Ethics and biosafety
The protocol for using achieved sera and patient data was performed in concordance with the recommendation of the Declaration of Helsinki. Documentary Proof of Exemption Review was obtained from the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University (MUTM-EXMPT2017-005). Sample anonymity was maintained and all samples were re-coded without name and hospital ID. Biosafety was approved by the institute Biosafety Committee (MU2019-002).
A retrospective study was carried out to assess the diagnostic performance of rGroEL1–524 IgM-ELISA and to evaluate commercial whole-cell antigen-based ELISAs performance in detecting specific antibodies using local Thai blood samples compared with reference culture and MAT methods. The laboratory investigations were conducted at the Faculty of Tropical Medicine, Mahidol University, in Bangkok.
Reference leptospirosis diagnosis
Leptospira isolation was performed at the day of patient hospitalization by culturing blood specimens in EMJH (Ellinghausen-McCullough-Jonson-Harris) medium, and incubation for 16 weeks5. Sera were tested by MAT assay with 20 reference Leptospira serovars, as described previously5,36. Patients’ specimens were investigated by cultivation and/or MAT. The reference diagnosis was conducted at Loei Provincial Hospital. Single leptospirosis sera were confirmed for MAT titers at the Faculty of Tropical Medicine, Mahidol University with reference strains recommended by the WHO. MAT-positive criteria were defined as single MAT titer of ³1:400 in a single specimen, sero-conversion from negative to titer ³1:400, or a four-fold rise in MAT titer using paired-sera. A MAT-negative sample was defined as MAT titer £1:5017,21,32,37-38.
Leptospirosis patientsand sera
A suspected leptospirosis case was clinically diagnosed based on WHO criteria, i.e., acute undifferentiated febrile illness (AUFI) patients (fever ³38 °C) with headache, myalgia with history of exposure to animal reservoirs or flooded environments17. A confirmed leptospirosis case was defined as a clinically diagnosed, suspected leptospirosis case combined with positive laboratory diagnosis by culture method and/or MAT (single MAT titer ³1:400 in single serum or 4-fold rise or seroconversion of paired-sera).
Confirmed leptospirosis sera (n=133) were obtained from patients during an epidemic outbreak at Loei provincial hospital (n=95), between July-October 2002, and sporadic cases at Nakhon Ratchasima and Sakhon Nakhon northeastern provinces (n=38) before 20095,36 (Fig. 1). The median time for onset of symptoms was 3 days (IQR: 1-10). Leptospirosis sera (n=133) were acute sera (n=52) collected on the first day of hospitalization during DPO1-10 and classified into DPO1-3 (n=31), and DPO 4-10 (n=12), and convalescent sera (n=45) collected 14 days later.
Among the sera, samples with a single MAT titer of 1:100-1:200 (n=25) were excluded. All confirmed leptospirosis sera (n=107) were classified according to reference tests into culture-positive samples (n=30) and MAT-positive samples, including single MAT titer of ³1:400 (n=28), and MAT-negative (n=38) of seroconversion paired-sera. The most prevalent serogroups (serovars) among the MAT-positive sera were Autumnalis (Autumnalis, New), Australis (Australis, Bangkok, Bratislava), Icterohemorrhagiae (Copenhageni), Sarmin (Sarmin), and Sejroe (Sejroe)5.
To assess the specificity of the ELISA tests, a panel of control samples (total no.=210) consisting of 60 non-endemic control plasma (seronegative and negative for leptospirosis IgM detection) and 150 laboratory-confirmed infectious diseases other than leptospirosis, were used. Control samples (n=21) were excluded from this study because of insufficient sample volumes and a limited number of commercial test kits (Fig. 1). Non-endemic plasma controls were collected from healthy volunteers and undifferentiated febrile plasma (n=60) at the hospital for Tropical Diseases, Bangkok, in 2014. Laboratory-confirmed other febrile illnesses were seropositive dengue paired-sera (n=20), collected from undifferentiated fever; classic dengue fever patients at Sisaket Provincial Hospital, Srisaket Province, in 2013; scrub typhus samples (n=20) were PCR positive, and sero-positive acute serum collected at Umphang Hospital, Tak Province, in 2018; influenza paired-sera (n=20) were collected from hemagglutination inhibition seroconversion of H1N1-infected patients; malaria plasma (n=20) was collected from malaria vivax-positive patients in Tak Province (n=20); and melioidosis sera (n=20) were collected from IgM-seropositive melioidosis patients from northeastern Thailand, before 2018. Whole blood samples (n=50) were collected from AUFI patients (total no.=50) admitted to the Hospital for Tropical Diseases, Bangkok, during the period 2013-2015. The samples were laboratory-confirmed murine typhus (n=15), dengue (n=30), and bacterial sepsis (n=5) caused by E. coli, Streptococcus agalactiae, Salmonella Typhi, and Viridans Streptococci infections (Fig. 1)14. The samples were collected in microtubes and stored at −70°C.
Production of a transformed E. coli carrying a recombinant GroEL1–524–pET23a(+) plasmid.
Briefly, DNA sequence encoding for GroEL1-524 was amplified by PCR reaction using specific primers GroEL-NdeI/F (5’-GGCCCATATGGCGAAAGATATTGAATAT-3’) and GroEL-BamHI/R (5’-TTGGATCC ATCTGGTTTGTCTGTGATTGT-3’). PCR reaction were performed in a volume of 25 mL of PCR super mix (Quantabio, MA, USA) containing MgCl2, dNTP, Taq DNA polymerase, each GroEL primer, and 100 ng of genomic DNA extracted from L. interrogans serovar Icterohemorrhagiae. Amplifications were performed according to the following conditions: one cycle of 94 °C for 5 min, 29 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1.30 min, followed by a final period of 72 °C for 5 min. The PCR product was analyzed under 1% agarose gel electrophoresis and visualized by Gel Documentation (Bio-Rad, California, USA).
The GroEL1-524 fragment was digested with NdeI and BamHI restriction endonucleases and ligated into a linearized plasmid backbone to produce a recombinant GroEL1–524-pET23a(+) plasmid, subsequently introduced into BL21(DE3) E. coli. Positive transformed E. coli were PCR-screened using universal T7 primers. The GroEL1–524 sequence was verified by standard sequencing (Bioneer, Daejeon, Republic of Korea). The genetic map of the GroEL1–524-pET23a(+) plasmid is illustrated in supplementary Fig. S1.
Production of recombinant GroEL1–524 protein.
Recombinant GroEL1–524 protein was produced under E. coli expression system using a transformed E. coli strain bearing the recombinant GroEL1–524 plasmid. Briefly, E. coli was grown in Luria-Bertani broth containing 100 µg/mL ampicillin at 37 °C with 200 rpm shaking until the culture reached an OD600nm of 0.5. Thereafter, isopropyl β-d-1-thiogalactopyranoside (IPTG, 1 mM) was added to induce rGroEL1-524 protein expression at 37 °C with 200 rpm shaking for 3 h.
A soluble fraction from the IPTG-induced bacteria containing rGroEL1-524 protein was prepared in phosphate-buffered saline (1×PBS, pH 7.4) using a French pressure cell press at 30 kilo-pounds per-square-inch, repeated 4 times. The rGroEL1-524 protein was purified from the soluble proteins by native affinity chromatography using Ni2+-sepharose (GE Healthcare, Uppsala, Sweden). The purified rGroEL1-524 protein was concentrated in 1×PBS (pH 7.4) using a 3-kDa cut-off Amicon Ultra filter (Merck Millipore, MA, USA) and was determined for protein concentration using a Bradford assay (Thermo Fisher Scientific, MA, USA). Aliquots of the protein (1 mg/mL) were lyophilized using the Labcono Freeze Dry system, and then kept at −70 °C.
SDS-PAGE and Western blotting
Protein was analyzed under 13% SDS-PAGE gel electrophoresis, denaturing condition, and Coomassie Brilliant Blue G250 stain. Antigenic specificity testing of the rGroEL1-524 protein was performed by probing the blotted membrane with anti-6×His-Tag monoclonal antibody (1:1,000) (R&D Systems, MN, USA) for 1 h at 25 °C, followed by HRP-conjugated goat anti-mouse IgG secondary antibody (1:2,000) (Jackson ImmunoResearch, PA, USA) for 1 h at 25 °C (Southern Biotechnology, AL, USA). The reactive band was developed using 3,3-diaminobenzidine (DAB) chromogenic substrate (Thermo Fisher Scientific, MA, USA).
In-house rGroEL1–524 IgM-ELISA.
Recombinant GroEL1–524 (1 mg) immobilized ELISA strips (Jet Biofil, Guangzhou, China) were prepared as follows: rGroEL1–524 protein in 100 µL of carbonate–bicarbonate buffer (pH 9.6) was immobilized on ELISA wells at 37 °C for 24 h and the antigen-coated wells were washed using washing buffer (300 µL/well of PBST; 0.05% Tween 20 in 1×PBS, pH 7.4). Washing was conducted by an automated microplate washer (Tecan Trading AG, Switzerland) three times to remove unbound material. The coated wells were then incubated with blocking reagent (300 µL of 1% BSA in 1×PBS) for 1 h at 37 °C, followed by incubating the pre-blocked wells with 300 µL of 2% sucrose solution at 25 °C for 1 h. The ELISA wells were washed after each incubation step, as described above, and then air-dried. The pre-blocked rGroEL1–524 ELISA strips were packed with desiccant in press-seal bags and stored at −20 °C until use.
To detect anti-GroEL1-524 IgM antibody, serum dilution (1:100, 100 µL) in a serum diluent (1×PBS containing 0.2% gelatin, 0.2% BSA), along with an internal positive control (pooled MAT-positive patient sera, where the adjusted actual optical density (AOD) exceeded 0.2) and a reagent control (serum diluent) were incubated in pre-blocked antigen-coated wells at 37 °C for 1 h, followed by washing three times with PBST. Thereafter, HRP-conjugated goat anti-human IgM antibody (100 mL, 1:2,000) (Southern Biotechnology, AL, USA) was added to ELISA wells at 37 °C for 1 h incubation. ABTS chromophore diammonium salt (EMD Millipore, Germany) substrate solution (1 mg/mL ABTS tablet in 0.1 M sodium citrate buffer) was added (100 µl), and the plate was incubated for 15 min at 37 °C, after which 100 µL of 1% SDS solution was added to stop reaction. The optical density (OD) was measured at wavelength 410 nm against the reference 650 nm (OD410nm/650nm) using a microplate reader (Bio-Tek Instruments, VT, USA). Sample AOD was calculated by subtracting the OD of the reagent blank. The IgM-ELISA assay is valid when the OD of the reagent blank is <0.2 and the positive AOD control is ³0.2. A rGroEL1-524 IgM-ELISA protocol was optimized and the optimal concentration of rGroEL1-524 was 1 µg/well; serum dilution was 1:100 and secondary antibody dilution was 1:1,000-1:3,000 dilutions.
Panbio Leptospira IgM-ELISA.
The diagnostic performance of the commercial Panbio Leptospira IgM-ELISA (Abbott Diagnostics, Illinois, USA) (Lot no. 02P10E001), using Leptospira genus-specific antigen, was assessed in Thai blood samples. The Panbio IgM-ELISA protocol was performed as per the manufacturer’s instructions, measuring absorbance at OD450nm/650nm. An index value was calculated by dividing the sample absorbance by the cut-off value. The result was expressed as Panbio units (index value multiplied by 10). Interpretation of the validity results was as follows: Panbio units (anti-Leptospira IgM) of <9 was a negative result, suggesting no evidence of recent infection, Panbio units ³9 to <11 was an equivocal result, suggesting possible recent infection, and Panbio units ³11 was positive by IgM detection and interpreted as a recent or current infection. An equivocal result was considered a positive result. The Panbio IgM-ELISA test performance showed 96.5% sensitivity and 98.5% specificity and has been validated to detect leptospira infections by serovars Pomona, Copenhageni, Australis, Canicola, Grippotyphosa, Tarsassovi, Hardjo, Madanesis, Kremastos, Nokolaevo, Cellodoni, Szwajizak, and Djasiman.
Virion-Serion Classic Leptospira IgG-ELISA.
Institute Virion-Serion ELISA Classic Leptospira IgG (Institut Virion/Serion GmbH, Warburg, Germany) (order no. ESR 125 G) was used to detect anti-Leptospira IgG, IgA, or IgM antibodies from serum or plasma using a crude membrane extract of L. biflexa serovar Patoc strain Patoc I, which contains genus-specific epitopes for all Leptospira spp. Virion-Serion IgG-ELISA procedure was performed as per the manufacturer’s instructions, with measuring absorbance at OD405nm/650nm. To interpret the qualitative results, the upper and lower cut-off range was calculated according to parameters provided with the kit. Actual OD (AOD) value (anti-Leptospira IgG) lower than the cut-off was a negative result suggesting no evidence of past exposure, an AOD value in the cut-off range was a borderline result, suggesting possible past exposure, and an AOD value higher than the upper cut-off was positive by IgG detection, suggesting previous exposure. Borderline was considered a positive result. The diagnostic performance of the Virion-Serion Leptospira IgG-ELISA was 96.7% sensitivity and 99.8% specificity.
Evaluation of diagnostic accuracy.
The Standards for Reporting of Diagnostic Accuracy studies (STARD 2015) checklist for reporting diagnostic accuracy is provided in supplementary Table S6.
Sample size was estimated as a minimum of 35 cases and control samples to achieve 90% sensitivity and specificity at 95% confidence interval (CI) and 7% precision. All sera were tested as anonymous samples. A total of 107 leptospirosis sera and 189 controls (Fig. 1) were randomly selected to evaluate the diagnostic performance of the following tests: (i) rGroEL1–524 IgM-ELISA, (ii) commercial Panbio Leptospira IgM-ELISA, and (iii) Virion-Serion Classic IgG-ELISA. The estimated diagnostic sensitivity and specificity with 95% CI were calculated by 2´2 cross-tabulation table. The hook effect (prozone effect) was determined in serial dilutions of 1:100 and 1:1,000 in MAT-positive leptospirosis sera and other febrile-illness patient sera.
Conservation of GroEL sequences of the selected in the genus Leptospira and among GroEL orthologs were determined using Clustal Omega multiple sequence alignment program interface39 and the results were analyzed using the BioEdit sequence alignment editor tool. Linear B-cell epitopes of the L. interrogans serovar Icterohemorrhagiae GroEL sequence were computationally predicted using a Bepipred-1.0 Linear Epitope Prediction tool40.
Data were collected in Microsoft Excel and were analyzed using MedCalc Statistical Software version 19.2.5 (MedCalc Software Ltd, Ostend, Belgium; https://www.medcalc.org; 2020). Cut-off value was determined using receiver operating characteristic (ROC) curve analysis using paired leptospirosis sera and non-endemic control and other febrile-illness control. Diagnostic parameters were calculated as follows: sensitivity = [(true positive (TP)/(TP + false negative (FN))] × 100; specificity = [(true negative (TN))/(TN + false positive (FP))] × 100. Normal distribution was tested using the Kolmogorov-Smirnov test. The Mann-Whitney test was used in non-normal distributed data. p<0.05 was considered statistically significant.