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

<p>Abstract</p> <p>Background</p> <p>The ability to culture <it>Mycobacterium tuberculosis </it>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 <it>M. tuberculosis </it>isolates prepared within the same laboratory over a two-week period of time.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>The application of MST has been demonstrated to serve as a rapid and efficient method to investigate cases of possible cross-contamination with <it>M. tuberculosis</it>.</p

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.Keywords:&nbsp;Leptospirosis &middot; zoonosis &middot; methods &middot; diagnosi

Pyrosequencing identification of Mycobacterium tuberculosis W-Beijing

<p>Abstract</p> <p>Background</p> <p>The worldwide expanding <it>Mycobacterium tuberculosis </it>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.</p> <p>Findings</p> <p>Pyrosequencing found a G/A substitution in the Rv0927c-pstS3 intergenic spacer and a RD105 deletion, identifying 8/104 <it>M. tuberculosis </it>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 (<it>p </it>= 0.2, X²test). Pyrosequencing correctly identified <it>M. tuberculosis </it>organisms in 26/26 sputum specimens exhibiting acid-fast bacilli. Pyrosequencing results were obtained within four hours, incurring an estimated cost of 1.86 €/test.</p> <p>Conclusion</p> <p>Pyrosequencing of the Rv0927c gene and adjacent intergenic spacer is an efficient, low-cost technique for the rapid identification of W-Beijing isolates.</p

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 M<8 IS6110 copies (P<0.0003). MST was successfully applied to 7/10 clinical specimens exhibiting a Cts <= 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)

A Single-Step Sequencing Method for the Identification of Mycobacterium tuberculosis Complex Species

The Mycobacterium tuberculosis complex (MTC) comprises several closely related species responsible for strictly human and zoonotic tuberculosis. Some of the species are restricted to Africa and were responsible for the high prevalence of tuberculosis. However, their identification at species level is difficult and expansive. Accurate species identification of all members is warranted in order to distinguish between strict human and zoonotic tuberculosis, to trace source exposure during epidemiological studies, and for the appropriate treatment of patients. In this paper, the Exact Tandem Repeat D (ETR-D) intergenic region was investigated in order to distinguish MTC species. The ETR-D sequencing unambiguously identified MTC species type strain except M. pinnipedii and M. microti, and the results agreed with phenotypic and molecular identification. This finding offers a new tool for the rapid and accurate identification of MTC species in a single sequencing reaction, replacing the current time-consuming polyphasic approach. Its use could assist public health interventions and aid in the control of zoonotic transmission in African countries, and could be of particular interest with the current emergence of multidrug-resistant and extended-resistance isolates

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

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

International audienceLeptospirosis 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

Rapid MALDI-TOF mass spectrometry identification of <em>Leptospira</em> organisms: A reply

International audienc

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 Reunion." 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