Dataset Information

Molecular detection and speciation of pathogenic Leptospira spp. in blood from patients with culture-negative leptospirosis.

ABSTRACT: Pathogenic Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR. A proportion of patients who are positive by PCR are negative by culture. Leptospira spp. are fastidious bacteria, and we hypothesized that a false-negative culture result may represent infection with a distinct bacterial subset that fail to grow in standard culture medium.We evaluated our hypothesis during a prospective study of 418 consecutive patients presenting to a hospital in northeast Thailand with an acute febrile illness. Admission blood samples were taken for Leptospira culture and PCR. A single tube nested PCR that amplified a region of the rrs gene was developed and applied, amplicons sequenced and a phylogenetic tree reconstructed.39/418 (9%) patients were culture-positive for Leptospira spp., and 81/418 (19%) patients were culture-negative but rrs PCR-positive. The species associated with culture-positive leptospirosis (37 L. interrogans and 2 L. borgpetersenii) were comparable to those associated with culture-negative, PCR-positive leptospirosis (76 L. interrogans, 4 L. borgpetersenii, 1 unidentified, possibly new species).Molecular speciation failed to identify a unique bacterial subset in patients with culture-negative, PCR-positive leptospirosis. The rate of false-negative culture was high, and we speculate that antibiotic pre-treatment is the most likely explanation for this.

SUBMITTER: Boonsilp S

PROVIDER: S-EPMC3297668 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Molecular detection and speciation of pathogenic Leptospira spp. in blood from patients with culture-negative leptospirosis.

Boonsilp Siriphan S Thaipadungpanit Janjira J Amornchai Premjit P Wuthiekanun Vanaporn V Chierakul Wirongrong W Limmathurotsakul Direk D Day Nicholas P NP Peacock Sharon J SJ

BMC infectious diseases 20111213

<h4>Background</h4>Pathogenic Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR. A proportion of patients who are positive by PCR are negative by culture. Leptospira spp. are fastidious bacteria, and we hypothesized that a false-negative culture result may represent infection with a distinct bacterial subset that fail to grow in standard culture medium.<h4>Methods</h4>We evaluated our hypothesis during a pro ...[more]

PMID: 22151687

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Project description:BackgroundAlthough previous data indicate that the overall incidence of human leptospirosis in the Peruvian Amazon is similar in urban and rural sites, severe leptospirosis has been observed only in the urban context. As a potential explanation for this epidemiological observation, we tested the hypothesis that concentrations of more virulent Leptospira would be higher in urban than in rural environmental surface waters.Methods and findingsA quantitative real-time PCR assay was used to compare levels of Leptospira in urban and rural environmental surface waters in sites in the Peruvian Amazon region of Iquitos. Molecular taxonomic analysis of a 1,200-bp segment of the leptospiral 16S ribosomal RNA gene was used to identify Leptospira to the species level. Pathogenic Leptospira species were found only in urban slum water sources (Fisher's exact test; p = 0.013). The concentration of pathogen-related Leptospira was higher in urban than rural water sources (approximately 10(3) leptospires/ml versus 0.5 x 10(2) leptospires/ml; F = 8.406, p < 0.05). Identical 16S rRNA gene sequences from Leptospira interrogans serovar Icterohaemorrhagiae were found in urban slum market area gutter water and in human isolates, suggesting a specific mode of transmission from rats to humans. In a prospective, population-based study of patients presenting with acute febrile illness, isolation of L. interrogans-related leptospires from humans was significantly associated with urban acquisition (75% of urban isolates); human isolates of other leptospiral species were associated with rural acquisition (78% of rural isolates) (chi-square analysis; p < 0.01). This distribution of human leptospiral isolates mirrored the distribution of leptospiral 16S ribosomal gene sequences in urban and rural water sources.ConclusionsOur findings data support the hypothesis that urban severe leptospirosis in the Peruvian Amazon is associated with higher concentrations of more pathogenic leptospires at sites of exposure and transmission. This combined quantitative and molecular taxonomical risk assessment of environmental surface waters is globally applicable for assessing risk for leptospiral infection and severe disease in leptospirosis-endemic regions.

Project description:Shedding of DNA of pathogenic Leptospira spp. has been documented in naturally infected cats in several countries, but urinary shedding of infectious Leptospira spp. has only recently been proven. The climate in Southern Chile is temperate rainy with high annual precipitations which represents ideal preconditions for survival of Leptospira spp., especially during spring and summer. The aims of this study were to investigate shedding of pathogenic Leptospira spp. in outdoor cats in Southern Chile, to perform molecular characterization of isolates growing in culture, and to assess potential risk factors associated with shedding. Urine samples of 231 outdoor cats from rural and urban areas in southern Chile were collected. Urine samples were investigated for pathogenic Leptospira spp. by 4 techniques: qPCR targeting the lipL32 gene, immunomagnetic separation (IMS)-coupled qPCR (IMS-qPCR), direct culture and IMS-coupled culture. Positive urine cultures were additionally confirmed by PCR. Multilocus sequence typing (MLST) was used to molecularly characterize isolates obtained from positive cultures. Overall, 36 urine samples (15.6%, 95% confidence interval (CI) 11.4-20.9) showed positive results. Eighteen (7.8%, 95% CI 4.9-12.1), 30 (13%, 95% CI 9.2-18), 3 (1.3%, 0.3-3.9) and 4 cats (1.7%; 95% CI 0.5-4.5) were positive in qPCR, IMS-qPCR, conventional culture, and IMS-coupled culture, respectively. MLST results of 7 culture-positive cats revealed sequences that could be assigned to sequence type 17 (6 cats) and sequence type 27 (1 cat) corresponding to L. interrogans (Pathogenic Leptospira Subgroup 1). Shedding of pathogenic Leptospira spp. by cats might be an underestimated source of infection for other species including humans. The present study is the first one reporting growth of leptospires from feline urine in culture in naturally infected cats in South-America and characterisation of culture-derived isolates. So far, very few cases of successful attempts to culture leptospires from naturally infected cats are described worldwide.

Project description:The suitability of incubated blood culture material for forensic molecular malaria diagnosis was assessed for non-endemic settings for cases in which the differential diagnosis malaria was initially overlooked. For the proof-of-principle assessment, residual blood culture materials from febrile patients from tropical Ghana were investigated by real-time PCR and compared with available historic microscopic results. In 2114 samples, for which microscopical results and real-time PCR results were available, microscopical results comprised 711 P. falciparum detections, 7 P. malariae detections, 1 microscopically not-further-discriminable Plasmodium spp. detection as well as 13 detections of mixed infections comprising 12 cases of P. falciparum/P. malariae co-infections and 1 case of a P. falciparum/P. ovale complex co-infection, while real-PCR indicated 558 P. falciparum detections, 95 P. malariae detections, 10 P. ovale complex detections, 1 P. vivax detection and 4 detected P. falciparum/P. malariae co-infections. Concordance of routine microscopy and real-time PCR was imperfect. Using routine microscopy as reference was associated with a seemingly low agreement of positive real-time PCR results of 90.9%. However, if positive samples, either by routine microscopy or real-time PCR or both, were applied as a combined reference, the agreement of positive results obtained with real-time PCR was increased from 74.0% to 77.9%, while the agreement of positive results obtained with routine microscopy was decreased from 100% to 85.3%. The predictive value of routine microscopy for negative results in the reference was slightly better with 90.9% compared to real-time PCR with 86.9%; the concordance between routine microscopy and real-time PCR was imperfect. In conclusion, even suboptimal sample materials such as incubated blood culture materials can be applied for forensic malaria diagnosis, if more suitable sample materials are not available, but the molecular detection rate of positive results in routine microscopy is much lower than previously reported for non-incubated blood.

Dataset Information

Molecular detection and speciation of pathogenic Leptospira spp. in blood from patients with culture-negative leptospirosis.

Publications

Molecular detection and speciation of pathogenic Leptospira spp. in blood from patients with culture-negative leptospirosis.

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