RESUMO
A Taqman assay for the detection of pathogenic Leptospira was modified to suit the LightCycler instrument. The modified assay was found to have an analytical sensitivity of 10 copies/reaction. The assay was then compared to the current gold standard for acute phase detection, culture, and to a commercially available Leptospira-specific IgM enzyme-linked immunosorbent assay (ELISA). Of the 236 samples including serum and ethylenediaminetetraacetic acid anticoagulated blood sample submitted for testing, polymerase chain reaction (PCR) was able to detect Leptospira DNA in 27 of the 28 culture positives and in 1 negative culture. Discrepant results were resolved by using the microscopic agglutination test on convalescent sera. The PCR was found to have a clinical specificity and sensitivity of 99.5% and 96.4%, respectively, when compared to the culture. Comparisons between culture and ELISA showed that the ELISA lack sensitivity (4.2%) and positive predictability (3.6%) for the detection of acute phase Leptospira infections. These results show that the modified LightCycler Taqman assay could be used as a replacement of culture for the detection of pathogenic Leptospira in a clinical setting.
Assuntos
Meios de Cultura , Imunoglobulina M/sangue , Leptospira/isolamento & purificação , Leptospirose/diagnóstico , Reação em Cadeia da Polimerase/métodos , Taq Polimerase , Anticorpos Antibacterianos/sangue , Técnicas Bacteriológicas , DNA Bacteriano/sangue , DNA Bacteriano/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Humanos , Leptospira/classificação , Leptospira/genética , Leptospira/imunologia , Leptospirose/microbiologia , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
The common brushtail possum (Trichosurus vulpecula) is indeed a common marsupial in major cities of Australia. This species is known to be susceptible to leptospirosis and often lives in close contact with humans, raising concerns about the potential for transmission of this disease in urban areas. A total of 192 brushtail possum blood samples were collected from 136 individuals in suburban areas of metropolitan Sydney from November 2002 to November 2004. Sera were screened against a reference panel of 21 Leptospira spp. using the microscopic agglutination test. Leptospiral antibodies were detected in 9.6% (13/136) of tested brushtail possums and represented two serovars; antibodies to Leptospira interrogans serovar Hardjo were most frequently identified (11/136). A representative of the exotic sero-group Ballum, most likely serovar Arborea, was found in two of 136 brushtail possums. Exposure to leptospirosis seemed to be associated with age, as older animals had a higher incidence, but there was no distinction in relation to gender. Antibody prevalence varied between the different sampling sites and seropositive animals were clustered and restricted to a few sites. These data support the possible role of brushtail possums as a maintenance host for Leptospira spp. in urban environments and also identified them as a previously unknown and potential source of serovar Arborea.
Assuntos
Anticorpos Antibacterianos/sangue , Leptospira/imunologia , Leptospirose/veterinária , Trichosurus/microbiologia , Animais , Austrália/epidemiologia , Transmissão de Doença Infecciosa/veterinária , Feminino , Humanos , Leptospirose/sangue , Leptospirose/epidemiologia , Leptospirose/transmissão , Masculino , Fatores de Risco , Estudos Soroepidemiológicos , ZoonosesRESUMO
BACKGROUND: Leptospira is the causative genus of the disease, leptospirosis. Species identification of pathogenic Leptospira in the past was generally performed by either DNA-DNA hybridisation or 16s rRNA gene sequencing. Both methods have inherent disadvantages such as the need for radio-labelled isotopes or significant homology between species. A conventional and real-time PCR amplification and sequencing method was developed for an alternate gene target: DNA gyrase subunit B (gyrB). Phylogenetic comparisons were undertaken between pathogenic Leptospira 16srRNA and gyrB genes using clustering and minimum evolution analysis. In addition 50 unidentified Leptospira isolates were characterised by gyrB sequencing and compared with conventional 16s rRNA sequencing. RESULTS: A conventional and real-time PCR methodology was developed and optimised for the amplification of the gyrB from pathogenic Leptospira species. Non pathogenic and opportunistic Leptospira species such as L. fainei and L. broomi were not amplified. The gyrB gene shows greater nucleotide divergence (3.5% to 16.1%) than the 16s rRNA gene (0.1% to 1.4%). Minimum evolution analysis reveals that the gyrB has a different evolution topology for L. kirschneri and L. interrogans. When the two genes were compared for the identification of the 50 unknown isolates there was 100% agreement in the results. CONCLUSION: This research has successfully developed a methodology for the identification of pathogenic Leptospira using an alternate gene to 16s rRNA. The gyrB encoding gene shows higher nucleotide/evolutionary divergence allowing for superior identification and also the potential for the development of DNA probe based identification.
Assuntos
DNA Girase/genética , Leptospira/isolamento & purificação , Leptospira/patogenicidade , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Leptospira/enzimologia , Leptospira/genética , FilogeniaRESUMO
In this study, an improved multiple-locus variable number of tandem repeats analysis (MLVA) method based upon a previously published method is described. Improvements to the method included redesigned primers and PCR conditions, combined with pooled capillary electrophoresis using multicolored dyes. Allele sizes were converted into an allele string, and each unique allele string was assigned a numerical MLVA type (MVT). The improved MLVA method was then applied to 96 previously characterized Leptospira interrogans serovar Australis isolates from human and animal sources. The improved MLVA was found to have between six and 13 alleles at each locus, compared with three to eight in the original. The mean Hunter-Gaston diversity index (HGDI) for the improved MLVA method was 0.654, compared with 0.599 in the original; this increase in diversity was largely due to changes in the analysis of the variable number of tandem repeat (VNTR) data. When the improved MLVA method was compared with the fluorescent amplified fragment length polymorphism (FAFLP) method, there was a high level of concordance between the profiles; however, the MLVA method produced an additional four unique profiles amongst the subset of 30 isolates tested. Given that the improved MLVA method was found to be superior to the original MLVA method, it was subsequently used to redefine the molecular epidemiology of L. interrogans serovar Australis in Queensland, Australia. Using cluster analysis, the authors were able to demonstrate clonal links amongst rodent isolates, rodent and human isolates, and rodent and canine isolates. These results highlight the role of rodents in the disease, and also the potential role of MLVA in defining the molecular epidemiology of L. interrogans.
Assuntos
Impressões Digitais de DNA/métodos , Leptospira interrogans serovar australis/genética , Leptospirose/microbiologia , Repetições Minissatélites , Epidemiologia Molecular , Animais , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese Capilar , Corantes Fluorescentes/química , Variação Genética , Humanos , Leptospira interrogans serovar australis/classificação , Leptospirose/epidemiologia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Queensland/epidemiologiaRESUMO
BACKGROUND: Leptospirosis is a zoonotic disease caused by the genus, Leptospira. Leptospira interrogans is the most common genomospecies implicated in the disease. Epidemiological investigations are needed to distinguish outbreak situations or to trace reservoirs of the organisms. Current methodologies used for typing Leptospira have significant drawbacks. The development of an easy to perform yet high resolution method is needed for this organism. METHODS: In this study we have searched the available genomic sequence of L. interrogans serovar Copenhageni strain Fiocruz L1-130 for the presence of tandem repeats. These repeats were evaluated against reference strains for diversity. Six loci were selected to create a Multiple Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) to explore the genetic diversity within L. interrogans serovar Australis clinical isolates from Far North Queensland. RESULTS: The 39 reference strains used for the development of the method displayed 39 distinct patterns. Diversity Indexes for the loci varied between 0.80 and 0.93 and the number of repeat units at each locus varied between less than one to 52 repeats. When the MLVA was applied to serovar Australis isolates three large clusters were distinguishable, each comprising various hosts including Rattus species, human and canines. CONCLUSION: The MLVA described in this report, was easy to perform, analyse and was reproducible. The loci selected had high diversity allowing discrimination between serovars and also between strains within a serovar. This method provides a starting point on which improvements to the method and comparisons to other techniques can be made.
Assuntos
Leptospira interrogans serovar australis/genética , Leptospira interrogans/genética , Repetições Minissatélites , Animais , Análise por Conglomerados , Marcadores Genéticos , HumanosRESUMO
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.
Assuntos
Leptospira/genética , Leptospira/patogenicidade , Reação em Cadeia da Polimerase/métodos , Coleta de Amostras Sanguíneas/efeitos adversos , Coleta de Amostras Sanguíneas/métodos , DNA Bacteriano/sangue , DNA Bacteriano/urina , Heparina/efeitos adversos , Heparina/análogos & derivados , Humanos , Leptospira/isolamento & purificação , Leptospirose/diagnóstico , Leptospirose/genética , Lítio/efeitos adversos , Reação em Cadeia da Polimerase/efeitos dos fármacos , Sensibilidade e Especificidade , Especificidade da Espécie , Taq PolimeraseRESUMO
Eastern grey kangaroos (Macropus giganteus) are one of the most abundant large macropodids sharing the landscape with humans. Despite this, little is known about the prevalence of Leptospira carriage within this species and the role that they may partake in the transmission of this disease in Australia. The sera of 87 free-ranging eastern grey kangaroos, captured in the Warragamba Catchment Area, Sydney, Australia, from June 2004 to November 2006, were screened against a reference panel of 22 Leptospira serovars using the microscopic agglutination test (MAT). Leptospiral antibodies were detected in 47% (41 of 87) of serum samples collected. Leptospira weilii Topaz, a newly emergent serovar in Australia, was detected in all seropositive kangaroos (41 of 41; 100%). The sex and tail-fat body condition index of kangaroos appeared to have no significant effect on the exposure to the disease. This serologic-based study is the first reported for L. weilii serovar Topaz in New South Wales, to our knowledge, having previously been isolated only in humans and two other animal species (bovine and long-nosed bandicoot [Perameles nasuta]) in Western Australia and Queensland. The potential role of eastern grey kangaroos in the maintenance and zoonotic spread of the disease to livestock and humans is discussed.
Assuntos
Anticorpos Antibacterianos/sangue , Leptospira/imunologia , Leptospirose/veterinária , Macropodidae/microbiologia , Animais , Animais Selvagens/microbiologia , Austrália/epidemiologia , Feminino , Leptospira/classificação , Leptospirose/epidemiologia , Leptospirose/transmissão , Masculino , Estudos Soroepidemiológicos , Sorotipagem/veterinária , ZoonosesRESUMO
Leptospira borgpetersenii serovar Arborea is an emerging cause of leptospirosis in Australia. It was not previously recognized as an endemic serovar before the 1990s, but at that point, human infections with the serovar increased significantly. Using fluorescent-amplified fragment-length polymorphism (FAFLP) molecular typing, human and rodent isolates were compared genetically. Typing revealed 11 unique profiles among the 23 isolates examined; however, there was no clonality revealed between the human and rodent isolates. There was clonality among rodent isolates from geographically related areas. This study highlights the utility of Leptospira culture combined with FAFLP for the examination of the epidemiology of this disease.
Assuntos
Leptospira/classificação , Leptospirose/epidemiologia , Animais , Análise por Conglomerados , Humanos , Leptospira/genética , Epidemiologia Molecular , Técnicas de Amplificação de Ácido Nucleico , Filogenia , Queensland/epidemiologia , Fatores de TempoRESUMO
It has been recognized that there is heterogeneity among Leptospira isolates in culture collections worldwide, causing confounding results for researchers utilizing these organisms; one such culture is Leptospira meyeri serovar Perameles. The serovar reference strain Bandicoot 343 was previously identified to the species level by DNA-DNA hybridization; however, subsequent published studies demonstrated results that contradicted the initial speciation. In this study, initial serological testing was performed with isolates from the culture collections of the Centers for Disease Control (CDC), Atlanta, USA (strain Lepto0214), and the WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis, Brisbane, Australia (strain Bandicoot 343), and the original serovar Perameles hyperimmune antiserum produced in 1964. The results indicated that strain Lepto0214 was not serologically reactive to the antiserum. However, further investigations revealed an alternative serovar Perameles strain held in the CDC collection (Lepto0213) that yielded titres against the antiserum. 16S rRNA gene sequencing of the three strains revealed that Lepto0214 had significant sequence similarity with previously sequenced L. meyeri strains; however, strains Lepto0213 and Bandicoot 343 had significant similarity with Leptospira interrogans strains. 16S rRNA gene sequencing results were confirmed by pulsed-field gel electrophoresis; Lepto0214 had a pattern similar to that of L. meyeri serovar Hardjo strain Went 5, and the pattern differed significantly from those of Lepto0213 and Bandicoot 343. This research provides evidence for the reclassification of serovar Perameles from L. meyeri to L. interrogans. This reclassification highlights a need for changes to how reference Leptospira serovars are identified, disseminated and stored, with the aim of reducing heterogeneity of reference strains between culture collections.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Leptospira interrogans/classificação , Leptospira/classificação , Leptospirose/microbiologia , Análise de Sequência de DNA , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/análise , Eletroforese em Gel de Campo Pulsado , Humanos , Leptospira/genética , Leptospira interrogans/genética , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Sorotipagem , Especificidade da EspécieRESUMO
A single Leptospira strain (designated Bejo-Iso9(T)) was isolated from a soil sample taken in Johor, Malaysia. The isolate showed motility and morphology typical of the genus Leptospira under dark-field microscopy. Cells were found to be 10-13 microm in length and 0.2 microm in diameter, with a wavelength of 0.5 microm and an amplitude of approximately 0.2 microm. Phenotypically, strain Bejo-Iso9(T) grew in Ellinghausen-McCullough-Johnson-Harris medium at 13, 30 and 37 degrees C, and also in the presence of 8-azaguanine. Serologically, strain Bejo-Iso9(T) produced titres towards several members of the Tarassovi serogroup, but was found to be serologically unique by cross-agglutinin absorption test and thus represented a novel serovar. The proposed name for this serovar is Malaysia. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the genus Leptospira, with sequence similarities within the range 90.4-99.5% with respect to recognized Leptospira species. DNA-DNA hybridization against the three most closely related Leptospira species was used to confirm the results of the 16S rRNA gene sequence analysis. The G+C content of the genome of strain Bejo-Iso9(T) was 36.2 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Bejo-Iso9(T) represents a novel species of the genus Leptospira, for which the name Leptospira kmetyi sp. nov. is proposed. The type strain is Bejo-Iso9(T) (=WHO LT1101(T)=KIT Bejo-Iso9(T)).
Assuntos
Leptospira/classificação , Leptospira/isolamento & purificação , Microbiologia do Solo , Composição de Bases , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Leptospira/genética , Leptospira/fisiologia , Locomoção , Malásia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
A prospective study in Thailand identified 106 patients with culture-proven leptospirosis. The accuracy of the microscopic agglutination test (MAT) in predicting the infecting serovar was evaluated in 78/106 (74%) patients with a diagnostic titer. MAT correctly determined the infecting serovar in 26 cases (33%), indicating that this assay is a poor predictor of infecting serovar in our setting.
Assuntos
Testes de Aglutinação/métodos , Leptospira/classificação , Leptospirose/diagnóstico , Leptospirose/microbiologia , Microscopia/métodos , Adolescente , Adulto , Idoso , Feminino , Humanos , Leptospirose/epidemiologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Tailândia/epidemiologia , Adulto JovemRESUMO
A single Leptospira strain (designated Khorat-H2(T)) was isolated from the urine of an adult male patient with suspected leptospirosis from the province of Nakornrachasima, Thailand. The isolate showed typical Leptospira motility and morphology under dark-field microscopy. Cells were 10-13 mum long and 0.2 mum in diameter, with a wavelength of 0.5 mum and an amplitude of approximately 0.3 mum. Phenotypically, strain Khorat-H2(T) did not grow at 13 degrees C but grew at 30 and 37 degrees C and in the presence of 8-azaguanine. Serological identification using the microscopic agglutination test revealed that strain Khorat-H2(T) had no cross-reaction with any recognized Leptospira serogroups. Phylogenetic analysis of the 16S rRNA gene sequence placed the novel strain within the radiation of the genus Leptospira, with sequence similarities of 88.1-97.7 % to recognized Leptospira species. DNA-DNA hybridization against the type strains of the three most closely related Leptospira species was used to confirm the results of the 16S rRNA sequence analysis. The G+C content of strain Khorat-H2(T) was 41.8 mol%. On the basis of phenotypic, serological and phylogenetic data, strain Khorat-H2(T) represents a novel species of the genus Leptospira, for which the name Leptospira wolffii sp. nov. is proposed. The type strain is Khorat-H2(T) (=WHO LT1686(T) =KIT Khorat-H2(T)).
Assuntos
Leptospira/classificação , Leptospira/genética , Leptospirose/microbiologia , Adulto , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Genes Bacterianos , Genes de RNAr , Humanos , Leptospira/isolamento & purificação , Leptospirose/urina , Masculino , Dados de Sequência Molecular , Fenótipo , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , TailândiaRESUMO
Leptospirosis is a zoonotic disease with a worldwide distribution. Leptospira weilii serovar (sv.) Topaz is a newly described serovar first isolated in the far north of Queensland, Australia. The epidemiology of L. weilii sv. Topaz infections in Australia was characterised through the use of surveillance questionnaires and molecular studies. There have been 24 human and 2 animal (bovine and bandicoot) L. weilii sv. Topaz infections diagnosed since 1991. The majority of these infections have occurred in Far North Queensland, with the remaining infections occurring in South East Queensland and in Western Australia. The majority of patients with L. weilii sv. Topaz infections presented with classical leptospirosis symptoms including; fever, headaches, sweats, chills and myalgia. The occupations of human cases of L. weilii sv. Topaz infection included banana farming, dairy and beef cattle production and tourist related activities. Fluorescent amplified fragment length polymorphism (FAFLP) was performed on 15 L. weilii sv. Topaz isolates including 2 animal isolates. Clustering analysis grouped the 15 isolates into 5 main clades with 13 unique FAFLP profiles. A high level of relatedness was demonstrated between 2 animal and 2 human isolates.
Assuntos
Leptospira/patogenicidade , Leptospirose/epidemiologia , Zoonoses/epidemiologia , Adolescente , Adulto , Animais , Austrália/epidemiologia , Feminino , Genótipo , Humanos , Leptospira/classificação , Leptospira/genética , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Filogenia , Vigilância de Evento Sentinela , SorotipagemRESUMO
We defined the positive predictive accuracy of a hospital-based clinical diagnosis of leptospirosis in 9 provinces across Thailand. Of 700 suspected cases, 143 (20%) were confirmed by laboratory testing. Accuracy of clinical diagnosis varied from 0% to 50% between the provinces and was highest during the rainy season. Most confirmed cases occurred in the north and northeast regions of the country.
Assuntos
Leptospirose/diagnóstico , Leptospirose/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estações do Ano , Tailândia/epidemiologiaRESUMO
A prospective study of 989 patients with acute febrile illness was performed in northeast Thailand to define the yield of Leptospira from four different types of blood sample. Based on a comparison of the yields from whole blood, surface plasma, deposit from spun plasma, and clotted blood samples from 80 patients with culture-proven leptospirosis, we suggest a sampling strategy in which culture is performed using whole blood and deposit from spun plasma.
Assuntos
Técnicas Bacteriológicas , Sangue/microbiologia , Leptospira/crescimento & desenvolvimento , Leptospira/isolamento & purificação , Leptospirose/microbiologia , Humanos , Sensibilidade e Especificidade , Manejo de Espécimes , TailândiaRESUMO
The diagnostic utility of immunochromatographic (Leptotek) and enzyme-linked immunosorbent assay (ELISA; Panbio) tests for the detection of Leptospira immunoglobulin M antibodies was assessed in febrile adults admitted in Vientiane, Laos. Both tests demonstrated poor diagnostic accuracy using admission serum (Leptotek sensitivity of 47.3% and specificity of 75.5%: ELISA sensitivity of 60.9% and specificity of 65.6%) compared to the Leptospira "gold standard" microscopic agglutination test.