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ABSTRACTA novel lateral flow immunochromatographic device (LFD) was evaluated in several veterinary diagnostic laboratories. It was confirmed to be specific for Mycobacterium bovisand M.capraecells. The performance of the novel LFD was assessed relative to the confirmatory tests routinely applied after culture (spoligotyping or quantitative PCR [qPCR]) in each laboratory; liquid (MGIT or BacT/Alert) and/or solid (Stonebrink, Coletsos, or Lowenstein-Jensen) cultures were tested. In comparison to spoligotyping of acid-fast-positive MGIT cultures, percent agreement between positive LFD and spoligotyping results was excellent in two United Kingdom laboratories (97.7 to 100%) but lower in the Spanish context (76%), where spoligotyping was applied to MGIT cultures previously confirmed to be positive for M. tuberculosiscomplex (MTBC) by qPCR. Certain spoligotypes of M. bovisand M. capraewere not detected by the LFD in Spanish MGIT cultures. Compared to qPCR confirmation, the agreement between positive LFD and qPCR results was 42.3% and 50% for BacT/Alert and MGIT liquid cultures, respectively, and for solid cultures, it ranged from 11.1 to 89.2%, depending on the solid medium employed (Coletsos, 11.1%; Lowenstein-Jensen, 55.6%; Stonebrinks, 89.2%). Correlation between the novel LFD and BD MGIT TBc Identification test results was excellent when 190 MGIT cultures were tested (r= 0.9791; P< 0.0001), with the added benefit that M. boviswas differentiated from another MTBC species in one MGIT culture by the novel LFD. This multilaboratory evaluation demonstrated the novel LFD's potential utility as a rapid test to confirm isolation of M. bovisand M. capraefrom veterinary specimens following culture.

ABSTRACTGenotyping of multidrug-resistant (MDR) Mycobacterium tuberculosisstrains isolated from tuberculosis (TB) patients in four South African provinces (Western Cape, Eastern Cape, KwaZulu-Natal, and Gauteng) revealed a distinct population structure of the MDR strains in all four regions, despite the evidence of substantial human migration between these settings. In all analyzed provinces, a negative correlation between strain diversity and an increasing level of drug resistance (from MDR-TB to extensively drug-resistant TB [XDR-TB]) was observed. Strains predominating in XDR-TB in the Western and Eastern Cape and KwaZulu-Natal Provinces were strongly associated with harboring an inhApromoter mutation, potentially suggesting a role of these mutations in XDR-TB development in South Africa. Approximately 50% of XDR-TB cases detected in the Western Cape were due to strains probably originating from the Eastern Cape. This situation may illustrate how failure of efficient health care delivery in one setting can burden health clinics in other areas.

ABSTRACTThe long-term persistence of Mycobacterium tuberculosisin communities with high tuberculosis prevalence is a serious problem aggravated by the presence of drug-resistant tuberculosis strains. Drug resistance in an individual patient is often discovered only after a long delay, particularly if the diagnosis is based on current culture-based drug sensitivity testing methods. During such delays, the patient may transmit tuberculosis to his or her contacts. Rapid diagnosis of drug resistance would be expected to reduce this transmission and hence to decrease the prevalence of drug-resistant strains. To investigate this quantitatively, a mathematical model was constructed, assuming a homogeneous population structure typical of communities in South Africa where tuberculosis incidence is high. Computer simulations performed with this model showed that current control strategies will not halt the spread of multidrug-resistant tuberculosis in such communities. The simulations showed that the rapid diagnosis of drug resistance can be expected to reduce the incidence of drug-resistant cases provided the additional measure of screening within the community is implemented.

ABSTRACTThe Latin American-Mediterranean (LAM) family of Mycobacterium tuberculosisis believed to be the cause of ~15% of tuberculosis cases worldwide. Previously, we defined a prevalent sublineage of the LAM family in Brazil by a single characteristic genomic deletion designated RDRio. Using the Brazilian strains, we pinpoint an Ag85C103single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism [RFLP] analysis) that correctly identified all LAM family strains. Importantly, all RDRiostrains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between “wild-type” and RDRiostrains, were then used to analyze an international collection of M. tuberculosisstrains. RDRioM. tuberculosiswas identified from four continents involving 11 countries. Phylogenetic analysis of the IS6110-RFLP patterns from representative RDRioand LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C103SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS6110element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RDRiostrains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RDRiosublineage.

ABSTRACTIn order to fully understand the global tuberculosis (TB) epidemic it is important to investigate the population structure and dissemination of the causative agent that drives the epidemic. Mycobacterium tuberculosisstrain family 11 (F11) genotype isolates (found in 21.4% of all infected patients) are at least as successful as the Beijing genotype family isolates (16.5%) in contributing to the TB problem in some Western Cape communities of South Africa. This study describes key molecular characteristics that define the F11 genotype. A data-mining approach coupled with additional molecular analysis showed that members of F11 can easily and uniquely be identified by PCR-based techniques such as spoligotyping and dot blot screening for a specific rrs491polymorphism. Isolates of F11 not only are a major contributor to the TB epidemic in South Africa but also are present in four different continents and at least 25 other countries in the world. Careful study of dominant compared to rare strains should provide clues to their success and possibly provide new ideas for combating TB.

ABSTRACTIn an ongoing molecular epidemiology study, human immunodeficiency virus-negative patients with first-time pulmonary tuberculosis from a high-incidence community were enrolled. Mycobacterium tuberculosisstrains were identified by restriction fragment length polymorphism analysis with two fingerprinting probes. Of 131 patients, 3 (2.3%) were shown to have a mixture of strains in one or two of their serial cultures. This study further investigated these cases with disease caused by multiple M. tuberculosisstrains in the context of the molecular epidemiology of the study setting.

ABSTRACTCorrect and rapid diagnosis is essential in the management of multidrug-resistant tuberculosis (MDR-TB). In this population-based study of 61 patients with drug-resistant tuberculosis, we evaluated the frequency of mutations and compared the performance of genotypic (mutation analysis by dot blot hybridization) and phenotypic (indirect proportion method) drug resistance tests. Three selected codons (rpoB531, rpoB526, and katG315) allowed identification of 90% of MDR-TB cases. Ninety percent of rifampin, streptomycin, and ethambutol resistance and 75% of isoniazid resistance were detected by screening for six codons: rpoB531, rpoB526, rrs-513, rpsL43, embB306, and katG315. The performance (reproducibility, sensitivity, and specificity) of the genotypic method was superior to that of the routine phenotypic method, with the exception of sensitivity for isoniazid resistance. A commercialized molecular genetic test for a limited number of target loci might be a good alternative for a drug resistance screening test in the context of an MDR “DOTS-plus” strategy.

ABSTRACTSequencing of the Mycobacterium tuberculosispncAgene allows for pyrazinamide susceptibility testing. We summarize data on pncApolymorphisms that do not confer resistance at a susceptibility breakpoint of 100 µg/ml pyrazinamide in MGIT within a cohort of isolates from South Africa and the U.S. Centers for Disease Control and Prevention.

ABSTRACTThe performance and cost of the Capilia TB assay were evaluated for use in a resource-limited setting. The sensitivity and specificity were 99.6% and 99.5%, respectively. The incremental costs of the Capilia test were estimated to be $1.46 and $1.84 when the test was added to liquid and solid culture processes, respectively. These findings suggest that the Capilia TB assay represents a rapid, simple, and inexpensive Mycobacterium tuberculosisidentification test that can be used in resource-limited settings.

ABSTRACTConcern has been raised about the efficacy of the heat killing of mycobacteria during the isolation of DNA. We demonstrate a method that allows for the efficient killing of Mycobacterium tuberculosiswithout compromising DNA integrity for subsequent molecular investigation. This method reduces the risk of infection to the research scientist.

ABSTRACTA mutation (C-to-T transition) at position 491 of therrsgene was identified in a Mycobacterium tuberculosisstrain family (n= 208 isolates) that was predominant in a suburb of Cape Town, South Africa. This nucleotide change is not involved in streptomycin resistance, and we suggest caution in assuming that all mutations in genes targeted by antituberculosis drugs confer drug resistance.

ABSTRACTWe developed a pyrazinamidase gene DNA-sequencing method to rapidly identify pyrazinamide resistance-causing mutations in GenoLyse-treated, smear-positive sputum specimens. The sensitivity and specificity were 90.9 and 100%, respectively, compared to those of MGIT drug susceptibility testing, after the exclusion of synonymous mutations and nonsynonymous mutations previously associated with susceptibility to pyrazinamide.