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Second line molecular diagnosis for bovine tuberculosis to improve diagnostic schemes.

ABSTRACT: Surveillance of bovine tuberculosis (bTB) is partly based on the sanitary inspection of carcasses at the abattoir to detect bTB-like lesions which, in compliance with EU recommendations, are analysed by bacteriology and histopathology to disclose Mycobacterium bovis (or M. caprae) infection. Moreover, since 2012, a PCR method with similar sensitivity and specificity values of histopathology and bacteriology respectively is additionally employed in France, partially compensating for the weaknesses of classical diagnostic methods. We analysed a collection of bTB-like lesions from cattle presenting positive histological results albeit with negative PCR results. We present here the results of these samples, recovered from 292 animals culled between 2013 and 2016, analysed with a second line molecular diagnosis approach that consists in a combination of PCRs targeting the M. tuberculosis-M. avium complexes as well as the Mycobacterium genus and sequencing of hsp65 gene. These molecular analyses disclosed to identify the presence of non-tuberculous bacteria which could be responsible for most of these non-specific TB lesions: non tuberculous mycobacteria (24%) or Actinomycetales (56%) such as Rhodococcus equi (53%); 24% of the samples were negative. M. bovis -or any other MTBC members- was neither detected by molecular methods nor isolated in any of them at the end of the 3 months of culture. In conclusion, these results highlight the lack of specificity of histopathology and the usefulness of a first line PCR with a second line molecular diagnostic test to circumvent it. This diagnostic strategy makes it possible to reduce the number of suspect bTB cases raised at the abattoir or shortening their lock-up periods. By simplifying diagnostic schemes, the use of this tool could improve bTB surveillance and make eradication programs more efficient in the future.

SUBMITTER: Michelet L

PROVIDER: S-EPMC6261039 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Second line molecular diagnosis for bovine tuberculosis to improve diagnostic schemes.

Michelet Lorraine L de Cruz Krystel K Karoui Claudine C Tambosco Jennifer J Moyen Jean-Louis JL Hénault Sylvie S Boschiroli María Laura ML

PloS one 20181126 11

Surveillance of bovine tuberculosis (bTB) is partly based on the sanitary inspection of carcasses at the abattoir to detect bTB-like lesions which, in compliance with EU recommendations, are analysed by bacteriology and histopathology to disclose Mycobacterium bovis (or M. caprae) infection. Moreover, since 2012, a PCR method with similar sensitivity and specificity values of histopathology and bacteriology respectively is additionally employed in France, partially compensating for the weaknesse ...[more]

PMID: 30475835

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Project description:Bovine tuberculosis (BTB) caused by Mycobacterium bovis remains a major problem in both the developed and developing countries. Control of BTB in the UK is carried out by test and slaughter of infected animals, based primarily on the tuberculin skin test (PPD). Vaccination with the attenuated strain of the M. bovis pathogen, BCG, is not used to control bovine tuberculosis in cattle at present, due to its variable efficacy and because it interferes with the PPD test. Diagnostic tests capable of Differentiating Infected from Vaccinated Animals (DIVA) have been developed that detect immune responses to M. bovis antigens absent in BCG; but these are too expensive and insufficiently sensitive to be used for BTB control worldwide. To address these problems we aimed to generate a synergistic vaccine and diagnostic approach that would permit the vaccination of cattle without interfering with the conventional PPD-based surveillance. The approach was to widen the pool of M. bovis antigens that could be used as DIVA targets, by identifying antigenic proteins that could be deleted from BCG without affecting the persistence and protective efficacy of the vaccine in cattle. Using transposon mutagenesis we identified genes that were essential and those that were non-essential for persistence in bovine lymph nodes. We then inactivated selected immunogenic, but non-essential genes in BCG Danish to create a diagnostic-compatible triple knock-out ?BCG TK strain. The protective efficacy of the ?BCG TK was tested in guinea pigs experimentally infected with M. bovis by aerosol and found to be equivalent to wild-type BCG. A complementary diagnostic skin test was developed with the antigenic proteins encoded by the deleted genes which did not cross-react in vaccinated or in uninfected guinea pigs. This study demonstrates the functionality of a new and improved BCG strain which retains its protective efficacy but is diagnostically compatible with a novel DIVA skin test that could be implemented in control programmes.

Project description:Rapid molecular diagnostics have great potential to limit the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) (M/XDR-TB). These technologies detect mutations in the Mycobacterium tuberculosis genome that confer phenotypic drug resistance. However, there have been few data published regarding the relationships between the detected M. tuberculosis resistance mutations and M/XDR-TB treatment outcomes, limiting our current ability to exploit the full potential of molecular diagnostics. We analyzed clinical, microbiological, and sequencing data for 451 patients and their clinical isolates collected in a multinational, observational cohort study to determine if there was an association between M. tuberculosis resistance mutations and patient mortality. The presence of an rrs 1401G mutation was associated with significantly higher odds of patient mortality (adjusted odds ratio [OR] = 5.72; 95% confidence interval [CI], 1.65 to 19.84]) after adjusting for relevant patient clinical characteristics and all other resistance mutations. Further analysis of mutations, categorized by the associated resistance level, indicated that the detection of mutations associated with high-level fluoroquinolone (OR, 3.99 [95% CI, 1.10 to 14.40]) and kanamycin (OR, 5.47 [95% CI, 1.64 to 18.24]) resistance was also significantly associated with higher odds of patient mortality, even after accounting for clinical site, patient age, reported smoking history, body mass index (BMI), diabetes, HIV, and all other resistance mutations. Specific gyrA and rrs resistance mutations, associated with high-level resistance, were associated with patient mortality as identified in clinical M. tuberculosis isolates from a diverse M/XDR-TB patient population at three high-burden clinical sites. These results have important implications for the interpretation of molecular diagnostics, including identifying patients at increased risk for mortality during treatment. (This study has been registered at ClinicalTrials.gov under registration no. NCT02170441.).

Dataset Information

Second line molecular diagnosis for bovine tuberculosis to improve diagnostic schemes.

Publications

Second line molecular diagnosis for bovine tuberculosis to improve diagnostic schemes.

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