The potential of a multiplex high-throughput molecular assay for early detection of first and second line tuberculosis drug resistance mutations to improve infection control and reduce costs: a decision analytical modeling study.

BACKGROUND: Molecular resistance detection (MRD) of resistance to second-line anti-tuberculous drugs provides faster results than phenotypic tests, may shorten treatment and allow earlier separation among patients with and without second-line drug resistance. METHODS: In a decision-analytical model we simulated a cohort of patients diagnosed with TB in a setting where drug resistant TB is highly prevalent and requires initial hospitalization, to explore the potential benefits of a high-throughput MRD-assay for reducing potential nosocomial transmission of highly resistant strains, and total costs for diagnosis of drug resistance, treatment and hospitalization. In the base case scenario first-line drug resistance was diagnosed with WHO-endorsed molecular tests, and second-line drug resistance with culture and phenotypic methods. Three alternative scenarios were explored, each deploying high-throughput MRD allowing either detection of second-line mutations in cultured isolates, directly on sputum, or MRD with optimized markers. RESULTS: Compared to a base case scenario, deployment of high-throughput MRD reduced total costs by 17-21 %. The period during which nosocomial transmission may take place increased by 15 % compared to the base case if MRD had currently reported suboptimal sensitivity and required cultured isolates; increased by 7 % if direct sputum analysis were possible including in patients with smear-negative TB, and reduced by 24 % if the assay had improved markers, but was still performed on cultured isolates. Improved clinical sensitivity of the assay (additional markers) by more than 35 % would be needed to avoid compromising infection control. CONCLUSIONS: Further development of rapid second-line resistance testing should prioritize investment in optimizing markers above investments in a platform for direct analysis of sputum.

Mycobacterium tuberculosis population structure determines the outcome of genetics-based second-line drug resistance testing.

The global emergence of multidrug-resistant tuberculosis has highlighted the need for the development of rapid tests to identify resistance to second-line antituberculosis drugs. Resistance to fluoroquinolones and aminoglycosides develops through nonsynonymous single nucleotide polymorphisms in the gyrA and gyrB genes and the rrs gene, respectively. Using DNA sequencing as the gold standard for the detection of mutations conferring resistance, in conjunction with spoligotyping, we demonstrated heteroresistance in 25% and 16.3% of Mycobacterium tuberculosis isolates resistant to ofloxacin and amikacin, respectively. Characterization of follow-up isolates from the same patients showed that the population structure of clones may change during treatment, suggesting different phases in the emergence of resistance. The presence of underlying mutant clones was identified in isolates which failed to show a correlation between phenotypic resistance and mutation in the gyrA or rrs gene. These clones harbored previously described mutations in either the gyrA or rrs gene, suggesting that rare mutations conferring resistance to ofloxacin or amikacin may not be as important as was previously thought. We concluded that the absence of a correlation between genotypic and phenotypic resistance implies an early phase in the emergence of resistance within the patient. Thus, the diagnostic utility of genetics-based drug susceptibility tests will depend on the proportion of patients whose bacilli are in the process of acquiring resistance in the study setting. These data have implications for the interpretation of molecular and microbiological diagnostic tests for patients with drug-susceptible and drug-resistant tuberculosis who fail to respond to treatment and for those with discordant results.

Identification and Genotyping of the Etiological Agent of Tuberculous Lymphadenitis in Ethiopia

Background: In Ethiopia; little has been done to assess how Mycobacterium bovis has contributed to human tuberculosis; though the population routinely consumes unpasteurized milk and raw meat. The aim of this study was to determine the proportion of M. tuberculosis and M. bovis as etiological agents of tuberculous lymphadenitis (TBLN). Methods: Patients with lymphadenopathy (n = 171) were included in a cross-sectional study at Butajira Hospital; Southern Ethiopia. Lymph node biopsies were cultured. Patients' HIV status was identified. DNA from positive cultures was tested by PCR to identify M. bovis and M. tuberculosis. Isolates were genotyped by multiplex ligation-dependent probe amplification (MLPA) assay. Results: Among 171 patients; 156 had culture results. Of these; 107 (69) were positive for M. tuberculosis complex (MTC). Six of the 10 HIV-positive patients were culture positive. M. tuberculosis specific sequences were identified in the DNA of each of 100 samples as assessed by RD10 targeted PCR; and each of the 95 isolates exhibited the M. tuberculosis specific TbD1 deletion by MLPA analysis. No M. bovis was identified. These results indicate that all the isolates were modern M. tuberculosis strains. Furthermore; MLPA studies confirmed that 42of the isolates showed the Haarlem genotype and 12displayed sequences compatible with INH resistance. No mutations conferring resistance to ethambutol or rifampicin were detected. Conclusions: Our data showed that M. tuberculosis strains had common characteristics with strains causing pulmonary TB; which appears to be the main etiological agent of TBLN

Development of multiplex assay for rapid characterization of Mycobacterium tuberculosis.

We have developed a multiplex assay, based on multiplex ligation-dependent probe amplification (MLPA), that allows simultaneous detection of multiple drug resistance mutations and genotype-specific mutations at any location in the Mycobacterium tuberculosis genome. The assay was validated on a reference panel of well-characterized strains, and the results show that M. tuberculosis can be accurately characterized by our assay. Eighteen discriminatory markers identifying drug resistance (rpoB, katG, inhA, embB), members of the M. tuberculosis complex (16S rRNA, IS6110, TbD1), the principal genotypic group (katG, gyrA), and Haarlem and Beijing strains (ogt, mutT2, mutT4) were targeted. A sequence specificity of 100% was reached for 16 of the 18 selected genetic targets. In addition, a panel of 47 clinical M. tuberculosis isolates was tested by MLPA in order to determine the correlation between phenotypic drug resistance and MLPA and between spoligotyping and MLPA. Again, all mutations present in these isolates that were targeted by the 16 functional probes were identified. Resistance-associated mutations were detected by MLPA in 71% of the identified rifampin-resistant strains and in 80% of the phenotypically isoniazid-resistant strains. Furthermore, there was a perfect correlation between MLPA results and spoligotypes. When MLPA is used on confirmed M. tuberculosis clinical specimens, it can be a useful and informative instrument to aid in the detection of drug resistance, especially in laboratories where drug susceptibility testing is not common practice and where the rates of multidrug-resistant and extensively drug resistant tuberculosis are high. The flexibility and specificity of MLPA, along with the ability to simultaneously genotype and detect drug resistance mutations, make MLPA a promising tool for pathogen characterization.