Leptospirosis is an emerging infectious disease 1. Leptospira spp. are endemic to feral and domestic animals that may serve as reservoirs, with rats and rodents recognised as the most important sources 23. Human infections result from contact with contaminated soil, vegetation or water, or with the body fluids of infected animals. The genus Leptospira comprises both pathogenic and non-pathogenic species: L. interrogans, L. borgpetersenii, L. weilii, L. noguchii, L. meyeri, L. fainei, L. alexanderi, L. santarosai, L. kirschneri, L. inadai; L. biflexa, and L. wolbachii1. There is some controversy about the classification or pathogenicity of some members within species such as L. meyeri and L. inadai245.

The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis 6. For example, patients presenting with severe fever and haemorrhage have symptoms which are difficult to distinguish from viral haemorrhagic fever 7.

There are over 230 known serovars in the genus Leptospira2. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which is able to detect specific antibody produced against the infecting leptospiral organism 2. The MAT relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. A specific antibody response detectable by the MAT generally occurs at around 8–10 days after onset of the illness 6. The MAT is generally performed by reference laboratories due to the inherent safety risks of handling cultures of live leptospiral organisms, the high cost of commercial media, and the need for ongoing maintenance of representative serovars or serogroups. Other techniques, such as the enzyme linked immunosorbent assay (ELISA) and slide agglutination test (SAT), can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT 2.

Isolation of leptospires from human specimens is possible for limited periods in the course of the infection. This may be due to limited survival of the organism in the collected primary tissue or body fluid sample, immune system responses, or exposure to administered antibiotics 6. Culture may take up to eight weeks and must be examined weekly. In the acute phase, which lasts up to about 10 days, leptospires may be cultured from the blood or cerebrospinal fluid (CSF), 2.

The polymerase chain reaction (PCR) has been used to detect a large number of microorganisms, including those of clinical significance 89101112. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. Moreover, using real-time PCR, it is possible to quantify the amount of template and therefore the number of target organisms. Such quantitation can be achieved using a number of approaches, for example employing TaqMan chemistry 13.

Attempts have been made to design PCR primers specifically for Leptospira spp. as reviewed by Levett 1. It has been difficult to identify all pathogenic species and, in some instances, to discriminate between non-pathogenic and pathogenic species. Although Woo et al., 1415 have employed TaqMan fluorogenic probes on leptospires, this non-clinically directed research concentrated on the use of TaqMan probes for the differentiation and identification of Leptospira species using pure isolates obtained from a reference culture collection.

In this paper, we report on the development of real-time detection of Leptospira spp. using a TaqMan fluorogenic probe. The assay is sensitive and appears able to differentiate between pathogenic and non-pathogenic species with highly sensitive limits of detection on clinical samples and without the need for prior isolation and culture.

We have received appropriate ethics approval for this work and have complied with the Helsinki declaration.

This report describes the development of a real-time PCR assay employing a TaqMan probe for the detection and quantitation of organisms belonging to the genus Leptospira. The assay detected pathogenic Leptospira species, but seemed not to detect non-pathogenic strains (see Table 2), nor a number of commonly-occurring non-leptospiral pathogens (see Table 1), including the spirochete Borrelia burdorferi. There is some contention about whether L. meyeri and L. inadai are pathogenic 24. We observed an amplification product for L. meyeri serovar perameles strain bandicoot 343, a strain considered by at least one group to be pathogenic 4. The amplification product obtained for our strain Feral, an inadai species like organism 5 but not L. inadai serovar lyme strain 10, a non-pathogen, suggests the PCR detects some members within this species but further evaluation of strain Feral will need to be undertaken.

The ability of the PCR to distinguish between pathogenic and non-pathogenic Leptospira species may be useful for the testing of environmental samples but this will require further evaluation. The non-pathogenic representative of L. biflexa used in this study is the most commonly-described environmental contaminant and frequently confuses the identification of pathogenic strains from environmental samples. Culture under specialized conditions facilitates discrimination between L. biflexa and other pathogenic species, it is however possible the PCR could provide an alternate or supporting role for identification.

This PCR assay provides the potential to detect pathogenic Leptospira spp. in a range of clinical specimens providing for earlier diagnosis and unequivocal evidence of active infection. As reported for the detection of meningococcal cells 22, the PCR assay developed in this work will provide an enhanced capability for confirmation of cases where early antibiotic treatment has precluded detection by culture. Opportunity also exists for better postmortem testing where samples are generally of poor quality and do not lend themselves to culturing or serology.

The limit of detection of leptospires in urine was approximately 10 cells and for serum approximately 2 cells. The reason for the lower sensitivity in urine samples is not clear, however, two possible reasons are that a component in the urine sample was extracted with the DNA and interfered with the PCR 2, or that extraction of DNA from cells in urine is not as efficient as that in serum. The assay of Merien et al. 23 reported very similar detection levels for seeded urine samples. Bal et al. 24 successfully detected leptospires in patient urine by conventional PCR analysis but recorded problems of reproducibility and sensitivity with different extraction methods caused by the presence of inhibitors. Further investigations of extraction methods for urine will need to be undertaken to monitor the impact of inhibitors.

Previously the diagnosis of leptospirosis has relied heavily on the detection of antibodies using either ELISA or MAT. In many cases, particularly in the early phase of infection, the lack of antibodies in patient sera results in an inability to confirm diagnosis. Patients presenting with clinical symptoms suggestive of leptospirosis will have an opportunity for early detection on a single specimen and the process has the potential to greatly enhance patient management through the timely commencement of medical treatment.

Because real-time PCR enables the quantitative monitoring of leptospiral cells, there exists the opportunity to monitor treatment efficacy, in particular the use of chemotherapeutics. For example, patients in intensive care management, where ineffective treatment is life-threatening, would greatly benefit from direct measurements of infectious organisms.

The PCR assay developed in this study can be completed in around 5 hours. This assay also lends itself to large-scale analysis due to the availability of high-throughput sequence detection systems.

The tube collection systems evaluation demonstrated that lithium heparin may inhibit the PCR. The observed increase in Ct value for the lithium heparin tubes suggests that relevant controls need to be included.

The assay will have application in the human and veterinary fields as both a research tool and supporting diagnostic test.

We have developed a real-time PCR assay for the detection of leptospires in clinical and environmental samples. This method appears to discriminate pathogenic from non-pathogenic strains and can detect as few as 2 cells in serum and 10 cells in urine. The PCR method has a clear advantage over other methods such as the microscopic agglutination test (MAT) which relies on the detection of antibodies, the presence of which cannot be detected until days after infection.

None declared

L. Smythe conducted the molecular studies, sequence alignments and the culture of bacterial species, and participated significantly in the writing and reviews of the manuscript. I. Smith contributed to the design of part of this study, assisted with sequence alignments and aspects of the molecular studies. G. Smith participated in the writing of the manuscript and design of part of this study. M. Dohnt provided laboratory support and expertise for culture and maintenance of reference cultures, provided blind trial samples and also participated in the writing and review of the manuscript. M. Symonds provided laboratory support, collection of sera for the inhibition component of the study, growth medium preparation, culture maintenance and assistance with the writing and review of the manuscript. L. Barnett participated in the design of the study, critical review and writing of the manuscript. D. McKay provided expert input for writing and construction of the manuscript and also participated in the design of part of this study. All authors have read and approved the manuscript.