Survey on neutralizing antibodies against Zika virus eighteen months post-outbreak in two southern Thailand communities

Survey on neutralizing antibodies against Zika virus eighteen months post-outbreak in two southern Thailand communities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Survey on neutralizing antibodies against Zika virus eighteen months post-outbreak in two southern Thailand communities Theerut Densathaporn, Rassamee Sangthong, Monvaris Sakolnapa, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-34709/v4 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Dec, 2020 Read the published version in BMC Infectious Diseases → Version 4 posted 2 You are reading this latest preprint version Show more versions Abstract Background: In 2016 and 2017, Zika virus (ZIKV) infection outbreaks occurred in two communities in southern Thailand. This re-immerging infection can widely spread by mosquito bites and cause serious complications in a central nervous system among children born to infected mothers. Thus, they should be protected. This study aims to (1) To determine the prevalence of neutralizing ZIKV antibodies in the post-outbreak areas among the general population and pregnancy women residing at various distances from the houses of the nearest index patients; (2) To examine the cross-neutralizing capacity of antibodies against ZIKV on other flaviviruses commonly found in the study areas; (3) To identify factors associated with the presence of neutralizing ZIKV antibodies. Methods: The two post-outbreak communities were visited at 18 months after the outbreaks. We enrolled (1) 18 confirmed ZIKV infected (index) cases, (2) sample of 554 neighbors in the outbreak areas who lived at various distances from the index patients’ houses, (3) 190 residents of non-outbreak areas, and (4) all pregnant women regardless of gestational age residing in the study areas (n = 805). All serum specimens underwent the plaque reduction neutralization test (PRNT). Ten randomly selected ZIKV seropositive and ten randomly selected seronegative specimens were tested for dengue virus serotypes 1-4 (DENV1-4) and Japanese encephalitis virus (JEV) antibodies using PRNT90. Serum titer above 1:10 was considered positive. Multiple logistic regression was used to assess factors associated with seropositivity. Results: Out of all 18 index cases, 9 remained seropositive. The seroprevalence (95% CI) in the two outbreak areas were 43.7% (35.9-51.6%) and 29.7% (23.3-36.0%) in general population, and 24.3% (20.1-28.8%) and 12.8% (9.7-16.5%) in pregnant women. Multivariate analysis showed that seropositivity was independent of the distance gradient from the index’s houses. However, being elderly was associated with seropositivity. DENV1-4 and JEV neutralizing antibodies were present in most ZIKV-positive and negative subsamples. Conclusion: Protective herd immunity for ZIKV infection is inadequate, especially among pregnant women in the two post-outbreak areas in southern Thailand. Infectious Diseases Zika virus seroprevalence survey cross-protection. Figures Figure 1 Figure 1 Figure 2 Figure 2 Background Zika virus (ZIKV) is a flavivirus that causes acute febrile illness. 1–3 Serious complications include congenital neurological syndrome from vertical transmission and Guillain-Barre syndrome. 4–8 In the last decade, ZIKV epidemics occurred in many Pacific islands, South America, and other countries around the world. Globally, 87 countries in 4 continents have reported ZIKV outbreaks with a total cumulative number of nearly one million cases since 2015. 9–11 In Southeast Asia, there were evidence of the existence of neutralization antibodies against ZIKV from the serological surveys in the population of the region between 1960 and 1980s. 12–14 Since then, no cases were reported in Thailand until 2013 when two foreigners visiting the country were found to have contracted ZIKV after they returned home. 15,16 Domestically, 7 confirmed ZIKV infected citizens were reported in Thailand during 2012-2014. 17 After the rising public awareness of ZIKV from the South American outbreaks in 2015-2016, 1,121 confirmed ZIKV infected cases were detected in 43 provinces in 2016 and 577 cases in 33 provinces in 2017. 18 These alarming figures raised concern on the population at risk for future outbreaks. Neutralizing antibodies are an important protective element against virus infection. Knowledge of their prevalence against ZIKV can allow epidemiologists to evaluate whether a population has enough immunity to prevent an outbreak. Theoretically, the proportion of immune population greater than 1-1/Basic reproduction number(R0) is required to eliminate the infection by maintaining reproduction number less than 1. 19 R0 of ZIKV in tropical areas varied from minimum of 1.22 to maximum of 6.9. 20 Taking the maximum value of 6.9, the prevalence needed to stop the transmission would be up to 85.5%. Such information on the prevalence can assist in the evaluation of the worthiness of developing a ZIKV vaccine for the country. The cross-reaction of antibodies against different flaviviruses has been well documented. It is, however, not known whether other endemic types of flavivirus in Thailand such as dengue and Japanese encephalitis contribute to the protection of the newly resurgent ZIKV. In 2016, two ZIKV outbreaks occurred in southern Thailand, one in Surat Thani province during September - November and the other in Narathiwat province, 500 km away from the first outbreak site, during November 2016 - January 2017. We took this opportunity to conduct a serological survey to find the answers to the abovementioned knowledge gaps. The objectives of this study were to determine the prevalence of neutralizing ZIKV antibodies among general population and pregnant women in the outbreak areas, examine the cross-neutralizing capacity of ZIKV antibodies against different types of flavivirus infection, and identify factors associated with the presence of neutralizing ZIKV antibodies. We hypothesized that increasing proximity to an index house would increase the likelihood of having neutralizing antibodies against ZIKV. Methods Study settings An outbreak district (or District A) in Surat Thani province has a total area of 835.1 km 2 . It is characterized by plain areas surrounded by hills, forests, and rubber plantations. Its population of 50,905 resided in 17,337 households. The other outbreak district (or District B) is located in Narathiwat province, one of the southernmost provinces of Thailand. It has a total area of 372.6 km 2 . Small rivers run from the mountains creating peat swamp forests in the area. The local population of 47,965 resided in 11,285 households. 21 During the outbreaks, local health officers followed the national guideline 22 to control the infection. All index patients underwent reverse transcriptase-polymerase chain reaction (RT-PCR) testing for disease confirmation. The guideline also included the screening of their household contacts, and all pregnant women in the outbreak districts for the infection by the same test. Furthermore, intensive space spraying of insecticide and mosquito surveillance were implemented in the whole affected village area. Our research team retrospectively reviewed the medical records at one month after the end of the outbreak and had the meetings with the health officers to plan our current study. The review and the meetings revealed that ZIKV infection were confirmed by RT-PCR in 24 and 18 patients in District A and B, respectively. The outbreak covered 12 villages in 6 subdistricts of District A and 5 villages in 3 subdistricts of District B. Figure 1 displays maps of both districts. Dark grey areas denote affected subdistricts with the number of confirmed ZIKV cases. Light grey areas are subdistricts adjacent to the outbreak districts and white areas denote non-adjacent and non-affected subdistricts. Study design A cross-sectional serological survey was conducted in the two affected districts approximately 18 months after each outbreak. In each study district, we recruited two study populations, non-pregnant adults and pregnant women. Details in sampling technique for each group are as follow. 1. Non-pregnant adults Based on the preceding outbreak records, we recruited all index cases, their household contacts, and a random sample of residents who lived varying distances from the nearest index case's house. All study subjects were at least 18 years old and lived in the area for more than 18 months. Exclusion criteria included pregnancy, immunodeficiency disease, and current use of immunosuppressive drugs. In order to test our hypothesis, we stratified the non-case population into five groups based on the distance from their household to the house of the nearest index case. Household members of the index cases. Other residents of the subdistrict where the number of the confirmed cases was highest, who lived within 100 meters from an index case Similar to #2 but the distance to the nearest index case was between 101 and 400 meters Similar to #2 but the distance to the nearest index case was between 401 and 1,000 meters Residents of a randomly selected village in a non-affected and non-adjacent subdistrict (white areas shown in Figure 1) Based on the limitation of a finite number of cases and the population, for each district, we planned to recruit 400 eligible non-pregnant including all of the index’s household members (i) and addition to a hundred of their neighbors who lived within 100 meters (ii), and 100 non-pregnant subjects in each other three distance stratum (iii, iv, and v). /..Having a significance level set to 0.05 and a power of 80%, this sample size would allow us to detect a 20% difference in the prevalence of ZIKV neutralizing antibodies among the distance strata. 2. Pregnant women All pregnant women aged 18 years or above, attending an antenatal care clinic, and living in one of the same districts as a case for more than 12 months were recruited. Exclusion criteria included known cases of ZIKV identified during the outbreak period, major psychiatric or physical illness, cognitive impairment, inability to communicate in Thai, immunocompromised, rheumatologic disorders, and autoimmune diseases. Four hundred pregnant women per district were recruited regardless of their gestational age and distance from their house to the house of the nearest index case. This sample size was calculated based on initial expected seroprevalence of 20%, +/- 4% and alpha = 0.05. They were treated as a separate stratum in the descriptive analysis, but we did not analyze for risk factors among pregnant women because the risk behavior information was not available. Data collection In the non-pregnant adult group, the survey was conducted in District A during March-May 2018 and District B during July-September 2018. A team of local health volunteers were trained as research assistants and instructed to recruit participants and conduct the interviews. The recruitment process involved visiting potential participants at their home, explaining to them the objectives of the study, and requesting their informed consent to participate in the study. Consenting participants were interviewed using a structured questionnaire to collect individual and household information. The distance between the center of each participant’s household and the nearest index case’s household was estimated using Google Maps ® . The participants were invited to the health centers for venipuncture at the end of the week where a 10-mL blood sample was taken by a local health officer. Between July 2018 and May 2019 in District A and B, consecutive pregnant women who attended the antenatal clinics were invited by the research team. Informed consent was obtained. Then a blood sample was taken for serological test. At least 30 minutes after venipuncture, each blood specimen was centrifuged at 3,200 revolutions per minute, divided into 4 aliquots and stored at -20◦C in a freezer at the district hospital, and finally shipped in lots to the Center of Vaccine Development (CVD), Institute of Molecular Biosciences, Mahidol University in Bangkok as it is the WHO-approved reference laboratory on serology and virology for arboviruses. Laboratory tests Plaque reduction neutralization tests (PRNT) for ZIKV-neutralizing antibodies were performed using the following procedures. Rhesus monkey kidney epithelial cells (LLC-MK2) were first seeded in 6-well plates at 1 × 10 5 cells/well for 7 days. The serum samples were four-fold serially diluted by phosphate buffer solution pH 7.5 with 30% fetal bovine serum, and then mixed with Zika virus strain MR766 at 50 plaque-forming unit (pfu)/well (for a final starting dilution of 1:10) for 1 hour. Following infection, cells were overlaid with Dulbecco's Modified Eagle Medium containing fetal bovine serum, 3.0% carboxymethyl cellulose, and neutral red. Plaques were visualized and counted at 7 days after infection. Probit analysis was used to determine the titer and interpreted as a PRNT50 and later on PRNT90 titer per a reviewer’s suggestion which is the reciprocal of the dilution showing a 50% and 90% reduction, respectively, in plaque count. A neutralization titer ≥1:10 by PRNT90 was considered as a seropositive. 23,24 Random samples of 10 positive (PRNT90 titers> 1:10) and 10 negative (PRNT90 titer < 1:10) serum samples were used to further test for neutralizing antibody against dengue virus (DENV) serotype 1-4 (strain 16007,16681 16562, and C036/06 respectively) and Japanese encephalitis virus (Beijing strain). Statistical analysis The main outcome variable was whether the subject had a positive neutralizing antibody defined by a PRNT90 titer above 1:10. The detailed titer was further analyzed against the titer of neutralizing antibodies against other types of flavivirus. The main independent variable was the distance from the participant's household to the nearest index case's house. Other independent variables were personal characteristics of the subjects such as age, occupation, behavior related to protective measures against mosquito bites such as the use of mosquito repellents, domestic garbage management, and self-reported history of dengue and chikungunya infection. Prevalence estimation, statistical tests, and the regression in non-pregnant data were computed using the ‘survey’ package to adjust the standard errors based on the sampling weights. 25 Variations in seroprevalence among different geographical locations were observed in previous studies, 26–28 thus the seroprevalence in the two districts were described separately. For non-pregnant participants, the estimated prevalence of seropositive cases was stratified by the distance band between the household of the participant and the household of the nearest index case. Chi-square test was used to initially determine whether there is a significant difference between seroprevalence of affected and non-affected subdistrict. Proportional trend test was used to determine whether there is a linear trend in the prevalence across gradient of distance from the affected subdistrict. In order to inquire more power to examine associated factors of seropositivity, the data of the two districts were combined and ‘district’ was handled as one of the independent variables. Predictors for seropositivity from non-pregnant adults were tested using the Rao-Scott Chi-Square test. Independent variables that showed an initial association with ZIKV seropositivity (P-value<0.2) were included in the multivariate logistic regression model to adjust for potential confounding effects. Likelihood ratio test (LR test) is used to test whether the model with that predictive factor is fit the data significantly better than the more restrictive model. Wald test is used to test whether removing of that level is substantially harm the fit of the model. For cross-neutralization, titers of neutralizing antibodies against ZIKV were plotted against those of the flaviviruses, one-by-one, on a logarithmic scale. Statistical significance was set at 0.05. All statistical analysis was undertaken using R software. Results The overall response rate of non-pregnant participants was 74.8% (377 out of 504 participants) and 77.2% (385 out of 499 participants) from District A and B, respectively. Half from a total of 18 index cases remained seropositive. Table1 [see Additional file 1] shows the seroprevalence by subgroup. The weighted prevalence [95% confidence interval] was 43.7% [35.9-51.6%] in the affected subdistricts of District A, which was not significantly different that of 29.7% [23.3-36.0%] in the affected subdistricts of District B. We detected no significant difference in the prevalence of neutralizing antibodies between the affected subdistricts and the non-affected subdistricts of both districts. The prevalence among pregnant women in both districts was significantly lower than most of all other subgroups. The prevalence of pregnant participants (24.3% [20.1-28.8%] in district A, and 12.8% [9.7-16.5%] in district B) were not significant difference from non-pregnant participants aged 18-40 years in the same district (30.7% [20.5-42.4%] in district A and 14.4% [8.9-21.6%] in district B). Table 2 [see Additional file 2] compares the prevalence of neutralizing antibodies among subgroups of non-pregnant participants. The prevalence were significantly associated with age and living near the natural water within 100m. The prevalence of neutralizing antibodies was 20.3% among young adults aged 18-40 years, 35.4% among those aged 41-60 years and 46.5% among those aged more than 60 years. Table 3 [see Additional file 3] shows the results of the multivariable logistic regression analysis. There was no significant effect of distance from the nearest index case’s house to the participant’s house. Only significant predictors for a subject having neutralizing antibodies included age more than 60 years Figure 2 illustrates the cross-distribution of titers of neutralizing antibodies against various flaviviruses (Y-axes) and ZIKV (X-axis). The black dots scattered on the right represent positive ZIKV tests. The crosses on the left side at a titer of 1:10 represent samples that tested negative. Nearly all samples had positive tests against the other viruses, indicating that the majority of subjects with ZIKV negative tests had positive test results for neutralizing antibodies against other flaviviruses. Thus, a high proportion of ZIKV negative cases were harboring neutralizing antibodies against all study flaviviruses. Discussion Approximately 18 months after the two ZIKV outbreaks in southern Thailand, one-third to nearly half of the population had neutralizing antibodies against the virus. The prevalence was not significantly different between outbreak and non-outbreak areas. Elderly groups were more likely to have this neutralizing antibody. Pregnant women had a significantly lower prevalence of neutralizing antibody than the non-pregnant group. The prevalence of seropositivity reported in this study was in the range of that found in the post-outbreak area of French Polynesia (49% measured at 18 months post-outbreak) and Nicaragua (56% measured at 1 year post-outbreak), French Guiana (23.3% measured at 2 years post-outbreak) and Suriname (35.1% measured at 1 year post-outbreak). 26–29 Our investigation was conducted 18 months after the outbreak when no active cases were detected. The immunity have developed in response to, or independent from, the ZIKV outbreak 18-month ago ZIKV, or is an artifact of the serological background of the ZIKV prior to the pandemic occurring. ZIKV was believed to be endemic in the Southeast Asia region for many years. 12,24,30,31 Regardless of the nature of the source, more than half of the population are at risk of infection. The level of seroprevalence did not reach the theoretical threshold of herd immunity of 85.5%. This scenario is comparable with situation of dengue virus in Thailand. Even through the seroprevalence of DENV was as high as 79.2%, but there were around 100,000 cases reported annually. 32 In contrast with contagious diseases, spreading ability of arboviral diseases depends on vector and environmental factors. Our results failed to demonstrate a dose-response relationship between seropositivity and distance to the nearest index case's house. There was also no significant difference in seropositivity between outbreak areas and adjacent non-outbreak areas suggesting that the outbreak did not produce significant immunity in the population. This may be because the sizes of the ZIKV outbreak were very small compared to those in the Pacific Islands and the Americas where the numbers of cases exceeded 900,000. 10 Our results, combined with those from a survey among healthy Thais in Central Thailand (seroprevalence of 70.4%[PRNT50≥10] and 20.2%[PRNT90≥20]), suggest that this Thai population were only partially protected by the antibody. 24 These levels of immunity may explain the low but sustained level of ZIKV transmission in Thailand as proposed by previous authors (Ruchusatsawat et al., 2019). 33 Our results showed that older age is associated with seropositivity. This result contrasts with those of studies from Nicaragua, French Guiana and Suriname, where the Zika virus had been believed to be a de novo pathogen in the Americas during the outbreak. 26–28 The association between older age and seropositivity was also observed in a serosurvey in Thailand of other endemic flaviviruses such as dengue and chigunkunya. 32,34 Therefore, this finding supports the theory that ZIKV has been circulating in the country for many year. The immunological cross-reactivity between Zika and other flaviviruses is well known. 35,36 In this study, we used PRNT, which reflects whether or not a person is protected against a particular virus. 23,37 Thus, we are concerned about cross-protection rather than cross-reaction. However, a high proportion of negative ZIKV PRNT cases with positive titers of other flaviviruses suggests that antibodies against those viruses may not completely protect individuals against ZIKV. Further studies in a greater cohort are needed to confirm the hypothesis that the endemic for other flaviviuses might not protect the population against ZIKV infection. The low prevalence of neutralizing antibodies among pregnant women in these two outbreak areas is of important public health concern. Apart from age group, pregnancy can reduce the immunity of women making them more susceptible to many infections. 38–42 A low immunity against ZIKV in endemic areas would increase the risk of both the women and the fetuses to develop an infection, which can cause serious consequences, especially neurological deficit and microcephaly. One main limitation of our study was that we examined the seroprevalence only 18 months after the outbreak. The initial and changing prevalence of neutralization in the population could, therefore, not be assessed. Our limited resources also allowed us to test neutralizing antibodies against other types of flavivirus in only a small number of subjects. Conclusions The fact that more than half of the general population and more than three-quarters of the pregnant women were seronegative indicates a sustained risk for future ZIKV outbreaks. The community will therefore benefit from efficacious ZIKV vaccine once it becomes available. List Of Abbreviations Adj OR Adjusted odds ratio CVD Center for Vaccine Development DENV Dengue virus JEV Japanese encephalitis virus OR Odds ratio PFU Plaque-forming unit PRNT Plaque reduction neutralization test RT-PCR Reverse transcriptase-polymerase chain reaction ZIKV Zika virus Declarations Ethics approval and consent to participate The study was approved by the Ethics Committee of the Faculty of Medicine, Prince of Songkla University for both general population and pregnant women studies (REC.60-362-18-1, and REC.61-092-18-1). All participants signed a written consent form before data collection. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interest The authors declare that they have no competing interests. Funding This study was supported by a research grant from the National Science and Technology Development Agency of Thailand (Project number FDA-C0-2561-5958-TH). The funder had no role in the design of this study and will did not have any role during its execution, analyses, interpretation of the data, or decision to submit results. Authors’ contribution VC conceived the study. TD and MS led the data collection. SS, MK, PM and SY contributed to the laboratory tests. TD did the analysis with the support of EM, RS and VC. TD wrote and RS, PM, EM, and VC revised the manuscript. All authors read and approved the final manuscript. Acknowledgement We are grateful to Somchai Nakthungtao and Khomkai Nakthungtao for their fieldwork support. 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Infect Dis Obstet Gynecol. 2013;2013:752852. Gilles HM, Lawson JB, Sibellas M, Voller A, Allan N. Malaria and pregnancy. Trans R Soc Trop Med Hyg. 1969;63(1):1. Brabin BJ. An analysis of malaria in pregnancy in Africa. Bull World Health Organ. 1983;61(6):1005–16. Goulet V, Hebert M, Hedberg C, Laurent E, Vaillant V, De Valk H, et al. Incidence of listeriosis and related mortality among groups at risk of acquiring listeriosis. Clin Infect Dis Off Publ Infect Dis Soc Am. 2012 Mar 1;54(5):652–60. R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing; 2019. Available from: https://www.R-project.org/ GADM [Internet]. [cited 2020 Nov 23]. Available from: https://gadm.org/ Supplementary Files ReviseiiAdditionalfile1.docx Additional file 1 – Table 1 Seroprevalence of ZIKV neutralizing antibodies among subgroups of participants in District A and B ReviseiiAdditionalfile1.docx Additional file 1 – Table 1 Seroprevalence of ZIKV neutralizing antibodies among subgroups of participants in District A and B ReviseiiAdditionalfile2.docx Additional file 2 - Table 2 Univariable analysis exploring factors associated with ZIKV seropositivity in non-pregnant participants in the two sites combined. (a multi-page table) ReviseiiAdditionalfile2.docx Additional file 2 - Table 2 Univariable analysis exploring factors associated with ZIKV seropositivity in non-pregnant participants in the two sites combined. (a multi-page table) ReviseiiAdditionalfile3.docx Additional file 3 - Table 3 Odds ratios for ZIKV seropositivity from the multivariate logistic regression analysis among non-pregnant participants in the two sites combined. (a multi-page table) ReviseiiAdditionalfile3.docx Additional file 3 - Table 3 Odds ratios for ZIKV seropositivity from the multivariate logistic regression analysis among non-pregnant participants in the two sites combined. (a multi-page table) Cite Share Download PDF Status: Published Journal Publication published 03 Dec, 2020 Read the published version in BMC Infectious Diseases → Version 4 posted Submission checks completed at journal 24 Nov, 2020 Editorial decision: Accept 23 Nov, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-34709","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":5296911,"identity":"88591478-7ef9-4681-b677-ec7413b16ac6","order_by":0,"name":"Theerut Densathaporn","email":"","orcid":"","institution":"The University of Manchester Faculty of Biology Medicine and Health","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Theerut","middleName":"","lastName":"Densathaporn","suffix":""},{"id":5296912,"identity":"ded7ad3b-8969-454b-be66-67b2dd25b958","order_by":1,"name":"Rassamee 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","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-34709/v4/ca53ad75b93855e7e610a327.png"},{"id":4027313,"identity":"1384929e-5d95-409c-bb18-95a87cfe1812","added_by":"auto","created_at":"2020-12-04 18:34:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":514876,"visible":true,"origin":"","legend":"Relationship between PRNT90 titers of various flaviviruses in ZIKV seropositives and seronegatives ","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-34709/v4/da8155bdbc56d0c0c23a9ad0.png"},{"id":13625862,"identity":"a6291849-3720-4432-bcc7-0f422381d0b4","added_by":"auto","created_at":"2021-09-17 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18:34:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16831,"visible":true,"origin":"","legend":"Additional file 1 – Table 1 Seroprevalence of ZIKV neutralizing antibodies among subgroups of participants in District A and B","description":"","filename":"ReviseiiAdditionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-34709/v4/98a450ce8a5a44bc018c2de0.docx"},{"id":4027320,"identity":"8b3ab7c0-d8c4-497f-b0ee-60c829cbb0c1","added_by":"auto","created_at":"2020-12-04 18:34:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":35767,"visible":true,"origin":"","legend":"Additional file 2 - Table 2 Univariable analysis exploring factors associated with ZIKV seropositivity in non-pregnant participants in the two sites combined. (a multi-page table)","description":"","filename":"ReviseiiAdditionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-34709/v4/63e2066c9475c1e78e4ad370.docx"},{"id":4027314,"identity":"71e340e5-9032-4d8b-8254-36723f1b813d","added_by":"auto","created_at":"2020-12-04 18:34:45","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":35767,"visible":true,"origin":"","legend":"Additional file 2 - Table 2 Univariable analysis exploring factors associated with ZIKV seropositivity in non-pregnant participants in the two sites combined. (a multi-page table)","description":"","filename":"ReviseiiAdditionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-34709/v4/acd66a51232b59198af350bb.docx"},{"id":4027321,"identity":"0c735c89-ab99-4012-9bdd-34ea93ec77d4","added_by":"auto","created_at":"2020-12-04 18:34:52","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":23289,"visible":true,"origin":"","legend":"Additional file 3 - Table 3 Odds ratios for ZIKV seropositivity from the multivariate logistic regression analysis among non-pregnant participants in the two sites combined. (a multi-page table)","description":"","filename":"ReviseiiAdditionalfile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-34709/v4/b78e73f55cb5232fbade3bcb.docx"},{"id":4027315,"identity":"4f64df82-dbf4-483c-b239-8c389492eb6e","added_by":"auto","created_at":"2020-12-04 18:34:45","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":23289,"visible":true,"origin":"","legend":"Additional file 3 - Table 3 Odds ratios for ZIKV seropositivity from the multivariate logistic regression analysis among non-pregnant participants in the two sites combined. (a multi-page table)","description":"","filename":"ReviseiiAdditionalfile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-34709/v4/7f28eff5aa1128322796a6f1.docx"}],"financialInterests":"","formattedTitle":"Survey on neutralizing antibodies against Zika virus eighteen months post-outbreak in two southern Thailand communities","fulltext":[{"header":"Background","content":"\u003cp\u003eZika virus (ZIKV) is a flavivirus that causes acute febrile illness.\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e Serious complications include congenital neurological syndrome from vertical transmission and Guillain-Barre syndrome.\u003csup\u003e4\u0026ndash;8\u003c/sup\u003e In the last decade, ZIKV epidemics occurred in many Pacific islands, South America, and other countries around the world. Globally, 87 countries in 4 continents have reported ZIKV outbreaks with a total cumulative number of nearly one million cases since 2015.\u003csup\u003e9\u0026ndash;11\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eIn Southeast Asia, there were evidence of the existence of neutralization antibodies against ZIKV from the serological surveys in the population of the region between 1960 and 1980s.\u003csup\u003e12\u0026ndash;14\u003c/sup\u003e Since then, no cases were reported in Thailand until 2013 when two foreigners visiting the country were found to have contracted ZIKV after they returned home.\u003csup\u003e15,16\u003c/sup\u003e Domestically, 7 confirmed ZIKV infected citizens were reported in Thailand during 2012-2014.\u003csup\u003e17\u003c/sup\u003e After the rising public awareness of ZIKV from the South American outbreaks in 2015-2016, 1,121 confirmed ZIKV infected cases were detected in 43 provinces in 2016 and 577 cases in 33 provinces in 2017.\u003csup\u003e18\u003c/sup\u003e These alarming figures raised concern on the population at risk for future outbreaks.\u003c/p\u003e\n\u003cp\u003eNeutralizing antibodies are an important protective element against virus infection. Knowledge of their prevalence against ZIKV can allow epidemiologists to evaluate whether a population has enough immunity to prevent an outbreak. Theoretically, the proportion of immune population greater than 1-1/Basic reproduction number(R0) is required to eliminate the infection by maintaining reproduction number less than 1.\u003csup\u003e19\u003c/sup\u003e R0 of ZIKV in tropical areas varied from minimum of 1.22 to maximum of 6.9.\u003csup\u003e20\u003c/sup\u003e Taking the maximum value of 6.9, the prevalence needed to stop the transmission would be up to 85.5%. Such information on the prevalence can assist in the evaluation of the worthiness of developing a ZIKV vaccine for the country.\u003c/p\u003e\n\u003cp\u003eThe cross-reaction of antibodies against different flaviviruses has been well documented. It is, however, not known whether other endemic types of flavivirus in Thailand such as dengue and Japanese encephalitis contribute to the protection of the newly resurgent ZIKV.\u003c/p\u003e\n\u003cp\u003eIn 2016, two ZIKV outbreaks occurred in southern Thailand, one in Surat Thani province during September - November and the other in Narathiwat province, 500 km away from the first outbreak site, during November 2016 - January 2017. We took this opportunity to conduct a serological survey to find the answers to the abovementioned knowledge gaps. The objectives of this study were to determine the prevalence of neutralizing ZIKV antibodies among general population and pregnant women in the outbreak areas, examine the cross-neutralizing capacity of ZIKV antibodies against different types of flavivirus infection, and identify factors associated with the presence of neutralizing ZIKV antibodies. We hypothesized that increasing proximity to an index house would increase the likelihood of having neutralizing antibodies against ZIKV.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy settings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn outbreak district (or District A) in Surat Thani province has a total area of 835.1\u0026nbsp;km\u003csup\u003e2\u003c/sup\u003e. It is characterized by plain areas surrounded by hills, forests, and rubber plantations. Its population of 50,905 resided in 17,337 households. The other outbreak district (or District B) is located in Narathiwat province, one of the southernmost provinces of Thailand. It has a total area of 372.6\u0026nbsp;km\u003csup\u003e2\u003c/sup\u003e. Small rivers run from the mountains creating peat swamp forests in the area. The local population of 47,965 resided in 11,285 households.\u003csup\u003e21\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eDuring the outbreaks, local health officers followed the national guideline\u003csup\u003e22\u003c/sup\u003e to control the infection. All index patients underwent reverse transcriptase-polymerase chain reaction (RT-PCR) testing for disease confirmation. The guideline also included the screening of their household contacts, and all pregnant women in the outbreak districts for the infection by the same test. Furthermore, intensive space spraying of insecticide and mosquito surveillance were implemented in the whole affected village area. Our research team retrospectively reviewed the medical records at one month after the end of the outbreak and had the meetings with the health officers to plan our current study.\u003c/p\u003e\n\u003cp\u003eThe review and the meetings revealed that ZIKV infection were confirmed by RT-PCR in 24 and 18 patients in District A and B, respectively. The outbreak covered 12 villages in 6 subdistricts of District A and 5 villages in 3 subdistricts of District B. Figure 1 displays maps of both districts. Dark grey areas denote affected subdistricts with the number of confirmed ZIKV cases. Light grey areas are subdistricts adjacent to the outbreak districts and white areas denote non-adjacent and non-affected subdistricts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA cross-sectional serological survey was conducted in the two affected districts approximately 18 months after each outbreak. In each study district, we recruited two study populations, non-pregnant adults and pregnant women. Details in sampling technique for each group are as follow.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e1. Non-pregnant adults\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBased on the preceding outbreak records, we recruited all index cases, their household contacts, and a random sample of residents who lived varying distances from the nearest index case's house. All study subjects were at least 18 years old and lived in the area for more than 18 months. Exclusion criteria included pregnancy, immunodeficiency disease, and current use of immunosuppressive drugs.\u003c/p\u003e\n\u003cp\u003eIn order to test our hypothesis, we stratified the non-case population into five groups based on the distance from their household to the house of the nearest index case.\u003c/p\u003e\n\u003col style=\"list-style-type: lower-roman;\"\u003e\n\u003cli\u003eHousehold members of the index cases.\u003c/li\u003e\n\u003cli\u003eOther residents of the subdistrict where the number of the confirmed cases was highest, who lived within 100 meters from an index case\u003c/li\u003e\n\u003cli\u003eSimilar to #2 but the distance to the nearest index case was between 101 and 400 meters\u003c/li\u003e\n\u003cli\u003eSimilar to #2 but the distance to the nearest index case was between 401 and 1,000 meters\u003c/li\u003e\n\u003cli\u003eResidents of a randomly selected village in a non-affected and non-adjacent subdistrict (white areas shown in \u003cbr /\u003e Figure 1)\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eBased on the limitation of a finite number of cases and the population, for each district, we planned to recruit 400 eligible non-pregnant including all of the index\u0026rsquo;s household members (i) and addition to a hundred of their neighbors who lived within 100 meters (ii), and 100 non-pregnant subjects in each other three distance stratum (iii, iv, and v). /..Having a significance level set to 0.05 and a power of 80%, this sample size would allow us to detect a 20% difference in the prevalence of ZIKV neutralizing antibodies among the distance strata.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2. Pregnant women\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll pregnant women aged 18 years or above, attending an antenatal care clinic, and living in one of the same districts as a case for more than 12 months were recruited. Exclusion criteria included known cases of ZIKV identified during the outbreak period, major psychiatric or physical illness, cognitive impairment, inability to communicate in Thai, immunocompromised, rheumatologic disorders, and autoimmune diseases. Four hundred pregnant women per district were recruited regardless of their gestational age and distance from their house to the house of the nearest index case. This sample size was calculated based on initial expected seroprevalence of 20%, +/- 4% and alpha = 0.05. They were treated as a separate stratum in the descriptive analysis, but we did not analyze for risk factors among pregnant women because the risk behavior information was not available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the non-pregnant adult group, the survey was conducted in District A during March-May 2018 and District B during July-September 2018. A team of local health volunteers were trained as research assistants and instructed to recruit participants and conduct the interviews. The recruitment process involved visiting potential participants at their home, explaining to them the objectives of the study, and requesting their informed consent to participate in the study. Consenting participants were interviewed using a structured questionnaire to collect individual and household information. The distance between the center of each participant\u0026rsquo;s household and the nearest index case\u0026rsquo;s household was estimated using Google Maps\u003csup\u003e\u0026reg;\u003c/sup\u003e. The participants were invited to the health centers for venipuncture at the end of the week where a 10-mL blood sample was taken by a local health officer.\u003c/p\u003e\n\u003cp\u003eBetween July 2018 and May 2019 in District A and B, consecutive pregnant women who attended the antenatal clinics were invited by the research team. Informed consent was obtained. Then a blood sample was taken for serological test. At least 30 minutes after venipuncture, each blood specimen was centrifuged at 3,200 revolutions per minute, divided into 4 aliquots and stored at -20◦C in a freezer at the district hospital, and finally shipped in lots to the Center of Vaccine Development (CVD), Institute of Molecular Biosciences, Mahidol University in Bangkok as it is the WHO-approved reference laboratory on serology and virology for arboviruses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLaboratory tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlaque reduction neutralization tests (PRNT) for ZIKV-neutralizing antibodies were performed using the following procedures. Rhesus monkey kidney epithelial cells (LLC-MK2) were first seeded in 6-well plates at 1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e\u0026nbsp;cells/well for 7 days. The serum samples were four-fold serially diluted by phosphate buffer solution pH 7.5 with 30% fetal bovine serum, and then mixed with Zika virus\u0026nbsp;strain MR766 at 50 plaque-forming unit (pfu)/well (for a final starting dilution of 1:10) for 1 hour.\u0026nbsp; Following infection, cells were overlaid with Dulbecco's Modified Eagle Medium containing fetal bovine serum, 3.0% carboxymethyl cellulose, and neutral red. Plaques were visualized and counted at 7 days after infection. Probit analysis was used to determine the titer and interpreted as a PRNT50 and later on PRNT90 titer per a reviewer\u0026rsquo;s suggestion which is the reciprocal of the dilution showing a 50% and 90% reduction, respectively, in plaque count. A neutralization titer \u0026ge;1:10 by PRNT90 was considered as a seropositive.\u003csup\u003e23,24\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eRandom samples of 10 positive (PRNT90 titers\u0026gt; 1:10) and 10 negative (PRNT90 titer \u0026lt; 1:10) serum samples were used to further test for neutralizing antibody against dengue virus (DENV) serotype 1-4 (strain 16007,16681 16562, and C036/06 respectively) and Japanese encephalitis virus (Beijing strain).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main outcome variable was whether the subject had a positive neutralizing antibody defined by a PRNT90 titer above 1:10. The detailed titer was further analyzed against the titer of neutralizing antibodies against other types of flavivirus.\u003c/p\u003e\n\u003cp\u003eThe main independent variable was the distance from the participant's household to the nearest index case's house. Other independent variables were personal characteristics of the subjects such as age, occupation, behavior related to protective measures against mosquito bites such as the use of mosquito repellents, domestic garbage management, and self-reported history of dengue and chikungunya infection. Prevalence estimation, statistical tests, and the regression in non-pregnant data were computed using the \u0026lsquo;survey\u0026rsquo; package to adjust the standard errors based on the sampling weights.\u003csup\u003e25\u003c/sup\u003eVariations in seroprevalence among different geographical locations were observed in previous studies,\u003csup\u003e26\u0026ndash;28\u003c/sup\u003e thus the seroprevalence in the two districts were described separately. For non-pregnant participants, the estimated prevalence of seropositive cases was stratified by the distance band between the household of the participant and the household of the nearest index case. Chi-square test was used to initially determine whether there is a significant difference between seroprevalence of affected and non-affected subdistrict. Proportional trend test was used to determine whether there is a linear trend in the prevalence across gradient of distance from the affected subdistrict.\u003c/p\u003e\n\u003cp\u003eIn order to inquire more power to examine associated factors of seropositivity, the data of the two districts were combined and \u0026lsquo;district\u0026rsquo; was handled as one of the independent variables. Predictors for seropositivity from non-pregnant adults were tested using the Rao-Scott Chi-Square test. Independent variables that showed an initial association with ZIKV seropositivity (P-value\u0026lt;0.2) were included in the multivariate logistic regression model to adjust for potential confounding effects. Likelihood ratio test (LR test) is used to test whether the model with that predictive factor is fit the data significantly better than the more restrictive model. Wald test is used to test whether removing of that level is substantially harm the fit of the model.\u003c/p\u003e\n\u003cp\u003eFor cross-neutralization, titers of neutralizing antibodies against ZIKV were plotted against those of the flaviviruses, one-by-one, on a logarithmic scale.\u003c/p\u003e\n\u003cp\u003eStatistical significance was set at 0.05.\u0026nbsp; All statistical analysis was undertaken using R software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe overall response rate of non-pregnant participants was 74.8% (377 out of 504 participants) and 77.2% (385 out of 499 participants) from District A and B, respectively. Half from a total of 18 index cases remained seropositive. Table1\u003c/p\u003e\n\u003cp\u003e[see Additional file 1] shows the seroprevalence by subgroup. The weighted prevalence [95% confidence interval] was 43.7% [35.9-51.6%] in the affected subdistricts of District A, which was not significantly different that of 29.7% [23.3-36.0%] in the affected subdistricts of District B. We detected no significant difference in the prevalence of neutralizing antibodies between the affected subdistricts and the non-affected subdistricts of both districts.\u003c/p\u003e\n\u003cp\u003eThe prevalence among pregnant women in both districts was significantly lower than most of all other subgroups. The prevalence of pregnant participants (24.3% [20.1-28.8%] in district A, and 12.8% [9.7-16.5%] in district B) were not significant difference from non-pregnant participants aged 18-40 years in the same district (30.7% [20.5-42.4%] in district A and 14.4% [8.9-21.6%] in district B).\u003c/p\u003e\n\u003cp\u003eTable 2 [see Additional file 2] compares the prevalence of neutralizing antibodies among subgroups of non-pregnant participants. The prevalence were significantly associated with age and living near the natural water within 100m. The prevalence of neutralizing antibodies was 20.3% among young adults aged 18-40 years, 35.4% among those aged 41-60 years and 46.5% among those aged more than 60 years.\u003c/p\u003e\n\u003cp\u003eTable 3 [see Additional file 3] shows the results of the multivariable logistic regression analysis. There was no significant effect of distance from the nearest index case\u0026rsquo;s house to the participant\u0026rsquo;s house. Only significant predictors for a subject having neutralizing antibodies included age more than 60 years\u003c/p\u003e\n\u003cp\u003eFigure 2 illustrates the cross-distribution of titers of neutralizing antibodies against various flaviviruses (Y-axes) and ZIKV (X-axis). The black dots scattered on the right represent positive ZIKV tests. The crosses on the left side at a titer of 1:10 represent samples that tested negative. Nearly all samples had positive tests against the other viruses, indicating that the majority of subjects with ZIKV negative tests had positive test results for neutralizing antibodies against other flaviviruses. Thus, a high proportion of ZIKV negative cases were harboring neutralizing antibodies against all study flaviviruses.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eApproximately 18 months after the two ZIKV outbreaks in southern Thailand, one-third to nearly half of the population had neutralizing antibodies against the virus. The prevalence was not significantly different between outbreak and non-outbreak areas. Elderly groups were more likely to have this neutralizing antibody. Pregnant women had a significantly lower prevalence of neutralizing antibody than the non-pregnant group.\u003c/p\u003e\n\u003cp\u003eThe prevalence of seropositivity reported in this study was in the range of that found in the post-outbreak area of French Polynesia (49% measured at 18 months post-outbreak) and Nicaragua (56% measured at 1 year post-outbreak), French Guiana (23.3% measured at 2 years post-outbreak) and Suriname (35.1% measured at 1 year post-outbreak).\u003csup\u003e26\u0026ndash;29\u003c/sup\u003e Our investigation was conducted 18 months after the outbreak when no active cases were detected. The immunity have developed in response to, or independent from, the ZIKV outbreak 18-month ago ZIKV, or is an artifact of the serological background of the ZIKV prior to the pandemic occurring. ZIKV was believed to be endemic in the Southeast Asia region for many years.\u003csup\u003e12,24,30,31\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eRegardless of the nature of the source, more than half of the population are at risk of infection. The level of seroprevalence did not reach the theoretical threshold of herd immunity of 85.5%. This scenario is comparable with situation of dengue virus in Thailand. Even through the seroprevalence of DENV was as high as 79.2%, but there were around 100,000 cases reported annually.\u003csup\u003e32\u003c/sup\u003e In contrast with contagious diseases, spreading ability of arboviral diseases depends on vector and environmental factors.\u003c/p\u003e\n\u003cp\u003eOur results failed to demonstrate a dose-response relationship between seropositivity and distance to the nearest index case's house. There was also no significant difference in seropositivity between outbreak areas and adjacent non-outbreak areas suggesting that the outbreak did not produce significant immunity in the population. This may be because the sizes of the ZIKV outbreak were very small compared to those in the Pacific Islands and the Americas where the numbers of cases exceeded 900,000.\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eOur results, combined with those from a survey among healthy Thais in Central Thailand (seroprevalence of 70.4%[PRNT50\u0026ge;10] and 20.2%[PRNT90\u0026ge;20]), suggest that this Thai population were only partially protected by the antibody.\u003csup\u003e24\u003c/sup\u003e These levels of immunity may explain the low but sustained level of ZIKV transmission in Thailand as proposed by previous authors (Ruchusatsawat et al., 2019).\u003csup\u003e33\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eOur results showed that older age is associated with seropositivity. This result contrasts with those of studies from Nicaragua, French Guiana and Suriname, where the Zika virus had been believed to be a de novo pathogen in the Americas during the outbreak.\u003csup\u003e26\u0026ndash;28\u003c/sup\u003e The association between older age and seropositivity was also observed in a serosurvey in Thailand of other endemic flaviviruses such as dengue and chigunkunya.\u003csup\u003e32,34\u003c/sup\u003e Therefore, this finding supports the theory that ZIKV has been circulating in the country for many year. The immunological cross-reactivity between Zika and other flaviviruses is well known.\u003csup\u003e35,36\u003c/sup\u003e In this study, we used PRNT, which reflects whether or not a person is protected against a particular virus.\u003csup\u003e23,37\u003c/sup\u003e Thus, we are concerned about cross-protection rather than cross-reaction. However, a high proportion of negative ZIKV PRNT cases with positive titers of other flaviviruses suggests that antibodies against those viruses may not completely protect individuals against ZIKV. Further studies in a greater cohort are needed to confirm the hypothesis that the endemic for other flaviviuses might not protect the population against ZIKV infection.\u003c/p\u003e\n\u003cp\u003eThe low prevalence of neutralizing antibodies among pregnant women in these two outbreak areas is of important public health concern. Apart from age group, pregnancy can reduce the immunity of women making them more susceptible to many infections.\u003csup\u003e38\u0026ndash;42\u003c/sup\u003e A low immunity against ZIKV in endemic areas would increase the risk of both the women and the fetuses to develop an infection, which can cause serious consequences, especially neurological deficit and microcephaly.\u003c/p\u003e\n\u003cp\u003eOne main limitation of our study was that we examined the seroprevalence only 18 months after the outbreak. The initial and changing prevalence of neutralization in the population could, therefore, not be assessed. Our limited resources also allowed us to test neutralizing antibodies against other types of flavivirus in only a small number of subjects.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe fact that more than half of the general population and more than three-quarters of the pregnant women were seronegative indicates a sustained risk for future ZIKV outbreaks. The community will therefore benefit from efficacious ZIKV vaccine once it becomes available.\u003c/p\u003e"},{"header":"List Of Abbreviations","content":"\u003cp\u003eAdj OR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Adjusted odds ratio\u003c/p\u003e\n\u003cp\u003eCVD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; Center for Vaccine Development\u003c/p\u003e\n\u003cp\u003eDENV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Dengue virus\u003c/p\u003e\n\u003cp\u003eJEV\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Japanese encephalitis virus\u003c/p\u003e\n\u003cp\u003eOR\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Odds ratio\u003c/p\u003e\n\u003cp\u003ePFU\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Plaque-forming unit\u003c/p\u003e\n\u003cp\u003ePRNT\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Plaque reduction neutralization test\u003c/p\u003e\n\u003cp\u003eRT-PCR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Reverse transcriptase-polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eZIKV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Zika virus\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of the Faculty of Medicine, Prince of Songkla University for both general population and pregnant women studies (REC.60-362-18-1, and REC.61-092-18-1). All participants signed a written consent form before data collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a research grant from the National Science and Technology Development Agency of Thailand (Project number FDA-C0-2561-5958-TH). The funder had no role in the design of this study and will did not have any role during its execution, analyses, interpretation of the data, or decision to submit results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVC conceived the study. TD and MS led the data collection. SS, MK, PM and SY contributed to the laboratory tests. TD did the analysis with the support of EM, RS and VC. TD wrote and RS, PM, EM, and VC revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Somchai Nakthungtao and Khomkai Nakthungtao for their fieldwork support. We thank the local health officers and staff at the local hospitals for their kind assistance during data collection. We also want to extend our thanks to all patients and community residents who kindly participated in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDuffy MR, Chen T-H, Hancock WT, Powers AM, Kool JL, Lanciotti RS, et al. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med. 2009 Jun 11;360(24):2536\u0026ndash;43.\u003c/li\u003e\n\u003cli\u003eMusso D, Bossin H, Mallet HP, Besnard M, Broult J, Baudouin L, et al. Zika virus in French Polynesia 2013-14: anatomy of a completed outbreak. Lancet Infect Dis. 2018;18(5):e172\u0026ndash;82.\u003c/li\u003e\n\u003cli\u003eCerbino-Neto J, Mesquita EC, Souza TML, Parreira V, Wittlin BB, Durovni B, et al. Clinical Manifestations of Zika Virus Infection, Rio de Janeiro, Brazil, 2015. Emerg Infect Dis. 2016 Jul;22(7):1318\u0026ndash;20.\u003c/li\u003e\n\u003cli\u003ePomar L, Vouga M, Lambert V, Pomar C, Hcini N, Jolivet A, et al. 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Analysis of Zika virus neutralizing antibodies in normal healthy Thais. Sci Rep. 2018 21;8(1):17193.\u003c/li\u003e\n\u003cli\u003eComplex Surveys: A Guide to Analysis Using R. 1st edition. Hoboken, N.J: Wiley; 2010. 296 p.\u003c/li\u003e\n\u003cli\u003eLangerak T, Brinkman T, Mumtaz N, Arron G, Hermelijn S, Baldewsingh G, et al. Zika Virus Seroprevalence in Urban and Rural Areas of Suriname, 2017. J Infect Dis. 2019 05;220(1):28\u0026ndash;31.\u003c/li\u003e\n\u003cli\u003eZambrana JV, Bustos Carrillo F, Burger-Calderon R, Collado D, Sanchez N, Ojeda S, et al. Seroprevalence, risk factor, and spatial analyses of Zika virus infection after the 2016 epidemic in Managua, Nicaragua. Proc Natl Acad Sci U S A. 2018 11;115(37):9294\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eImpact of Zika Virus Emergence in French Guiana: A Large General Population Seroprevalence Survey. - PubMed - NCBI [Internet]. [cited 2020 Apr 17]. Available from: https://www.ncbi.nlm.nih.gov/pubmed/31418012\u003c/li\u003e\n\u003cli\u003eAubry M, Teissier A, Huart M, Merceron S, Vanhomwegen J, Roche C, et al. Zika Virus Seroprevalence, French Polynesia, 2014-2015. Emerg Infect Dis. 2017;23(4):669\u0026ndash;72.\u003c/li\u003e\n\u003cli\u003ePastorino B, Sengvilaipaseuth O, Chanthongthip A, Vongsouvath M, Souksakhone C, Mayxay M, et al. Low Zika Virus Seroprevalence in Vientiane, Laos, 2003-2015. Am J Trop Med Hyg. 2019;100(3):639\u0026ndash;42.\u003c/li\u003e\n\u003cli\u003eSam I-C, Montoya M, Chua CL, Chan YF, Pastor A, Harris E. Low seroprevalence rates of Zika virus in Kuala Lumpur, Malaysia. Trans R Soc Trop Med Hyg. 2019 01;113(11):678\u0026ndash;84.\u003c/li\u003e\n\u003cli\u003eVongpunsawad S, Intharasongkroh D, Thongmee T, Poovorawan Y. Seroprevalence of antibodies to dengue and chikungunya viruses in Thailand. PloS One. 2017;12(6):e0180560.\u003c/li\u003e\n\u003cli\u003eRuchusatsawat K, Wongjaroen P, Posanacharoen A, Rodriguez-Barraquer I, Sangkitporn S, Cummings DAT, et al. Long-term circulation of Zika virus in Thailand: an observational study. Lancet Infect Dis. 2019 Apr;19(4):439\u0026ndash;46.\u003c/li\u003e\n\u003cli\u003eTsai TF. New initiatives for the control of Japanese encephalitis by vaccination: minutes of a WHO/CVI meeting, Bangkok, Thailand, 13-15 October 1998. Vaccine. 2000 May 26;18 Suppl 2:1\u0026ndash;25.\u003c/li\u003e\n\u003cli\u003eLanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, et al. Genetic and Serologic Properties of Zika Virus Associated with an Epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis. 2008 Aug;14(8):1232\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eTsai W-Y, Youn HH, Brites C, Tsai J-J, Tyson J, Pedroso C, et al. Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests. Clin Infect Dis Off Publ Infect Dis Soc Am. 2017 Dec 1;65(11):1829\u0026ndash;36.\u003c/li\u003e\n\u003cli\u003eLindsey NP, Staples JE, Powell K, Rabe IB, Fischer M, Powers AM, et al. Ability To Serologically Confirm Recent Zika Virus Infection in Areas with Varying Past Incidence of Dengue Virus Infection in the United States and U.S. Territories in 2016. J Clin Microbiol. 2018;56(1).\u003c/li\u003e\n\u003cli\u003eKourtis AP, Read JS, Jamieson DJ. Pregnancy and Infection. N Engl J Med. 2014 Jun 5;370(23):2211\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eSappenfield E, Jamieson DJ, Kourtis AP. Pregnancy and susceptibility to infectious diseases. Infect Dis Obstet Gynecol. 2013;2013:752852.\u003c/li\u003e\n\u003cli\u003eGilles HM, Lawson JB, Sibellas M, Voller A, Allan N. Malaria and pregnancy. Trans R Soc Trop Med Hyg. 1969;63(1):1.\u003c/li\u003e\n\u003cli\u003eBrabin BJ. An analysis of malaria in pregnancy in Africa. Bull World Health Organ. 1983;61(6):1005\u0026ndash;16.\u003c/li\u003e\n\u003cli\u003eGoulet V, Hebert M, Hedberg C, Laurent E, Vaillant V, De Valk H, et al. Incidence of listeriosis and related mortality among groups at risk of acquiring listeriosis. Clin Infect Dis Off Publ Infect Dis Soc Am. 2012 Mar 1;54(5):652\u0026ndash;60.\u003c/li\u003e\n\u003cli\u003eR Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing; 2019. Available from: https://www.R-project.org/\u003c/li\u003e\n\u003cli\u003eGADM [Internet]. [cited 2020 Nov 23]. Available from: https://gadm.org/\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Zika virus, seroprevalence survey, cross-protection.","lastPublishedDoi":"10.21203/rs.3.rs-34709/v4","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-34709/v4","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eIn 2016 and 2017, Zika virus (ZIKV) infection outbreaks occurred in two communities in southern Thailand. This re-immerging infection can widely spread by mosquito bites and cause serious complications in a central nervous system among children born to infected mothers. Thus, they should be protected. This study aims to\u003cstrong\u003e \u003c/strong\u003e(1) To determine the prevalence of neutralizing ZIKV antibodies in the post-outbreak areas among the general population and pregnancy women residing at various distances from the houses of the nearest index patients; (2) To examine the cross-neutralizing capacity of antibodies against ZIKV on other flaviviruses commonly found in the study areas; (3) To identify factors associated with the presence of neutralizing ZIKV antibodies.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe two post-outbreak communities were visited at 18 months after the outbreaks. We enrolled (1) 18 confirmed ZIKV infected (index) cases, (2) sample of\u0026nbsp;554 neighbors in the outbreak areas who lived at various distances from the index patients’ houses, (3) 190 residents of non-outbreak areas, and (4) all pregnant women regardless of gestational age residing in the study areas (n = 805). All serum specimens underwent the plaque reduction neutralization test (PRNT). Ten randomly selected ZIKV seropositive and ten randomly selected seronegative specimens were tested for dengue virus serotypes 1-4 (DENV1-4) and Japanese encephalitis virus (JEV) antibodies using PRNT90. Serum titer above 1:10 was considered positive. Multiple logistic regression was used to assess factors associated with seropositivity.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eOut of all 18 index cases, 9 remained seropositive. The seroprevalence (95% CI) in the two outbreak areas were 43.7% (35.9-51.6%) and 29.7% (23.3-36.0%) in general population, and 24.3% (20.1-28.8%) and 12.8% (9.7-16.5%) in pregnant women. Multivariate analysis showed that seropositivity was independent of the distance gradient from the index’s houses. However, being elderly was associated with seropositivity. DENV1-4 and JEV neutralizing antibodies were present in most ZIKV-positive and negative subsamples.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u0026nbsp;\u003c/strong\u003eProtective herd immunity for ZIKV infection is inadequate, especially among pregnant women in the two post-outbreak areas in southern Thailand.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Survey on neutralizing antibodies against Zika virus eighteen months post-outbreak in two southern Thailand communities","msid":"","msnumber":"","nonDraftVersions":[{"code":4,"date":"2020-12-04 18:34:43","doi":"10.21203/rs.3.rs-34709/v4","editorialEvents":[{"type":"communityComments","content":0},{"type":"checksComplete","content":"","date":"2020-11-25T00:01:43+00:00","index":"","fulltext":""},{"type":"decision","content":"Accept","date":"2020-11-24T00:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":3,"date":"2020-11-02 23:30:12","doi":"10.21203/rs.3.rs-34709/v3","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2020-11-18T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-11-17T00:00:00+00:00","index":1,"fulltext":"Recommendation: Accept without revision\nForm responses:\n---\n\nComments to Author:\n---\nThe author have appropriately addressed the issues previously raised and the manuscript have been accordingly revised.* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **I declare that I have no competing interests.**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"editorAssigned","content":"","date":"2020-10-28T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-10-28T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-10-28T12:00:00+00:00","index":1,"fulltext":""},{"type":"checksComplete","content":"","date":"2020-10-27T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-10-27T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2020-10-12 20:42:24","doi":"10.21203/rs.3.rs-34709/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2020-10-21T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable pending editorial decision\n"},{"type":"reviewerAgreed","content":"","date":"2020-10-05T12:00:00+00:00","index":2,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-10-01T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-10-01T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-10-01T12:00:00+00:00","index":1,"fulltext":""},{"type":"checksComplete","content":"","date":"2020-09-30T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-09-30T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-06-13 00:03:53","doi":"10.21203/rs.3.rs-34709/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2020-08-01T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-07-31T12:00:00+00:00","index":1,"fulltext":"Recommendation: Accept after minor essential revisions\nForm responses:\n---\n\nComments to Author:\n---\nThis is a nicely structured article that reports interesting results on an important topic. This article is generally well written, but could use a light but complete review (with editing) by a native English speaker.\n\n\nAbstract:\n\nIn the abstract, the authors mention that ZIKV can cause \"serious complications in a central nervous system, especially in babies\". This sounds like it is when babies are infected they can have serious neurological problems. Please reword this to reflect that it is fetal neurological development that is impacted if the woman is infected during pregnancies.\n\n\nBackground:\n\n- on page 3, line 60. This statement about one serological survey providing evidence of ZIKV in the 1950s makes it sound like this is the only indication of ZIKV in Southeast Asia until 2013. However, there were many reports/hints of ZIKV throughout the region between 1950 and 1980s (although laboratory techniques for assessing presence of ZIKV were not necessarily the most accurate). Previously, I've found Posen et al, 2016 -Epidemiology of Zika virus, 1947-2007 - BMJ Global Health as well as Kindhauser et al., 2016 - Zika: the origin and spread of a mosquito-borne virus - Bull World Health Organ thorough in trying to understand pre ~2014 spread in Asia. It would be nice if the authors made clear that they are taking into account other Southeast Asian reports from that era.\n- on page 3, line 67. The authors mention herd immunity and the need for epidemiologists to understand what is the level of population immunity needed to prevent further outbreaks. Is there yet any literature providing estimates of what level of herd immunity is needed for limiting the spread of Zika specifically? If not do we have this for DENV or CHIKV? If so it would be important to mention this background info here. This ties into my other comments on the results and conclusions when the authors refer to an insufficient level of ZIKV immunity in the community.\n\n\nMethods:\n- on page 4, line 86-88. It would be nice to understand, in a sentence or two, a general summary of what were the national guidelines to control the ZIKV outbreak. Is the testing of household contacts and pregnant women included in this national guideline?\n- On page 4, line 99-100. Could the random sampling techniques be described in a sentence or two?\n- On page 5 line 117-118. I agree with not including pregnant women in the model for risk factors in the general population, however, why did the authors not do a separate model for risk factors in pregnant women?\n- On page 5 line 119-120, the sentence referring to sample size calculation. Do you consider that there are 5 distance stratum as laid out in your point by point description above? I would leave this statement as is but also just mention here the total also for the ease of the reader - if I understand correctly this means 500 non-pregnant participants and 400 pregnant participants per district?\n- In data collection, there are two paragraphs repeated - starting from line 140 is a repeat of what was starting in line 126.\n- The 'variables' subsection should instead be included in the statistical analysis section, the latter of which should have a detailed description of the model used to generate ORs and the variables considered within it. There are multiple types of p-values presented throughout the manuscript, these and their significance should be described first in the statistical analysis section. Please also mention the rubber plantation inclusion when discussing the model, and the reason for this (rubber plantation proximity is associated with increased mosquito and associated virus diversity as far as I understand?)\n- Why choose distance band as the primary independent variable? I understand that there was a hypothesis about this, but is there another reason that is more practical or public health based? Would there be an intervention implemented based on or informed by this risk factor?\n\nResults:\n- line 195. Please give the response rate by district.\n- Line 202. The authors state that the prevalence among pregnant women was lower in both districts than in all other subgroups. How did the prevalence in women compare to the prevalence in non-pregnant women of the same age range (i.e. or at least, how did it compare to 18-40 year old non-pregnant participants). Please present this by district.\n- Line 211. The association of seropositivity with the absence of a rubber plantation needs to be explained/discussed/hypothesized about in the discussion.\n\n\nDiscussion: a few general comments\n- It is interesting to see the results of testing for antibody titers through PRNT for other flaviviruses in addition to ZIKV, but it seems that the authors are trying to imply that they could possibly make some conclusions ('suggests' etc, around line 248) that detecting more than one flavivirus may indicate cross-protectivity. This study is not designed to answer that question, and the number of tests done for other flavivirus (10 for each ZIKV + and ZIKV-?) is very few. The authors cannot make any conclusions or suggestions about cross protectivity from these findings, they can simply state what they found and suggest further research (if desired).\n- Discuss the finding of absence of rubber plantations being associated with seropositivity.\n- There is mention that the level of immunity in the population is not enough to prevent further outbreaks. What level is needed? What literature do the authors have to discuss this and back up this conclusion?\n\n\nTables and Figures:\n\nFigure 1 - this is personal preference, but I find that in graphics of infectious disease prevalence or incidence it is often the most highly affected areas that are coloured in the darkest, with moving to 'white' or lightest when the area is the least affected. This figure is currently the opposite. The authors may want to reverse this. Further, would it be possible to show the location of rubber plantations on the graphic (or are there too many?)? The eventual apparent protective effect of living close to a rubber plantation is quite confusing - see my further comments on this in that section.\nTable 2 - it is not necessary to present both seropositivity and seronegativity (just the former) as the second measure is inferred by the first. Instead, it would be informative to see the results by district (as presented throughout the manuscript).\nTable 2 and 3: describe the many different types of p-values used in the methods section and/or in a footnote.\n\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **I declare that I have no competing interests**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **No**\n* Are the methods sufficiently described to allow the study to be repeated?: **No**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"editorInvitedReview","content":"","date":"2020-07-28T12:00:00+00:00","index":2,"fulltext":"Recommendation: Major revisions required\nForm responses:\n---\n\nComments to Author:\n---\nThis manuscript by Densathaporn and colleagues addresses an important issue for flavivirus endemic areas: to investigate the prevalence of individuals carrying ZIKV neutralizing antibodies (nAb). The authors have examined antibody neutralization by using plaque reduction test (PRNT) among a general population and pregnant women residing a post-outbreak area in Thailand for investigating whether the ZIKV nAb prevalence is enough to dampen the transmission of the virus.\n\nUnfortunately, the results obtained do not support the conclusions drawn, and some particular aspects within the dataset remain to be questioned. This renders the manuscripts to be lacking impact.\n\nHere are my comments:\n\n1. The interpretation of the serological tests may be especially difficult for ZIKV at flavivirus endemic areas. It has been demonstrated that sera of individuals with a previous history of infection from other flaviviruses (especially dengue, yellow fever and West Nile) can cross-react in these tests. Although PRNT offers a greater specificity in the detection of neutralizing antibodies, cross-reactions have also been documented.\nFor instance the WHO recommendation for DENV PRNT analysis suggest that in flavivirus endemic areas a PRNT90 cutoff value is preferred, and several studies have used a 90% reduction in virus titer as the criteria for PRNT of ZIKV antibodies in such areas. Some of these studies have even used a 20 fold initial dilution, and thus positivity was defined as PRNT90 ≥ 20.\nFor greater stringency, the present study should have used (at minimum) PRNT90 to determine the endpoint of neutralization activity.\n\n2. Table 2: there is no data showing the geometric mean titers (GMT) and 95% CI obtained by PRNT in each group and subgroup. Is there any relevant difference on GMT among the groups/subgroups?\n\n3. Methods section (lines 140-153): these paragraphs are duplicated.\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons upon publication of the manuscript. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **I declare that I have no competing interests.**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **No**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"reviewerAgreed","content":"","date":"2020-07-10T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-07-09T12:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-06-12T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-06-11T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-06-11T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-06-11T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-06-09T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"infd","sideBox":"Learn more about [BMC Infectious Diseases](http://bmcinfectdis.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/infd","title":"BMC Infectious Diseases","twitterHandle":"#bmcinfectdis","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b94345ad-7f83-4ca6-b570-5fcde7cf0ef1","owner":[],"postedDate":"December 4th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":959402,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2020-12-06T15:02:30+00:00","versionOfRecord":{"articleIdentity":"rs-34709","link":"https://doi.org/10.1186/s12879-020-05654-8","journal":{"identity":"bmc-infectious-diseases","isVorOnly":false,"title":"BMC Infectious Diseases"},"publishedOn":"2020-12-03 15:00:39","publishedOnDateReadable":"December 3rd, 2020"},"versionCreatedAt":"2020-12-04 18:34:43","video":"","vorDoi":"10.1186/s12879-020-05654-8","vorDoiUrl":"https://doi.org/10.1186/s12879-020-05654-8","workflowStages":[]},"version":"v4","identity":"rs-34709","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-34709","identity":"rs-34709","version":["v4"]},"buildId":"pf3fE39SIOqb-0xH_OWvX","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}