Leptospirosis Serodiagnosis by the Microscopic Agglutination Test

Marga G.A. Goris1, Rudy A. Hartskeerl1

1 Royal Tropical Institute (KIT), KIT Biomedical Research, WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis, Amsterdam
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12E.5
DOI:  10.1002/9780471729259.mc12e05s32
Online Posting Date:  February, 2014
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Abstract

The microscopic agglutination test (MAT) is the gold standard for sero‐diagnosis of leptospirosis because of its unsurpassed diagnostic specificity. It uses panels of live leptospires, ideally recent isolates, representing the circulating serovars from the area where the patient became infected. A dilution series of the patient's serum is mixed with a suspension of live leptospires in microtiter plates. After incubating for about 2 hr at 30°C, results are read under the dark‐field microscope. The titer is the last dilution in which ≥50% of the leptospires have remained agglutinated. Seroconversion or ≥4‐fold titer rise in paired sera is consistent with current leptospirosis. The significance of a titer in a single sample depends on the frequency of residual titers due to past infections and cross‐reacting other diseases in the population. Full standardization of the MAT is not possible, but quality assurance can be achieved by participation in the international MAT proficiency testing scheme. Curr. Protoc. Microbiol. 32:12E.5.1‐12E.5.18. © 2014 by John Wiley & Sons, Inc.

Keywords: MAT; agglutination; leptospirosis; antibodies; serology

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Microscopic Agglutination Test (MAT), Quantitative, Indirect Reading
  • Basic Protocol 2: Microscopic Agglutination Test, Screening, Indirect Reading
  • Alternate Protocol 1: Microscopic Agglutination Test (MAT), Direct Reading
  • Support Protocol 1: Growth of Leptospira Strains (Antigens) for the Microscopic Agglutination Test
  • Support Protocol 2: Counting Leptospires in a Helber Counting Chamber
  • Support Protocol 3: Using Optical Density to Determine Number of Leptospires
  • Support Protocol 4: The McFarland Nephelometric Method for Determining the Density of Cultures
  • Support Protocol 5: Estimating Culture Density by Eye
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Microscopic Agglutination Test (MAT), Quantitative, Indirect Reading

  Materials
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.2
  • Serum or plasma (it is recommended to heat‐inactivate serum for 30 min in a water bath at 56°C to reduce infection risks; plasma samples cannot be heat‐inactivated)
  • Live Leptospira cultures at ∼2 × 108 leptospires/ml of each of the required serovars
  • 96% ethanol
  • 96‐well microtiter plate, V‐bottom, flat‐bottom or U‐bottom
  • Calibrated pipets: 2 to 20 μl and 20 to 200 μl (optional: 50‐μl multichannel pipet and 50‐μl multistepper pipet)
  • Microtiter plate cover
  • Microshaker, optional
  • 30°C incubator
  • Microscope slides
  • Wire loop
  • Dark‐field microscope

Basic Protocol 2: Microscopic Agglutination Test, Screening, Indirect Reading

  Materials
  • Serum or plasma (it is recommended to heat‐inactivate serum for 30 min in a water bath at 56°C to reduce infection risks; plasma samples cannot be heat‐inactivated)
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.2
  • Live Leptospira cultures at approximately 2 × 108 cells/ml of each of the required serovars
  • 96% ethanol
  • Small tubes
  • 96‐well microtiter plate, V‐bottom, flat‐bottom or U‐bottom
  • Calibrated pipets: 20 to 200 μl (optional: 50‐μl multistepper pipet)
  • Microtiter plate cover
  • Microshaker, optional
  • 30°C incubator
  • Microscope slides
  • Wire loop
  • Dark‐field microscope

Alternate Protocol 1: Microscopic Agglutination Test (MAT), Direct Reading

  Materials
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.2
  • Serum and plasma (it is recommended to heat‐inactivate serum for 30 min in a water bath at 56°C to reduce infection risks; plasma samples cannot be heat‐inactivated)
  • Live Leptospira cultures at ∼2 × 108 leptospires/ml of each of the required serovars
  • 96‐well microtiter plate, flat‐bottom (Greiner Crystal‐Clear, cat. no. 655101)
  • Calibrated pipets: 2 to 20 μl and 20 to 200 μl (optional: 50‐μl multichannel pipet and 50‐μl multistepper pipet)
  • Microtiter plate cover
  • Microshaker, optional
  • 30°C incubator
  • Microscope slides
  • Wire loop
  • Dark‐field microscope suitable for direct reading

Support Protocol 1: Growth of Leptospira Strains (Antigens) for the Microscopic Agglutination Test

  Materials
  • Leptospira strains in liquid medium
  • Liquid medium in tubes (5 to 10 ml; e.g., EMJH; see unit 12.1)
  • Laminar flow cabinet
  • Microscope slide
  • Wire loops, sterile
  • Pipets, sterile
  • Dark‐field microscope
  • 29°C incubator

Support Protocol 2: Counting Leptospires in a Helber Counting Chamber

  Materials
  • Phosphate‐buffered saline (PBS; see recipe), pH 7.2
  • Formalin (37% formaldehyde)
  • Suspension of leptospires to be counted
  • 70% (v/v) ethanol
  • Distilled water
  • 2‐ to 20‐μl and 20‐ to 200‐μl micropipets
  • Small tubes
  • Vortex mixer
  • Helber bacteria counting chamber with special slide cover
  • Dark‐field microscope
  • Tally counter, optional

Support Protocol 3: Using Optical Density to Determine Number of Leptospires

  Materials
  • Distilled water
  • Culture medium
  • Leptospires
  • Cuvettes
  • Spectrophotometer with visible light at 420 nm

Support Protocol 4: The McFarland Nephelometric Method for Determining the Density of Cultures

  Materials
  • Barium chloride (BaCl 2; anhydrous)
  • Distilled water
  • Sulfuric acid (H 2SO 4; 95% to 97%)
  • Leptospiral culture
  • Fume hood
  • Tubes, appropriate size for nephelometer
  • Nephelometer
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Figures

Videos

Literature Cited

  Adler, B. and de la Pena, M. A. 2010. Leptospira and leptospirosis. Vet. Microbiol. 140:287‐296.
  Anonymous. 2011. International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Leptospiraceae. Minutes of the closed meeting, 22 September 2009, Cochin, India. Int. J. Syst. Evol. Microbiol. 61:698‐699.
  Borg‐Petersen, C. and Fagraeus, A. 1949. The influence of the antigen density and other factors on the serum titer in the agglutination‐lysis‐test for leptospirosis. Acta Pathol. Microbiol. Scand. 26:555‐567.
  Carbrey, E.A. 1960. The relative importance of variable factors in the agglutination‐lysis‐test. 64th Annu. Proc. U.S. Livestock Sanit. Assoc.
  Chappel, R.J., Goris, M., Palmer, M.F., and Hartskeerl, R.A. 2004. Impact of proficiency testing on results of the microscopic agglutination test for diagnosis of leptospirosis. J. Clin. Microbiol. 42:5484‐5488.
  Cole, J.R., Sulzer, C.R., and Pursell, A.R. 1973. Improved microtechnique for the leptospiral microscopic agglutination test. Appl. Microbiol. 25:976‐980.
  Faine, S., Adler, B., Bolin, C.A., and Perolat, P. 1999. Leptospira and Leptospirosis. MediSci, Melbourne, Australia.
  Goris, M.G.A., Leeflang, M.M.G., Boer, K.R., Goeijenbier, M., van Gorp, E.C.M., Wagenaar, J.F.P., and Hartskeerl, R.A. 2011. Establishment of valid laboratory cases definition of human leptospirosis. J. Bacteriol. Parasitol. 3:132. doi:10.4172/2155‐9597.1000132.
  ILRI. 2012. Mapping of poverty and likely zoonoses hotspots. Zoonoses Project 4. Report to Department for International Development, U.K. pp. 1‐119.
  Kmety, E. 1958. Paradox reactions and their significance in serodiagnosis of some types of leptospirosis. Zentralbl. Bakteriol. Orig. 170:597‐608.
  Levett, P.N. 2003. Usefulness of serologic analysis as a predictor of the infecting serovar in patients with severe leptospirosis. Clin. Infect. Dis. 36:447‐452.
  Limmathurotsakul, D., Turner, E.L., Wuthiekanun, V., Thaipadungpanit, J., Suputtamongkol, Y., Chierakul, W., Smythe, L.D., Day, N.P., Cooper, B., and Peacock, S.J. 2012. Fool's gold: Why imperfect reference tests are undermining the evaluation of novel diagnostics: A reevaluation of 5 diagnostic tests for leptospirosis. Clin. Infect. Dis. 55:322‐331.
  Martin, L. and Pettit, A. 1918. Sero‐diagnostic de la spirochetose icterohemorrhagique. Bull. Mem. Soc. Med. Hopitaux Paris 672‐675.
  OIE. 2008. OIE manual of diagnostic tests and vaccines for terrestrial animals.6th edition. OIE, Paris.
  Ryu, E. 1970. Rapid microscopic agglutination test for Leptospira without non‐specific reaction. Bull. Off. Int. Epizoot. 73:49‐58.
  Schuffner, W. and Mochtar, A. 1926. Experiments on the differential characters of Leptospira strains with introductory remarks on the process of agglutination and lysis. Proceedings of the Section of Sciences. K. Nederlandse Akademie von Wetenschappen, 30:25‐32.
  Shimabukuro, F.H., Costa, V.M., Silva, R.C., Langoni, H., Silva, A.V., Carvalho, L.R., and Domingues, P.F. 2013. Prozone effects in microscopic agglutination tests for leptospirosis in the sera of mice infected with the pathogenic Leptospira interrogans serovar Canicola. Mem. Inst. Oswaldo Cruz 108:668‐670.
  Wolff, J. 1954. The laboratory diagnosis of leptospirosis. Thomas, Springfield, Illinois.
  World Health Organization. 2003. Human leptospirosis: Guidance for diagnosis, surveillance and control.
  World Health Organization. 2011. Report of the Second Meeting of the Leptospirosis Burden Epidemiology Reference Group.
Key References
  Faine et al., 1999. See above.
  This book provides comprehensive coverage of all aspects of leptospirosis.
  World Health Organization. 2003. See above.
  This book provides basic information on human leptospirosis and details diagnostic methods and public health related issues.
  OIE manual. See above.
  This book provides comprehensive information on veterinary leptospirosis, diagnosis and vaccine requirements.
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