Multiplex PCR-based detection of Leptospira in environmental water samples obtained from a slum settlement

Vital-Brazil, Juliana Magalhães; Balassiano, Ilana Teruszkin; Oliveira, Fabiano Sutter de; Costa, Alberto Dias de Souza; Hillen, Leandro; Pereira, Martha María

doi:10.1590/s0074-02762010000300020

Cited by 31 publications

(21 citation statements)

References 17 publications

(17 reference statements)

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“…While such assays may prove useful in other contexts, such as testing environmental samples [28], [36], [37], the results of this evaluation highlight concerns regarding such a testing approach for human specimens. Of the clinical samples that were successfully sequenced, 49/50 yielded sequence consistent with pathogenic Leptospira species.…”

Section: Discussionmentioning

confidence: 99%

Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

et al. 2014

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BackgroundBacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species.Methodology/Principal FindingsFor the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay.Conclusions/SignificanceThe pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.

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Section: Discussionmentioning

confidence: 99%

Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

et al. 2014

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“…and has been highly conserved amongst these species (Guerreiro et al 2001;Haake et al 2000;Stoddard et al 2009). The ampli fication of the LipL32 gene has proved to be a valuable tool for identifying pathogenic leptospires in water samples (Vital-Brazil et al 2010). Cheema et al (2007) used PCR based on LipL21 and LipL32 genes for detection of pathogenic Leptospira in the serum and tissue samples of cattle and buffaloes and found that both genes are useful for diagnosis of leptospirosis.…”

Section: Discussionmentioning

confidence: 99%

“…The amplified prod ucts were analysed by electrophoresis on ethidium bromide-stained 2% agarose gels and the results were observed using Ultraviolet (UV) light. A sample was considered positive when the 474 bp DNA band was obtained (Vital-Brazil et al 2010). …”

Section: Polymerase Chain Reactionmentioning

confidence: 99%

Evaluation of ‘white-spotted kidneys’ associated with leptospirosis by polymerase chain reaction based <i>LipL32</i> gene in slaughtered cows

Azizi

Tajbakhsh

Hajimirzaei

et al. 2012

J. S. Afr. Vet. Assoc.

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The presence of white spots in the kidneys of cattle at slaughter (so-called white-spotted kidneys) can be an indication of infection with Leptospira, a spirochaete of public health concern because it causes zoonotic disease. In this study, 24 kidneys of 180 slaughtered cows (13.3%) showed focal to multifocal white spots at inspection. These kidneys, together with matching urine (n = 18) and blood (n = 24) samples, were examined by polymerase chain reaction (PCR) targeting the LipL32 gene. Leptospiral deoxyribonucleic acid (DNA) was detected in 19 (79.2%) out of 24 kidneys, as well as 7 (29.2%) blood and 10 (55.5%) urine samples of cows with white spots in their kidneys. Histopathological findings revealed multifocal infiltration of mononuclear cells, including lymphocytes and a few plasma cells in the renal interstitial tissues. In addition, 14 apparently normal kidneys and associated urine and blood samples were similarly examined by PCR but did not provide any positive results. In this study, high detection of leptospirosis in kidneys with interstitial nephritis suggests that Leptospira spp. are associated with white spotted kidneys. The present findings indicate that white spotted kidneys can be due to leptospirosis in this region in southwestern Iran, which indicates an increased risk of zoonotic disease. The data show that LipL32-based primers are useful for PCR-based diagnosis of leptospirosis.

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“…PCR has been successfully applied as a rapid, sensitive, and specific tool for the detection of several microorganisms, including Leptospira, 11 in a variety of specimens from different hosts. [12][13][14] The rapidity and reproducibility of pulsed-field gel electrophoresis (PFGE) makes it a very useful technique for typing Leptospira strains. 15 PFGE is able to discriminate between closely related serovars of the Leptospira spp., which may aid in the identification of the different strains at serovar level, 16 and this identification is important in understanding the epidemiology of leptospirosis in both humans and animals in any geographic region.…”

Section: Introductionmentioning

confidence: 99%

Isolation and Molecular Characterization of Leptospira interrogans and Leptospira borgpetersenii Isolates from the Urban Rat Populations of Kuala Lumpur, Malaysia

Benacer

Zain²,

Amran³

et al. 2013

The American Journal of Tropical Medicine and Hygiene

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Abstract. Rats are considered the principal maintenance hosts of Leptospira. The objectives of this study were isolation and identification of Leptospira serovars circulating among urban rat populations in Kuala Lumpur. Three hundred urban rats (73% Rattus rattus and 27% R. norvegicus) from three different sites were trapped. Twenty cultures were positive for Leptospira using dark-field microscopy. R. rattus was the dominant carrier (70%). Polymerase chain reaction (PCR) confirmed that all isolates were pathogenic Leptospira species. Two Leptospira serogroups, Javanica and Bataviae, were identified using microscopic agglutination test (MAT). Pulsed-field gel electrophoresis (PFGE) identified two serovars in the urban rat populations: L. borgpetersenii serovar Javanica (85%) and L. interrogans serovar Bataviae (15%). We conclude that these two serovars are the major serovars circulating among the urban rat populations in Kuala Lumpur. Despite the low infection rate reported, the high pathogenicity of these serovars raises concern of public health risks caused by rodent transmission of leptospirosis.

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Multiplex PCR-based detection of Leptospira in environmental water samples obtained from a slum settlement

Abstract: Detection of Leptospira in water • Juliana Magalhães Vital-Brazil et al.

Cited by 31 publications

References 17 publications

Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

Evaluation of ‘white-spotted kidneys’ associated with leptospirosis by polymerase chain reaction based <i>LipL32</i> gene in slaughtered cows

Isolation and Molecular Characterization of Leptospira interrogans and Leptospira borgpetersenii Isolates from the Urban Rat Populations of Kuala Lumpur, Malaysia

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