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 ), nor a number of commonly-occurring non-leptospiral pathogens (see Table ), including the spirochete Borrelia burdorferi
. There is some contention about whether L. meyeri
and L. inadai
are pathogenic [2
]. 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.
] 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.