Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene.

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.

Analysis of Bordetella pertussis pertactin and pertussis toxin types from Queensland, Australia, 1999-2003.

BACKGROUND: In Australia two acellular Bordetella pertussis vaccines have replaced the use of a whole cell vaccine. Both of the licensed acellular vaccines contain the following three components; pertussis toxoid, pertussis filamentous haemagglutinin and the 69 kDa pertactin adhesin. One vaccine also contains pertussis fimbriae 2 and 3. Various researchers have postulated that herd immunity due to high levels of pertussis vaccination might be influencing the makeup of endemic B. pertussis populations by selective pressure for strains possessing variants of these genes, in particular the pertactin gene type. Some publications have suggested that B. pertussis variants may be contributing to a reduced efficacy of the existing vaccines and a concomitant re-emergence of pertussis within vaccinated populations. This study was conducted to survey the pertactin and pertussis toxin subunit 1 types from B. pertussis isolates in Queensland, Australia following the introduction of acellular vaccines. METHODS: Forty-six B. pertussis isolates recovered from Queensland patients between 1999 and 2003 were examined by both DNA sequencing and LightCycler real time PCR to determine their pertactin and pertussis toxin subunit 1 genotypes. RESULTS: Pertactin typing showed that 38 isolates possessed the prn1 allele, 3 possessed the prn2 allele and 5 possessed the prn3 allele. All forty-six isolates possessed the pertussis toxin ptxS1A genotype. Amongst the circulating B. pertussis population in Queensland, 82.5% of the recovered clinical isolates therefore possessed the prn1/ptxS1A genotype. CONCLUSION: The results of this study compared to historical research on Queensland isolates suggest that B. pertussis pertactin and pertussis toxin variants are not becoming more prevalent in Queensland since the introduction of the acellular vaccines. Current prevalences of pertactin variants are significantly different to that described in a number of other countries with high vaccine coverage. Relative paucity of recovered isolates compared to notified infections, due primarily to non culture based pertussis diagnostics is however a confounding factor in the assessment of variant prevalence.

Development of a Multiple-Locus Variable Number of Tandem Repeat Analysis (MLVA) for Leptospira interrogans and its application to Leptospira interrogans serovar Australis isolates from Far North Queensland, Australia.

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.

Molecular epidemiology of Leptospira borgpetersenii serovar Arborea, Queensland, Australia, 1998-2005.

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.

Reclassification of Leptospira meyeri serovar Perameles to Leptospira interrogans serovar Perameles through serological and molecular analysis: evidence of a need for changes to current procedures in Leptospira taxonomy.

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.

Leptospira kmetyi sp. nov., isolated from an environmental source in Malaysia.

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)).

The microscopic agglutination test (MAT) is an unreliable predictor of infecting Leptospira serovar in Thailand.

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.

Leptospira wolffii sp. nov., isolated from a human with suspected leptospirosis in Thailand.

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)).

Epidemiology of Leptospira weilii serovar Topaz infections in Australia.

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.

Clinical diagnosis and geographic distribution of leptospirosis, Thailand.

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.

Optimization of culture of Leptospira from humans with leptospirosis.

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.

A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand.

BACKGROUND: A sustained outbreak of leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown. METHODS AND FINDINGS: A prospective study was conducted between 2000 and 2005 to identify patients with leptospirosis presenting to Udon Thani Hospital in northeast Thailand, and to isolate the causative organisms from blood. A multilocus sequence typing scheme was developed to genotype these pathogenic Leptospira. Additional typing was performed for Leptospira isolated from human cases in other Thai provinces over the same period, and from rodents captured in the northeast during 2004. Sequence types (STs) were compared with those of Leptospira drawn from a reference collection. Twelve STs were identified among 101 isolates from patients in Udon Thani. One of these (ST34) accounted for 77 (76%) of isolates. ST34 was Leptospira interrogans, serovar Autumnalis. 86% of human Leptospira isolates from Udon Thani corresponded to ST34 in 2000/2001, but this figure fell to 56% by 2005 as the outbreak waned (p = 0.01). ST34 represented 17/24 (71%) of human isolates from other Thai provinces, and 7/8 (88%) rodent isolates. By contrast, 59 STs were found among 76 reference strains, indicating a much more diverse population genetic structure; ST34 was not identified in this collection. CONCLUSIONS: Development of an MLST scheme for Leptospira interrogans revealed that a single ecologically successful pathogenic clone of L. interrogans predominated in the rodent population, and was associated with a sustained outbreak of human leptospirosis in Thailand.