Rapid isolation and susceptibility testing of Leptospira spp. using a new solid medium, LVW agar.

Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO(2) incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO(2) for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC(90) values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 μg/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research

Diagnostic accuracy of real-time PCR assays targeting 16S rRNA and lipL32 genes for human leptospirosis in Thailand: a case-control study.

BACKGROUND: Rapid PCR-based tests for the diagnosis of leptospirosis can provide information that contributes towards early patient management, but these have not been adopted in Thailand. Here, we compare the diagnostic sensitivity and specificity of two real-time PCR assays targeting rrs or lipL32 for the diagnosis of leptospirosis in northeast Thailand. METHODS/PRINCIPAL FINDINGS: A case-control study of 266 patients (133 cases of leptospirosis and 133 controls) was constructed to evaluate the diagnostic sensitivity and specificity (DSe and DSp) of both PCR assays. The median duration of illness prior to admission of cases was 4 days (IQR 2-5 days; range 1-12 days). DSe and DSp were determined using positive culture and/or microscopic agglutination test (MAT) as the gold standard. The DSe was higher for the rrs assay than the lipL32 assay (56%, (95% CI 47-64%) versus 43%, (95% CI 34-52%), p<0.001). No cases were positive for the lipL32 assay alone. There was borderline evidence to suggest that the DSp of the rrs assay was lower than the lipL32 assay (90% (95% CI 83-94%) versus 93%, (95%CI 88-97%), p = 0.06). Nine controls gave positive reactions for both assays and 5 controls gave a positive reaction for the rrs assay alone. The DSe of the rrs and lipL32 assays were high in the subgroup of 39 patients who were culture positive for Leptospira spp. (95% and 87%, respectively, p = 0.25). CONCLUSIONS/SIGNIFICANCE: Early detection of Leptospira using PCR is possible for more than half of patients presenting with leptospirosis and could contribute to individual patient care

<i>Leptospira</i> spp. used during this study.

@<p>WHO/FOA/OIE/ Collaborating Center for Reference and Research on Leptospirosis, Australia;</p><p>*Bureau of Emerging Infection Disease, Ministry of Public Health, Thailand;</p>#<p>American Type Culture Collection, USA;</p>$<p>Royal Tropical Institute (KIT), Netherland;</p>a<p>Dr Thareerat Kalambaheti, Mahidol University, Thailand.</p><p>+ and − indicate a positive or negative result in the relevant PCR assay, respectively. (+) indicates a positive PCR result at a high Cq.</p

Diagnostic sensitivity and specificity of real-time PCR assays targeting <i>rrs</i> or <i>lipL32.</i>

<p>Diagnostic sensitivity and specificity of real-time PCR assays targeting <i>rrs</i> or <i>lipL32.</i></p

Study design and patient recruitment.

<p>Study design and patient recruitment.</p