Advances in the Diagnosis of Leptospirosis
ACVIM 2008
Richard E. Goldstein, DVM, DACVIM, DECVIM (CA)
Ithaca, NY, USA

Introduction

Leptospirosis is an important world-wide zoonosis, caused by an infection with a pathogenic species of the genus Leptospira. These are highly motile obligate aerobic spirochetes that share features of both gram-negative and gram-positive bacteria. Dark field or phase contrast microscopy is necessary to visualize these bacteria since they stain so poorly. This genus is classified today based on genetic determinations. Most of the commonly diagnosed canine pathogenic serovars are still classified (as before) as belonging to the L interrogans species.1 This talk will begin with a brief overview of the pros and cons of the diagnostic options currently available to practitioners, including recommendations for their practical use. The majority of the time will then be spent presenting new and exciting data regarding future diagnostics on the horizon that should greatly enhance our ability to obtain a rapid and accurate diagnosis for this important canine and human disease.

How Can We Diagnose Leptospirosis Today?

The diagnosis of canine leptospirosis is not definitive in most cases in veterinary practice today. It is based on a combination of appropriate clinical signs, clinico-pathologic and imaging data as well as serology. The actual identification of the organism in blood, urine or tissue is uncommon because of the technical difficulties of culture and direct visualization of the organism and the high sensitivity of the organism to antibiotics.

Clinical Signs

The clinical signs may depend on the virulence of the infecting strain, the immune/vaccinal status of the patient and the organ targeted by the bacteria. Although most cases diagnosed today in dogs are associated with acute renal failure other syndromes exist as well, and will be presented in other presentations at the 2008 Forum. Data will be presented in this talk showing the most common clinical signs of anorexia, lethargy and vomiting, PU/PD and abdominal pain study performed in NY State.2 In that same study there was no difference between suspected infecting serovars as to the type of clinical signs diagnosed although a large number of cases for retrospective evaluation were only available from suspected serovars Pomona, Grippotyphosa and Autumnalis. Dogs also presenting with unexplained vasculitis like signs, fever and even polyarthritis should be considered for testing as well.

Clinico-Pathologic Data

Leukocytosis, thrombocytopenia, azotemia, mild hyperbilirubinemia, decreased urine specific gravity, mild proteinuria and glycosuria are common findings in dogs suffering from Leptospirosis.2 A wide index of suspicion is necessary as there are little pathognomonic hints to when to test for this disease.

Culture

Although ideal from a purist standpoint this is not a commercially available option. It is extremely expensive, time consuming, difficult to do and raises human health questions.

Visualization of the Organism in Clinical Samples

Direct fluorescent antibodies (FA) and dark field microscopy can be useful on tissue and occasionally urine. The organisms though tend to disappear within a day or two of therapy causing a high number of false negative results.

Urine PCR

Urine PCR has been reported and is currently commercially available in a limited fashion. PCR identification of Leptospiral DNA in urine, blood, CSF aqueous humor and tissue has been used in human medicine and veterinary research.3,4 The problem with this assay in veterinary clinical cases appears to have been a low sensitivity or high false positive rate. This is the case because of inconsistent tubular shedding of organisms as well as, in our experience, a low rate of positivity once dogs have received antibiotics. PCR (ideally quantitative, see below) does remain the method of choice for identifying active shedding dogs in a kennel, working dog or multi-dog household situation.

Serology

The microscopic agglutination test (MAT) is the most commonly used test in veterinary medicine. The highest dilution of serum that agglutinates 50% of the leptospira organism is reported as the titer. The serum is tested against multiple serovars in different wells. A titer >1:800 against a serovar that the dog has not been vaccinated for or a titer > 1:3,200 against a serovar that the dog has been vaccinated for in the past are thought to be true positives. A detailed description of this test including its pros and cons will appear elsewhere in the symposium. In short the main problems with this test are:

It tends to be negative early on in the disease process when the dogs are first showing clinical signs. The same is true for humans, in fact, the vast majority of people that die from acute leptospirosis, die with negative MAT titers. This is an important limitation of this test in veterinary medicine because as the practitioner's index of suspicion is raised against this disease and the testing is done earlier, more dogs will test negative. This problem can be solved by repeating the titer 10-14 days after the initial titer was drawn. Waiting longer (the typical 3 weeks for a convalescent titer) is not recommended as the titer may rise and then fall again in that time frame especially in a treated dog.

The test does not differentiate between antibodies produced against the currently available vaccines and natural exposure. This of course is a very important limitation in areas where dogs are well vaccinated. The magnitude of the titer (see above) and a 4 fold rise in a convalescent titer are ways to try and identify true infection but are still not ideal.

If a titer against the correct serovar is not requested this could cause only a low positive (because of cross reactivity) or false negative result despite active infection.

The test is cumbersome, requires keeping cultures of multiple serovars, therefore is only performed in a few labs, expensive and slow turn-around time.

One of the possible advantages of this test is that it is thought to some degree to be serovar specific. In actuality it is at best serogroup specific but we will not go into that degree of relatively unimportant detail here. How specific is this test for serovars? There is a large degree of cross reactivity, we assume that the serovar with the highest titer is the one causing the infection. Disappointingly, this was found to be true only in about 50% of the cases in a human study comparing MAT titers to cultures.5 Luckily, the knowledge of what serovar is actually causing the infection, although important epidemiologically and from a vaccination/prevention strategy, is not crucial information regarding the treatment of an individual case.

ELISA testing is the most commonly used test for screening in humans, although to date not in veterinary medicine. The human test ELISA testing can be performed for IgM and IgG. The IgM ELISA may be positive prior to the MAT titer in an acute infection, although because of a lack of specificity it is often confirmed by MAT.

So What Would We Be Looking For to Improve Our Diagnostic Testing? This Is My Wish-List

 A reliable test that would diagnose a dog with an active leptospirosis infection early on in the disease, as clinical signs occur, before the currently available serologic tests are positive.

 A test with a rapid turn-around, either in-clinic or available at many reference laboratories.

 A test that would differentiate a vaccinated dog from one that is truly actively infected.

 A test that would be positive for exposure to all pathogenic serovars. It does not have to be serovar specific.

 A test that would remain positive despite antibiotics, as long as there was active infection, but would be negative a relatively short time after clearance of the organisms.

Is There That Single Perfect Test on the Horizon?

Probably not. But--I believe we will get a lot closer to it within the next few years. Exciting new data will be presented regarding the use of new testing methods on blood, urine and tissue in naturally and experimentally infected dogs. These data will include the use of:

 Quantitative PCR

 Western and immuno-blotting

 A novel approach to develop new ELISA methods

 FISH--fluorescent in-situ hybridization techniques

References

1.  Bharti AR, et al. Lancet Infect Dis 2003 ;3 :757.

2.  Goldstein RE, et al. J Vet Intern Med 2006;20(3):489-94.

3.  Harkin KR, et al. J Am Vet Med Assoc 2003 ;222(9) :1224.

4.  Wagenaar J, et al. Am J Vet Res. 2000; 61(3):316-20.

5.  Levett PN, et al. Clin Inf Dis 2003;36(4):447-552.

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

Richard Goldstein, DVM, DACVIM, DECVIM-CA
Cornell University
Ithaca, NY


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