Overview of laboratory methods to diagnose Leptospirosis and to identify and to type leptospires

Leptospirosis is a virulent zoonosis with a global distribution. Pathogenic spirochetes of the genus Leptospira are responsible for this disease, and the primary animal reservoirs are rodentvvvs. Direct and indirect contact with infected urine constitutes the main route of transmission. Renal failure and advanced abortions are frequently observed in animals affected by leptospirosis, causing serious problems for farms. In humans, there is a high rate of mortality (10 percent), and farmers and persons in contact with water are frequently exposed. However, vaccines and strict prevention measures confer protection against leptospirosis. Serological tests facilitate the detection and identification of leptospire strains. Such tests are based on specific surface antigen recognition and are used for clinical analyses. To determine which serovars circulate in the environment, leptospires must be typed. Molecular methods, such as restriction enzyme-based techniques and the sequencing of specific regions, permit serovar identification. Unfortunately, although there are numerous techniques, they are not very efficient, and thus, new methods must be developed. With the advent of genomic sequencing, a substantial amount of information regarding leptospire genomes is now available, facilitating the selection of regions to discriminate between strains. Typing is important for both epidemiologic purposes and clinical analyses

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Overview of laboratory methods to diagnose Leptospirosis and to identify and to type leptospires.

Leptospirosis is a virulent zoonosis with a global distribution. Pathogenic spirochetes of the genus Leptospira are responsible for this disease, and the primary animal reservoirs are rodentvvvs. Direct and indirect contact with infected urine constitutes the main route of transmission. Renal failure and advanced abortions are frequently observed in animals affected by leptospirosis, causing serious problems for farms. In humans, there is a high rate of mortality (10 percent), and farmers and persons in contact with water are frequently exposed. However, vaccines and strict prevention measures confer protection against leptospirosis. Serological tests facilitate the detection and identification of leptospire strains. Such tests are based on specific surface antigen recognition and are used for clinical analyses. To determine which serovars circulate in the environment, leptospires must be typed. Molecular methods, such as restriction enzyme-based techniques and the sequencing of specific regions, permit serovar identification. Unfortunately, although there are numerous techniques, they are not very efficient, and thus, new methods must be developed. With the advent of genomic sequencing, a substantial amount of information regarding leptospire genomes is now available, facilitating the selection of regions to discriminate between strains. Typing is important for both epidemiologic purposes and clinical analyses.

First Observation of Leptospira interrogans in the Lungs of Rattus norvegicus.

We report the first two cases of pulmonary presence of leptospires in apparently healthy rats captured in a city park in Lyon (France). Only renal carriage of Leptospira has been described in the literature. Blood serology was performed in parallel with molecular and histological analyses of the kidney and lung samples. We isolated leptospires from the kidneys of two out of three seropositive wild rats. These results were confirmed by specific detection of pathogenic Leptospira by real-time PCR. Moreover, Leptospira DNA was detected in lung tissues. Immunohistochemistry and Warthin-Starry staining revealed that leptospires were present on the surface of the ciliated epithelium of the bronchi. Using PCR of the rrs (16S) gene and Multispacer Sequence Typing, DNA extracts of the kidney and lung were identified as belonging to Leptospira interrogans serovar Icterohaemorrhagiae "CHU Réunion." This first observation of the presence Leptospira in the lung with simultaneous renal carriage will require further study in future on several target organs to gain a better understanding of the Leptospira infection in wild rat.

Hedgehogs and Mustelid Species: Major Carriers of Pathogenic Leptospira, a Survey in 28 Animal Species in France (20122015).

Human leptospirosis is a zoonotic and potentially fatal disease that has increasingly been reported in both developing and developed countries, including France. However, our understanding of the basic aspects of the epidemiology of this disease, including the source of Leptospira serogroup Australis infections in humans and domestic animals, remains incomplete. We investigated the genetic diversity of Leptospira in 28 species of wildlife other than rats using variable number tandem repeat (VNTR) and multispacer sequence typing (MST). The DNA of pathogenic Leptospira was detected in the kidney tissues of 201 individuals out of 3,738 tested individuals. A wide diversity, including 50 VNTR profiles and 8 MST profiles, was observed. Hedgehogs and mustelid species had the highest risk of being infected (logistic regression, OR = 66.8, CI95% = 30.9-144 and OR = 16.7, CI95% = 8.7-31.8, respectively). Almost all genetic profiles obtained from the hedgehogs were related to Leptospira interrogans Australis, suggesting the latter as a host-adapted bacterium, whereas mustelid species were infected by various genotypes, suggesting their interaction with Leptospira was different. By providing an inventory of the circulating strains of Leptospira and by pointing to hedgehogs as a potential reservoir of L. interrogans Australis, our study advances current knowledge on Leptospira animal carriers, and this information could serve to enhance epidemiological investigations in the future.

Comparison of Mucosal, Subcutaneous and Intraperitoneal Routes of Rat Leptospira Infection.

Leptospirosis is a zoonosis found worldwide that is caused by a spirochete. The main reservoirs of Leptospira, which presents an asymptomatic infection, are wild rodents, including the brown rat (Rattus norvegicus). Experimental studies of the mechanisms of its renal colonization in rats have previously used an intraperitoneal inoculation route. However, knowledge of rat-rat transmission requires the use of a natural route of inoculation, such as a mucosal or subcutaneous route. We investigated for the first time the effects of subcutaneous and mucosal inoculation routes compared to the reference intraperitoneal route during Leptospira infection in adult rats. Infection characteristics were studied using Leptospira renal isolation, serology, and molecular and histological analyses. Leptospira infection was asymptomatic using each inoculation route, and caused similar antibody production regardless of renal colonization. The observed renal colonization rates were 8 out of 8 rats, 5 out of 8 rats and 1 out of 8 rats for the intraperitoneal, mucosal and subcutaneous inoculation routes, respectively. Thus, among the natural infection routes studied, mucosal inoculation was more efficient for renal colonization associated with urinary excretion than the subcutaneous route and induced a slower-progressing infection than the intraperitoneal route. These results can facilitate understanding of the infection modalities in rats, unlike the epidemiological studies conducted in wild rats. Future studies of other natural inoculation routes in rat models will increase our knowledge of rat-rat disease transmission and allow the investigation of infection kinetics.

Distribution of Leptospira interrogans by Multispacer Sequence Typing in Urban Norway Rats (Rattus norvegicus): A Survey in France in 2011-2013.

BACKGROUND: Urban leptospirosis has increasingly been reported in both developing and developed countries. The control of the disease is limited because our understanding of basic aspects of the epidemiology, including the transmission routes of leptospires among rat populations, remains incomplete. Through the ability to distinguish among Leptospira strains in rats, multispacer sequence typing (MST) could provide a modern understanding of Leptospira epidemiology; however, to our knowledge, the distribution of Leptospira strains among urban rat colonies has not been investigated using MST. AIMS AND METHODOLOGY: The objective of this study was to identify the Leptospira strains present in rats (Rattus norvegicus) in Lyon (France) using MST and to characterize their spatial distribution. Kidneys and urine were collected from rats trapped live in seven locations in the city and in one suburban location. Each location was considered to represent a rat colony. Bacterial cultures and quantitative polymerase chain reaction (qPCR) assays were performed, and the L. interrogans DNA identified was then genotyped using MST. The distributions of Leptospira strains were spatially described. KEY RESULTS: Among 84 wild rats, MST profiles were obtained in 35 of 37 rats that had a positive result for L. interrogans by bacterial culture and/or qPCR analyses. All of the MST profiles were related to reference strains previously isolated from human patients that belong to the serogroup Icterohaemorrhagiae and the serovars [strain(s)] Copenhageni [Wijinberg or M20] (n = 26), Icterohaemorrhagiae [CHU Réunion] (n = 7), Icterohaemorrhagiae [R1] (n = 1) and Copenhageni [Shibaura 9] (n = 1). Each colony was infected with leptospires having the same MST profile. MAJOR CONCLUSIONS: This study demonstrated that MST could be used for the purpose of field studies, either on culture isolates or on DNA extracted from kidneys and urine, to distinguish among L. interrogans isolates in rats. MST could thus be used to monitor their distributions in urban rats from the same city, thereby providing new knowledge that could be applied to explore the circulation of L. interrogans infection in rat colonies. Because the strains are related to those previously found in humans, this application of MST could aid in the source tracking of human leptospirosis, and the findings would be relevant for public health purposes according to the One Health principle.

High-resolution typing of Leptospira interrogans strains by multispacer sequence typing.

Leptospirosis is a worldwide zoonosis which is responsible for the typical form of Weil's disease. The epidemiological surveillance of the Leptospira species agent is important for host prevalence control. Although the genotyping methods have progressed, the identification of some serovars remains ambiguous. We investigated the multispacer sequence typing (MST) method for genotyping strains belonging to the species Leptospira interrogans, which is the main agent of leptospirosis worldwide. A total of 33 DNA samples isolated from the reference strains of L. interrogans serogroups Icterohaemorrhagiae, Australis, Canicola, and Grippotyphosa, which are the most prevalent serogroups in France, were analyzed by both the variable-number tandem-repeat (VNTR) and MST methods. An MST database has been constructed from the DNA of these reference strains to define the MST profiles. The MST profiles corroborated with the VNTR results. Moreover, the MST analysis allowed the identification at the serovar level or potentially to the isolate level for strains belonging to L. interrogans serovar Icterohaemorrhagiae, which then results in a higher resolution than VNTR (Hunter-Gaston index of 0.94 versus 0.68). Regarding L. interrogans serogroups Australis, Canicola, and Grippotyphosa, the MST and VNTR methods similarly identified the genotype. The MST method enabled the acquisition of simple and robust results that were based on the nucleotide sequences. The MST identified clinical isolates in correlation with the reference serovar profiles, thus permitting an epidemiological surveillance of circulating L. interrogans strains, especially for the Icterohaemorrhagiae serogroup, which includes the most prevalent strains of public health interest.

Rapid MALDI-TOF mass spectrometry identification of Leptospira organisms.

Leptospirosis is a worldwide deadly zoonotic disease. Accurate identification of the causative Leptospira spp. spirochetes ascertains the pathogenic status of the isolates, identifies potential source of infection and recognises outbreaks. Species identification is currently based on technically demanding, time and resources consuming serological and molecular methods. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) recently emerged as a first-line method for the accurate identification of bacteria, yet no data issued for Leptospira spp. We investigated the potential of MALDI-TOF-MS for the rapid identification of Leptospira isolates. Starting from a 10(5)organisms/mL suspension, MALDI-TOF-MS yielded an unique protein profile for each one of 19 Leptospira species reference isolates with a 100% reproducibility over 12 repeats, allowing to create a Leptopsira database. MALDI-TOF-MS further accurately identified 20/21 additional reference isolates representative of various serogroups at the species level as Leptospira interrogans (n=12), Leptospira kirschneri (n=5), Leptospira borgpetersenii (n=3), Leptospira noguchii (n=1) with identification score value of 2-2.5. Furthermore, six clinical isolates previously identified by rpoB sequencing, were correctly identified by MALDI-TOF-MS as L. interrogans (n=5) and L. borgpetersenii (n=1) with identification score value of 2-2.6. Identification was achieved in 40 min starting from the Leptospira suspension. MALDI-TOF-MS could complement serological and sequencing-based methods for the first line, rapid identification of Leptospira isolates in the clinical microbiology laboratory.

Palaeogenomics of Mycobacterium tuberculosis: epidemic bursts with a degrading genome.

Genome-scale analysis suggests that the last common ancestor of the Mycobacterium tuberculosis complex and Mycobacterium leprae diverged 36 million years ago, and members of the Mycobacterium tuberculosis complex differentiated 40,000 years ago. Analysis of palaeomicrobiological data from a 17,000-year-old sample from a bison and a 9000-year-old sample from a human being suggested that M tuberculosis preceded Mycobacterium bovis and related species. Whole-genome comparisons show that members of the M tuberculosis complex form a unique bacterial species with distinct ecotypes that are transmissible from any infected mammalian species to several others. Genomic deletions identified several M tuberculosis lineages that could be placed on a phylogeographical map, suggesting adaptation to local host populations. The degrees of transmissibility and virulence vary between M tuberculosis clones, with increased virulence mainly linked to gene loss in regulatory pathways. Such data suggest that most M tuberculosis clones have a restricted spreading capacity between the host population, allowing unpredictable bursts of highly transmissible, virulent, and successful clones, such as the east Asian (Beijing) clone. Advances in genomics have helped the development of molecular techniques for accurate identification of species and clones in the M tuberculosis complex, which is essential for tracing the source of infections.

Pyrosequencing identification of Mycobacterium tuberculosis W-Beijing.

BACKGROUND: The worldwide expanding Mycobacterium tuberculosis W-Beijing family is associated with treatment failure and relapse. Its identification currently relies on spoligotyping and conventional sequencing. We developed pyrosequencing as an alternative method for its identification. FINDINGS: Pyrosequencing found a G/A substitution in the Rv0927c-pstS3 intergenic spacer and a RD105 deletion, identifying 8/104 M. tuberculosis isolates as W-Beijing isolates. In addition, pyrosequencing found a previously unreported TGC deletion in the Rv0927c gene of W-Beijing isolates. Total concordance was found between the pyrosequencing data and conventional sequencing, as well as reference molecular identification. Multispacer Sequence Typing assigned the W-Beijing isolates to the Asian lineage and the 96 non-W-Beijing isolates to the Euro-American lineage (P < 10-5). The W-Beijing isolates were all susceptible to streptomycin, rifampin, isoniazid, ethambutol, and pyrazinamide; no resistance-associated mutations were detected in these eight W-Beijing isolates. There were no statistically significant differences in the antibiotic susceptibility of W-Beijing and non-W-Beijing isolates (p = 0.2, X2 test). Pyrosequencing correctly identified M. tuberculosis organisms in 26/26 sputum specimens exhibiting acid-fast bacilli. Pyrosequencing results were obtained within four hours, incurring an estimated cost of 1.86 euro/test. CONCLUSION: Pyrosequencing of the Rv0927c gene and adjacent intergenic spacer is an efficient, low-cost technique for the rapid identification of W-Beijing isolates.

Rapid detection of laboratory cross-contamination with Mycobacterium tuberculosis using multispacer sequence typing.

BACKGROUND: The ability to culture Mycobacterium tuberculosis from clinical specimens serves as the gold standard for the diagnosis of tuberculosis. However, a number of false-positive diagnoses may be due to cross-contamination of such specimens. We herein investigate such episode of cross-contamination by using a technique known as multispacer sequence typing (MST). This technique was applied to six M. tuberculosis isolates prepared within the same laboratory over a two-week period of time. RESULTS: MST analysis indicated a unique and common sequence profile between a strain isolated from a patient with proven pulmonary tuberculosis and a strain isolated from a patient diagnosed with lung carcinoma. Using this approach, we were able to provide a clear demonstration of laboratory cross-contamination within just four working days. Further epidemiological investigations revealed that the two isolates were processed for culture on the same day. CONCLUSION: The application of MST has been demonstrated to serve as a rapid and efficient method to investigate cases of possible cross-contamination with M. tuberculosis.

A single-step sequencing method for the identification of Mycobacterium tuberculosis complex species.

BACKGROUND: The Mycobacterium tuberculosis complex (MTC) comprises closely related species responsible for strictly human and zoonotic tuberculosis. Accurate species determination is useful for the identification of outbreaks and epidemiological links. Mycobacterium africanum and Mycobacterium canettii are typically restricted to Africa and M. bovis is a re-emerging pathogen. Identification of these species is difficult and expensive. METHODOLOGY/PRINCIPAL FINDINGS: The Exact Tandem Repeat D (ETR-D; alias Mycobacterial Interspersed Repetitive Unit 4) was sequenced in MTC species type strains and 110 clinical isolates, in parallel to reference polyphasic identification based on phenotype profiling and sequencing of pncA, oxyR, hsp65, gyrB genes and the major polymorphism tandem repeat. Inclusion of M. tuberculosis isolates in the expanding, antibiotic-resistant Beijing clone was determined by Rv0927c gene sequencing. The ETR-D (780-bp) sequence unambiguously identified MTC species type strain except M. pinnipedii and M. microti thanks to six single nucleotide polymorphisms, variable numbers (1-7 copies) of the tandem repeat and two deletions/insertions. The ETR-D sequencing agreed with phenotypic identification in 107/110 clinical isolates and with reference polyphasic molecular identification in all isolates, comprising 98 M. tuberculosis, 5 M. bovis BCG type, 5 M. canettii, and 2 M. africanum. For M. tuberculosis isolates, the ETR-D sequence was not significantly associated with the Beijing clone. CONCLUSIONS/SIGNIFICANCE: ETR-D sequencing allowed accurate, single-step identification of the MTC at the species level. It circumvented the current expensive, time-consuming polyphasic approach. It could be used to depict epidemiology of zoonotic and human tuberculosis, especially in African countries where several MTC species are emerging.

Multispacer sequence typing for Mycobacterium tuberculosis genotyping.

BACKGROUND: Genotyping methods developed to survey the transmission dynamics of Mycobacterium tuberculosis currently rely on the interpretation of restriction and amplification profiles. Multispacer sequence typing (MST) genotyping is based on the sequencing of several intergenic regions selected after complete genome sequence analysis. It has been applied to various pathogens, but not to M. tuberculosis. METHODS AND FINDINGS: In M. tuberculosis, the MST approach yielded eight variable intergenic spacers which included four previously described variable number tandem repeat loci, one single nucleotide polymorphism locus and three newly evaluated spacers. Spacer sequence stability was evaluated by serial subculture. The eight spacers were sequenced in a collection of 101 M. tuberculosis strains from five phylogeographical lineages, and yielded 29 genetic events including 13 tandem repeat number variations (44.82%), 11 single nucleotide mutations (37.93%) and 5 deletions (17.24%). These 29 genetic events yielded 32 spacer alleles or spacer-types (ST) with an index of discrimination of 0.95. The distribution of M. tuberculosis isolates into ST profiles correlated with their assignment into phylogeographical lineages. Blind comparison of a further 93 M. tuberculosis strains by MST and restriction fragment length polymorphism-IS6110 fingerprinting and mycobacterial interspersed repetitive units typing, yielded an index of discrimination of 0.961 and 0.992, respectively. MST yielded 41 different profiles delineating 16 related groups and proved to be more discriminatory than IS6110-based typing for isolates containing < 8 IS6110 copies (P<0.0003). MST was successfully applied to 7/10 clinical specimens exhibiting a Cts < or = 42 cycles in internal transcribed spacer-real time PCR. CONCLUSIONS: These results support MST as an alternative, sequencing-based method for genotyping low IS6110 copy-number M. tuberculosis strains. The M. tuberculosis MST database is freely available (http://ifr48.timone.univ-mrs.fr/MST_MTuberculosis/mst).