Enzyme immunoassays (EIA) for serodiagnosis of human leptospirosis: specific IgG3/IgG1 isotyping may further inform diagnosis of acute disease.

The laborious microscopic agglutination test (MAT) is the gold standard serologic test for laboratory diagnosis of leptospirosis. We developed EIA based serologic assays using recombinant proteins (rLigA, rLigB, rLipL32) and whole-cell extracts from eight Leptospira serovars as antigen and assessed the diagnostic performance of the new assay within each class, against MAT positive (MAT+) human sera panels from Portugal/PT (n = 143) and Angola/AO (n = 100). We found that a combination of recombinant proteins rLigA, rLigB and rLipL32 correctly identified antigen-specific IgG from patients with clinical and laboratory confirmed leptospirosis (MAT+) with 92% sensitivity and ~ 97% specificity (AUC 0.974) in serum from the provinces of Luanda (LDA) and Huambo (HBO) in Angola. A combination of whole cell extracts of L. interrogans sv Copenhageni (LiC), L. kirschneri Mozdok (LkM), L. borgpetersenii Arborea (LbA) and L. biflexa Patoc (LbP) accurately identified patients with clinical and laboratory confirmed leptospirosis (MAT+) with 100% sensitivity and ~ 98% specificity for all provinces of Angola and Portugal (AUC: 0.997 for AO/LDA/HBO, 1.000 for AO/HLA, 0.999 for PT/AZ and 1.000 for PT/LIS). Interestingly, we found that MAT+ IgG+ serum from Angola had a significantly higher presence of IgD and that IgG3/IgG1 isotypes were significantly increased in the MAT+ IgG+ serum from Portugal. Given that IgM/IgD class and IgG3/IgG1 specific isotypes are produced in the earliest course of infection, immunoglobulin G isotyping may be used to inform diagnosis of acute leptospirosis. The speed, ease of use and accuracy of EIA tests make them excellent alternatives to the laborious and expensive MAT for screening acute infection in areas where circulating serovars of pathogenic Leptospira are well defined.

Introduction

Leptospirosis is a neglected emerging zoonotic disease with worldwide distribution that affects essentially all vertebrates, mostly in resource-poor and developing countries [1]. It affects vulnerable populations such as rural subsistence farmers and urban slum dwellers. Urban epidemics are reported mostly in cities of developing countries and will likely increase as the world’s slum population doubles to 2 billion by 2030 [1]. In developed countries, unexpected deadly outbreaks have been reported in New York City in 2017 [2] and 2021 [3]. However, outbreaks of leptospirosis are expected in grain-growing rainy regions of Australia when reservoir host populations of house mice skyrocket [4]. Sub-Saharan African countries lack notification surveillance systems for leptospirosis and in most cases the laboratory diagnosis is not done [5]. The estimated prevalence from countries that report the disease is high (75–102 per 100,000 population in Tanzania) compared with occidental countries [6,7]. Laboratory diagnosis of leptospirosis is not established in many Sub-Saharan countries despite the need for differential diagnosis with malaria, dengue, yellow fever and other common febrile illnesses [8,9], as is the case in Angola [10].

Human leptospirosis ranges in severity from a mild, self-limited febrile illness to a fulminant life-threatening disease [11]. A review of published cases estimated that leptospirosis causes ~1 million cases a year [1], resulting in ~5–10% death rate [1,12]. A number of organs is involved, reflecting the systemic nature of the infection. As a result, the symptoms of leptospirosis are frequently mistaken for other causes of acute febrile syndrome such as dengue, hepatitis [11], and malaria, depending on the overlap of endemic geographic areas. Unlike other spirochetal diseases that are characterized by signs and symptoms that aid in clinical diagnosis (ex. the bull’s eye erythema migrans in Lyme disease, and the chancre in primary syphilis), diagnosis of leptospirosis is tentatively based on evaluation of fever and myalgia in patients presenting at the hospital in areas of endemicity, and it is rarely confirmed in most parts of the world due to lack of affordable diagnostic assays.

The microscopic agglutination test (MAT) is the current gold standard serologic test for laboratory diagnosis of leptospirosis. However, it cannot be used for immediate case identification because it is insensitive in early infection [11] and it can only be performed in specialized laboratories with highly trained personnel and specific conditions such as reference collections of live Leptospira serovars, darkfield microscopy, among others. Thus, it is rarely performed by clinical diagnostic laboratories [13]. Less cumbersome, accurate, affordable, and accessible serologic tests are needed to better inform leptospirosis management [14]. We developed Enzyme ImmunoAssays (EIA) using either recombinant proteins or Leptospira whole-cell extracts as antigen and assessed the diagnostic performance of the new assay using MAT+ sera panels from Portugal and Angola.

Methods

Ethics statements

Humans

The present study followed international ethical guidelines and was evaluated and approved for use of human serum by the Health Ethics Committee of the Hospital do Divino Espírito Santo de Ponta Delgada, Azores, EPER (HDES/CES/159/2009), by the National Data Protection Commission authorization granted according to WHO guidelines to the reference laboratory for human leptospirosis diagnosis in Portugal (Leptospirosis and Lyme Borreliosis Laboratory—LLB-Lab) at the Instituto de Higiene e Medicina Tropical/IHMT (no.55/2000), and by the scientific board of the National Ethical Committee of Health of Angola (Ref. MINSA/CES/04/2011). The involvement of human subjects in this study falls under exemption 4 as outlined in the US HHS regulations (45 CFR Part 46). A total of 286 de-identified human serum samples were used. Serum was collected between 2009–2015 and was kept at -20°C without glycerol until use in 2018/2019. IRB was approved under FWA00021769 by Integreview/Advarra Ethical Review Board #2.

Animals

Eight-week-old female C3H/HeJ mice (Charles River, Boston) were acclimatized for one week in the pathogen-free environment in the UTHSC LACU before infection with L. interrogans FioCruz. Experimental animals were used per the guide for Care and Use of Laboratory Animals of the National Institutes of Health under approved protocol (19–0062) of the University of Tennessee Health Science Center Institutional Animal Care and Use Committee.

Geographic areas

Human serum samples from an African country (Southern Hemisphere, Angola—AO, provinces of Luanda—LDA, Huambo—HBO, and Huila—HLA) and from a Western European country (Northern Hemisphere, Portugal–PT, mostly from the Azores islands–AZ, and from the mainland—LIS) were used in this study. These different geographic areas are quite distinct in economic development and climate.

Serum panels

AO (LDA/HBO)

A panel of 50 human serum samples from individuals with fever and laboratory-confirmed leptospirosis (MAT+ titer > 1:100) collected between 2010 and 2012 in the provinces of Luanda and Huambo in Angola.

AO (HLA)

A panel of 50 human serum samples from individuals with fever and laboratory-confirmed leptospirosis (MAT+ titer > 1:100) collected between 2012 and 2013 in the province of Huila in Angola.

PT (AZ)

A panel of 43 human serum samples from individuals with clinical symptoms (fever, myalgia) and laboratory-confirmed leptospirosis (MAT+ titer > 1:100) collected between 2009 and 2015 at the HDES, São Miguel, Azores, Portugal.

PT (LIS)

A panel of 100 human serum samples from individuals with clinical symptoms (fever, myalgia) and laboratory-confirmed leptospirosis (MAT+ titer > 1:100) collected at the LLB-Lab/IHMT, Lisbon, Portugal. This panel contains samples collected between 2009 and 2015 in several regions of mainland Portugal (Coimbra, Lisbon, and Évora), and from the Terceira Island in the Azores.

Healthy

A panel of 43 human serum samples from healthy individuals from a non-endemic area for leptospirosis from the US (Florida) acquired in 2018.

Murine serum: a panel of 12 serum samples from 6 mice infected intraperitoneally with 107 L. interrogans FioCruz and from 6 non-infected controls.

Antigens

We used a commercial service (GenScript Biotech, Piscataway, NJ) to clone ten L. interrogans genes (flaB, flgC, flgJ, fliE, ligA, ligB, loa22, lemA, lipL32, tolC) in pET plasmid vectors containing 6xHis-tags. We used the following eight serovars of Leptospira obtained from different sources: L. interrogans serovar (sv) Copenhageni L1-130 (human isolate from Brazil, a gift from Dr. D. Haake), L. interrogans Pomona sv Pomona (human isolate from Australia, ATCC 23478), L. kirschneri Pomona sv Mozdok 61H (human isolate from Brazil, a gift from Dr. O. Dellagostin), L. kirschneri sv Cynopteri 3522C (bat isolate from Indonesia, LLB-Lab/IHMT collection), L. borgpetersenii sv Castellonis (wood mouse isolate from Spain, ATCC #23580), L. borgpetersenii sv Hardjo Type Bovis (bovine isolate, a gift from Dr. J. Nally), L. borgpetersenii Ballum sv Arborea (wood mouse isolate from Italy, LLB-Lab/IHMT collection), L. biflexa sv Patoc (soil isolate from France, ATCC #23582).

Purification of recombinant proteins

Plasmids were used to transform E. coli BL21(DE3)pLysS competent cells (New England Biolabs, Ispwich, MA) containing vector specific selective antibiotic markers (Kan+ or Amp+) and the plates were incubated overnight at 37°C; individual colonies cultured in TBY supplemented with antibiotic were induced with IPTG, cells were harvested by centrifugation, digested with BugBuster reagent (Millipore, Billerica, MA) in the presence of protease K inhibitor and the following recombinant proteins (rFlaB, rFlgC, rFlgJ, rFliE, rLigA7-13, rLigB1-6, rLoa22, rLemA, rLipL32, rTolC) were purified by affinity chromatography using HisPur Cobalt Resin (Thermo Fisher Scientific, Waltham, MA). Purity was checked by molecular weight size (KDa) on 10% denaturing polyacrylamide gel stained with Coomassie Blue and after electrotransfer to PVDF (Millipore, Billerica, MA) followed by western blot analysis using a mouse antigen-specific polyclonal antibody (1:100) and anti-mouse secondary antibody (1:1000) labeled with alkaline phosphatase. Examples of purified rLigA7-13, rLigB1-6 and rLipL32 are provided in .

Production of whole-cell extract antigens

Leptospira spp previously maintained in EMJH liquid medium were sub-cultured in EMJH semi-solid medium supplemented with 10% Difco Leptospira enrichment (Thermo Fisher Scientific, Waltham, MA), 1% of 5-Fluorouracil, and 2% inactivated rabbit serum (56°C, 1h) and grown for 15–30 days at 29°C shaking (100 rpm) in the dark. The individual cultures successfully recovered were grown in 25mL EMJH liquid for 5–10 days (to 108 Cells/mL) and then centrifuged at 9,000 rpm for 15 min at room temperature (RT); the supernatant was discarded leaving an aliquot of 1mL which was centrifuged at 20,000 rpm (3 min) and the supernatant was discarded. The pellet was incubated with 1mL of BugBuster solution (protein extraction reagent containing nuclease and lysozyme) at RT in a shaking incubator (100 rpm) for 20 min, homogenized by vortex, and stocks were saved at -20°C.

Serologic testing on Enzyme ImmunoAssay (EIA)

EIA was performed according to a comprehensive published method (Protocol 3.3.1. in CPM [15]) with the following modifications (a step-by-step protocol is provided in ). Briefly, purified recombinant proteins quantified by the Lowry protein assay kit (Thermo Fisher Scientific, Waltham, MA) or whole-cell extract of Leptospira diluted in 1X sodium carbonate coating buffer was used to coat Nunc MaxiSorp flat-bottom EIA plates (eBioscience, San Diego, CA) at 0.5–1 μg/ml (protein) or 105-108cells/well (Leptospira extract) overnight at 4°C. The following day the plates were washed with 1XPBS, blocked with 1% BSA for 2h, washed again, and incubated with human or murine serum diluted at 1:50 or 1:100. Goat anti-mouse or goat anti-human IgG conjugated to HRP diluted at 1:10000 (Jackson ImmunoResearch, West Grove, PA) was used as the secondary antibody. The OD450 was read on a SpectraMax Plus EIA reader (Molecular Devices). For determination of Ig class and IgG isotypes the secondary antibody conjugated to HRP was diluted as follows: anti-human -IgG1, -IgG2, -IgG3, -IgG4 (1:1000, Invitrogen), anti-human IgA (1:8000, Southern Biotech), anti-human IgD (1:1200, Southern Biotech) and IgM (1:10,000, Jackson ImmunoResearch Laboratories, Inc.). The EIA cutoff was set at three standard deviations above the average OD450 of all healthy control samples.

Statistical analysis

For analysis of diagnostic performance, the OD cutoff for each antigen was determined for each leptospirosis panel versus healthy control by constructing a receiver-operating characteristic (ROC) analysis with area under the curve (AUC), and selecting the OD cutoff value which resulted in the maximum sensitivity given a minimum of 97% specificity. Furthermore, Ordinary One-Way ANOVA was used to assess the significance of differences between each of the leptospirosis panels and the healthy control. For the combined algorithm, we chose the recombinant protein or whole cell extract with the highest sensitivity and then included the negative samples that tested positive against any other biomarker candidate (a positive in 1 of the 3 or 4 biomarkers was considered positive). For analysis of differences between two IgG+ panels for Ig class and IgG isotyping the Welch’s t test was used.

Results

Screening recombinant protein candidates for specificity and potential sensitivity

To evaluate EIA specificity of the 10 candidate biomarkers we tested purified recombinant proteins using L. interrogans whole cell extract (WCE) as control, against serum (1:50) from healthy individuals from an area (FL/US) that is not endemic for leptospirosis (). We found that seven recombinant proteins (rFlaB, rFlgC, rLigA7-13, rLigB1-6, rLemA, rLipL32 and rTolC) were more specific (less cross-reactive) to the healthy human serum than the WCE control. Of these seven, FlaB, LigA, LigB and LipL32 were shown to be reactive with serum from leptospirosis patients [16], and three (LigA, LigB, LipL32) had 95–99% homology between all pathogenic Leptospira serovars [17]. To confirm the potential sensitivity of the three leads in comparison with other candidates, we tested purified rLigA, rLigB, rLipL32, rTolC and rFliE using serum from C3H-HeJ mice infected with L. interrogans sv Copenhageni FioCruz. We confirmed that unlike TolC and FliE, the three recombinant proteins LigA, LigB and LipL32 were highly immunogenic in mice ().

Specificity analysis of candidate serodiagnostic biomarkers.

Human IgG specific to purified L. interrogans recombinant proteins compared to L. interrogans whole cell extract (WCE) using a serum panel from healthy individuals (n = 12 samples, diluted 1:50) from a non-endemic area for leptospirosis (FL, US). Statistics by Mann-Whitney test (unpaired, exact): comparison between each recombinant protein and WCE, * p<0.005, ** p<0.0001, ns, not significant.

Diagnostic performance of recombinant proteins and Leptospira whole-cell extracts against human leptospirosis serum panels from two distinct geographic regions

Four MAT+ leptospirosis serum panels from two countries, representing Northern and Southern Hemispheres geographic regions, were used to evaluate the diagnostic potential of each antigen (recombinant protein and whole-cell extract) by measuring their sensitivity and specificity. We tested the three lead recombinant proteins (rLigA, rLigB and rLipL32) selected after our initial specificity analysis against four distinct serum panels by EIA and found that if keeping a specificity >96%, each recombinant protein detected each serum panel with different sensitivities ( and ). For Angola (AO), rLigA and rLipL32 performed better than rLigB: rLigA detected MAT+ serum with 88% sensitivity for LDA/HBO (AUC 0.958) and rLipL32 detected MAT+ serum with 76% sensitivity for HLA (AUC 0.917). For Portugal (PT), rLigB and LipL32 fared much better than rLigA: rLigB detected MAT+ serum with 72% and 73% sensitivity for AZ and LIS (AUC 0.937 and 0.942, respectively) and rLipL32 detected MAT+ serum with 87% sensitivity for LIS (AUC 0.944).

Diagnostic performance of each L. interrogans recombinant protein.

Human IgG specific to rLigA, rLigB and rLipL32 in four MAT+ leptospirosis sera panels, AO (LDA/HBO) n = 49/50, AO (HLA) n = 49/50, PT (AZ) n = 43, PT (LIS) n = 100 and one control panel, Healthy n = 33/34. Statistics by Ordinary One-Way ANOVA between each leptospirosis panel and healthy control and by ROC Area Under the Curve (AUC).

We did a side-by-side analysis of each serum sample against each of the three recombinant antigens to assess if an improvement in sensitivity could be measured when screening the MAT+ samples against one, two or the three proteins. A sample that was positive in 1 of the 3 biomarkers was considered positive. We found that this combination dramatically increased the diagnostic performance of the assay ( and ). While each recombinant protein detected the Angola AO (LDA/HBO) and AO (HLA) serum panels with sensitivities ranging from 35% to 88% and 32% to 76%, respectively, inclusion of positive samples to one of the three biomarkers (LigA or LigB or LipL32) detected the same panels with 92% (AUC 0.974) and 76% sensitivity (AUC 0.883). The same trend was observed for the Portugal (PT) panels: single recombinant protein detection ranged from 44% to 72% (PT AZ) and 67% to 87% (PT LIS), whereas inclusion of positive samples to one of the three biomarkers (LigA or LigB or LipL32) resulted in 74% (AUC 0.849) and 88% (AUC 0.938) sensitivities for the same panels.

Diagnostic performance of combined recombinant proteins.

Human IgG specific for rLigA or LigB or LipL32 in four MAT+ leptospirosis sera panels, AO (LDA/HBO) n = 50, AO (HLA) n = 50, PT (AZ) n = 43, PT (LIS) n = 100 and one control panel, Healthy n = 33. A. Scatter plot of all sera panels and B. ROC curve of each sera panel. Statistics by Ordianry One-Way ANOVA and ROC AUC.

Next, we tested whole cells extracts from eight Leptospira serovars against the same serum panels by EIA and found that keeping a specificity >97%, each extract detected the four panels with different sensitivities (, Tables and and ). For Angola, two whole-cell extracts performed well (sensitivity >90%) only against the sera panel from HLA: L. interrogans sv Copenhageni and L. borgpetersenii sv Arborea detected MAT+ serum with 97.67% and 93% sensitivity (AUC 0.995 and 0.976, respectively). For Portugal, L. interrogans sv Copenhageni was the best whole cell extract detecting MAT+ sera from AZ and LIS with 97.67% and 93% sensitivity (AUC 0.997 and 0.993, respectively).

Diagnostic performance of each Leptospira serovar extract.

Human IgG specific to L. interrogans Copenhageni FioCruz L1-130 (LiC), L. kirschneri Mozdok 61H (LkM), L. borgpetersenii Arborea (LbA) and L. biflexa Patoc 1 (LbP) in four MAT+ leptospirosis sera panels, AO (LDA/HBO) n = 43, AO (HLA) n = 43, PT (AZ) n = 42–43, PT (LIS) n = 40–43 and one control panel, Healthy n = 43. Statistics by Ordinary One-Way ANOVA between each leptospirosis panel and healthy control and by ROC Area Under the Curve (AUC).

We did a side-by-side analysis of each serum sample against each of the four Leptospira extracts to assess if an improvement in sensitivity could be measured when screening the MAT+ samples against one, two, three or four whole cell extracts. A sample that was positive to 1 of the 4 biomarkers (LiC or LbA or LkM or LbP) was considered positive. We found that this combination improved the diagnostic performance of the assay for all 4 sera panels tested ( and ) with the largest gain in the AO (LDA/HBO) sensitivity which increased from 62.69% to 100% (AUC 0.997).

Diagnostic performance of combined extracts of Leptospira serovars.

Human IgG specific to L. interrogans Copenhgeni FioCruz L1-130 (LiC), L. borgpetersenii Arborea (LbA), L. kirschneri Mozdoc 61H (LkM) and L. biflexa Patoc 1 (LbP) in four MAT+ leptospirosis sera panels, AO (LDA/HBO) n = 36, AO (HLA) n = 42, PT (AZ) n = 43, PT (LIS) n = 43 and one control panel, Healthy n = 43. A. Scatter plot of all sera panels and B. ROC curve of each sera panel. Statistics by Ordinary One-Way ANOVA and ROC AUC.

Lastly, we analyzed IgM, IgD and IgA class, and IgG isotypes between two MAT+, LiC IgG+ serum panels, one from Portugal (PT-AZ) and one from Angola (AO-HLA) ( and ). We found that MAT+ IgG+ serum from Angola had a significantly higher presence of IgD than MAT+ IgG+ serum from Portugal; and that IgG3/IgG1 isotypes were significantly increased in the MAT+ IgG+ serum from Portugal in contrast MAT+IgG+ serum from Angola. As expected, there were no significant differences in IgM or IgA between the two panels which were included based on IgG positivity to LiC.

Analysis of immunoglobulin class M, D and A, and IgG isotypes in MAT+ LiC IgG+ sera panels (AUC>0.995).

Scatter plots of L. interrogans specific IgM, IgD, IgA and IgG3, IgG1, IgG2, IgG4 in a serum panel from Portugal (PT-AZ, n = 43) and a panel from Angola (AO-HLA, n = 29). Statistics by Welch’s t test.

Discussion

The current definitive serologic test for leptospirosis is the microscopic agglutination test (MAT) which has been used for 100 years. Infection can be confirmed if paired acute and convalescent samples from the same patient show a 4X increase in antibody titer [11]. In addition, MAT allows for identification of the serogroup of the infecting serovar. This is important information to acquire in areas where a medley of pathogenic, intermediate and non-pathogenic Leptospira strains circulate. Thus, MAT is the assay of choice to confirm active infection. However, the following major limitations have been noted: MAT cannot be used for immediate case identification because it is insensitive in early infection [11]; it is not suitable for epidemiologic studies [18]; it is unable to distinguish between IgM and IgG [19]; it requires maintenance of large collections of different Leptospira serovars that can only be cultured in expensive specialized reference laboratories managed and operated by highly trained personnel. Furthermore, MAT is subjective to human interpretation and cross-reactivity among several Leptospira serovars and false positive reactions due to auto-agglutination have also been reported [11,12,20]. Given these weaknesses, several molecular techniques are currently being used for diagnosis of acute infection. These techniques can detect the etiologic agent itself in blood or urine by PCR amplification of Leptospira DNA or RNA [21-26]. However, there still is a need for versatile and affordable serologic assays that can be performed routinely in clinical laboratories in endemic areas where circulating strains are well known.

A recent study showing high incidence of leptospirosis in Vanuatu highlighted the need for improved diagnostic capabilities in developing countries [27]. Interest in redefining the gold standard testing for laboratory diagnosis of leptospirosis has been mounting recently and led to the proposal of several options: combinations of molecular tests and MAT [28,29], as well as molecular tests and EIA [30-32] [25,33-38]. Molecular tests have dramatically improved patient care and reduced deaths due to leptospirosis in a developed country [25]. However, molecular tests do not offer information on the immunity status of the individual. This is important as it has been recently shown that infection of mice with L. interrogans (sv Copenhageni, sv Manila, sv Icterohaemorrhagiae) induce an antibody based immune response that protects against homologous re-infection [39]. This opens the field of Leptospirosis diagnostics to applications of easy to deploy serologic assays. We and others [18,29,40] explored the use of a ubiquitous and highly sensitive technique—EIA—to develop an in-house serologic test for laboratory diagnosis of leptospirosis.

We found that in Angola where the predominant circulating Leptospira serovars are L. interrogans Copenhageni and L. borgpetersenii Ballum Arborea a combination of the L. interrogans recombinant proteins LigA, LigB and LipL32 correctly identified antigen-specific IgG from patients with clinical and laboratory confirmed leptospirosis (MAT+) with 92% sensitivity and specificity ~ 97% (AUC 0.974) in the province of Luanda and Huambo (AO LDA/HBO), whereas a combination of whole cell extracts of L. interrogans sv Copenhageni (LiC), L. kirschneri Mozdok (LkM), L. borgpetersenii Arborea (LbA) and L. biflexa Patoc (LbP) accurately identified patients with clinical and laboratory confirmed leptospirosis (MAT+) with 100% sensitivity and specificity ~ 98% for both provinces (AUC 0.997 for LDA/HBO and 1.000 for HLA). Others have shown that combinations of recombinant proteins containing rLigA, rLigB and rLipL32 on EIA detected antigen-specific IgM and IgG in serum from leptospirosis patients from Peru with 82% sensitivity and 86% specificity compared to MAT [41]. Further, others found that LigA-IgM EIA was more sensitive, but not more specific, than whole-cell based IgM EIA for the early diagnosis of leptospirosis in the Philippines [42]. Another study done recently using a commercial, whole-Leptospira-based IgM EIA, reported low accuracy [43]. However, a weakness of that study was the comparison between different classes of tests using a molecular test as the gold standard to gauge sensitivity and specificity of a serologic test. These two different classes of tests complement each other given that a molecular test is expected to detect the infectious agent early in the course of infection, whereas a serologic test should be positive 7–14 days post a new infection. Furthermore, it was recently found that long term production of IgM to Leptospira interrogans sv Copenhageni and sv Manilae correlate with colonization of the kidney, in contrast to sv Icteroheamorrhagiae which induced a classical temporary IgM response without kidney colonization [44]. These data further support development of EIA based serologic assays for research purposes.

For Portugal, the best diagnostic performance was achieved with a combination of whole cell extracts of L. interrogans sv Copenhageni (LiC), L. kirschneri sv Mozdok (LkM), L. borgpetersenii sv Arborea (LbA) and L. biflexa sv Patoc (LbP) which accurately identified patients with clinical and laboratory confirmed leptospirosis (MAT+) with 100% sensitivity and ~98% specificity (AUC 1.000). In-house ELISAs done by others using whole cell extracts of the most prevalent serovar in Germany (Leptospira kirschneri sv Grippotyphosa) performed with a clinical sensitivity of 83% and clinical specificity of 98.5% in MAT+ serum [18], which is equivalent to our results in regards to specificity (98%). However, in our study, whole-cell in-house EIAs made with L. interrogans sv Copenhageni produced higher sensitivities in MAT+ serum (93%-98%) than the study in Germany, which could be attributed to the very high prevalence of this serovar in Portugal. Our data also suggests that sensitivity of the EIA can be improved by adding extracts from other prevalent serovars in the region such as the leptospires isolated from the Angola specimens [10]. Our data further supports [40] the development of in-house IgG EIAs using Leptospira serovars known to circulate in the region/country [29] as an alternative method for the diagnosis of Leptospirosis. Overall, the ease of use and accuracy of the EIA make them excellent serologic alternatives to the laborious MAT for diagnostic screening and for epidemiologic studies in resource-limited countries, as we offer an example for Angola.

Interestingly, we found that MAT+ IgG+ serum from Angola had a significantly higher presence of IgD and that IgG3/IgG1 isotypes were significantly increased in the MAT+ IgG+ serum from Portugal. As expected, there were no significant differences in IgM or IgA between the two panels given that both sera panels used had equivalent levels of IgG as this was the criteria to do the IgG subtyping analysis. Circulating IgD is found at low levels in serum, it has a short half-life and its function is not clear. Because there is a FcR for IgD in CD3 T cells, it has been proposed that IgD might serve as a bridge for antigen presentation by B cells to T cells [45]. Presence of transient IgD in serum may be indicative of a pre-adaptive immune response ongoing in acute leptospirosis when mature B cells producing IgM+/IgD+ reach the spleen. Increased IgD in the Angola panel suggests a very early stage of disease. This is corroborated by the clinical inclusion criteria for patients in the Angola study, which was fever. IgG3 is the first IgG to appear in serum as switching from IgM/D to IgG takes place and it is an early effector, independent of T cell help [46]. IgG1 soon follows after T cell help has been engaged. Presence of IgG3/IgG1 in the Azores panel suggests that these patients also presented at the hospital with acute leptospirosis. Production of IgG2 and IgG4 is associated with long exposure to antigen and switching to IgG4 is associated with induction of tolerance [46,47]. Absence of IgG2 and IgG4, two isotypes commonly associated with later stages of disease, further corroborate the clinical characterization of the patients included as acute leptospirosis. Thus, immunoglobulin G isotyping provides another measure that can aide in clinical characterization and should be further tested to discriminate infection from reinfection.

The increased sensitivity and specificity of this assay may be due to the use of Bugbuster reagent in our protein extraction protocol. This reagent breaks up the Leptospira membrane gently but preserves integrity of the proteins. The Leptospira LPS induces specific immune responses to each serovar leading to low cross-reactivity/specificity between strains, and thus it defines the serovars. Thus, the high specificity of our assay might be associated to the protein extracted by the Bugbuster reagent. This is further corroborated by the detection of IgG3/IgG1 rather than IgG2/IgG4 given that IgG1 and IgG3 are generally induced by protein antigens, whereas IgG2/IgG4 are generally induced by polysaccharide antigens (in this case, Leptospira LPS) [45,46].

One limitation of this study was that we did not have access to serum from healthy individuals from the endemic areas used in this study (Portugal and Angola). Use of serum from healthy individuals from an endemic area may lower the overall specificity of the assay. Another limitation is that serum from other diseases used in differential diagnosis of leptospirosis in co-endemic areas (e.g. malaria) was not available for cross-reactivity testing.

In conclusion, in areas where circulating serovars of pathogenic Leptospira are well defined, accurate assays such as EIA which can be operated at a fraction of the cost of MAT should be explored as effective serodiagnostic tools for leptospirosis applicable to humans and animals. Addition of IgG3/IgG1 isotyping further increases likelihood that the samples tested represent acute human leptospirosis.

Purified recombinant proteins rLigA, rLigB and rLipL32.

The SDS-PAGE gel was stained by coomassie blue to confirm the molecular weight of the protein and antigenicity was confirmed by western blot of proteins electrotransferred into PVDF membranes against antigen-specific mouse polyclonal antibodies.

(EPS)

Click here for additional data file.

Potential sensitivity of the lead recombinant protein candidates.

IgG specific to 5 L. interrogans recombinant proteins was determined in serum from 6 C3H-heJ mice infected with 107 L. interrogans Copenhageni by ELISA; serum from 6 non-infected controls were tested against LigA to establish the cutoff.

(EPS)

Click here for additional data file.

Diagnostic performance of other Leptospira extracts.

LDA/HBO, Luanda/Huambo; HLA, Huila; AZ, Azores, LIS, Lisbon; ROC(AUC), receiver-operating characteristic (ROC) analysis with area under the curve (AUC).

(DOCX)

Click here for additional data file.

Excel file containing all the data used to make the figure plots and tables.

(XLSX)

Click here for additional data file.

Step-by-step EIA protocol for detection of anti-Leptospira antibody in human serum.

(DOCX)

Click here for additional data file.

3 Jan 2022

Dear Dr. Gomes-Solecki,

Thank you very much for submitting your manuscript "Enzyme immunoassays (EIA) for serodiagnosis of human leptospirosis: IgG3/IgG1 can help define acute disease" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations. I apologize for the long review process for this manuscript; it was difficult to recruit reviewers and one who agreed was so late that I have provided only two reviews.

One significant point raised in the reviews is the age of the sera used, and how the collection and storage conditions might affect the results. Please address this point in the text by either performing some tests of your ELISA-based diagnostics with fresh sera or by acknowledgint the possible limitations of using sera collected years ago.

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Jenifer Coburn, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Richard Phillips

Deputy Editor

PLOS Neglected Tropical Diseases

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? yes

-Is the study design appropriate to address the stated objectives? yes

-Is the population clearly described and appropriate for the hypothesis being tested? yes

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? yes

-Were correct statistical analysis used to support conclusions? yes

-Are there concerns about ethical or regulatory requirements being met? no

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: Does the analysis presented match the analysis plan? yes

-Are the results clearly and completely presented? yes

-Are the figures (Tables, Images) of sufficient quality for clarity? yes

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: -Are the conclusions supported by the data presented? yes

-Are the limitations of analysis clearly described? methodology needs more detail, see comments above

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study? yes

-Is public health relevance addressed? yes

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: Line 245. There is a extra % in the end of the text.

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The paper reported affordable in-house enzyme immunoassays using Leptospira serovars whole protein extracts and recombinant proteins, individually and in combination. The test can be performed for laboratory confirmation of leptospirosis to replace the laborious microscopic agglutination test (MAT). Interestingly, the authors showed increased sensitivities when a combination of antigens were used. This approach has been used previously to increase diagnostic sensitivity in many diseases. In general, the paper addresses an important issue, since the gold standard test (MAT) has several limitations. However, there are some concerns which should be considered.

• The study lake sera of healthy individual from same endemic areas/countries. These sera are better to assess specificity of the test.

• Purity of the recombinant proteins and their molecular sizes are not shown in Figures.

• It is not clear why the authors used sera of infected mice.

• In line 30: put the acronym (AO) in full when come first.

• Fig 1 could be omitted.

• In line 261: it is stated that whole cell extract from eight Leptospira serovars were used, however, fig 5, table 2 and table S1 show only results of 4 serovars. Some results are probably missing!

• In EIA protocol, please include Ph of the coating buffer (sod carbonate) and content/nature of the washing buffer.

• Line 718: please indicate whether substrate was incubated at dark?

• For figs 3-6 and S1, it is not clear what do the horizontal lines represent?

Reviewer #2: Fortes-Gabriel et al, describe an Enzyme immunoassays (EIA) for serodiagnosis of human leptospirosis and describe that IgG3/IgG1 can help define acute disease. This is a great effort to improve human serological diagnosis in Leptospirosis as an alternative to the laborious and expensive MAT for screening acute infections in areas where circulating serovars of pathogenic Leptospira are well defined.

Although the article is clearly described, there is room for improvements such as the following:

1. The serum collection is old (2010 and therefore the quality of these sera can be affected by the preservation methods. The text does not indicate whether they were preserved at -20 ° C or -80 ° C with or without glycerol. This maintenance directly affects the quality and preservation of the immunoglobulins, which can give variable results. This should be included in the methodology and discussed in the results.

2. Date of collection of the serum are not mentioned for PT (AZ), PT (LIS) and healthy patients from Florida

3. Line 61-62. Brucellosis is another zoonotic disease common as differential diagnosis in the African continent. Please consider this differential to be include it.

4. It is recommended to include animal reservoirs in the countries that participate and sources of contagion in the description of the disease so the One Health concept is fully addressed.

5. Could this immunoassay be adapted and be useful in animals in these regions? this can be included in the discussion

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Reviewer #1: Yes: Elfadil Abass

Reviewer #2: Yes: Gabriela Hernandez-Mora

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References

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Submitted filename: Comments.docx

Click here for additional data file.

4 Feb 2022

Submitted filename: Response to reviewers 020122.docx

Click here for additional data file.

9 Feb 2022

Dear Dr Gomes-Solecki,

We are pleased to inform you that your manuscript 'Enzyme immunoassays (EIA) for serodiagnosis of human leptospirosis: specific IgG3/IgG1 isotyping can further inform diagnosis of acute disease' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Jenifer Coburn, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Richard Phillips

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

17 Feb 2022

Dear Dr Gomes-Solecki,

We are delighted to inform you that your manuscript, "Enzyme immunoassays (EIA) for serodiagnosis of human leptospirosis: specific IgG3/IgG1 isotyping may further inform diagnosis of acute disease," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Table 1

Specificity and Sensitivity of Recombinant Proteins.

	SPECIFICITY	SENSITIVITY
	Healthy	Angola (LDA/HBO)	Angola (HLA)	Portugal (AZ)	Portugal (LIS)
rLigA	96.97%	88.00%	46.94%	58.14%	67.00%
rLigB	97.06%	34.69%	32.00%	72.09%	73.00%
rLipL32	97.06%	73.47%	76.00%	44.19%	87.00%
rLigA/rLigB/rLipL33	96.97%	92.00%	76.00%	74.42%	88.00%

LDA/HBO, Luanda/Huambo; HLA, Huila; AZ, Azores, LIS, Lisbon.

Table 2

Specificity and Sensitivity of Leptospira Whole Cell Extracts.

	SPECIFICITY	SENSITIVITY
	Healthy	Angola (LDA/HBO)	Angola (HLA)	Portugal (AZ)	Portugal (LIS)
L. interrogans Copenhagenii L1-130	97.73%	53.49%	97.67%	97.67%	93.02%
L. kirschneri Mozdok	97.67%	46.51%	72.09%	67.44%	76.74%
L. borgpetersenii Arborea	97.67%	62.79%	93.02%	73.81%	87.50%
L. biflexa Patoc1	97.67%	37.21%	48.84%	62.79%	67.44%
LiC or LbA or LkM or LbP	97.67%	100.00%	100.00%	100.00%	100.00%

LiC, L. interrogans Copenhageni; LbA, L. borgpetersenii Arborea, LkM, L. kirschneri Mozdoc; LbP, L. biflexa Patoc.