Comprehensive Case-Control Study of Protective and Risk Factors for Buruli Ulcer, Southeastern Australia.

To examine protective and risk factors for Buruli ulcer (BU), we conducted a case-control study of 245 adult BU cases and 481 postcode-matched controls across BU-endemic areas of Victoria, Australia. We calculated age- and sex-adjusted odds ratios for socio-environmental, host, and behavioral factors associated with BU by using conditional logistic regression. Odds of BU were >2-fold for persons with diabetes mellitus and persons working outdoors who had soil contact in BU-endemic areas (compared with indoor work) but were lower among persons who had bacillus Calmette-Guérin vaccinations. BU was associated with increasing numbers of possums and with ponds and bore water use at residences. Using insect repellent, covering arms and legs outdoors, and immediately washing wounds were protective; undertaking multiple protective behaviors was associated with the lowest odds of BU. Skin hygiene/protection behaviors and previous bacillus Calmette-Guérin vaccination might provide protection against BU in BU-endemic areas.

Novel Hendra Virus Variant Circulating in Black Flying Foxes and Grey-Headed Flying Foxes, Australia.

A novel Hendra virus variant, genotype 2, was recently discovered in a horse that died after acute illness and in Pteropus flying fox tissues in Australia. We detected the variant in flying fox urine, the pathway relevant for spillover, supporting an expanded geographic range of Hendra virus risk to horses and humans.

Ecological dynamics of emerging bat virus spillover.

Viruses that originate in bats may be the most notorious emerging zoonoses that spill over from wildlife into domestic animals and humans. Understanding how these infections filter through ecological systems to cause disease in humans is of profound importance to public health. Transmission of viruses from bats to humans requires a hierarchy of enabling conditions that connect the distribution of reservoir hosts, viral infection within these hosts, and exposure and susceptibility of recipient hosts. For many emerging bat viruses, spillover also requires viral shedding from bats, and survival of the virus in the environment. Focusing on Hendra virus, but also addressing Nipah virus, Ebola virus, Marburg virus and coronaviruses, we delineate this cross-species spillover dynamic from the within-host processes that drive virus excretion to land-use changes that increase interaction among species. We describe how land-use changes may affect co-occurrence and contact between bats and recipient hosts. Two hypotheses may explain temporal and spatial pulses of virus shedding in bat populations: episodic shedding from persistently infected bats or transient epidemics that occur as virus is transmitted among bat populations. Management of livestock also may affect the probability of exposure and disease. Interventions to decrease the probability of virus spillover can be implemented at multiple levels from targeting the reservoir host to managing recipient host exposure and susceptibility.

Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5.

BACKGROUND: Australian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined. METHODS: ABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol. RESULTS: Here we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry. CONCLUSIONS: The predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.

First Detection of Benign Rabbit Caliciviruses in Chile.

Pathogenic lagoviruses (Rabbit hemorrhagic disease virus, RHDV) are widely spread across the world and are used in Australia and New Zealand to control populations of feral European rabbits. The spread of the non-pathogenic lagoviruses, e.g., rabbit calicivirus (RCV), is less well studied as the infection results in no clinical signs. Nonetheless, RCV has important implications for the spread of RHDV and rabbit biocontrol as it can provide varying levels of cross-protection against fatal infection with pathogenic lagoviruses. In Chile, where European rabbits are also an introduced species, myxoma virus was used for localised biocontrol of rabbits in the 1950s. To date, there have been no studies investigating the presence of lagoviruses in the Chilean feral rabbit population. In this study, liver and duodenum rabbit samples from central Chile were tested for the presence of lagoviruses and positive samples were subject to whole RNA sequencing and subsequent data analysis. Phylogenetic analysis revealed a novel RCV variant in duodenal samples that likely originated from European RCVs. Sequencing analysis also detected the presence of a rabbit astrovirus in one of the lagovirus-positive samples.

mAb therapy controls CNS-resident lyssavirus infection via a CD4 T cell-dependent mechanism.

Infections with rabies virus (RABV) and related lyssaviruses are uniformly fatal once virus accesses the central nervous system (CNS) and causes disease signs. Current immunotherapies are thus focused on the early, pre-symptomatic stage of disease, with the goal of peripheral neutralization of virus to prevent CNS infection. Here, we evaluated the therapeutic efficacy of F11, an anti-lyssavirus human monoclonal antibody (mAb), on established lyssavirus infections. We show that a single dose of F11 limits viral load in the brain and reverses disease signs following infection with a lethal dose of lyssavirus, even when administered after initiation of robust virus replication in the CNS. Importantly, we found that F11-dependent neutralization is not sufficient to protect animals from mortality, and a CD4 T cell-dependent adaptive immune response is required for successful control of infection. F11 significantly changes the spectrum of leukocyte populations in the brain, and the FcRγ-binding function of F11 contributes to therapeutic efficacy. Thus, mAb therapy can drive potent neutralization-independent T cell-mediated effects, even against an established CNS infection by a lethal neurotropic virus.

Utilizing Molecular Epidemiology and Citizen Science for the Surveillance of Lagoviruses in Australia.

Australia has multiple lagoviruses with differing pathogenicity. The circulation of these viruses was traditionally determined through opportunistic sampling events. In the lead up to the nationwide release of RHDVa-K5 (GI.1aP-GI.1a) in 2017, an existing citizen science program, RabbitScan, was augmented to allow members of the public to submit samples collected from dead leporids for lagovirus testing. This study describes the information obtained from the increased number of leporid samples received between 2015 and 2022 and focuses on the recent epidemiological interactions and evolutionary trajectory of circulating lagoviruses in Australia between October 2020 and December 2022. A total of 2771 samples were tested from January 2015 to December 2022, of which 1643 were lagovirus-positive. Notable changes in the distribution of lagovirus variants were observed, predominantly in Western Australia, where RHDV2-4c (GI.4cP-GI.2) was detected again in 2021 after initially being reported to be present in 2018. Interestingly, we found evidence that the deliberately released RHDVa-K5 was able to establish and circulate in wild rabbit populations in WA. Overall, the incorporation of citizen science approaches proved to be a cost-efficient method to increase the sampling area and enable an in-depth analysis of lagovirus distribution, genetic diversity, and interactions. The maintenance of such programs is essential to enable continued investigations of the critical parameters affecting the biocontrol of feral rabbit populations in Australia, as well as to enable the detection of any potential future incursions.

Cross-protection, infection and case fatality rates in wild European rabbits experimentally challenged with different rabbit haemorrhagic disease viruses.

Rabbit haemorrhagic disease virus 2 (RHDV2) is now the dominant calicivirus circulating in wild rabbit populations in Australia. This study compared the infection and case fatality rates of RHDV2 and two RHDVs in wild rabbits, as well as their ability to overcome immunity to the respective other strains. Wild rabbits were allocated to groups either blindly or based on pre-screening for RHDV/RHDV2 antibodies at capture. Rabbits were monitored regularly until their death or humane killing at 7 days post infection. Liver and eyeball samples were collected for lagovirus testing and aging rabbits, respectively. At capture, rabbits showed high seroprevalence to RHDV2 but not to RHDV. In RHDV/RHDV2 seronegative rabbits at capture, infection rates were highest in those inoculated with RHDV2 (81.8%, 18 out of 22), followed by K5 (53.8%, seven out of 13) and CZECH (40.0%, two out of five), but these differences were not statistically significant. In rabbits with previous exposure to RHDV2 at capture, infection rates were highest when inoculated with K5 (59.6%, 31 out of 52) followed by CZECH (46.0%, 23 out of 50), with infection rates higher in younger rabbits for both viruses. In RHDV/RHDV2 seronegative rabbits at capture, case fatality rates were highest for those inoculated with K5 (71.4%), followed by RHDV2 (50.0%) and CZECH (50.0%). In rabbits with previous exposure to RHDV2 at capture, case fatality rates were highest in rabbits inoculated with K5 (12.9%) followed by CZECH (8.7%), with no case fatalities following RHDV2 inoculation. Case fatality rates did not differ significantly between inoculums in either serostatus group at capture. Based on multivariable modelling, time to death post RHDV inoculation increased in rabbits with recent RHDV2 exposure compared with seronegative rabbits and with age. The results suggest that RHDV2 may cause higher mortalities than other variants in seronegative rabbit populations but that K5 may be more effective in reducing rabbit populations in an RHDV2-dominant landscape.

Co-circulation of alpha- and beta-coronaviruses in Pteropus vampyrus flying foxes from Indonesia.

Bats are important reservoirs for alpha- and beta-coronaviruses. Coronaviruses (CoV) have been detected in pteropodid bats from several Southeast Asian countries, but little is known about coronaviruses in the Indonesian archipelago in proportion to its mammalian biodiversity. In this study, we screened pooled faecal samples from the Indonesian colonies of Pteropus vampyrus with unbiased next-generation sequencing. Bat CoVs related to Rousettus leschenaultii CoV HKU9 and Eidolon helvum CoV were detected. The 121 faecal samples were further screened using a conventional hemi-nested pan-coronavirus PCR assay. Three positive samples were successfully sequenced, and phylogenetic reconstruction revealed the presence of alpha- and beta-coronaviruses. CoVs belonging to the subgenera Nobecovirus, Decacovirus and Pedacovirus were detected in a single P. vampyrus roost. This study expands current knowledge of coronavirus diversity in Indonesian flying foxes, highlighting the need for longitudinal surveillance of colonies as continuing urbanization and deforestation heighten the risk of spillover events.

Isolation and Characterization of Cross-Reactive Human Monoclonal Antibodies That Potently Neutralize Australian Bat Lyssavirus Variants and Other Phylogroup 1 Lyssaviruses.

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, administration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.39 and 6.25 µg/mL and 0.19 and 0.39 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, effectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest that A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections.

Erratum: Johnson et al. Alston Virus, a Novel Paramyxovirus Isolated from Bats Causes Upper Respiratory Tract Infection in Experimentally Challenged Ferrets. Viruses 2018, 10, 675.

The authors wish to make the following corrections to this paper [...].

Establishment of a longitudinal pre-clinical model of lyssavirus infection.

Traditional mouse models of lyssavirus pathogenesis rely on euthanizing large groups of animals at various time points post-infection, processing infected tissues, and performing histological and molecular analyses to determine anatomical sites of infection. While powerful by some measures, this approach is limited by the inability to monitor disease progression in the same mice over time. In this study, we established a novel non-invasive mouse model of lyssavirus pathogenesis, which consists of longitudinal imaging of a luciferase-expressing Australian bat lyssavirus (ABLV) reporter virus. In vivo bioluminescence imaging (BLI) in mice revealed viral spread from a peripheral site of inoculation into the central nervous system (CNS), with kinetically and spatially distinct foci of replication in the footpad, spinal cord, and hindbrain. Detection of virus within the CNS was associated with onset of clinical disease. Quantification of virus-derived luminescent signal in the brain was found to be a reliable measure of viral replication, when compared to traditional molecular methods. Furthermore, we demonstrate that in vivo imaging of ABLV infection is not restricted to the use of albino strains of mice, but rather strong BLI signal output can be achieved by shaving the hair from the heads and spines of pigmented strains, such as C57BL/6. Overall, our data show that in vivo BLI can be used to rapidly and non-invasively identify sites of lyssavirus replication and to semi-quantitatively determine viral load without the need to sacrifice mice at multiple time points.

Safety, tolerability, pharmacokinetics, and immunogenicity of a human monoclonal antibody targeting the G glycoprotein of henipaviruses in healthy adults: a first-in-human, randomised, controlled, phase 1 study.

BACKGROUND: The monoclonal antibody m102.4 is a potent, fully human antibody that neutralises Hendra and Nipah viruses in vitro and in vivo. We aimed to investigate the safety, tolerability, pharmacokinetics, and immunogenicity of m102.4 in healthy adults. METHODS: In this double-blind, placebo-controlled, single-centre, dose-escalation, phase 1 trial of m102.4, we randomly assigned healthy adults aged 18-50 years with a body-mass index of 18·0-35·0 kg/m2 to one of five cohorts. A sentinel pair for each cohort was randomly assigned to either m102.4 or placebo. The remaining participants in each cohort were randomly assigned (5:1) to receive m102.4 or placebo. Cohorts 1-4 received a single intravenous infusion of m102.4 at doses of 1 mg/kg (cohort 1), 3 mg/kg (cohort 2), 10 mg/kg (cohort 3), and 20 mg/kg (cohort 4), and were monitored for 113 days. Cohort 5 received two infusions of 20 mg/kg 72 h apart and were monitored for 123 days. The primary outcomes were safety and tolerability. Secondary outcomes were pharmacokinetics and immunogenicity. Analyses were completed according to protocol. The study was registered on the Australian New Zealand Clinical Trials Registry, ACTRN12615000395538. FINDINGS: Between March 27, 2015, and June 16, 2016, 40 (52%) of 77 healthy screened adults were enrolled in the study. Eight participants were assigned to each cohort (six received m102.4 and two received placebo). 86 treatment-emergent adverse events were reported, with similar rates between placebo and treatment groups. The most common treatment-related event was headache (12 [40%] of 30 participants in the combined m102.4 group, and three [30%] of ten participants in the pooled placebo group). No deaths or severe adverse events leading to study discontinuation occurred. Pharmacokinetics based on those receiving m102.4 (n=30) were linear, with a median half-life of 663·3 h (range 474·3-735·1) for cohort 1, 466·3 h (382·8-522·3) for cohort 2, 397·0 h (333·9-491·8) for cohort 3, and 466·7 h (351·0-889·6) for cohort 4. The elimination kinetics of those receiving repeated dosing (cohort 5) were similar to those of single-dose recipients (median elimination half-time 472·0 [385·6-592·0]). Anti-m102.4 antibodies were not detected at any time-point during the study. INTERPRETATION: Single and repeated dosing of m102.4 were well tolerated and safe, displayed linear pharmacokinetics, and showed no evidence of an immunogenic response. This study will inform future dosing regimens for m102.4 to achieve prolonged exposure for systemic efficacy to prevent and treat henipavirus infections. FUNDING: Queensland Department of Health, the National Health and Medical Research Council, and the National Hendra Virus Research Program.

Enhanced Autophagy Contributes to Reduced Viral Infection in Black Flying Fox Cells.

Bats are increasingly implicated as hosts of highly pathogenic viruses. The underlying virus⁻host interactions and cellular mechanisms that promote co-existence remain ill-defined, but physiological traits such as flight and longevity are proposed to drive these adaptations. Autophagy is a cellular homeostatic process that regulates ageing, metabolism, and intrinsic immune defense. We quantified basal and stimulated autophagic responses in black flying fox cells, and demonstrated that although black flying fox cells are susceptible to Australian bat lyssavirus (ABLV) infection, viral replication is dampened in these bat cells. Black flying fox cells tolerated prolonged ABLV infection with less cell death relative to comparable human cells, suggesting post-entry mechanisms interference with virus replication. An elevated basal autophagic level was observed and autophagy was induced in response to high virus doses. Pharmacological stimulation of the autophagy pathway reduced virus replication, indicating autophagy acts as an anti-viral mechanism. Enhancement of basal and virus-induced autophagy in bat cells connects related reports that long-lived species possess homeostatic processes that dampen oxidative stress and macromolecule damage. Exemplifying the potential that evolved cellular homeostatic adaptations like autophagy may secondarily act as anti-viral mechanisms, enabling bats to serve as natural hosts to an assortment of pathogenic viruses. Furthermore, our data suggest autophagy-inducing drugs may provide a novel therapeutic strategy for combating lyssavirus infection.

Operational trials of remote mosquito trap systems for Japanese encephalitis virus surveillance in the Torres Strait, Australia.

Japanese encephalitis virus (JEV) appears nearly annually in the Torres Strait in far northern Queensland, Australia, and is a threat to invade the Australian mainland. Surveillance has involved the use of sentinel pigs that develop detectable viremias and antibody titers to JEV. However, pigs are amplifying hosts for JEV, and thus pose a health risk to the public and to pig handlers who bleed the pigs. A remote mosquito trap system would not have these risks. We report on trials using a remote mosquito trap system for the surveillance of JEV in the Torres Strait. The Mosquito Magnet (MM) Pro, MM Liberty Plus, and a novel updraft trap, the NAQS Mozzie Trap, were run at Badu and Moa islands in the Torres Strait and at Bamaga in the northern Cape York Peninsula from 2002-2005. TaqMan real-time polymerase chain reaction (PCR) was used to detect JEV nucleic acid in weekly mosquito collections. Sentinel pigs located at Badu were also bled and the serum processed by reverse transcriptase (RT)-PCR for JEV antigen and enzyme-linked immunosorbent assay (ELISA) for anti-JEV antibodies. JEV was detected in mosquito collections each year but not in each trap. No JEV was detected in trapped mosquitoes before detection in sentinel pigs. The mosquito trap system cost ca. AU$10,000 per site, about AU$5,000 less than a pig-based system. However, trap failures caused by mosquito-clogged motors, electrical faults, and blocked gas lines reduced the efficacy of some mosquito traps. Nonetheless, a remote mosquito trap system, employing stand alone traps and PCR for viral antigen detection, can be a safe, economical way to detect arbovirus activity in remote areas.

Development and evaluation of real-time polymerase chain reaction assays to identify mosquito (Diptera: Culicidae) bloodmeals originating from native Australian mammals.

Real-time TaqMan polymerase chain reaction (PCR) assays were developed for the identification of mosquito (Diptera: Culicidae) bloodmeals originating from three groups of native Australian mammals. Primers and probes were designed to amplify a partial fragment of the cytochrome b gene of the agile wallaby, Macropus agilis (Gould); brushtail possum, Trichosurus vulpecula (Kerr); and the consensus sequence of the four species of Australian flying fox: Pteropus alecto Temminck, Pteropus conspicillatus Gould, Pteropus poliocephalus Temminck, and Pteropus scapulatus Peters. When tested on DNA extracted from whole tissue, each assay was shown to be specific for the vertebrate host that it was designed to identify. To evaluate the TaqMan assays, 137 field-collected blood-fed mosquitoes were analyzed, from which 128 (93.4%) were identified using one of the assays. Compared with other PCR-based techniques for bloodmeal identification, the TaqMan assays are sensitive, specific, and provide a rapid result without the need for post-PCR manipulation and visualization of products.

Short report: the first isolation of Japanese encephalitis virus from mosquitoes collected from mainland Australia.

In response to an incursion of Japanese encephalitis virus (JEV) on Cape York Peninsula, Australia, in 2005, 23,144 Culex mosquitoes were processed for virus detection. A single isolate of JEV was obtained from a pool of Culex sitiens subgroup mosquitoes. This is the first reported mosquito isolate of JEV from the Australian mainland.

Detection of Australian bat lyssavirus using a fluorogenic probe.

BACKGROUND: Australian bat lyssavirus (ABLV) has been transmitted to humans following a scratch or bite from an infected bat in two cases. Following a scratch or bite to a person, the bat is usually submitted for testing and diagnosis is made using a direct fluorescent antibody test on a brain smear. A nested RT-PCR assay has also been utilised to confirm diagnosis. If positive for lyssavirus, post-exposure prophylaxis is administered. OBJECTIVES: The TaqMan assay was developed to improve the diagnosis of ABLV infection, following problems encountered with the generation of spurious PCR products in the nested RT-PCR and also to reduce the high risk of contamination inherent with nested PCRs. STUDY DESIGN: RNA was extracted from 161 bat brains and the samples were compared using a conventional RT-PCR and the TaqMan based assay. Samples from a patient with an ABLV infection collected antemortem and postmortem were also tested. RESULTS: The sensitivity of the new TaqMan based PCR assay compared favourably with the nested PCR previously in use in our laboratory. This assay was able to detect RNA in samples collected antemortem and postmortem for the diagnosis of a human case of ABLV. CONCLUSIONS: The major advantage of the TaqMan based assay was the speed of diagnosis with a result within minutes of completing the PCR (a result within 4 h of receiving the specimen). This test greatly reduces the chance of false positives through the elimination of second-round PCR and the requirement for agarose gels. The assay is sensitive and specific and should be invaluable for future antemortem and postmortem diagnosis of ABLV infection in humans.

Detection of Australasian Flavivirus encephalitic viruses using rapid fluorogenic TaqMan RT-PCR assays.

The development of single, sensitive, fluorogenic reverse transcriptase-polymerase chain reaction (TaqMan) assays were required for the rapid and specific detection of three encephalitic viruses found in the Australasian region, namely; Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), and Kunjin virus (KUNV). Primers and a fluorogenic probe were individually designed to be complementary to a nucleotide region encompassing the 3' terminus of the nonstructural (NS) 5 gene and a portion of the 3' untranslated region (NS5-3'UTR) of each of the viral genomes respectively. Synthetically produced primer and probe controls were developed to minimize the likelihood of contamination and generation of false positives. Viral RNA from singly infected mosquitoes could be detected in pools of 1000 mosquitoes and positive mosquito pools collected from the field have been identified using each assay, indicating a high level of sensitivity and suitability for use in mosquito surveillance programs. In addition, the JEV TaqMan assay has been used to detect successfully viral RNA in sentinel pig serum samples. These assays potentially offer superior and timely detection of encephalitic viruses from surveillance samples, which is essential for the rapid implementation of vector control measures and continued monitoring of virus activity in the Australasian region.

A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp.

BACKGROUND: Leptospirosis is an emerging infectious disease. 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. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. 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. METHODS: The polymerase chain reaction (PCR) has been used to detect a large number of microorganisms, including those of clinical significance. 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. We employed real-time (quantitative) PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. RESULTS AND CONCLUSIONS: The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.