Novel flaB gene variants of Leptospira interrogans detected in leptospirosis patient samples from the Western province of Sri Lanka

Novel flaB gene variants of Leptospira interrogans detected in leptospirosis patient samples from the Western province of Sri Lanka | 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 Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel flaB gene variants of Leptospira interrogans detected in leptospirosis patient samples from the Western province of Sri Lanka Shiroma Handunnetti, Yomendra De Silva, Jagathpriya Weerasena, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4764619/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background Leptospirosis caused by pathogenic spirochaetes of Leptospira spp remains the most widespread zoonotic disease in the world. Clinical status in leptospirosis patients varies from asymptomatic infection to mild illness to severe/fatal outcomes. The objective of this study was to determine the species of Leptospira that cause mild and severe infections, from patient blood samples obtained from the Western Province in Sri Lanka. Methods DNA extracted from 60 blood samples of previously confirmed leptospirosis patients by Lipl32 based Real Time PCR, and clinically characterized as mild and severe (n = 30 each) were used to perform nested PCR with primers designed from fla B gene sequence. Results Of the 45 samples that gave a 725 bp fragment in fla B nested PCR, 23 and 22 were from severe and mild leptospirosis patients respectively. Of these fla B PCR positive samples, 16 which exhibited strong bands (8 severe cases and 8 mild) were selected for Sanger’s dideoxy sequencing. The sequences obtained from 16 samples were deposited in GenBank. Those partial fla B gene sequences showed highest similarity with L. interrogans sequences available in GenBank database. Phylogenetic analysis was performed with 82 other randomly selected Leptospira fla B gene sequences (from the GenBank) by Neighbor-Joining method using MEGA 11 tool. According to dendrogram, 16 partial fla B gene sequences obtained in this study clustered together with L. interrogans . Three sequences, one from a severe leptospirosis patient and two from mild leptospirosis patients exhibited novel mutations. Computational analysis of the predicted amino acid substitutions in these three variants deduced from mutant DNA sequences with that of the wild type FlaB indicate possible functional changes in the flagellar protein. However, the impact of these novel mutations on the flagella assembly and motility could not be determined without functional confirmation. This highlights the need for further functional conformational studies to elucidate possible impact on motility which may influence the pathogenicity of different isolates of L. interrogans. Leptospirosis Leptospira interrogans flaB gene single nucleotide mutation Sri Lanka Figures Figure 1 Background Leptospirosis is a neglected, re-emerging zoonotic disease, prevalent worldwide, caused by the pathogenic spirochetes belonging to the genus Leptospira . The estimated global occurrence of leptospirosis is more than 1 million cases annually, leading to approximately 58,900 reported deaths. Its prevalence is notably higher among males aged 20–49 years [ 1 ]. In Sri Lanka, leptospirosis has emerged as a major public health threat and leptospirosis became a notifiable disease in 1991 in Sri Lanka [ 2 ]. It is prevalent in 22 out of 24 districts, and the estimated annual incidence is 52.1 per 100,000 and 730 annual deaths [ 2 ]. Large outbreaks of leptospirosis in Sri Lanka were recorded in 2008 with 7423 cases and in 2020 with 8579 cases [ 3 ]. Human leptospirosis primarily impacts low-income communities in both urban and rural areas within tropical regions [ 4 , 5 ]. Recently, there has been a growing number of reports of human leptospirosis in industrialized nations and temperate zones, potentially due to increasing ambient temperatures and humidity levels [ 6 ]. Multiple studies have shown that leptospires are harboured in chronic carrier hosts, mainly rodents and insectivores [ 7 , 8 ]. Bats and other small mammals have also been reported to harbour Leptospira species [ 9 , 10 ]. Typically, Leptospira do not induce illness in reservoir hosts. However, humans can contract infections through contact with infected animals, their tissues or excretions, or via abrasions, cuts, or exposure of the conjunctiva to contaminated water and food [ 10 ]. Human leptospirosis exhibits a spectrum of clinical symptoms, spanning from mild asymptomatic cases to severe, potentially life-threatening conditions [ 11 ]. Identification of criteria that predict disease severity is important for clinicians in disease management and for timely and appropriate interventions. The common clinical manifestations of mild disease include fever, myalgia, malaise, conjunctivitis, anorexia, nausea, vomiting, and abdominal pain. The initial signs of severe disease include jaundice, hemorrhage, oliguria, shortness of breath, and hepatosplenomegaly leading to single or multiple organ involvement [ 11 ]. Hence in our previous studies, a combination of clinical symptoms, and biochemical and other parameters were used to define the disease severity as mild and severe to clinically categorize the laboratory-confirmed leptospirosis patients [ 11 – 13 ]. According to a recent re-definition, the genus Leptospira contains 68 different species that include more than 300 serovars [ 14 ]. The latest classification of Leptospira is derived from whole genome sequencing. Leptospira is categorized into two primary clades: pathogenic (P) and saprophytic (S). Within the pathogenic clade, there are two subclades P1 and P2: P1, formerly referred to as the pathogenic group, comprises 19 species and P2, formerly referred to as the intermediate group, comprises 21 species [ 15 ]. The saprophytic (S) clade also comprises two subclades, S1 and S2. S1 comprises 21 species, and the new subclade S2 comprises five new saprophytic species identified from environmental samples [ 15 ]. Distinguishing pathogenic Leptospira species is important for epidemiological and taxonomical classification purposes [ 15 ]. Further, identifying the species and serovars that cause severe disease would be important for patient management as well as contribute to vaccine development [ 16 – 18 ]. To date, four species of Leptospira have been identified based on molecular studies on Leptospirosis patient samples from Sri Lanka where the majority of cases were caused by Leptospira interrogans , followed by L. kirschneri, L. borgpetersenii and L. weilli [ 18 – 24 ]. All these four species belong to the P1subclade. The objective of this study was to determine the Leptospira species in laboratory confirmed, clinically characterized mild and severe leptospirosis patients in the Western province of Sri Lanka. PCR amplification and direct sequencing was conducted using DNA extracted from patients’ blood samples and fla B primers specific for pathogenic Leptospira . The amino acid sequences deduced from the three mutants Leptospira interrogans DNA sequences were subjected to a computational analysis to predict possible functional changes that may result from the mutant proteins. Materials and Methods Clinical Samples Blood samples obtained from patients who were clinically suspected of having leptospirosis and had been admitted to the medical wards at The National Hospital of Sri Lanka (NHSL) between 1st of August 2012 to 28th February 2014 were used for this study. The samples were obtained from patients with acute febrile illness within 10 days of onset of fever. Clinical characterisation of these patients retrieved from the database as mild leptospirosis and severe leptospirosis patients had been performed based on previously defined criteria [ 11 , 13 ]. Laboratory confirmation for Leptospirosis was performed using Lipl32 based Real-Time PCR using Genesig Standard Real-time PCR detection kit for Leptospirosis (Primer Design Ltd, UK) [ 24 , 25 ]. Sixty Real-Time PCR-positive samples (n = 30 each for severe and mild cases) were selected for bacterial species identification using fla B gene amplification nested PCR followed by amplicon sequencing. Venous blood samples (1.0 ml) collected in EDTA tubes and stored frozen at -80 o C were used for this study. Extraction of genomic DNA and nested-PCR for fla B gene amplification Genomic DNA was extracted from blood samples which had been frozen at -80 o C using BactoSpin D™ genomic DNA extraction kit (CEYGEN Biotech (Pvt) Ltd, Sri Lanka) according to the manufacturer’s instructions with some modifications. Blood was mixed with red cell lysis buffer (CEYGEN Biotech (Pvt) Ltd, Sri Lanka), centrifuged at 7,500 g for 20 min at 4 o C and the pellet was used for purification of Leptospira DNA as per manufacturer’s instructions. Extracted DNA was quantified using Biospec Nano spectrophotometer (Shimadzu Corporation, Japan). Nested PCR was carried out to amplify Flagellin B ( fla B2) gene as described previously [ 26 – 28 ]. The PCR was performed in a total volume 25 µl reaction containing 200 µM of dNTPs, 2.5 mM of MgCl 2 , 0.2 µM of each primer, 1 U of Taq DNA Polymerase and 1X Green GoTaq buffer (Promega, USA) for both PCR reactions. For the first and second PCR amplifications, 50 ng of DNA and 2 µl of the first PCR product were used as the template, respectively. Primer sequences and amplicon sizes are shown in Table S1 . First PCR amplification was performed using following conditions: nitial denaturation at 94 ℃ for 5 min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 50 ℃ for 30 s, extension at 72 ℃ for 1 min. The final extension was at 72 ℃ for 10 min. For the second PCR amplification, nested primers were used and conditions were as follows: initial denaturation at 94 ℃ for 5 min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min then final extension at 72 ℃ for 10 min. PCR products were separated and visualised by 2% agarose gel electrophoresis. PCR product purification and DNA sequencing Amplicons were purified using SpinClean Gel Band and PCR Products Purification Kit (CEYGEN Biotech (Pvt) Ltd, Sri Lanka) and sequenced using Sanger dideoxy sequencing method (Genelabs Medical Ltd, Sri Lanka). The fla B sequences were deposited in GenBank database. The dendrogram was constructed with 16 partial fla B gene sequences and 82 fla B gene sequences which show high similarity with Leptospira from Sri Lanka and other countries randomly selected from the GenBank. The dendrogram was constructed using MEGA 11 [ 29 ] by aligning the sequences with MUSCLE and phylogenetic distances were calculated using Tamura-3-parameter model, sequences were clustered by Neighbor-Joining method. The statistical stability of the nodes was assessed by bootstrapping with 1000 replicates. Prediction of amino acid sequences and computational analysis of the three FlaB variants Partial fla B nucleotide sequences of the three fla B variants (NHSL13ML092, NHSL13ML506 and NHSL13SL215) were translated to amino acid (AA) sequences using Open Reading Frame Finder ( https://www.ncbi.nlm.nih.gov/orffinder/ ) and deposited in GenBank with following accession numbers: WKR38874, WKR38873 and WKR44734 respectively. Considering the FlaB AA sequences obtained from the NCBI database, the most abundant FlaB AA sequence with accession number WP_000586170 was used as the reference for comparison. Variations in the above AA sequences were identified using BLAST2 tool ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ) and the wild-type FlaB AA sequence (WP_000586170). Protein structure homology-modelling of the wild type and variant AA sequences was performed using SWISS-MODEL web server ( https://swissmodel.expasy.org/ ). Conserved region in the protein sequence of the wild type and variant sequences were identified by the ConSurf web server ( https://consurf.tau.ac.il/consurf_index.php ). Thermodynamic stability and interatomic interaction predictions of FlaB protein variants were carried out by the Dynamut web server ( https://biosig.lab.uq.edu.au/dynamut/ ) and compared with that of the wild-type FlaB protein. Results Leptospira species identification using partial fla B sequences Out of 60 selected samples, 45 amplified 732 bp amplicon in the fla B nested PCR, including 23 severe and 22 mild patient samples. Of these 45 fla B PCR positive samples, 16 which exhibited strong bands were selected for sequencing. The demographic and other criteria of these 16 patients (8 severe and 8 mild cases) are given in Table 1 . The resultant fla B sequences of all 16 samples showed the highest similarity with L. interrogans sequences in the GenBank (Table 2 ). Table 1 Demographic and disease complications of patient samples used for sequencing partial fla B gene fragment Sample no. Age (years) Gender (M/F) Days of illness MAT titer District Clinical category Disease complication NHSL12SL012 36 M 6 3200 Gampaha Severe Acute kidney injury NHSL12SL059 45 M 7 800 Colombo Severe Acute kidney injury, Thrombocytopenia NHSL12SL150 27 M 7 3200 Colombo Severe Acute kidney injury, Myocarditis, NHSL13SL215 55 M 6 800 Gampaha Severe Acute kidney injury NHSL13SL442 37 M 6 1600 Colombo Severe Acute kidney injury NHSL13SL537 52 M 10 3200 Colombo Severe Acute kidney injury NHSL13SL580 42 F 5 800 Colombo Severe Acute kidney injury NHSL13SL581 33 M 5 400 Gampaha Severe Acute kidney injury, Thrombocytopenia NHSL12ML051* 24 M 7 1600 Colombo Severe Thrombocytopenia NHSL13ML092 27 M 5 NA Gampaha Mild - NHSL12ML180 51 M 10 3200 Colombo Mild - NHSL12ML185 30 M 6 800 Gampaha Mild - NHSL13ML506 40 M 8 1600 Colombo Mild Thrombocytopenia NHSL13ML520 52 M 9 3200 Gampaha Mild - NHSL13ML713 65 F 4 NA Gampaha Mild - NHSL14ML769* 36 M 3 400 Gampaha Mild - SL – Severe Leptospirosis, ML – Mild Leptospirosis, F – female, M – male, NA – not available Sources of exposure by rat infected water or recent skin injury for all patients, except for those marked with (*) who had exposure by sources other than rat-infested water/ recent skin injury/infected by animals. Table 2 Leptospira sequence identity and there predicted amino acid sequences Sample no. Nucleotide Accession no. Leptospira species and serovar identified Sequence Similarity (%) Mutations identified Amino Acid Accession no. Amino acid substitutions† NHSL12SL012 OQ575709 L. interrogans Bataviae 100 - WKE35572 - NHSL12SL059 OQ408277 L. interrogans Bataviae, Canicola 100 - WJJ80047 - NHSL12SL150 OR480416 L. interrogans Lai 100 - WNO24712 - NHSL13SL215 OQ689693 L. interrogans Bataviae, Canicola 99 A1974716G* WKR44734 Y164C† A1974742G* - NHSL13SL442 OR459953 L. interrogans Canicola 100 - WNO24711 - NHSL13SL537 OQ536446 L. interrogans Copenhageni 100 - WJJ80049 - NHSL13SL580 OQ408278 L. interrogans Lai 100 - WJJ80048 - NHSL13SL581 OQ085075 L. interrogans Bataviae, Canicola 100 - WJJ80046 - NHSL12ML051 OQ547310 L. interrogans Lai 100 - WJJ80050 - NHSL13ML092 OQ682751 L. interrogans Bataviae, Canicola 99 A1974376G # WKR38874 D42G† A1974645G # K132E† T1974745C # I165T† NHSL12ML180 OR480417 L. interrogans Lai 100 - WNO24714 - NHSL12ML185 OR492662 L. interrogans Lai 100 - WNO24714 - NHSL13ML506 OQ658513 L. interrogans Copenhageni, Icterohaemorrhagiae 99 T2286802C # WKR38873 L175S† T2286920C # - C2286921T # - NHSL13ML520 OQ559482 L. interrogans Bataviae, Canicola 100 - WKD80855 - NHSL13ML713 OQ553816 L. interrogans Bataviae, Canicola 100 - WJJ80051 - NHSL14ML769 OR451931 L. interrogans Canicola 100 - WNO11800 - SL – Severe Leptospirosis, ML – Mild Leptospirosis, DNA sequences with mutations given in bold font. * with reference to LC505621; # with reference to: CP039258; † with reference to: WP_000586170 Three partial fla B gene sequences with novel mutations were identified from one severe (NHSL13SL215: OQ689693) and two mild (NHSL13ML092: OQ682751; NHSL13ML506: OQ658513) patients during this study referred to as variants. A > G mutations were identified at nucleotide positions 1974716 and 1974742 in the partial fla B gene sequence of one severe patient sample (NHSL13SL215) compared to the reference sequence LC505621 (Table 2 ). Similarly, among two mild patient samples, three mutations each were identified compared to reference sequence CP039258 (in NHSL13ML092, A > G mutations at 1974376 and 1974645 and T > C mutation at 1974745 and in NHSL13ML506, T > C mutations at 2286802 and 2286920, and C > T mutation at 2286921). Phylogenetic analysis The 16 partial fla B gene sequences used for the phylogenetic analysis showed 100% sequence identity with L. interrogans . Four clusters were observed in the dendrogram including these sequences (Fig. 1 ), ie, L. interrogans, L. kirschneri, L. weilli and L. borgpetersenii. These fla B sequences were aligned with sequences previously reported from countries in Asia, such as China, Japan, and Thailand, Latin American countries such as Brazil, and temperate countries like the USA indicating that these 16 sequences of L. interrogans identified from Sri Lanka are also found in other geographical regions. According to the dendrogram, all 98 sequences were clustered into four groups: L. interrogans (Cluster 1), L. kirschneri (Cluster 2), L. weilli (Cluster 3), and L. borgpetersenii (Cluster 4). Predicted amino acid sequences in the three variants Patient no NHSL13SL215, NHSL13ML092, and NHSL13ML506 showed point mutations that are predicted to alter the AA sequences (Table 2 & Table S2 ). In sample no. NHSL13SL215, the translated AA sequence shows substitution of Tyrosine (polar AA) by Cysteine (non-polar AA; Y164C) found in the variable region of the FlaB protein (Figure S1 ). The replacement of this AA may alter the carbon composition and disrupt interatomic interactions leading to destabilization of the FlaB protein (Table S2 and Figure S2 ). Similarly, in sample NHSL13ML506, the translated AA sequence shows substitution of Leucine (non-polar AA) by Serine (polar AA; L175S) found in the variable region of the FlaB protein. Due to this AA substitution, the carbon molecules and interatomic interactions may lead to destabilizing the FlaB protein. Although there are three substitutions in the translated AA sequence in sample NHSL13ML092, namely Aspartic acid (polar acidic AA) replaced by Glycine (non-polar AA; D42G), Lysine (polar basic AA) replaced by Glutamic acid (polar acidic AA; K132E) and Isoleucine (non-polar AA) replaced by Threonine (polar AA; I165T), substitution D42G lies in a highly conserved region while other two substitutions (K132E and I165T) are located in variable regions. Further, the two substitutions, D42G and I165T are predicted to be contributed to significant change in destabilizing the FlaB protein (Table S2 and Figure S2 ). Discussion The prevalence of Leptospira species causing different clinical outcomes in a locality is critical for various aspects including patient management strategies, treatment options and vaccine development against leptospirosis [ 16 ]. The present study was focused on Leptospira species identification using blood samples of clinically characterized, mild and severe leptospirosis patients who were hospitalized in two hospitals in the Western Province of Sri Lanka. The 16 sequences obtained in the present study included 8 severe cases and 8 mild cases aligned with L. interrogans , indicating that both categories had L. interrogans as the infecting species. However, the small sample size is a limitation of this study, and this needs further investigation with an adequate sample size. It is also noteworthy that there may be factors other than the infecting species (serovar), such as leptospiremia, host immune and genetic factors that may contribute to the clinical severity in leptospirosis infections. However, a previous study using quantitative PCR has indicated that leptospiremia in serum/whole blood samples did not directly correlate with the clinical manifestations [ 30 ] It is interesting to note that previous molecular studies conducted with patient samples from Sri Lanka have identified L. interrogans as the most prevalent species, with 73.9% (76 out of 111) of the cases being caused due to L. interrogans whereas the other species were less prevalent, i.e., L. kirschneri (22.5%), L. borgpetersenii (5.4%) and L. weilii (2.7%) [ 17 – 23 ]. Hence, the results from the present study are consistent with the previous findings on L. interrogans being a more prevalent species in Sri Lanka. Further, a previous study conducted with patient isolates from wet zone regions such as Western, Southern and Central Provinces of Sri Lanka had shown that L. interrogans was the most predominant species prevalent, and was associated with renal failure [ 17 , 19 , 21 , 23 , 31 ]. Acute renal failure was the most common disease complication observed among the severe leptospirosis patients in the present study. Although L. interrogans has been reported as the most prevalent species in Sri Lanka, recent studies focused on specific epidemics have reported other species such as L. kirschneri as the most prevalent species in 2011 [ 32 ]. The reason for this variation has mainly been attributed to the circulating species in different provinces in Sri Lanka and certain seasonal changes observed with a flood-associated outbreak in 2011 in the Anuradhapura District in North-Central Province in the dry zone, whereas most of the previous studies have sampled from leptospirosis patients from the wet zone districts and provinces [ 21 ]. Interestingly, in the phylogenetic analysis, the majority of sequences in this L. interrogans Cluster 1 comprised of samples derived from various Asian countries. However, the other sequences included in this phylogenetic analysis comprised of samples derived from diverse geographic regions, including the Brazil, Puerto Rico and United States. Several genes such as rrs , sec Y, lip L32, fla B, lfb 1, lig A, and lig B2 have been used for molecular diagnosis Leptospira [ 33 ]. Of these genes, a majority of studies have used lipL32 as the target gene which has sensitivity and specificity of 0.42 and 0.95 respectively whereas the gene used in the present study was fla B which had shown sensitivity and specificity of 0.41 and 0.90 respectively [ 33 , 34 ]. The fla B gene has been used previously to discriminate between Leptospira species in the majority of studies conducted in Sri Lanka [ 17 , 22 , 23 , 32 ]. It encodes a flagellum protein, and four isotypes of FlaB (FlaB1, FlaB2, FlaB3 and FlaB4) have been described [ 35 ]. Of the four genes encoding these isotypes, most of the studies have used fla B2 gene as their target [ 26 , 28 ]. The flagellar filaments of Leptospira , display a complex structure consisting of a central core made of FlaB protein (281 to 285 amino acids) surrounded by a sheath of FlaA protein [ 36 ]. The motility of leptospires depends on the presence of two endoflagella, which could play a critical role as a virulence factor. It is noteworthy that FlaB mutants of L. biflexa were shown to be non-motile and deficient in endoflagella, but the cell body of the mutants remained intact and retained its helical morphology [ 37 ]. In the present study, we have observed three novel fla B2 variants that are predicted to result in changes in the AA sequence which may result in possible changes in the FlaB protein. However, without functional confirmation, we could not determine the impact of these novel mutations on the flagella assembly and motility. In conclusion, the present study has shown novel as well as previously reported fla B gene sequences in the samples derived from clinically characterized mild and severe leptospirosis patients from the Western Province in Sri Lanka. All 16 sequences were identified as L. interrogans and grouped in 4 sub-clusters except for the three samples which comprised novel mutations that are predicted to result in amino acid changes in the FlaB protein. Declarations Declarations Ethics approval and consent to participate This study was carried out with the approved obtained from the Ethics Review Committee of Faculty of Medicine, University of Colombo, Sri Lanka (EC-12-056). Written, informed consent was obtained from all the patients prior to recruitment to the study. Clinical trial number Not applicable Consent for publication Not applicable. Competing interests Authors declare that there are no competing interests. Funding This study was supported by grant no. NRC-17-098, awarded to SH from the National Research Council, Sri Lanka ( http://www.nrc.gov.lk/ ). Data collection component of this study was funded by grant no. NRC-12-077, National Research Council, Sri Lanka and grant no. RG/ 2011/HS/19, National Science Foundation, Sri Lanka ( http://www.nsf.ac.lk/ ), awarded to SH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contribution Conceived and designed the experiments: SH NF SP SR. Sample selection, retrieval of demographic data and analysis: NF SH NC. Performed molecular biological experiments, computational analysis and data analysis: YS JW SH NF. Funding acquisition: SH. Wrote first draft of the paper: SH YS. Critically reviewed and revised the draft, and approved the final manuscript: SH YS JW SP NF HS NC SR. Acknowledgement Consultants and other Staff of the National Hospital of Sri Lanka; Consultant Bacteriologist and staff of the Medical Research Institute, Colombo, Sri Lanka; for help with data collection and clinical categorization. Data Availability The datasets supporting the results and conclusions are available from the GenBank (NCBI) database and the accession numbers are available in the main text. References Costa F, Hagan JE, Calcagno J, Kane M, Torgerson P, Martinez-Silveira MS, et al. Global morbidity and mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis. 2015;9:9. Warnasekara J, Koralegedara I, Agampodi S. Estimating the burden of leptospirosis in Sri Lanka; a systematic review. BMC Infect Dis. 2019;19:1–2. Warnasekara J, Agampodi S. Neglecting the neglected during the COVID-19 pandemic: the case of leptospirosis in Sri Lanka. Epidemiol Health epiH. 2022;44. Haake DA, Levett PN. Leptospirosis in humans. Curr Top Microbiol Immunol. 2015;387:65–97. Goarant C, Leptospirosis. Risk factors and management challenges in developing countries. Rep Trop Med. 2016;7:49–62. Dupouey J, Faucher B, Edouard S, Richet H, Kodjo A, Drancourt M, Davoust B. Human leptospirosis: An emerging risk in Europe? Comp. Immunol Microbiol Infect Dis. 2014;37:77–83. Ko AI, Goarant C, Picardeau M, Leptospira. The dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol. 2009;7:736–47. Rahelinirina S, Leon A, Harstskeerl RA, Sertour N, Ahmed A, Raharimanana C, et al. First isolation and direct evidence for the existence of large small mammal reservoirs of Leptospira spp . in Madagascar. PLoS ONE. 2010;5:11. Zhao M, Xiao X, Han HJ, Wang LJ, Lei SC, Liu JW, et al. Leptospira in bats from Hubei Province, China, 2018. J Wildl Dis. 2019;55(4):940–3. Antima BS. Modeling the dynamics of leptospirosis in India. Sci Rep. 2023;13(1):19791. Rajapakse S, Weeratunga P, Niloofa MR, Fernando N, Rodrigo C, Maduranga S, et al. Clinical and laboratory associations of severity in a Sri Lankan cohort of patients with serologically confirmed leptospirosis: a prospective study. Trans R Soc Trop Med Hyg. 2015;109(11):710–6. Kalugalage T, Rodrigo C, Vithanage T, Somaratne P, De Silva HJ, Handunnetti S, et al. Low serum total nitrite and nitrate levels in severe leptospirosis. BMC Infect Dis. 2013;13(1):206. Fernando N, Wickremesinghe S, Niloofa R, Rodrigo C, Karunanayake L, de Silva HJ, et al. Protein carbonyl as a biomarker of oxidative stress in severe leptospirosis, and its usefulness in differentiating leptospirosis from dengue infections. PLoS ONE. 2016;11:6. Thibeaux R, Girault D, Bierque E, Soupé-Gilbert M-E, Rettinger A, et al. Biodiversity of environmental Leptospira : improving identification and revisiting the diagnosis. Front Microbiol. 2018;9:816. Vincent AT, Schiettekatte O, Goarant C, Neela VK, Bernet E, Thibeaux R, et al. Revisiting the taxonomy and evolution of pathogenicity of the genus Leptospira through the prism of genomics. PLoS Negl Trop Dis. 2019;13:5. Azevedo IR, Amamura TA, Isaac L. Human leptospirosis: In search for a better vaccine. Scand J Immunol. 2023;98(5):e13316. Bandara KK, Weerasekera M, Gunasekara CP, Ranasinghe N, Marasinghe C, Fernando N. Molecular characterization and disease severity of leptospirosis in Sri Lanka. Mem Inst Oswaldo Cruz. 2015;110(4):485–91. Koizumi N, Gamage CD, Muto M, Kularatne SA, Budagoda SB, Rajapakse JR, et al. Serological and genetic analysis of leptospirosis in patients with acute febrile illness in Kandy, Sri Lanka. Jpn J Inf Dis. 2009;62(6):474–5. Nwafor-Okoli C, Koizumi N, Kularatne SA, Rajapakse J, Gamage CD, Muto M, et al. Leptospira infection at the University of Peradeniya Teaching Hospital, Sri Lanka: clinical and laboratory investigations. Southeast Asian J Trop Med Public Health. 2012;43(4):943–50. Agampodi SB, Peacock SJ, Thevanesam V, Nugegoda DB, Smythe L, Thaipadungpanit J, et al. Leptospirosis outbreak in Sri Lanka in 2008: lessons for assessing the global burden of disease. ASTMH. 2011;85(3):471. Agampodi SB, Dahanayaka NJ, Bandaranayaka AK, Perera M, Priyankara S, Weerawansa P, et al. Regional differences of leptospirosis in Sri Lanka: observations from a flood-associated outbreak in 2011. PLoS Negl Trop Dis. 2014;8(1):e2626. Nisansala GG, Muthusinghe D, Gunasekara TD, Weerasekera MM, Fernando SS, Ranasinghe KN, et al. Isolation and characterization of Leptospira interrogans from two patients with leptospirosis in Western Province, Sri Lanka. J Med Microbiol. 2018;67(9):1249–52. Karunanayake L, Gamage CD, Gunasekara CP, De Silva S, Izumiya H, Morita M, et al. Multilocus sequence typing reveals diverse known and novel genotypes of Leptospira spp . circulating in Sri Lanka. PLoS Negl Trop Dis. 2020;14(8):e0008573. Niloofa MJR. Studies on comparing rapid immunodiagnostic methods and immune status of leptospirosis patients in Western Province Sri Lanka: PhD Thesis. Institute of Biochemistry, Molecular Biology and Biotechnology: University of Colombo, Sri Lanka; 2016. Villumsen S, Pedersen R, Borre MB, Ahrens P, Jensen JS, Krogfelt KA. Novel TaqMan® PCR for detection of Leptospira species in urine and blood: pit-falls of in silico validation. J Microbiol Methods. 2012;91(1):184–90. Kawabata H, Dancel LA, Villanueva SY, Yanagihara Y, Koizumi N, Watanabe H. fla B-Polymerase chain reaction ( fla B‐PCR) and its restriction fragment length polymorphism (RFLP) analysis are an efficient tool for detection and identification of Leptospira spp . Microbiol Immunol. 2001;45(6):491–6. Koizumi N, Muto M, Yamamoto S, Baba Y, Kudo M, Tamae Y, et al. Investigation of reservoir animals of Leptospira in the northern part of Miyazaki prefecture. Jpn J Infect Dis. 2008;61:465–8. Haghnazari. Sepideh and khaki, Pejvak and Moradi Bidhendi, Soheila and Esmaelizad, Majid and Tebianian, Majid and Gharakhani, Mehdi, 1402, Molecular characterization of the flaB2 gene of pathogenic Leptospira vaccinal serovars, 24th International Congress of Microbiology of Iran, Tehran. https://civilica.com/doc/1922326 [accessed on 2024-12-10. Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38(7):3022–7. Agampodi SB, Matthias MA, Moreno AC, Vinetz JM. Utility of quantitative polymerase chain reaction in leptospirosis diagnosis: association of level of leptospiremia and clinical manifestations in Sri Lanka. Clin Infect Dis. 2012;54(9):1249–55. Jayasundara D, Senavirathna I, Warnasekara J, Gamage C, Siribaddana S, Kularatne SA et al. 12 Novel clonal groups of Leptospira infecting humans in multiple contrasting epidemiological contexts in Sri Lanka. PLoS Negl Trop Dis. 2021 18;15(3):e0009272. Naotunna C, Agampodi SB, Agampodi TC. Etiological agents causing leptospirosis in Sri Lanka: A review. Asian Pac J Trop Med. 2016;9(4):390–4. Lam JY, Low GK, Chee HY. Diagnostic accuracy of genetic markers and nucleic acid techniques for the detection of Leptospira in clinical samples: A meta-analysis. PLoS Negl Trop Dis. 2020;14(2):e0008074. Di Azevedo MI, Lilenbaum W. An overview on the molecular diagnosis of animal leptospirosis. Lett Appl Microbiol. 2021;72(5):496–508. Malmström J, Beck M, Schmidt A, Lange V, Deutsch EW, Aebersold R. Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans . Nature. 2009;460(7256):762–5. Lambert A, Picardeau M, Haake DA, Sermswan RW, Srikram A, Adler B, et al. FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath. Infect Immun. 2012;80(6):2019–25. Picardeau M, Brenot A, Saint Girons I. First evidence for gene replacement in Leptospira spp. Inactivation of L. biflexa fla B results in non-motile mutants deficient in endoflagella. Mol Microbiol. 2001;40(1):189–99. Abbreviations AA Amino Acid A Adenine Bp base pair BLAST Basic Local Alignment Search Tool C Cytosine flaB gene flagellin B gene G Guanine MEGA Molecular Evolutionary Genetics Analysis version MUSCLE Multiple Sequence Comparison by Log-Expectation NCBI National Center for Biotechnology Information NHSL National Hospital of Sri Lanka PCR Polymerase chain reaction T Thymine Supporting Information Additional File 1: Table S1 : Sequences of primers and product sizes for nested-PCR for fla B gene amplification. Additional File 1: Table S2: Predicted changes in the FlaB protein of the 3 variants compared to the wild type FlaB protein of L. interorgans Additional File 3: Figure S1 : FlaB protein of L. interrogans (A) (WP_000586170, reference), variant protein sequences of NHSL13SL215 (B) (WKR44734) NHSL13ML092 (C) (WKR38874), and NHSL13ML506 (D) (WKR38873). Additional File 4: Figure S2: Predicted protein stability for the 3 variants compared to the wild type FlaB protein of L. interrogans Additional Declarations No competing interests reported. Supplementary Files AdditionalFile1TableS1.docx AdditionalFile2TableS2.docx AdditionalFile3FigureS1.pdf AdditionalFile4FigureS2.pdf Cite Share Download PDF Status: Posted Version 2 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board 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-4764619","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":398307617,"identity":"a37fbc17-b3cb-463e-8261-c235ff31a32f","order_by":0,"name":"Shiroma Handunnetti","email":"","orcid":"","institution":"Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka","correspondingAuthor":false,"prefix":"","firstName":"Shiroma","middleName":"","lastName":"Handunnetti","suffix":""},{"id":398307618,"identity":"3d7c4fce-5f75-4b2f-89dd-3dd83fb9e5f4","order_by":1,"name":"Yomendra De Silva","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYNACNiA+3gAkDCxI0XLmAEiLBClabiSAWERoMTjenfi4oOyePN/N51c3/CiQYOBv707Ar+XM2c3GM84VG868nVN2swfoMIkzZzfg13Ijd5s0b1sC44bbOWk3eIBaDCRyCWrZ/huoxX7DzTNpN/8QqWUbM1BL4oYb7MduE2WLJNAv0jznEpJnnslhuy1jIMFD0C98x3s3fuYpS7DtO3782c03f2zk+Nt78WtROABn8hiASbzKQUC+Ac5kf0BQ9SgYBaNgFIxMAAD04U6MVdw5SAAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka","correspondingAuthor":true,"prefix":"","firstName":"Yomendra","middleName":"","lastName":"De Silva","suffix":""},{"id":398307620,"identity":"3350f0bf-8153-4a49-9632-3c73dc96ed0b","order_by":2,"name":"Jagathpriya Weerasena","email":"","orcid":"","institution":"Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka","correspondingAuthor":false,"prefix":"","firstName":"Jagathpriya","middleName":"","lastName":"Weerasena","suffix":""},{"id":398307621,"identity":"ea3008f3-ed72-462a-bdda-9ffb3a676049","order_by":3,"name":"Narmada Fernando","email":"","orcid":"","institution":"Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka","correspondingAuthor":false,"prefix":"","firstName":"Narmada","middleName":"","lastName":"Fernando","suffix":""},{"id":398307622,"identity":"3ab94e02-cfd8-4f17-afe8-652bb2d60542","order_by":4,"name":"Harindra Sathkumara","email":"","orcid":"","institution":"Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka","correspondingAuthor":false,"prefix":"","firstName":"Harindra","middleName":"","lastName":"Sathkumara","suffix":""},{"id":398307623,"identity":"721e43e2-07a9-4d05-8ddd-61ce0a5c335c","order_by":5,"name":"Nadeema Chandrapadma","email":"","orcid":"","institution":"Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka","correspondingAuthor":false,"prefix":"","firstName":"Nadeema","middleName":"","lastName":"Chandrapadma","suffix":""},{"id":398307625,"identity":"59f49f9e-73d9-4be9-a4c0-679df0c49b68","order_by":6,"name":"Sunil Premawansa","email":"","orcid":"","institution":"Departments of Zoology and Environment Sciences, Faculty of Science, University of Colombo","correspondingAuthor":false,"prefix":"","firstName":"Sunil","middleName":"","lastName":"Premawansa","suffix":""},{"id":398307627,"identity":"5cc104c8-d001-456a-b255-1f108b2f32a9","order_by":7,"name":"Senaka Rajapakse","email":"","orcid":"","institution":"Department of Clinical Medicine, Faculty of Medicine, University of Colombo, Colomb 08, Sri Lanka.","correspondingAuthor":false,"prefix":"","firstName":"Senaka","middleName":"","lastName":"Rajapakse","suffix":""}],"badges":[],"createdAt":"2024-07-18 18:36:12","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-4764619/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-4764619/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73077967,"identity":"90355096-9fcd-43b8-ba5b-710c2fc85f24","added_by":"auto","created_at":"2025-01-06 13:45:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":175972,"visible":true,"origin":"","legend":"\u003cp\u003eDendrogram based on \u003cem\u003efla\u003c/em\u003eB gene of \u003cem\u003eLeptospira\u003c/em\u003e species prevalent worldwide.\u003cstrong\u003e \u003c/strong\u003eDendrogram constructed by Neighbor-Joining method using Tamura-3-parameter model and bootstrapping with 1000 replicates. Bootstrap value is given next to the branches. The sequences derived from this study are in black and labelled with (red diamond).\u003c/p\u003e","description":"","filename":"Screenshot20250106at8.34.51AM.png","url":"https://assets-eu.researchsquare.com/files/rs-4764619/v2/d6d26af7cae9c6d909f34a2e.png"},{"id":74002128,"identity":"feb7ac29-74e8-4b91-96f9-67d2f4c0df4b","added_by":"auto","created_at":"2025-01-16 22:16:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1197032,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4764619/v2/fa28b981-242e-4deb-845a-f0abc97e0461.pdf"},{"id":73079090,"identity":"f2090c6d-9a29-4846-b659-06aecbc7a684","added_by":"auto","created_at":"2025-01-06 13:53:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14086,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile1TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4764619/v2/ff4fee12b652882d179856fb.docx"},{"id":73076516,"identity":"84e784c8-cf2a-4b9b-8277-e2a3b0167c0c","added_by":"auto","created_at":"2025-01-06 13:37:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19608,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile2TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4764619/v2/fc7868acfa52bc96b4740493.docx"},{"id":73079092,"identity":"92144dc7-76f9-4e20-b7e9-a2ee244c3fb9","added_by":"auto","created_at":"2025-01-06 13:54:00","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":498061,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile3FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4764619/v2/19590bcbf6cdea161a63da30.pdf"},{"id":73076524,"identity":"3a80010c-d0fd-42cc-914a-8e1c33bc9ece","added_by":"auto","created_at":"2025-01-06 13:37:59","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":592135,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFile4FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4764619/v2/01b2c93ff56a4a26e621b21a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel flaB gene variants of Leptospira interrogans detected in leptospirosis patient samples from the Western province of Sri Lanka","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLeptospirosis is a neglected, re-emerging zoonotic disease, prevalent worldwide, caused by the pathogenic spirochetes belonging to the genus \u003cem\u003eLeptospira\u003c/em\u003e. The estimated global occurrence of leptospirosis is more than 1\u0026nbsp;million cases annually, leading to approximately 58,900 reported deaths. Its prevalence is notably higher among males aged 20\u0026ndash;49 years [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In Sri Lanka, leptospirosis has emerged as a major public health threat and leptospirosis became a notifiable disease in 1991 in Sri Lanka [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is prevalent in 22 out of 24 districts, and the estimated annual incidence is 52.1 per 100,000 and 730 annual deaths [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Large outbreaks of leptospirosis in Sri Lanka were recorded in 2008 with 7423 cases and in 2020 with 8579 cases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Human leptospirosis primarily impacts low-income communities in both urban and rural areas within tropical regions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Recently, there has been a growing number of reports of human leptospirosis in industrialized nations and temperate zones, potentially due to increasing ambient temperatures and humidity levels [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Multiple studies have shown that leptospires are harboured in chronic carrier hosts, mainly rodents and insectivores [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Bats and other small mammals have also been reported to harbour \u003cem\u003eLeptospira\u003c/em\u003e species [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Typically, \u003cem\u003eLeptospira\u003c/em\u003e do not induce illness in reservoir hosts. However, humans can contract infections through contact with infected animals, their tissues or excretions, or via abrasions, cuts, or exposure of the conjunctiva to contaminated water and food [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHuman leptospirosis exhibits a spectrum of clinical symptoms, spanning from mild asymptomatic cases to severe, potentially life-threatening conditions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Identification of criteria that predict disease severity is important for clinicians in disease management and for timely and appropriate interventions. The common clinical manifestations of mild disease include fever, myalgia, malaise, conjunctivitis, anorexia, nausea, vomiting, and abdominal pain. The initial signs of severe disease include jaundice, hemorrhage, oliguria, shortness of breath, and hepatosplenomegaly leading to single or multiple organ involvement [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Hence in our previous studies, a combination of clinical symptoms, and biochemical and other parameters were used to define the disease severity as mild and severe to clinically categorize the laboratory-confirmed leptospirosis patients [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to a recent re-definition, the genus \u003cem\u003eLeptospira\u003c/em\u003e contains 68 different species that include more than 300 serovars [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The latest classification of \u003cem\u003eLeptospira\u003c/em\u003e is derived from whole genome sequencing. \u003cem\u003eLeptospira\u003c/em\u003e is categorized into two primary clades: pathogenic (P) and saprophytic (S). Within the pathogenic clade, there are two subclades P1 and P2: P1, formerly referred to as the pathogenic group, comprises 19 species and P2, formerly referred to as the intermediate group, comprises 21 species [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The saprophytic (S) clade also comprises two subclades, S1 and S2. S1 comprises 21 species, and the new subclade S2 comprises five new saprophytic species identified from environmental samples [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDistinguishing pathogenic \u003cem\u003eLeptospira\u003c/em\u003e species is important for epidemiological and taxonomical classification purposes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Further, identifying the species and serovars that cause severe disease would be important for patient management as well as contribute to vaccine development [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To date, four species of \u003cem\u003eLeptospira\u003c/em\u003e have been identified based on molecular studies on Leptospirosis patient samples from Sri Lanka where the majority of cases were caused by \u003cem\u003eLeptospira interrogans\u003c/em\u003e, followed by \u003cem\u003eL. kirschneri, L. borgpetersenii\u003c/em\u003e and \u003cem\u003eL. weilli\u003c/em\u003e [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. All these four species belong to the P1subclade.\u003c/p\u003e \u003cp\u003eThe objective of this study was to determine the \u003cem\u003eLeptospira\u003c/em\u003e species in laboratory confirmed, clinically characterized mild and severe leptospirosis patients in the Western province of Sri Lanka. PCR amplification and direct sequencing was conducted using DNA extracted from patients\u0026rsquo; blood samples and \u003cem\u003efla\u003c/em\u003eB primers specific for pathogenic \u003cem\u003eLeptospira\u003c/em\u003e. The amino acid sequences deduced from the three mutants \u003cem\u003eLeptospira interrogans\u003c/em\u003e DNA sequences were subjected to a computational analysis to predict possible functional changes that may result from the mutant proteins.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical Samples\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBlood samples obtained from patients who were clinically suspected of having leptospirosis and had been admitted to the medical wards at The National Hospital of Sri Lanka (NHSL) between 1st of August 2012 to 28th February 2014 were used for this study. The samples were obtained from patients with acute febrile illness within 10 days of onset of fever. Clinical characterisation of these patients retrieved from the database as mild leptospirosis and severe leptospirosis patients had been performed based on previously defined criteria [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Laboratory confirmation for Leptospirosis was performed using Lipl32 based Real-Time PCR using Genesig Standard Real-time PCR detection kit for Leptospirosis (Primer Design Ltd, UK) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Sixty Real-Time PCR-positive samples (n\u0026thinsp;=\u0026thinsp;30 each for severe and mild cases) were selected for bacterial species identification using \u003cem\u003efla\u003c/em\u003eB gene amplification nested PCR followed by amplicon sequencing. Venous blood samples (1.0 ml) collected in EDTA tubes and stored frozen at -80 \u003csup\u003eo\u003c/sup\u003eC were used for this study.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExtraction of genomic DNA and nested-PCR for\u003c/b\u003e \u003cb\u003efla\u003c/b\u003e\u003cb\u003eB gene amplification\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenomic DNA was extracted from blood samples which had been frozen at -80 \u003csup\u003eo\u003c/sup\u003eC using BactoSpin D\u0026trade; genomic DNA extraction kit (CEYGEN Biotech (Pvt) Ltd, Sri Lanka) according to the manufacturer\u0026rsquo;s instructions with some modifications. Blood was mixed with red cell lysis buffer (CEYGEN Biotech (Pvt) Ltd, Sri Lanka), centrifuged at 7,500 \u003cem\u003eg\u003c/em\u003e for 20 min at 4 \u003csup\u003eo\u003c/sup\u003eC and the pellet was used for purification of \u003cem\u003eLeptospira\u003c/em\u003e DNA as per manufacturer\u0026rsquo;s instructions. Extracted DNA was quantified using Biospec Nano spectrophotometer (Shimadzu Corporation, Japan).\u003c/p\u003e \u003cp\u003eNested PCR was carried out to amplify Flagellin B (\u003cem\u003efla\u003c/em\u003eB2) gene as described previously [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The PCR was performed in a total volume 25 \u0026micro;l reaction containing 200 \u0026micro;M of dNTPs, 2.5 mM of MgCl\u003csub\u003e2\u003c/sub\u003e, 0.2 \u0026micro;M of each primer, 1 U of Taq DNA Polymerase and 1X Green GoTaq buffer (Promega, USA) for both PCR reactions. For the first and second PCR amplifications, 50 ng of DNA and 2 \u0026micro;l of the first PCR product were used as the template, respectively. Primer sequences and amplicon sizes are shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. First PCR amplification was performed using following conditions: nitial denaturation at 94 ℃ for 5 min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 50 ℃ for 30 s, extension at 72 ℃ for 1 min. The final extension was at 72 ℃ for 10 min. For the second PCR amplification, nested primers were used and conditions were as follows: initial denaturation at 94 ℃ for 5 min, followed by 35 cycles of denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min then final extension at 72 ℃ for 10 min. PCR products were separated and visualised by 2% agarose gel electrophoresis.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePCR product purification and DNA sequencing\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAmplicons were purified using SpinClean Gel Band and PCR Products Purification Kit (CEYGEN Biotech (Pvt) Ltd, Sri Lanka) and sequenced using Sanger dideoxy sequencing method (Genelabs Medical Ltd, Sri Lanka). The \u003cem\u003efla\u003c/em\u003eB sequences were deposited in GenBank database.\u003c/p\u003e \u003cp\u003eThe dendrogram was constructed with 16 partial \u003cem\u003efla\u003c/em\u003eB gene sequences and 82 \u003cem\u003efla\u003c/em\u003eB gene sequences which show high similarity with \u003cem\u003eLeptospira\u003c/em\u003e from Sri Lanka and other countries randomly selected from the GenBank. The dendrogram was constructed using MEGA 11 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] by aligning the sequences with MUSCLE and phylogenetic distances were calculated using Tamura-3-parameter model, sequences were clustered by Neighbor-Joining method. The statistical stability of the nodes was assessed by bootstrapping with 1000 replicates.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePrediction of amino acid sequences and computational analysis of the three FlaB variants\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePartial \u003cem\u003efla\u003c/em\u003eB nucleotide sequences of the three \u003cem\u003efla\u003c/em\u003eB variants (NHSL13ML092, NHSL13ML506 and NHSL13SL215) were translated to amino acid (AA) sequences using Open Reading Frame Finder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and deposited in GenBank with following accession numbers: WKR38874, WKR38873 and WKR44734 respectively. Considering the FlaB AA sequences obtained from the NCBI database, the most abundant FlaB AA sequence with accession number WP_000586170 was used as the reference for comparison. Variations in the above AA sequences were identified using BLAST2 tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the wild-type FlaB AA sequence (WP_000586170). Protein structure homology-modelling of the wild type and variant AA sequences was performed using SWISS-MODEL web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Conserved region in the protein sequence of the wild type and variant sequences were identified by the ConSurf web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://consurf.tau.ac.il/consurf_index.php\u003c/span\u003e\u003cspan address=\"https://consurf.tau.ac.il/consurf_index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Thermodynamic stability and interatomic interaction predictions of FlaB protein variants were carried out by the Dynamut web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://biosig.lab.uq.edu.au/dynamut/\u003c/span\u003e\u003cspan address=\"https://biosig.lab.uq.edu.au/dynamut/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and compared with that of the wild-type FlaB protein.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eLeptospira\u003c/b\u003e \u003cb\u003especies identification using partial\u003c/b\u003e \u003cb\u003efla\u003c/b\u003e\u003cb\u003eB sequences\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOut of 60 selected samples, 45 amplified 732 bp amplicon in the \u003cem\u003efla\u003c/em\u003eB nested PCR, including 23 severe and 22 mild patient samples. Of these 45 \u003cem\u003efla\u003c/em\u003eB PCR positive samples, 16 which exhibited strong bands were selected for sequencing. The demographic and other criteria of these 16 patients (8 severe and 8 mild cases) are given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The resultant \u003cem\u003efla\u003c/em\u003eB sequences of all 16 samples showed the highest similarity with \u003cem\u003eL. interrogans\u003c/em\u003e sequences in the GenBank (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic and disease complications of patient samples used for sequencing partial \u003cem\u003efla\u003c/em\u003eB gene fragment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003cp\u003e(years)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003cp\u003e(M/F)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDays of illness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMAT titer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDistrict\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eClinical category\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eDisease complication\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12SL012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGampaha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAcute kidney injury\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12SL059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eColombo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAcute kidney injury, Thrombocytopenia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12SL150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eColombo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAcute kidney injury, Myocarditis,\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13SL215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGampaha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAcute kidney injury\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13SL442\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eColombo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAcute kidney injury\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13SL537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eColombo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAcute kidney injury\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13SL580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eColombo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAcute kidney injury\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13SL581\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGampaha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAcute kidney injury, Thrombocytopenia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12ML051*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eColombo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSevere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eThrombocytopenia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13ML092\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGampaha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12ML180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eColombo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12ML185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGampaha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13ML506\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eColombo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eThrombocytopenia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13ML520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGampaha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13ML713\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGampaha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL14ML769*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGampaha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMild\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSL \u0026ndash; Severe Leptospirosis, ML \u0026ndash; Mild Leptospirosis, F \u0026ndash; female, M \u0026ndash; male, NA \u0026ndash; not available\u003c/p\u003e \u003cp\u003eSources of exposure by rat infected water or recent skin injury for all patients, except for those marked with (*) who had exposure by sources other than rat-infested water/ recent skin injury/infected by animals.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eLeptospira\u003c/em\u003e sequence identity and there predicted amino acid sequences\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNucleotide Accession no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eLeptospira\u003c/em\u003e species \u003c/p\u003e \u003cp\u003eand serovar identified\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSequence Similarity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMutations identified\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAmino Acid Accession no.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAmino acid substitutions\u0026dagger;\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12SL012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOQ575709\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Bataviae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWKE35572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12SL059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOQ408277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Bataviae, Canicola\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWJJ80047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12SL150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR480416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Lai\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWNO24712\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eNHSL13SL215\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eOQ689693\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Bataviae, Canicola\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA1974716G*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eWKR44734\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eY164C\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA1974742G*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13SL442\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR459953\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Canicola\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWNO24711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13SL537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOQ536446\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Copenhageni\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWJJ80049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13SL580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOQ408278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Lai\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWJJ80048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13SL581\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOQ085075\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Bataviae, Canicola\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWJJ80046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12ML051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOQ547310\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Lai\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWJJ80050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eNHSL13ML092\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eOQ682751\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Bataviae, Canicola\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA1974376G\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eWKR38874\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eD42G\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA1974645G\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eK132E\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT1974745C\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eI165T\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12ML180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR480417\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Lai\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWNO24714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL12ML185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR492662\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Lai\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWNO24714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eNHSL13ML506\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eOQ658513\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Copenhageni, Icterohaemorrhagiae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT2286802C\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eWKR38873\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eL175S\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT2286920C\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC2286921T\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13ML520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOQ559482\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Bataviae, Canicola\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWKD80855\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL13ML713\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOQ553816\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Bataviae, Canicola\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWJJ80051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNHSL14ML769\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR451931\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. interrogans\u003c/em\u003e Canicola\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWNO11800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSL \u0026ndash; Severe Leptospirosis, ML \u0026ndash; Mild Leptospirosis, DNA sequences with mutations given in bold font.\u003c/p\u003e \u003cp\u003e* with reference to LC505621; \u003csup\u003e#\u003c/sup\u003e with reference to: CP039258; \u0026dagger; with reference to: WP_000586170\u003c/p\u003e \u003cp\u003eThree partial \u003cem\u003efla\u003c/em\u003eB gene sequences with novel mutations were identified from one severe (NHSL13SL215: OQ689693) and two mild (NHSL13ML092: OQ682751; NHSL13ML506: OQ658513) patients during this study referred to as variants. A\u0026thinsp;\u0026gt;\u0026thinsp;G mutations were identified at nucleotide positions 1974716 and 1974742 in the partial \u003cem\u003efla\u003c/em\u003eB gene sequence of one severe patient sample (NHSL13SL215) compared to the reference sequence LC505621 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, among two mild patient samples, three mutations each were identified compared to reference sequence CP039258 (in NHSL13ML092, A\u0026thinsp;\u0026gt;\u0026thinsp;G mutations at 1974376 and 1974645 and T\u0026thinsp;\u0026gt;\u0026thinsp;C mutation at 1974745 and in NHSL13ML506, T\u0026thinsp;\u0026gt;\u0026thinsp;C mutations at 2286802 and 2286920, and C\u0026thinsp;\u0026gt;\u0026thinsp;T mutation at 2286921).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePhylogenetic analysis\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe 16 partial \u003cem\u003efla\u003c/em\u003eB gene sequences used for the phylogenetic analysis showed 100% sequence identity with \u003cem\u003eL. interrogans\u003c/em\u003e. Four clusters were observed in the dendrogram including these sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), ie, \u003cem\u003eL. interrogans, L. kirschneri, L. weilli\u003c/em\u003e and \u003cem\u003eL. borgpetersenii.\u003c/em\u003e These \u003cem\u003efla\u003c/em\u003eB sequences were aligned with sequences previously reported from countries in Asia, such as China, Japan, and Thailand, Latin American countries such as Brazil, and temperate countries like the USA indicating that these 16 sequences of \u003cem\u003eL. interrogans\u003c/em\u003e identified from Sri Lanka are also found in other geographical regions. According to the dendrogram, all 98 sequences were clustered into four groups: \u003cem\u003eL. interrogans\u003c/em\u003e (Cluster 1), \u003cem\u003eL. kirschneri\u003c/em\u003e (Cluster 2), \u003cem\u003eL. weilli\u003c/em\u003e (Cluster 3), and \u003cem\u003eL. borgpetersenii\u003c/em\u003e (Cluster 4).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePredicted amino acid sequences in the three variants\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePatient no NHSL13SL215, NHSL13ML092, and NHSL13ML506 showed point mutations that are predicted to alter the AA sequences (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u0026amp; Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). In sample no. NHSL13SL215, the translated AA sequence shows substitution of Tyrosine (polar AA) by Cysteine (non-polar AA; Y164C) found in the variable region of the FlaB protein (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The replacement of this AA may alter the carbon composition and disrupt interatomic interactions leading to destabilization of the FlaB protein (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSimilarly, in sample NHSL13ML506, the translated AA sequence shows substitution of Leucine (non-polar AA) by Serine (polar AA; L175S) found in the variable region of the FlaB protein. Due to this AA substitution, the carbon molecules and interatomic interactions may lead to destabilizing the FlaB protein. Although there are three substitutions in the translated AA sequence in sample NHSL13ML092, namely Aspartic acid (polar acidic AA) replaced by Glycine (non-polar AA; D42G), Lysine (polar basic AA) replaced by Glutamic acid (polar acidic AA; K132E) and Isoleucine (non-polar AA) replaced by Threonine (polar AA; I165T), substitution D42G lies in a highly conserved region while other two substitutions (K132E and I165T) are located in variable regions. Further, the two substitutions, D42G and I165T are predicted to be contributed to significant change in destabilizing the FlaB protein (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe prevalence of \u003cem\u003eLeptospira\u003c/em\u003e species causing different clinical outcomes in a locality is critical for various aspects including patient management strategies, treatment options and vaccine development against leptospirosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The present study was focused on \u003cem\u003eLeptospira\u003c/em\u003e species identification using blood samples of clinically characterized, mild and severe leptospirosis patients who were hospitalized in two hospitals in the Western Province of Sri Lanka. The 16 sequences obtained in the present study included 8 severe cases and 8 mild cases aligned with \u003cem\u003eL. interrogans\u003c/em\u003e, indicating that both categories had \u003cem\u003eL. interrogans\u003c/em\u003e as the infecting species. However, the small sample size is a limitation of this study, and this needs further investigation with an adequate sample size. It is also noteworthy that there may be factors other than the infecting species (serovar), such as leptospiremia, host immune and genetic factors that may contribute to the clinical severity in leptospirosis infections. However, a previous study using quantitative PCR has indicated that leptospiremia in serum/whole blood samples did not directly correlate with the clinical manifestations [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eIt is interesting to note that previous molecular studies conducted with patient samples from Sri Lanka have identified \u003cem\u003eL. interrogans\u003c/em\u003e as the most prevalent species, with 73.9% (76 out of 111) of the cases being caused due to \u003cem\u003eL. interrogans\u003c/em\u003e whereas the other species were less prevalent, i.e., \u003cem\u003eL. kirschneri\u003c/em\u003e (22.5%), \u003cem\u003eL. borgpetersenii\u003c/em\u003e (5.4%) and \u003cem\u003eL. weilii\u003c/em\u003e (2.7%) [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Hence, the results from the present study are consistent with the previous findings on \u003cem\u003eL. interrogans\u003c/em\u003e being a more prevalent species in Sri Lanka. Further, a previous study conducted with patient isolates from wet zone regions such as Western, Southern and Central Provinces of Sri Lanka had shown that \u003cem\u003eL. interrogans\u003c/em\u003e was the most predominant species prevalent, and was associated with renal failure [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Acute renal failure was the most common disease complication observed among the severe leptospirosis patients in the present study.\u003c/p\u003e \u003cp\u003eAlthough \u003cem\u003eL. interrogans\u003c/em\u003e has been reported as the most prevalent species in Sri Lanka, recent studies focused on specific epidemics have reported other species such as \u003cem\u003eL. kirschneri\u003c/em\u003e as the most prevalent species in 2011 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The reason for this variation has mainly been attributed to the circulating species in different provinces in Sri Lanka and certain seasonal changes observed with a flood-associated outbreak in 2011 in the Anuradhapura District in North-Central Province in the dry zone, whereas most of the previous studies have sampled from leptospirosis patients from the wet zone districts and provinces [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, in the phylogenetic analysis, the majority of sequences in this \u003cem\u003eL. interrogans\u003c/em\u003e Cluster 1 comprised of samples derived from various Asian countries. However, the other sequences included in this phylogenetic analysis comprised of samples derived from diverse geographic regions, including the Brazil, Puerto Rico and United States.\u003c/p\u003e \u003cp\u003eSeveral genes such as \u003cem\u003errs\u003c/em\u003e, \u003cem\u003esec\u003c/em\u003eY, \u003cem\u003elip\u003c/em\u003eL32, \u003cem\u003efla\u003c/em\u003eB, \u003cem\u003elfb\u003c/em\u003e1, \u003cem\u003elig\u003c/em\u003eA, and \u003cem\u003elig\u003c/em\u003eB2 have been used for molecular diagnosis \u003cem\u003eLeptospira\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Of these genes, a majority of studies have used lipL32 as the target gene which has sensitivity and specificity of 0.42 and 0.95 respectively whereas the gene used in the present study was \u003cem\u003efla\u003c/em\u003eB which had shown sensitivity and specificity of 0.41 and 0.90 respectively [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The \u003cem\u003efla\u003c/em\u003eB gene has been used previously to discriminate between \u003cem\u003eLeptospira\u003c/em\u003e species in the majority of studies conducted in Sri Lanka [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. It encodes a flagellum protein, and four isotypes of FlaB (FlaB1, FlaB2, FlaB3 and FlaB4) have been described [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Of the four genes encoding these isotypes, most of the studies have used \u003cem\u003efla\u003c/em\u003eB2 gene as their target [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The flagellar filaments of \u003cem\u003eLeptospira\u003c/em\u003e, display a complex structure consisting of a central core made of FlaB protein (281 to 285 amino acids) surrounded by a sheath of FlaA protein [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The motility of leptospires depends on the presence of two endoflagella, which could play a critical role as a virulence factor. It is noteworthy that FlaB mutants of \u003cem\u003eL. biflexa\u003c/em\u003e were shown to be non-motile and deficient in endoflagella, but the cell body of the mutants remained intact and retained its helical morphology [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study, we have observed three novel \u003cem\u003efla\u003c/em\u003eB2 variants that are predicted to result in changes in the AA sequence which may result in possible changes in the FlaB protein. However, without functional confirmation, we could not determine the impact of these novel mutations on the flagella assembly and motility.\u003c/p\u003e \u003cp\u003eIn conclusion, the present study has shown novel as well as previously reported \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003efla\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eB\u003c/span\u003e gene sequences in the samples derived from clinically characterized mild and severe leptospirosis patients from the Western Province in Sri Lanka. All 16 sequences were identified as \u003cem\u003eL. interrogans\u003c/em\u003e and grouped in 4 sub-clusters except for the three samples which comprised novel mutations that are predicted to result in amino acid changes in the FlaB protein.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e \u003c/h2\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003ch2\u003eDeclarations\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003c/p\u003e\u003cp\u003eThis study was carried out with the approved obtained from the Ethics Review Committee of Faculty of Medicine, University of Colombo, Sri Lanka (EC-12-056). Written, informed consent was obtained from all the patients prior to recruitment to the study.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003eClinical trial number\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003eConsent for publication\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eAuthors declare that there are no competing interests.\u003c/p\u003e \u003cp\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by grant no. NRC-17-098, awarded to SH from the National Research Council, Sri Lanka (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nrc.gov.lk/\u003c/span\u003e\u003cspan address=\"http://www.nrc.gov.lk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Data collection component of this study was funded by grant no. NRC-12-077, National Research Council, Sri Lanka and grant no. RG/ 2011/HS/19, National Science Foundation, Sri Lanka (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.nsf.ac.lk/\u003c/span\u003e\u003cspan address=\"http://www.nsf.ac.lk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), awarded to SH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceived and designed the experiments: SH NF SP SR. Sample selection, retrieval of demographic data and analysis: NF SH NC. Performed molecular biological experiments, computational analysis and data analysis: YS JW SH NF. Funding acquisition: SH. Wrote first draft of the paper: SH YS. Critically reviewed and revised the draft, and approved the final manuscript: SH YS JW SP NF HS NC SR.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eConsultants and other Staff of the National Hospital of Sri Lanka; Consultant Bacteriologist and staff of the Medical Research Institute, Colombo, Sri Lanka; for help with data collection and clinical categorization.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets supporting the results and conclusions are available from the GenBank (NCBI) database and the accession numbers are available in the main text.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCosta F, Hagan JE, Calcagno J, Kane M, Torgerson P, Martinez-Silveira MS, et al. Global morbidity and mortality of leptospirosis: a systematic review. PLoS Negl Trop Dis. 2015;9:9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarnasekara J, Koralegedara I, Agampodi S. Estimating the burden of leptospirosis in Sri Lanka; a systematic review. BMC Infect Dis. 2019;19:1\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarnasekara J, Agampodi S. Neglecting the neglected during the COVID-19 pandemic: the case of leptospirosis in Sri Lanka. Epidemiol Health epiH. 2022;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaake DA, Levett PN. Leptospirosis in humans. Curr Top Microbiol Immunol. 2015;387:65\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoarant C, Leptospirosis. Risk factors and management challenges in developing countries. Rep Trop Med. 2016;7:49\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDupouey J, Faucher B, Edouard S, Richet H, Kodjo A, Drancourt M, Davoust B. Human leptospirosis: An emerging risk in Europe? Comp. Immunol Microbiol Infect Dis. 2014;37:77\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKo AI, Goarant C, Picardeau M, Leptospira. The dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol. 2009;7:736\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahelinirina S, Leon A, Harstskeerl RA, Sertour N, Ahmed A, Raharimanana C, et al. First isolation and direct evidence for the existence of large small mammal reservoirs of \u003cem\u003eLeptospira spp\u003c/em\u003e. in Madagascar. PLoS ONE. 2010;5:11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao M, Xiao X, Han HJ, Wang LJ, Lei SC, Liu JW, et al. \u003cem\u003eLeptospira\u003c/em\u003e in bats from Hubei Province, China, 2018. J Wildl Dis. 2019;55(4):940\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAntima BS. Modeling the dynamics of leptospirosis in India. Sci Rep. 2023;13(1):19791.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajapakse S, Weeratunga P, Niloofa MR, Fernando N, Rodrigo C, Maduranga S, et al. Clinical and laboratory associations of severity in a Sri Lankan cohort of patients with serologically confirmed leptospirosis: a prospective study. Trans R Soc Trop Med Hyg. 2015;109(11):710\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalugalage T, Rodrigo C, Vithanage T, Somaratne P, De Silva HJ, Handunnetti S, et al. Low serum total nitrite and nitrate levels in severe leptospirosis. BMC Infect Dis. 2013;13(1):206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernando N, Wickremesinghe S, Niloofa R, Rodrigo C, Karunanayake L, de Silva HJ, et al. Protein carbonyl as a biomarker of oxidative stress in severe leptospirosis, and its usefulness in differentiating leptospirosis from dengue infections. PLoS ONE. 2016;11:6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThibeaux R, Girault D, Bierque E, Soup\u0026eacute;-Gilbert M-E, Rettinger A, et al. Biodiversity of environmental \u003cem\u003eLeptospira\u003c/em\u003e: improving identification and revisiting the diagnosis. Front Microbiol. 2018;9:816.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVincent AT, Schiettekatte O, Goarant C, Neela VK, Bernet E, Thibeaux R, et al. Revisiting the taxonomy and evolution of pathogenicity of the genus \u003cem\u003eLeptospira\u003c/em\u003e through the prism of genomics. PLoS Negl Trop Dis. 2019;13:5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzevedo IR, Amamura TA, Isaac L. Human leptospirosis: In search for a better vaccine. Scand J Immunol. 2023;98(5):e13316.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBandara KK, Weerasekera M, Gunasekara CP, Ranasinghe N, Marasinghe C, Fernando N. Molecular characterization and disease severity of leptospirosis in Sri Lanka. Mem Inst Oswaldo Cruz. 2015;110(4):485\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoizumi N, Gamage CD, Muto M, Kularatne SA, Budagoda SB, Rajapakse JR, et al. Serological and genetic analysis of leptospirosis in patients with acute febrile illness in Kandy, Sri Lanka. Jpn J Inf Dis. 2009;62(6):474\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNwafor-Okoli C, Koizumi N, Kularatne SA, Rajapakse J, Gamage CD, Muto M, et al. Leptospira infection at the University of Peradeniya Teaching Hospital, Sri Lanka: clinical and laboratory investigations. Southeast Asian J Trop Med Public Health. 2012;43(4):943\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgampodi SB, Peacock SJ, Thevanesam V, Nugegoda DB, Smythe L, Thaipadungpanit J, et al. Leptospirosis outbreak in Sri Lanka in 2008: lessons for assessing the global burden of disease. ASTMH. 2011;85(3):471.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgampodi SB, Dahanayaka NJ, Bandaranayaka AK, Perera M, Priyankara S, Weerawansa P, et al. Regional differences of leptospirosis in Sri Lanka: observations from a flood-associated outbreak in 2011. PLoS Negl Trop Dis. 2014;8(1):e2626.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNisansala GG, Muthusinghe D, Gunasekara TD, Weerasekera MM, Fernando SS, Ranasinghe KN, et al. Isolation and characterization of \u003cem\u003eLeptospira interrogans\u003c/em\u003e from two patients with leptospirosis in Western Province, Sri Lanka. J Med Microbiol. 2018;67(9):1249\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarunanayake L, Gamage CD, Gunasekara CP, De Silva S, Izumiya H, Morita M, et al. Multilocus sequence typing reveals diverse known and novel genotypes of \u003cem\u003eLeptospira spp\u003c/em\u003e. circulating in Sri Lanka. PLoS Negl Trop Dis. 2020;14(8):e0008573.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiloofa MJR. Studies on comparing rapid immunodiagnostic methods and immune status of leptospirosis patients in Western Province Sri Lanka: PhD Thesis. Institute of Biochemistry, Molecular Biology and Biotechnology: University of Colombo, Sri Lanka; 2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillumsen S, Pedersen R, Borre MB, Ahrens P, Jensen JS, Krogfelt KA. Novel TaqMan\u0026reg; PCR for detection of \u003cem\u003eLeptospira\u003c/em\u003e species in urine and blood: pit-falls of \u003cem\u003ein silico\u003c/em\u003e validation. J Microbiol Methods. 2012;91(1):184\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKawabata H, Dancel LA, Villanueva SY, Yanagihara Y, Koizumi N, Watanabe H. \u003cem\u003efla\u003c/em\u003eB-Polymerase chain reaction (\u003cem\u003efla\u003c/em\u003eB‐PCR) and its restriction fragment length polymorphism (RFLP) analysis are an efficient tool for detection and identification of \u003cem\u003eLeptospira spp\u003c/em\u003e. Microbiol Immunol. 2001;45(6):491\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoizumi N, Muto M, Yamamoto S, Baba Y, Kudo M, Tamae Y, et al. Investigation of reservoir animals of \u003cem\u003eLeptospira\u003c/em\u003e in the northern part of Miyazaki prefecture. Jpn J Infect Dis. 2008;61:465\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaghnazari. Sepideh and khaki, Pejvak and Moradi Bidhendi, Soheila and Esmaelizad, Majid and Tebianian, Majid and Gharakhani, Mehdi, 1402, Molecular characterization of the flaB2 gene of pathogenic \u003cem\u003eLeptospira\u003c/em\u003e vaccinal serovars, 24th International Congress of Microbiology of Iran, Tehran. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://civilica.com/doc/1922326\u003c/span\u003e\u003cspan address=\"https://civilica.com/doc/1922326\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e [accessed on 2024-12-10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38(7):3022\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgampodi SB, Matthias MA, Moreno AC, Vinetz JM. Utility of quantitative polymerase chain reaction in leptospirosis diagnosis: association of level of leptospiremia and clinical manifestations in Sri Lanka. Clin Infect Dis. 2012;54(9):1249\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJayasundara D, Senavirathna I, Warnasekara J, Gamage C, Siribaddana S, Kularatne SA et al. 12 Novel clonal groups of \u003cem\u003eLeptospira\u003c/em\u003e infecting humans in multiple contrasting epidemiological contexts in Sri Lanka. PLoS Negl Trop Dis. 2021 18;15(3):e0009272.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaotunna C, Agampodi SB, Agampodi TC. Etiological agents causing leptospirosis in Sri Lanka: A review. Asian Pac J Trop Med. 2016;9(4):390\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLam JY, Low GK, Chee HY. Diagnostic accuracy of genetic markers and nucleic acid techniques for the detection of Leptospira in clinical samples: A meta-analysis. PLoS Negl Trop Dis. 2020;14(2):e0008074.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Azevedo MI, Lilenbaum W. An overview on the molecular diagnosis of animal leptospirosis. Lett Appl Microbiol. 2021;72(5):496\u0026ndash;508.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalmstr\u0026ouml;m J, Beck M, Schmidt A, Lange V, Deutsch EW, Aebersold R. Proteome-wide cellular protein concentrations of the human pathogen \u003cem\u003eLeptospira interrogans\u003c/em\u003e. Nature. 2009;460(7256):762\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLambert A, Picardeau M, Haake DA, Sermswan RW, Srikram A, Adler B, et al. FlaA proteins in \u003cem\u003eLeptospira interrogans\u003c/em\u003e are essential for motility and virulence but are not required for formation of the flagellum sheath. Infect Immun. 2012;80(6):2019\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePicardeau M, Brenot A, Saint Girons I. First evidence for gene replacement in \u003cem\u003eLeptospira\u003c/em\u003e spp. Inactivation of \u003cem\u003eL. biflexa fla\u003c/em\u003eB results in non-motile mutants deficient in endoflagella. Mol Microbiol. 2001;40(1):189\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAA Amino Acid\u003c/p\u003e\u003cp\u003eA Adenine\u003c/p\u003e\u003cp\u003eBp base pair\u003c/p\u003e\u003cp\u003eBLAST Basic Local Alignment Search Tool\u003c/p\u003e\u003cp\u003eC Cytosine\u003c/p\u003e\u003cp\u003e \u003cem\u003eflaB\u003c/em\u003e gene flagellin B gene\u003c/p\u003e\u003cp\u003eG Guanine\u003c/p\u003e\u003cp\u003eMEGA Molecular Evolutionary Genetics Analysis version\u003c/p\u003e\u003cp\u003eMUSCLE Multiple Sequence Comparison by Log-Expectation\u003c/p\u003e\u003cp\u003eNCBI National Center for Biotechnology Information\u003c/p\u003e\u003cp\u003eNHSL National Hospital of Sri Lanka\u003c/p\u003e\u003cp\u003ePCR Polymerase chain reaction\u003c/p\u003e\u003cp\u003eT Thymine\u003c/p\u003e"},{"header":"Supporting Information","content":"\u003cp\u003e \u003cstrong\u003eAdditional File 1: Table S1\u003c/strong\u003e \u003c/p\u003e\u003cp\u003e: Sequences of primers and product sizes for nested-PCR for \u003cem\u003efla\u003c/em\u003eB gene amplification.\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eAdditional File 1: Table S2:\u003c/strong\u003e \u003c/p\u003e\u003cp\u003ePredicted changes in the FlaB protein of the 3 variants compared to the wild type FlaB protein of \u003cem\u003eL. interorgans\u003c/em\u003e\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eAdditional File 3: Figure S1\u003c/strong\u003e \u003c/p\u003e\u003cp\u003e: FlaB protein of \u003cem\u003eL. interrogans\u003c/em\u003e (A) (WP_000586170, reference), variant protein sequences of NHSL13SL215 (B) (WKR44734) NHSL13ML092 (C) (WKR38874), and NHSL13ML506 (D) (WKR38873).\u003c/p\u003e\u003cp\u003e \u003cstrong\u003eAdditional File 4: Figure S2:\u003c/strong\u003e \u003c/p\u003e\u003cp\u003ePredicted protein stability for the 3 variants compared to the wild type FlaB protein of \u003cem\u003eL. interrogans\u003c/em\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Leptospirosis, Leptospira interrogans, flaB gene, single nucleotide mutation, Sri Lanka","lastPublishedDoi":"10.21203/rs.3.rs-4764619/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4764619/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLeptospirosis caused by pathogenic spirochaetes of \u003cem\u003eLeptospira spp\u003c/em\u003e remains the most widespread zoonotic disease in the world. Clinical status in leptospirosis patients varies from asymptomatic infection to mild illness to severe/fatal outcomes. The objective of this study was to determine the species of \u003cem\u003eLeptospira\u003c/em\u003e that cause mild and severe infections, from patient blood samples obtained from the Western Province in Sri Lanka.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eDNA extracted from 60 blood samples of previously confirmed leptospirosis patients by Lipl32 based Real Time PCR, and clinically characterized as mild and severe (n\u0026thinsp;=\u0026thinsp;30 each) were used to perform nested PCR with primers designed from \u003cem\u003efla\u003c/em\u003eB gene sequence.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOf the 45 samples that gave a 725 bp fragment in \u003cem\u003efla\u003c/em\u003eB nested PCR, 23 and 22 were from severe and mild leptospirosis patients respectively. Of these \u003cem\u003efla\u003c/em\u003eB PCR positive samples, 16 which exhibited strong bands (8 severe cases and 8 mild) were selected for Sanger\u0026rsquo;s dideoxy sequencing. The sequences obtained from 16 samples were deposited in GenBank. Those partial \u003cem\u003efla\u003c/em\u003eB gene sequences showed highest similarity with \u003cem\u003eL. interrogans\u003c/em\u003e sequences available in GenBank database. Phylogenetic analysis was performed with 82 other randomly selected \u003cem\u003eLeptospira fla\u003c/em\u003eB gene sequences (from the GenBank) by Neighbor-Joining method using MEGA 11 tool. According to dendrogram, 16 partial \u003cem\u003efla\u003c/em\u003eB gene sequences obtained in this study clustered together with \u003cem\u003eL. interrogans\u003c/em\u003e. Three sequences, one from a severe leptospirosis patient and two from mild leptospirosis patients exhibited novel mutations. Computational analysis of the predicted amino acid substitutions in these three variants deduced from mutant DNA sequences with that of the wild type FlaB indicate possible functional changes in the flagellar protein. However, the impact of these novel mutations on the flagella assembly and motility could not be determined without functional confirmation. This highlights the need for further functional conformational studies to elucidate possible impact on motility which may influence the pathogenicity of different isolates of \u003cem\u003eL. interrogans.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Novel flaB gene variants of Leptospira interrogans detected in leptospirosis patient samples from the Western province of Sri Lanka","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2025-01-06 13:37:55","doi":"10.21203/rs.3.rs-4764619/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2024-08-12 19:32:55","doi":"10.21203/rs.3.rs-4764619/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1cef34e-66db-477e-b076-468a16cefe60","owner":[],"postedDate":"January 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-16T22:08:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-06 13:37:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-4764619","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4764619","identity":"rs-4764619","version":["v2"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}