Leptospirosis is an acute bacterial infection caused by spirochetes of the pathogenic species Leptospira interrogans and Leptospira kirschneri.1,2 It is considered one of the most important reemerging zoonoses worldwide, with increasing seroprevalence and prevalence of clinical illness in humans and dogs over the past several years.1,3–6,a The wide spectrum of clinical manifestations in infected dogs varies from subclinical infection to peracute fatal multi-organ involvement dominated by renal, hepatic, hematologic, and pulmonary failure.6,7 Given that early and accurate diagnosis of affected dogs is essential to alter the course of the disease with appropriate antimicrobial treatment, the availability of validated diagnostic tools is necessary to confirm a clinical suspicion.8,9
The agglutinating antibodies induced in infected animals by leptospiral outer membrane proteins with variable O-side chain sugars are responsible for the phenotypic classification of leptospires in serogroups and serovars by use of the MAT.10,11 In both dogs and humans, leptospiral antibodies are usually detectable 7 to 10 days after the onset of disease.9,12 To prove an active disease and the etiologic role of leptospires in an animal with AKI, a convalescent and occasionally a third serum sample must be obtained at 7- to 14-day intervals, revealing seroconversion with at least a 4-fold increase in titers.6,8 Detection of seroconversion against specific serovars in animals with compatible clinical signs is still considered the reference method for the clinical diagnosis of leptospirosis.3,6,12,13 Although it delays a definitive diagnosis, paired serologic testing is essential and is recommended in the Consensus Statement of the American College of Veterinary Internal Medicine because a single MAT may fail to detect antibodies in the early phase of the disease or may give false-positive results after vaccination or exposure in endemic areas.6
Paired serologic testing may be impossible in some animals, such as those that die quickly from the severe acute form of the disease, and lack of inclusion of such cases may substantially bias clinical studies toward a conclusion that leptospirosis is a more benign disease than it truly is. In the absence of an agreement on an ideal single-sample cutoff titer as the best compromise between the high sensitivity of a low titer and the high specificity of a high titer, widely different cutoffs have been used in clinical practice and in research studies, ranging from 1:100 to 1:12,800.12,14–17 The World Health Organization and the International Leptospirosis Society recommend that the ideal cutoff for a single specimen be determined individually in a particular area on the basis of the seroprevalence of persistent antibodies in relation to the occurrence of other diseases that induce production of cross-reacting antibodies.12 However, the validity of these recommendations is limited when it comes to diagnosis of the disease in dogs because of the problems relating to vaccine titers. The sensitivity and specificity of a single initial MAT titer of ≥ 1:800 in dogs have been reported in 1 studyb to be 22% to 67% and 69% to 100%, respectively, and marked interlaboratory variations have been found.13 These limitations of serologic testing and its poor ability to predict the infecting serogroup, its cost, and the tedious laboratory methodology have favored the development of other reliable diagnostic methods, especially for early diagnosis.6
A diagnosis of leptospirosis based on pathogen detection has the potential advantage of early confirmation, independent of the time necessary for a serologic response to occur. The mere presence of leptospiral DNA would still need to be interpreted in the clinical context, considering the possibility of subclinical carriers shedding organisms in their urine. Polymerase chain reaction assays have been recommended for their high intrinsic detection sensitivity. One assay was sensitive but not always specific for the diagnosis of leptospirosis in a small group of affected dogs.18 Various PCR assays have been increasingly offered by commercial veterinary diagnostic laboratories worldwide, with variable analytic validation but typically with minimal or absent clinical validation. Preanalytic variables including patient and disease characteristics, sampling conditions and timing, and nature and preparation of the samples have, to the authors’ knowledge, not been evaluated in a clinical setting of dogs with severe AKI, where an accurate and timely diagnosis is critical. On the basis of data from experimental infections, blood is the recommended substrate in the first 10 days of infection and urine thereafter.19 However, ideal preanalytic conditions are rarely encountered in referral institutions, where most animals are treated with antimicrobials prior to referral and many dogs are anuric.a
The goals of the study reported here were thus to determine the diagnostic value of early and late serologic microagglutination testing, compared with reference paired serologic testing, and to evaluate the diagnostic value of a commercially available Leptospira LipL32 nested PCR assay on urine and blood obtained from dogs with AKI under the conditions of a referral hospital.
Acute kidney injury
Receiver operating characteristic
Francey T. Clinical features and epidemiology of presumptive canine leptospirosis in western Switzerland, 2003–2005 (abstr). J Vet Intern Med 2006;20:1530–1531.
Miller MD, Annis KM, Lappin MR, et al. Sensitivity and specificity of the microagglutination test for the diagnosis of leptospirosis in dogs (abstr). J Vet Intern Med 2008;22:787.
IVD Gesellschaft für Innovative Veterinärdiagnostik mbH, Hannover, Germany.
NCSS, version 8, NCSS LLC, Kaysville, Utah.
Hugonnard M, Djelouadji Z, Pouzot-Nevoret C, et al. Evaluation of polymerase chain reaction in the diagnosis of canine leptospirosis: comparison with serologic testing in 33 dogs (abstr). J Vet Intern Med 2011;25:1501.
2. Greene CE, Sykes JE, Brown CAet al., Leptospirosis. Greene CE, Infectious diseases of the dog and cat. 3rd ed. St Louis: Saunders Elsevier, 2006; 402–417.
3. Hartskeerl RA, Collares-Pereira M, Ellis WA, Emergence, control and re-emerging leptospirosis: dynamics of infection in the changing world. Clin Microbiol Infect. 2011; 17:494–501.
4. Bharti AR, Nally JE, Ricaldi JNet al., Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis. 2003; 3:757–771.
5. Alton GD, Berke O, Reid-Smith Ret al., Increase in seroprevalence of canine leptospirosis and its risk factors, Ontario 1998–2006. Can J Vet Res. 2009; 73:167–175.
6. Sykes JE, Hartmann K, Lunn KFet al., 2010 ACVIM small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment, and prevention. J Vet Intern Med. 2011; 25:1–13.
8. van de Maele I, Claus A, Haesebrouck Fet al., Leptospirosis in dogs: a review with emphasis on clinical aspects. Vet Rec. 2008; 163:409–413.
9. Ooteman MC, Vago AR, Koury MC, Evaluation of MAT, IgM ELISA and PCR methods for the diagnosis of human leptospirosis. J Microbiol Methods. 2006; 65:247–257.
12. World Health Organization, International Leptospirosis Society. Human leptospirosis: guidance for diagnosis, surveillance and control. NLM classification: WC 420. Geneva: World Health Organization, 2003.
13. Miller MD, Annis KM, Lappin MRet al., Variability in results of the microscopic agglutination test in dogs with clinical leptospirosis and dogs vaccinated against leptospirosis. J Vet Intern Med. 2011; 25:426–432.
14. Dey S, Mohan CM, Ramadass Pet al., Recombinant antigen-based dipstick ELISA for the diagnosis of leptospirosis in dogs. Vet Rec. 2007; 160:186–188.
15. Mastrorilli C, Dondi F, Agnoli Cet al., Clinicopathologic features and outcome predictors of Leptospira interrogans Australis serogroup infection in dogs: a retrospective study of 20 cases (2001–2004). J Vet Intern Med. 2007; 21:3–10.
16. Geisen V, Stengel C, Brem Set al., Canine leptospirosis infections—clinical signs and outcome with different suspected Leptospira serogroups (42 cases). J Small Anim Pract. 2007; 48:324–328.
17. Raghavan R, Brenner K, Higgins Jet al., Evaluations of land cover risk factors for canine leptospirosis: 94 cases (2002–2009). Prev Vet Med. 2011; 101:241–249.
18. Harkin KR, Roshto YM, Sullivan JT, Clinical application of a polymerase chain reaction assay for diagnosis of leptospirosis in dogs. J Am Vet Med Assoc. 2003; 222:1224–1229.
19. Greenlee JJ, Alt DP, Bolin CAet al., Experimental canine leptospirosis caused by Leptospira interrogans serovars Pomona and Bratislava. Am J Vet Res. 2005; 66:1816–1822.
20. Bossuyt PM, Reitsma JB, Bruns DEet al., Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD initiative. BMJ. 2003; 326:41–44.
21. Cowgill LD, Francey T, Acute uremia. Ettinger SJ, Feldman EC, Textbook of veterinary internal medicine. 6th ed. St Louis: Elsevier Saunders, 2004; 1731–1751.
22. Adin CA, Cowgill LD, Treatment and outcome of dogs with leptospirosis: 36 cases (1990–1998). J Am Vet Med Assoc. 2000; 216:371–375.
23. Newcombe RG, Two-sided confidence intervals for the single proportion: comparison of seven methods. Stat Med. 1998; 17:857–872.
24. Greiner M, Sohr D, Gobel P, A modified ROC analysis for the selection of cut-off values and the definition of intermediate results of serodiagnostic tests. J Immunol Methods. 1995; 185:123–132.
25. Langston C, Acute uremia. Ettinger SJ, Feldman EC, Textbook of veterinary internal medicine. 7th ed. St Louis: Saunders Elsevier, 2010; 1969–1985.
26. Segev G, Kass PH, Francey Tet al., A novel clinical scoring system for outcome prediction in dogs with acute kidney injury managed by hemodialysis. J Vet Intern Med. 2008; 22:301–308.
27. Doudier B, Garcia S, Quennee Vet al., Prognostic factors associated with severe leptospirosis. Clin Microbiol Infect. 2006; 12:299–300.
28. Cowgill LD, Francey T, Hemodialysis and extracorporeal blood purification. DiBartola SP, Fluid, electrolyte and acid-base disorders in small animal practice. 4th ed. St Louis: Saunders Elsevier, 2011; 680–713.
29. Barmettler R, Schweighauser A, Bigler Set al., Assessment of exposure to Leptospira serovars in veterinary staff and dog owners in contact with infected dogs. J Am Vet Med Assoc. 2011; 238:183–188.
31. Evangelista KV, Coburn J, Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses. Future Microbiol. 2010; 5:1413–1425.
32. Harkin KR, Roshto YM, Sullivan JTet al., Comparison of polymerase chain reaction assay, bacteriologic culture, and serologic testing in assessment of prevalence of urinary shedding of leptospires in dogs. J Am Vet Med Assoc. 2003; 222:1230–1233.
33. Hazart G, Hugonnard M, Kodjo Aet al., La leptospirose canine en France: étude rétrospective de 37 cas. Prat Med Chir Anim Comp. 2004; 45:59–64.
34. Ghneim GS, Viers JH, Chomel BBet al., Use of a case-control study and geographic information systems to determine environmental and demographic risk factors for canine leptospirosis. Vet Res. 2007; 38:37–50.
35. Kohn B, Steinicke K, Arndt Get al., Pulmonary abnormalities in dogs with leptospirosis. J Vet Intern Med. 2010; 24:1277–1282.