keeling) Islands Colombia Comoros Congo Cook Islands Costa Rica Cote d'Ivoire Croatia Cuba Cyprus Czech Republic Democratic Republic of the Congo Denmark Djibouti Dominica Dominican Republic Ecuador Egypt El Salvador Equatorial Guinea Eritrea Estonia Ethiopia Falkland Islands Faroe Islands Federated States of Micronesia Fiji Finland France French Guiana French Polynesia French Southern Territories Gabon Georgia Germany Ghana Gibraltar Greece Greenland Grenada Guadeloupe Guam Guatemala Guernsey Guinea Guinea-Bissau Guyana Haiti Heard Island and Mcdonald Islands Holy See (Vatican City State) Honduras Hong Kong Hungary Iceland India Indonesia Iran Iraq Ireland Israel Italy Jamaica Japan Jersey Jordan Kazakhstan Kenya Kiribati Kosovo (Serbia and Montenegro) Kuwait Kyrgyzstan Lao People's Democratic Republic Latvia Lebanon Lesotho Liberia Libya Liechtenstein Lithuania Luxembourg Macao Madagascar Malawi Malaysia Maldives Mali Malta Marshall Islands Martinique Mauritania Mauritius Mayotte Mexico Minor Outlying Islands of the United States Moldova Monaco Mongolia Montenegro Montserrat Morocco Mozambique Myanmar Namibia Nauru Nepal Netherlands Antilles New Caledonia New Zealand Nicaragua Niger Nigeria Niue Norfolk Island North Korea North Macedonia Northern Mariana Islands Norway Oman Pakistan Palau Palestinian Territory Panama Papua New Guinea Paraguay Peru Philippines Pitcairn Poland Portugal Puerto Rico Qatar Reunion Romania Russian Federation Rwanda Saint Helena Saint Kitts and Nevis Saint Lucia Saint Pierre and Miquelon Saint Vincent and the Grenadines Samoa San Marino Sao Tome and Principe Saudi Arabia Senegal Serbia Seychelles Sierra Leone Singapore Slovakia Slovenia Solomon Islands Somalia South Africa South Georgia and the South Sandwich Is South Korea South Sudan Spain Sri Lanka Sudan Suriname Svalbard and Jan Mayen Swaziland Sweden Switzerland Syria Taiwan Tajikistan Tanzania Thailand The Gambia The Netherlands Timor-Leste Togo Tokelau Tonga Trinidad and Tobago Tunisia Turkey Turkmenistan Turks and Caicos Islands Tuvalu UK USA Uganda Ukraine United Arab Emirates United States Virgin Islands Uruguay Uzbekistan Vanuatu Venezuela Vietnam Wallis and Futuna West Bank and Gaza Strip Western Sahara Yemen Zambia Zimbabwe Please select your country/region. You must enter a comment. Competing Interests Please disclose any competing interests that might be construed to influence your judgment of the article's or peer review report's validity or importance. Competing Interests Policy Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality. Consider the following examples, but note that this is not an exhaustive list: Examples of 'Non-Financial Competing Interests' Within the past 4 years, you have held joint grants, published or collaborated with any of the authors of the selected paper. You have a close personal relationship (e.g. parent, spouse, sibling, or domestic partner) with any of the authors. You are a close professional associate of any of the authors (e.g. scientific mentor, recent student). You work at the same institute as any of the authors. You hope/expect to benefit (e.g. favour or employment) as a result of your submission. You are an Editor for the journal in which the article is published. Examples of 'Financial Competing Interests' You expect to receive, or in the past 4 years have received, any of the following from any commercial organisation that may gain financially from your submission: a salary, fees, funding, reimbursements. You expect to receive, or in the past 4 years have received, shared grant support or other funding with any of the authors. You hold, or are currently applying for, any patents or significant stocks/shares relating to the subject matter of the paper you are commenting on. Please state your competing interests The comment has been saved. An error has occurred. Please try again. Cancel Post var lTitle = "Investigation of mutations in the partial...".replace("'", ''); var linkedInUrl = "http://www.linkedin.com/shareArticle?url=https://f1000research.com/articles/12-1232/v4" + "&title=" + encodeURIComponent(lTitle) + "&summary=" + encodeURIComponent('Read the article by '); var deliciousUrl = "https://del.icio.us/post?url=https://f1000research.com/articles/12-1232/v4&title=" + encodeURIComponent(lTitle); var redditUrl = "http://reddit.com/submit?url=https://f1000research.com/articles/12-1232/v4" + "&title=" + encodeURIComponent(lTitle); linkedInUrl += encodeURIComponent('Modise L et al.'); var offsetTop = /chrome/i.test( navigator.userAgent ) ? 4 : -10; var addthis_config = { ui_offset_top: offsetTop, services_compact : "facebook,twitter,www.linkedin.com,www.mendeley.com,reddit.com", services_expanded : "facebook,twitter,www.linkedin.com,www.mendeley.com,reddit.com", services_custom : [ { name: "LinkedIn", url: linkedInUrl, icon:"/img/icon/at_linkedin.svg" }, { name: "Mendeley", url: "http://www.mendeley.com/import/?url=https://f1000research.com/articles/12-1232/v4/mendeley", icon:"/img/icon/at_mendeley.svg" }, { name: "Reddit", url: redditUrl, icon:"/img/icon/at_reddit.svg" }, ] }; var addthis_share = { url: "https://f1000research.com/articles/12-1232", templates : { twitter : "Investigation of mutations in the partial sequences of the surface.... Modise L et al., published by " + "@F1000Research" + ", https://f1000research.com/articles/12-1232/v4" } }; if (typeof(addthis) != "undefined"){ addthis.addEventListener('addthis.ready', checkCount); addthis.addEventListener('addthis.menu.share', checkCount); } $(".f1r-shares-twitter").attr("href", "https://twitter.com/intent/tweet?text=" + addthis_share.templates.twitter); $(".f1r-shares-facebook").attr("href", "https://www.facebook.com/sharer/sharer.php?u=" + addthis_share.url); $(".f1r-shares-linkedin").attr("href", addthis_config.services_custom[0].url); $(".f1r-shares-reddit").attr("href", addthis_config.services_custom[2].url); $(".f1r-shares-mendelay").attr("href", addthis_config.services_custom[1].url); function checkCount(){ setTimeout(function(){ $(".addthis_button_expanded").each(function(){ var count = $(this).text(); if (count !== "" && count != "0") $(this).removeClass("is-hidden"); else $(this).addClass("is-hidden"); }); }, 1000); } close How to cite this report {{reportCitation}} Cancel Copy Citation Details $(function(){R.ui.buttonDropdowns('.dropdown-for-downloads');}); $(function(){R.ui.toolbarDropdowns('.toolbar-dropdown-for-downloads');}); $.get("/articles/acj/132498/186606") new F1000.Clipboard(); new F1000.ThesaurusTermsDisplay("articles", "article", "186606"); $(document).ready(function() { $( "#frame1" ).on('load', function() { var mydiv = $(this).contents().find("div"); var h = mydiv.height(); console.log(h) }); var tooltipLivingFigure = jQuery(".interactive-living-figure-label .icon-more-info"), titleLivingFigure = tooltipLivingFigure.attr("title"); tooltipLivingFigure.simpletip({ fixed: true, position: ["-115", "30"], baseClass: 'small-tooltip', content:titleLivingFigure + " " }); tooltipLivingFigure.removeAttr("title"); $("body").on("click", ".cite-living-figure", function(e) { e.preventDefault(); var ref = $(this).attr("data-ref"); $(this).closest(".living-figure-list-container").find("#" + ref).fadeIn(200); }); $("body").on("click", ".close-cite-living-figure", function(e) { e.preventDefault(); $(this).closest(".popup-window-wrapper").fadeOut(200); }); $(document).on("mouseup", function(e) { var metricsContainer = $(".article-metrics-popover-wrapper"); if (!metricsContainer.is(e.target) && metricsContainer.has(e.target).length === 0) { $(".article-metrics-close-button").click(); } }); var articleId = $('#articleId').val(); if($("#main-article-count-box").attachArticleMetrics) { $("#main-article-count-box").attachArticleMetrics(articleId, { articleMetricsView: true }); } }); var figshareWidget = $(".new_figshare_widget"); if (figshareWidget.length > 0) { window.figshare.load("f1000", function(Widget) { // Select a tag/tags defined in your page. In this tag we will place the widget. _.map(figshareWidget, function(el){ var widget = new Widget({ articleId: $(el).attr("figshare_articleId") //height:300 // this is the height of the viewer part. [Default: 550] }); widget.initialize(); // initialize the widget widget.mount(el); // mount it in a tag that's on your page // this will save the widget on the global scope for later use from // your JS scripts. This line is optional. //window.widget = widget; }); }); } close Error Close Add Reset F1000.MICROSERVICES.AFFILIATION = ''; $(document).ready(function () { $('.js-affiliations-form').each((index, form) => { new AffiliationForm({ formId: form.id, institutionErrorSelector: '.comment-enter-institution', departmentErrorSelector: '.comment-enter-department', placeSelector: '.js-add-comment-place', stateSelector: '.js-add-comment-state', zipCodeSelector: '.js-add-comment-zipcode', countrySelector: '.js-add-comment-country', countryErrorSelector: '.comment-enter-country', }); }); }); $(document).ready(function () { var reportIds = { "383013": 0, "383012": 0, "383015": 0, "383014": 0, "210983": 0, "383009": 0, "210982": 0, "383008": 0, "210981": 0, "383011": 0, "210980": 0, "383010": 0, "384813": 0, "384815": 0, "384814": 0, "383017": 0, "383016": 0, "384821": 0, "384820": 0, "384822": 0, "384817": 0, "384816": 0, "384819": 0, "384818": 0, "322164": 0, "322163": 0, "388733": 0, "391295": 0, "388735": 0, "407676": 10, "388734": 0, "407677": 0, "407674": 0, "407675": 0, "391301": 0, "388741": 0, "391300": 0, "388740": 0, "391303": 0, "391302": 0, "388742": 0, "391297": 0, "388737": 0, "391296": 0, "388736": 0, "391299": 0, "388739": 0, "391298": 0, "388738": 0, "391304": 0, "224159": 0, "224158": 0, "224157": 0, "224156": 0, "224163": 40, "224162": 0, "224161": 0, "224160": 0, "224165": 0, "224164": 0, "407982": 0, "407983": 0, "407980": 0, "407981": 0, "380329": 0, "407978": 0, "407979": 0, "380330": 5, "407977": 0, "407986": 0, "407984": 0, "407985": 0, "412350": 0, "412351": 0, "412348": 0, "412349": 0, "412346": 0, "412347": 0, "412354": 0, "412355": 0, "412352": 0, "412353": 0, "393934": 0, "393935": 24, "393932": 0, "393933": 0, "415702": 0, "415703": 0, "393940": 0, "415700": 0, "393941": 0, "415701": 0, "393938": 0, "393939": 0, "415699": 0, "393936": 0, "393937": 0, "386527": 0, "386526": 0, "415706": 0, "415707": 0, "415704": 0, "415705": 0, "386533": 0, "386532": 0, "386535": 0, "386534": 0, "386529": 0, "386528": 0, "386531": 12, "386530": 0, "284916": 54, "284915": 13, "380925": 0, "380924": 0, "380927": 0, "380926": 0, "211199": 0, "380921": 0, "211198": 0, "380920": 0, "211197": 44, "380923": 0, "380922": 0, }; $(".referee-response-container,.js-referee-report").each(function(index, el) { var reportId = $(el).attr("data-reportid"), reportCount = reportIds[reportId] || 0; $(el).find(".comments-count-container,.js-referee-report-views").html(reportCount); }); var uuidInput = $("#article_uuid"), oldUUId = uuidInput.val(), newUUId = "62fbba35-30f6-4f49-9508-03c0f3f08d68"; uuidInput.val(newUUId); $("a[href*='article_uuid=']").each(function(index, el) { var newHref = $(el).attr("href").replace(oldUUId, newUUId); $(el).attr("href", newHref); }); }); An innovative open access publishing platform offering rapid publication and open peer review, whilst supporting data deposition and sharing. Browse Gateways Collections How it Works Contact For Developers Cookie Notice Privacy Notice RSS Submit Your Research Follow us © 2012-2026 F1000 Research Ltd. ISSN 2046-1402 | Legal | Partner of Research4Life • CrossRef • ORCID • FAIRSharing R.templateTests.simpleTemplate = R.template(' $text $text $text $text $text '); R.templateTests.runTests(); var F1000platform = new F1000.Platform({ name: "f1000research", displayName: "F1000Research", hostName: "f1000research.com", id: "1", editorialEmail: "[email protected]", infoEmail: "[email protected]", usePmcStats: true }); $(function(){R.ui.dropdowns('.dropdown-for-authors, .dropdown-for-about, .dropdown-for-myresearch');}); // $(function(){R.ui.dropdowns('.dropdown-for-referees');}); $(document).ready(function () { if ($(".cookie-warning").is(":visible")) { $(".sticky").css("margin-bottom", "35px"); $(".devices").addClass("devices-and-cookie-warning"); } $(".cookie-warning .close-button").click(function (e) { $(".devices").removeClass("devices-and-cookie-warning"); $(".sticky").css("margin-bottom", "0"); }); $("#tweeter-feed .tweet-message").each(function (i, message) { var self = $(message); self.html(linkify(self.html())); }); $(".partner").on("mouseenter mouseleave", function() { $(this).find(".gray-scale, .colour").toggleClass("is-hidden"); }); }); Sign In Remember me Forgotten your password? Sign In Cancel Email or password not correct. Please try again Please wait... $(function(){ // Note: All the setup needs to run against a name attribute and *not* the id due the clonish // nature of facebox... $("a[id=googleSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("GOOGLE"); $("form[id=oAuthForm]").submit(); }); $("a[id=facebookSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("FACEBOOK"); $("form[id=oAuthForm]").submit(); }); $("a[id=orcidSignInButton]").click(function(event){ event.preventDefault(); $("input[id=oAuthSystem]").val("ORCID"); $("form[id=oAuthForm]").submit(); }); }); If you've forgotten your password, please enter your email address below and we'll send you instructions on how to reset your password. The email address should be the one you originally registered with F1000. Email address not valid, please try again You registered with F1000 via Google, so we cannot reset your password. To sign in, please click here . If you still need help with your Google account password, please click here . You registered with F1000 via Facebook, so we cannot reset your password. To sign in, please click here . If you still need help with your Facebook account password, please click here . Code not correct, please try again Reset password Cancel Email us for further assistance. Server error, please try again. If your email address is registered with us, we will email you instructions to reset your password. If you think you should have received this email but it has not arrived, please check your spam filters and/or contact for further assistance. Please wait... Register $(document).ready(function () { signIn.createSignInAsRow($("#sign-in-form-gfb-popup")); $(".target-field").each(function () { var uris = $(this).val().split("/"); if (uris.pop() === "login") { $(this).val(uris.toString().replace(",","/")); } }); });