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Leptospira interrogans insoluble fraction as a potential antigen source for lateral flow immunochromatography

Abstract

BACKGROUND

Leptospirosis is an emerging zoonosis that affects humans and animals. Immunochromatography rapid test is widely used for early diagnosis of leptospirosis, but with low sensitivity and specificity.

OBJECTIVES

To evaluate Leptospira interrogans insoluble fraction as a potential antigen source for lateral flow immunochromatography.

METHODS

Insoluble fraction derived from the crude bacterial extract was obtained by serial centrifugation. The polypeptide profile was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immune reactivity of this fraction was assessed by Western Blotting and lateral flow immunochromatography (LFI). It was tested 160 microagglutination test (MAT)-positive sera from patients in the acute phase, 100 MAT-negative sera from patients with acute febrile illness, and 45 patients with other infectious diseases.

FINDINGS

There was a predominance of low molecular mass-polypeptide bands, ranging from 2 to 37 kDa. The antibody reactivity of theses polypeptides was found to range from 13-50%, especially between 10 and 38 kDa. Among MAT-positive sera of patients with leptospirosis in the acute phase, 97% were also positive in LFI, indicating high sensitivity. Among MAT-negative sera, all were negative in LFI, indicating high specificity. Only 2% of cross-reactivity was detected.

CONCLUSION

The insoluble fraction can be a valuable antigen source for development of point-of-care diagnosis test for leptospirosis.

Key words:
leptospirosis; lateral flow; immunochromatography; diagnosis


Leptospirosis, in humans, is acquired through contact with animals, which are reservoirs for Leptospira, or an environment contaminated with their urine. The infection can be identified by general clinical manifestations, such as fever, headache, and myalgia in mild cases, which may progress to renal failure, jaundice, and pulmonary haemorrhage.11. Ko AI, Reis MG, Dourado CMR, Johnson WD, Riley LW. Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet. 1999; 354(9181): 820-5.,22. Levett PN. Leptospirosis. Clin Microbiol Rev. 2001; 14(2): 296-326. In the acute phase, the bacterial lysis releases the glycolipoprotein fraction (GLP), one of the most important Leptospira endotoxins. Although this fraction is involved in the main pathophysiological events in leptospirosis, however, it had not been identified until 1995, when our team described the association of GLP fraction and Na+K+ ATPase, including manifestation in the kidney, liver, and lung.33. Younes-Ibrahim M, Burth P, Faria MV, Buffin-Meyer B, Marsy S, Barlet-Bas C, et al. Inhibition of Na,K-ATPase by an endotoxin extracted from Leptospira interrogans: a possible mechanism for the physiopathology of leptospirosis. C R Acad Sci III. 1995; 318(5): 619-25.,44. Burth P, Younes-Ibrahim M, Gonçalez FH, Costa ER, Faria MV. Purification and characterization of a Na+, K+ ATPase inhibitor found in an endotoxin of Leptospira interrogans. Infect Immun. 1997; 65(4): 1557-60.

Due to unspecific clinical symptoms and the prevalence of other bacterial infections, particularly in endemic areas, it is important to provide an accurate diagnosis.55. Bandara AGNMK, Kalaivarny G, Pereira N, Indrakumar J. Aseptic meningitis as the initial presentation of Leptospira borgpetersenii serovar Tarassovi: two case reports and a literature review. BMC Infect Dis. 2021; 21(1): 488. The microagglutination test (MAT) is the gold standard and it is based on the agglutination of immune components that reacts against Leptospira strains with antibody titres visually determined by a dark-field microscope.66. Turner LH. Leptospirosis. II. Serology. Transs R Soc Trop Med Hyg. 1968; 62(6): 880-99. The enzyme-linked immunosorbent assay (ELISA) can also detect specific IgM or IgG antibodies to Leptospira antigens with higher sensitivity, but reasonably low specificity.77. Smith CR, Ketterer PJ, McGowan MR, Corney BG. A review of laboratory techniques and their use in the diagnosis of Leptospira interrogans serovar hardjo infection in cattle. Aust Vet J. 1994; 71(9): 290-4.. It is often carried out using L. interrogans or L. biflexa antigens and results can be obtained in a few hours.22. Levett PN. Leptospirosis. Clin Microbiol Rev. 2001; 14(2): 296-326.,88. Mcbride AJA, Atanázio DA, Reis MG, Ko AI. Leptospirosis. Curr Opin Infect Dis. 2005; 18(5): 376-86. Both procedures are difficult to execute, requiring specialised professionals and equipped laboratories.

For point-of-care diagnosis, immunochromatography-based tests detect IgM and IgG and provide an additional advantage by not requiring robust equipment.99. Haake DA, Levett PN. Leptospirosis in humans. Curr Top Microbiol Immunol. 2015; 387: 65-97. For leptospirosis, in addition to the lateral flow immunoassay (LFI), a double-path platform test (DPP) can also detect specific antibodies in few minutes.1010. Nabity SA, Ribeiro GS, Lessa Aquino C, Takahashi D, Damião AO, Gonçalves AHO, et al. Accuracy of a dual path platform (DPP) assay for the rapid point-of-care diagnosis of human leptospirosis. PLoS Negl Trop Dis. 2012; 6(11): e1878.,1111. Smits HL, Hartskeerl RA, Terpstra WJ. International multi-centre evaluation of a dipstick assay for human leptospirosis. Trop Med Int Health. 2000; 5(2): 124-8. Both tests are based on a cassette containing a 0.05-12 μm pore size nitrocellulose membrane associated to other polymers and conjugated molecules.1212. Posthuma-Trumpie GA, Korf J, Van Amerongen A. Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal Bioanal Chemy. 2009; 393(2): 569-82. The DPP assay for leptospirosis was developed by Chembio Diagnostic Systems (Medford, New York, USA) and it is manufactured and distributed in Brazil by Bio-Manguinhos/Fiocruz through a technology transfer agreement with the Brazilian Ministry of Health. In this test, high concentrations of recombinant Leptospira immunoglobulin-like proteins (rLig) were used as an antigen source. It is based on IgM and IgG detection, highly sensitive for the acute phase compared to ELISA, but with 73% sensitivity for mild disease. Within seven days of disease onset, sensitivity drops to 77% and 60% for severe and mild diseases, respectively.1010. Nabity SA, Ribeiro GS, Lessa Aquino C, Takahashi D, Damião AO, Gonçalves AHO, et al. Accuracy of a dual path platform (DPP) assay for the rapid point-of-care diagnosis of human leptospirosis. PLoS Negl Trop Dis. 2012; 6(11): e1878. Additionally, Smits et al.1313. Smits HL, Eapen CK, Sugathan S, Kuriakose M, Gasem MH, Yersin C, et al. Lateral-flow assay for rapid serodiagnosis of human leptospirosis. Clin Diagn Labo Immunol. 2001; 8(1): 166-9. described an LFI assay for the detection of Leptospira-specific IgM antibodies in human sera, using a broadly reactive Leptospira antigen recognised by anti-human IgM/colloidal gold, with 85% and 94% sensitivity and specificity, respectively. Despite using recombinant protein and total bacterial extract in a solid phase, no other antigen fraction has been assessed as a potential tool for a rapid diagnosis platform. Thus, we aimed to evaluate an insoluble fraction derived from the Leptospira crude extract as an alternative antigen source on LFI for detecting specific IgM antibodies.

SUBJECTS AND METHODS

Subjects - The study was approved by the local ethics committee, in affiliation with the Brazilian Ministry of Health (protocol 24215713.7.0000.00.40). Sera (n = 305) were characterised by Instituto Gonçalo Muniz/Fiocruz and Bio-Manguinhos/Fiocruz, and included in groups 1, 2, 3, and 4, based on their immune profile, as described in Table I. Additionally, eight sera (seven positive and one negative) obtained from a serological panel previously characterised by MAT and DPP for leptospirosis were used for screening membrane test.

TABLE I
Immunological profile of sera samples

Leptospira interrogans fraction - L. interrogans (serovar Canicola) strain was kindly provided by the National Reference Laboratory for Leptospirosis, Instituto Oswaldo Cruz (IOC/Fiocruz), being maintained and expanded as described elsewhere.1414. Ellinghausen Jr HC, Mccullough WG. Nutrition of leptospira pomona and growth of 13 other serotypes: fractionation of oleic albumin complex and a medium of bovine albumin and polysorbate 80. Am J Vet Res. 1965; 26: 45-51. For bacterial lysis, the suspension was kept in the ice bath and submitted to sonication, three cycles of 20 s, 25 s intervals, 22% amplitude. The lysate was centrifuged at 14,000 x g for 30 min and the insoluble fraction (IN) was stored at -80ºC until the tests were carried out. The protein content was determined by the BCA method (ThermoFisher Scientific, Rockford, USA) in polystyrene microplates (Nunc, ThermoFisher), according to the manufacturer’s instructions.

SDS-PAGE - The polypeptide profile of the IN fraction was determined by polyacrylamide gel electrophoresis in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (NuPAGE, ThermoFisher) with 7 µg protein/well in sample buffer (10% glycerol, 0.6% SDS, 0.25 M Tris-HCL, pH 6,8), added to 10% β-mercaptoethanol, 0.04% bromophenol blue and 0.25 mM dithiothreitol (DTT), denatured at 95ºC for 5 min and centrifuged at 1,000 x g for 1 min. Polypeptide molecular masses were calculated by Gel Doc XR+, version 4.1 (Bio-Rad, Hercules, USA), with SeeBlue Plus2 (ThermoFisher) as reference.

Western Blotting - Western Blotting was carried out to assess the immunological reactivity of Leptospira-positive sera and control individuals against IN.1515. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 1979; 76(9): 4350-4. Samples were prepared for SDS-PAGE, and polypeptides were transferred to a nitrocellulose membrane and blocked with 2% skimmed milk (Sigma). Sera were diluted in phosphate-buffered saline (PBS)-1% Tween 20 (Sigma) (PBS-T) added of 0.1% skimmed milk and loaded onto the membrane, incubated by 30 min, at 37ºC, under slow agitation. Subsequently, the membrane was washed three times with TBS-T, under moderate agitation, at 37ºC, for 5 min, followed by anti-μ/HRP antibody (Bio-Manguinhos/Fiocruz). The reaction was developed with 0.2% 3,3’-diaminobenzidine (DAB) and 0.01% H2O2 in PBS. Polypeptide molecular masses were calculated as previously described.

Membrane screening test - DPP Leptospirosis, DPP Syphilis, and HIV1/2 Rapid Test diagnosis kits (Bio-Manguinhos/Fiocruz) were used as a model for LFI. The assay was conducted according to the manufacturer’s instructions. All steps, from cassette assembly to production of the running buffer and colloidal gold-labelled human anti-IgM conjugate (anti-IgM/gold) were conducted according to internal documents.

Sample printing and cassette assembling were performed with fixed values for filling rate (8 µL/s), IN fraction volume (10 µL), syringe volume (25 µL), and membrane length (190 mm). Three pore size conditions were used, as follows: 3 µm, 5 µm, and 10 µm. Both IN and anti-IgM/colloidal gold were printed by the IsoFlow Flatbed Dispenser (Imagene Technology, Hanover, USA). Initially, seven homogeneously pooled sera (n = 7) Leptospira-positive and one control serum (Leptospira-negative) from a serological panel were tested. Once the best condition was defined, serum samples were increased as described in details in Table I.

LFI assay - Small and large-scale LFI assays, as well as interpretation of results, were performed according to standard protocols for rapid test manufactured by Bio-Manguinhos/Fiocruz. To perform the text, 5 μL of each serum sample was applied to the appropriate well in the cassette, followed by the addition of three drops of the running buffer. After 10 min, the result was visually interpreted in the designated region of the cassette for line visualisation. A positive test result was indicated by the appearance of a pink/purple line in both T (test) and C (control) tags, while a negative result was indicated by the reactivity of only the C line. An invalid result was considered when the line C was not reactive.

Statistical parameters - All statistical analysis were performed by Graphpad Prism 6.0 (GraphPad). Sensitivity, specificity, and positive/negative predictive values of the LFI test using IN fraction were determined using MAT as golden standard. Differences between the positive and negative sera for LFI was evaluated by the χ2 method or Fisher’s Exact Test, when applicable. Results were significant at a significance level of p < 0.05.

RESULTS

Immune reactivity to polypeptide bands - A broad spectrum of low molecular mass polypeptides was found IN fraction, as shown in SDS-PAGE (Fig. 1). In total, 8, 11, 13, 18, 25, and 37 kDa bands were detected with 700, 100, 50, 600, 10, and 10 i relative intensity values, respectively (Fig. 2).

Fig. 1:
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) polypeptide profile of Leptospira interrogans insoluble fraction (IN) (Lane 2) and molecular mass standard (Lane 1), with respective values; pink lines indicate the main polypeptide bands; silver staining.

Fig. 2:
signal intensity of polypeptide bands from Leptospira interrogans insoluble fraction (IN). Relative mobility (Rf) is represented in association with molecular mass values and intensity.

The Western Blotting assay revealed a consistent recognition profile of the polypeptide bands in sera with high antibody titres. In the IN fraction (Fig. 3), there was a clear immunodominance towards low molecular mass polypeptides, particularly the 10 and 17 kDa bands, although other bands ranging from 11 to 28 kDa were also detected. Notably, over 50% of the sera recognised the 10 kDa band, while 38% of the sera the 11, 14 and 17 kDa bands and the others were recognised by 13% of the sera (Fig. 4).

Fig. 3:
Western Blotting IgM reactivity profile of leptospirosis positive (1-8) and a negative serum against Leptospira interrogans insoluble fraction (IN). ST: molecular mass standard; pink lines indicate the main polypeptide bands.

Fig. 4:
polypeptides recognised by microagglutination test (MAT)-positive sera against Leptospira interrogans insoluble fraction (IN) in Western Blotting.

LFI assay - In parallel, three nitrocellulose membranes were assembled to the cassette and assay were developed at the same conditions, using positive and negative control sera. Control line (C) was present in all membranes although prominent in 3 µm (Fig. 5). Test line (T) was present when using positive sera and absent in negative with better resolution in 3 µm, which has been chosen as the standard. After determining the optimal conditions for membrane and running buffer (Table II), the IN fraction was printed in a 3 µm nitrocellulose membrane and devices were assembled, constituting a lateral flow (LFI) platform for IgM detection. Positivity rate was visually labelled high (+++), medium (++), and weak (+). The negative result (-) was described by the absence of the T test line. In groups 2, 3, and 4, that include MAT-negative patients, convalescent phase, and other diseases, respectively, only 13 sera were positive to IN fraction, although poorly. Among the Leptospira-positive sera (group 1), 97 (97%) were also positive to IN fraction, and only 3 sera (3%) had a negative result (p < 0.0001), indicating considerable importance of using this fraction for LFI. Among the acute phase negative sera (group 2), the majority (97%, p < 0.0001) reacted similarly to LFI loaded with IN fraction (Table III). The positive and negative predictive values reached 97% [95% confidence interval (CI) = 91% - 99%] for all parameters. For cross-reactivity assessment, when positive sera to other infectious diseases were tested, a serum (2%) of a patient with syphilis was also positive for LFI.

Fig. 5:
representative image of cassettes on nitrocellulose membrane test for lateral flow immunochromatography (LFI) using 3 µm, 5 µm, and 10 µm pore sizes loaded with Leptospira interrogans insoluble fraction (IN) associated with positive (+, n = 7, pooled) and negative (-, n = 1) sera.

TABLE II
Analytical profile of lateral flow immunochromatography (LFI) using insoluble fraction (IN) obtained from Leptospira interrogans lysate

TABLE III
Immunological profile of sera from groups 1, 2, 3, and 4 against the insoluble fraction (IN) derived from Leptospira interrogans lysate, determined by lateral flow immunochromatography (LFI)

DISCUSSION

Our study found that an insoluble fraction, obtained by serial centrifugations of L. interrogans extract, can be used in an immunochromatography assay for the diagnosis of leptospirosis. However, one of the main challenges in our study was to obtaining a sufficient yield after cultivating Leptospira bacterial mass in Leptospira Enrichment EMJH culture medium, which is known to have a low bacterial growth rate, as also described by others.1616. Adler B, Moctezuma AP. Leptospira and leptospirosis. Vet Microbiol. 2010; 140(3-4): 287-96.

Bacterial proteins constitute the main antigen basis for recognition by the immune system and this recognition pattern plays a crucial role in disease diagnosis. In SDS-PAGE, we observed prominent polypeptide bands at 8, 11, 13, 18, 25 and 37 kDa, especially from 11 to 25 kDa, which were also detected by previous studies.1717. Doungchawee G, Kositanont U, Niwetpathomwat A, Inwisai T, Sagarasaeranee P, Haake D. Early diagnosis of leptospirosis by immunoglobulin M immunoblot testing. Clin Vaccine Immunol. 2008; 15(3): 492-8.,1818. Widiyanti D, Koizumi N, Fukui T, Muslich LT, Segawa T, Villanueva SYAM, et al. Development of immunochromatography-based methods for detection of leptospiral lipopolysaccharide antigen in urine. Clin Vaccine Immunol. 2013; 20(5): 683-90.

For LFI screening, it has been tested different pore sizes of nitrocellulose membranes using sera from a standard panel. To date, no study has used any protein fraction isolated from Leptospira as a solid phase in LFI. In addition to obtaining an insoluble fraction, our group has suggested an analytical profile of these proteins associated to immune reactivity to provide an accurate diagnosis test. Posthuma-Trumpie et al.1212. Posthuma-Trumpie GA, Korf J, Van Amerongen A. Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal Bioanal Chemy. 2009; 393(2): 569-82. have reported that membranes screening is crucial to ensure proper flow of immune and chemical components through the pores. Since IFL results rely on visual interpretation, the lines revealed by antigen-antibody binding must be clear and well-defined. Based on these factors, three membranes were assessed, and we found that 3 µm-pore size membrane yielded the best results.

Furthermore, 305 sera were tested, including those from patients positive to other pathogens. This step is important for detecting potential cross-reactivity between Leptospira and other in the same endemic area, as reported by Smits et al.,1111. Smits HL, Hartskeerl RA, Terpstra WJ. International multi-centre evaluation of a dipstick assay for human leptospirosis. Trop Med Int Health. 2000; 5(2): 124-8. which used positive sera for HIV, hepatitis A, hepatitis B, syphilis, malaria, toxoplasmosis, meningitis, meningococcal meningitis, reporting cross-reactivity with later. Amran et al.1919. Amran F, Liow YL, Halim NAN. Evaluation of a commercial immuno-chromatographic assay kit for rapid detection of IgM antibodies against Leptospira antigen in human serum. J Korean Med Sci. 2018; 33(17): e131. also described cross-reactivity with dengue, syphilis, and typhoid. In the platform used in our study, although evidence of this cross-reactivity has also been found indicating a potential homology with Treponema pallidum components, these findings must be explored to clear the molecular mechanisms involved in this immune reactivity.

DPP immunochromatography assay has been used for leptospirosis diagnosis although low sensitivity has been reported as a limitation. A study reported 73% sensitivity with samples from patients with leptospirosis who have been submitted to DPP with recombinant Lig proteins.1010. Nabity SA, Ribeiro GS, Lessa Aquino C, Takahashi D, Damião AO, Gonçalves AHO, et al. Accuracy of a dual path platform (DPP) assay for the rapid point-of-care diagnosis of human leptospirosis. PLoS Negl Trop Dis. 2012; 6(11): e1878. However, we showed that IN fraction derived from Leptospira lysate on a rapid test platform can increase sensitivity to 93%.

The discordance rate in groups 1 and 2 was only 3%. The high reactivity found in acute samples compared to convalescent samples was an expected result, since the conjugate used in the IFL was specific for IgM detection. Our study evaluated sensitivity, specificity, and predictive values, with consistent results. The high sensitivity (97%) indicates a wide range of reactivity, and the specificity is also expressive, indicating few false positive samples. Our results demonstrate an advantage in relation to others that achieved < 95% specificity and 73% sensitivity in patients with mild disease.1010. Nabity SA, Ribeiro GS, Lessa Aquino C, Takahashi D, Damião AO, Gonçalves AHO, et al. Accuracy of a dual path platform (DPP) assay for the rapid point-of-care diagnosis of human leptospirosis. PLoS Negl Trop Dis. 2012; 6(11): e1878. Smits et al. obtained 86% sensitivity and 94% specificity while Alamuri et al., using a recombinant surface antigen Lsa27, revealed 90% and 91% sensitivity and specificity, respectively.1313. Smits HL, Eapen CK, Sugathan S, Kuriakose M, Gasem MH, Yersin C, et al. Lateral-flow assay for rapid serodiagnosis of human leptospirosis. Clin Diagn Labo Immunol. 2001; 8(1): 166-9.,1919. Amran F, Liow YL, Halim NAN. Evaluation of a commercial immuno-chromatographic assay kit for rapid detection of IgM antibodies against Leptospira antigen in human serum. J Korean Med Sci. 2018; 33(17): e131.,2020. Doungchawee G, Sutdan D, Niwatayakul K, Inwisai T, Sitthipunya A, Boonsathorn N, et al. Development and evaluation of an immunochromatographic assay to detect serum anti-leptospiral lipopolysaccharide IgM in acute leptospirosis. Sci Rep. 2017; 7(1): 2309.,2121. Temeiam N, Jareinpituk S, Phinyo P, Patumundond J, Srisawatid N. Development and validation of a simple score for diagnosis of leptospirosis at outpatient departments. PLoS Negl Trop Dis. 2020; 14(1): e0007977.,2222. Alamuri A, Kumar KV, Varghese B, Palkhade R, Mahadeviah S, Chaudhari S, et al. Evaluation of recombinant leptospiral surface antigen (Lsa27) lipoprotein for serodiagnosis of human leptospirosis by latex agglutination test. Indian J Med Microbiol. 2021; 39(2): 212-7. In other study, Alia et al. reported 40% sensitivity and 89% specificity, with immunochromatography tests carried out with samples previously characterised by MAT, while others revealed 91% sensitivity and 83% specificity.2323. Alia S, José N, Filipe N, Azhar N, Garba B, Masri S, et al. Diagnostic accuracy of rapid diagnostic tests for the early detection of leptospirosis. J Infect Public Health. 2019; 12(2): 263-9.,2424. Eugene E, Handunnetti SH, Wickramasinghe SA, Kalugalage TL, Rodrigo C, Wickremesinghe H, et al. Evaluation of two immunodiagnostic tests for early rapid diagnosis of leptospirosis in Sri Lanka: a preliminary study. BMC Infect Dis. 2015; 15: 319.,2525. Goris MGA, Leeflang MMG, Loden M, Wagenaar JFP, Klatser PR, Hartskeerl RA, et al. Prospective evaluation of three rapid diagnostic tests for diagnosis of human leptospirosis. PLoS Negl Trop Dis. 2013; 7(7): e2290.,2626. Rao M, Amran F, Aqilla N. Evaluation of a rapid kit for detection of IgM against Leptospira in human. Can J Infect Dis Med Microbiol. 2019; 2019: 5763595. Dinhuzen et al.2727. Dinhuzen J, Limothai U, Tachabon S, Krairojananan P, Laosatiankit B, Boonprasong S, et al. A prospective study to evaluate the accuracy of rapid diagnostic tests for diagnosis of human leptospirosis: result from THAI-LEPTO AKI study. PLoS Negl Trop Dis. 2021; 15(2): e0009159. described 40% sensitivity and 89% specificity using the same test. Comparative pattern among these studies is presented in Table IV.

TABLE IV
Sensitivity and specitivity values for leptospirosis diagnosis achieved in the current study (Campos et al.) and other reports

Overall, despite of the small sample size, our study showed that the IN fraction is a useful antigen source for LFI, as it achieved high specificity and sensitivity (97%). These findings suggest that this fraction can be a reliable alternative for developing a robust diagnostic platform for leptospirosis, which is critical for addressing public health concerns.

ACKNOWLEDGEMENTS

To Dr Shubhashish Mukopahadhay for reviewing the English language. This work is dedicated to the memory of Dr Hilton Jorge Nascimento.

REFERENCES

  • 1
    Ko AI, Reis MG, Dourado CMR, Johnson WD, Riley LW. Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet. 1999; 354(9181): 820-5.
  • 2
    Levett PN. Leptospirosis. Clin Microbiol Rev. 2001; 14(2): 296-326.
  • 3
    Younes-Ibrahim M, Burth P, Faria MV, Buffin-Meyer B, Marsy S, Barlet-Bas C, et al. Inhibition of Na,K-ATPase by an endotoxin extracted from Leptospira interrogans: a possible mechanism for the physiopathology of leptospirosis. C R Acad Sci III. 1995; 318(5): 619-25.
  • 4
    Burth P, Younes-Ibrahim M, Gonçalez FH, Costa ER, Faria MV. Purification and characterization of a Na+, K+ ATPase inhibitor found in an endotoxin of Leptospira interrogans. Infect Immun. 1997; 65(4): 1557-60.
  • 5
    Bandara AGNMK, Kalaivarny G, Pereira N, Indrakumar J. Aseptic meningitis as the initial presentation of Leptospira borgpetersenii serovar Tarassovi: two case reports and a literature review. BMC Infect Dis. 2021; 21(1): 488.
  • 6
    Turner LH. Leptospirosis. II. Serology. Transs R Soc Trop Med Hyg. 1968; 62(6): 880-99.
  • 7
    Smith CR, Ketterer PJ, McGowan MR, Corney BG. A review of laboratory techniques and their use in the diagnosis of Leptospira interrogans serovar hardjo infection in cattle. Aust Vet J. 1994; 71(9): 290-4..
  • 8
    Mcbride AJA, Atanázio DA, Reis MG, Ko AI. Leptospirosis. Curr Opin Infect Dis. 2005; 18(5): 376-86.
  • 9
    Haake DA, Levett PN. Leptospirosis in humans. Curr Top Microbiol Immunol. 2015; 387: 65-97.
  • 10
    Nabity SA, Ribeiro GS, Lessa Aquino C, Takahashi D, Damião AO, Gonçalves AHO, et al. Accuracy of a dual path platform (DPP) assay for the rapid point-of-care diagnosis of human leptospirosis. PLoS Negl Trop Dis. 2012; 6(11): e1878.
  • 11
    Smits HL, Hartskeerl RA, Terpstra WJ. International multi-centre evaluation of a dipstick assay for human leptospirosis. Trop Med Int Health. 2000; 5(2): 124-8.
  • 12
    Posthuma-Trumpie GA, Korf J, Van Amerongen A. Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal Bioanal Chemy. 2009; 393(2): 569-82.
  • 13
    Smits HL, Eapen CK, Sugathan S, Kuriakose M, Gasem MH, Yersin C, et al. Lateral-flow assay for rapid serodiagnosis of human leptospirosis. Clin Diagn Labo Immunol. 2001; 8(1): 166-9.
  • 14
    Ellinghausen Jr HC, Mccullough WG. Nutrition of leptospira pomona and growth of 13 other serotypes: fractionation of oleic albumin complex and a medium of bovine albumin and polysorbate 80. Am J Vet Res. 1965; 26: 45-51.
  • 15
    Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 1979; 76(9): 4350-4.
  • 16
    Adler B, Moctezuma AP. Leptospira and leptospirosis. Vet Microbiol. 2010; 140(3-4): 287-96.
  • 17
    Doungchawee G, Kositanont U, Niwetpathomwat A, Inwisai T, Sagarasaeranee P, Haake D. Early diagnosis of leptospirosis by immunoglobulin M immunoblot testing. Clin Vaccine Immunol. 2008; 15(3): 492-8.
  • 18
    Widiyanti D, Koizumi N, Fukui T, Muslich LT, Segawa T, Villanueva SYAM, et al. Development of immunochromatography-based methods for detection of leptospiral lipopolysaccharide antigen in urine. Clin Vaccine Immunol. 2013; 20(5): 683-90.
  • 19
    Amran F, Liow YL, Halim NAN. Evaluation of a commercial immuno-chromatographic assay kit for rapid detection of IgM antibodies against Leptospira antigen in human serum. J Korean Med Sci. 2018; 33(17): e131.
  • 20
    Doungchawee G, Sutdan D, Niwatayakul K, Inwisai T, Sitthipunya A, Boonsathorn N, et al. Development and evaluation of an immunochromatographic assay to detect serum anti-leptospiral lipopolysaccharide IgM in acute leptospirosis. Sci Rep. 2017; 7(1): 2309.
  • 21
    Temeiam N, Jareinpituk S, Phinyo P, Patumundond J, Srisawatid N. Development and validation of a simple score for diagnosis of leptospirosis at outpatient departments. PLoS Negl Trop Dis. 2020; 14(1): e0007977.
  • 22
    Alamuri A, Kumar KV, Varghese B, Palkhade R, Mahadeviah S, Chaudhari S, et al. Evaluation of recombinant leptospiral surface antigen (Lsa27) lipoprotein for serodiagnosis of human leptospirosis by latex agglutination test. Indian J Med Microbiol. 2021; 39(2): 212-7.
  • 23
    Alia S, José N, Filipe N, Azhar N, Garba B, Masri S, et al. Diagnostic accuracy of rapid diagnostic tests for the early detection of leptospirosis. J Infect Public Health. 2019; 12(2): 263-9.
  • 24
    Eugene E, Handunnetti SH, Wickramasinghe SA, Kalugalage TL, Rodrigo C, Wickremesinghe H, et al. Evaluation of two immunodiagnostic tests for early rapid diagnosis of leptospirosis in Sri Lanka: a preliminary study. BMC Infect Dis. 2015; 15: 319.
  • 25
    Goris MGA, Leeflang MMG, Loden M, Wagenaar JFP, Klatser PR, Hartskeerl RA, et al. Prospective evaluation of three rapid diagnostic tests for diagnosis of human leptospirosis. PLoS Negl Trop Dis. 2013; 7(7): e2290.
  • 26
    Rao M, Amran F, Aqilla N. Evaluation of a rapid kit for detection of IgM against Leptospira in human. Can J Infect Dis Med Microbiol. 2019; 2019: 5763595.
  • 27
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Publication Dates

  • Publication in this collection
    12 May 2023
  • Date of issue
    2023

History

  • Received
    18 Nov 2022
  • Accepted
    03 Apr 2023
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