1474Genotypic Characterization of pncA Gene in Patients with Multidrug-resistant Tuberculosis from South Africa

Abstract

Rationale: Pyrazinamide (PZA) is an essential component of empiric firstand second-line tuberculosis (TB) treatment regimens. However, PZA drug susceptibility testing (DST) is difficult to perform and not routinely available. Genetic mutations in the pncA gene of Mycobacterium tuberculosis (Mtb) commonly confer PZA resistance, but limited data on the prevalence and diversity of these mutations are available from clinical populations of TB patients. We sequenced pncA to estimate PZA resistance among MDR and XDR TB patients in KwaZulu-Natal province, South Africa. Methods: We prospectively enrolled 206 MDR TB and 377 XDR TB patients from September 2011 to September 2014. Sputum collected from each participant underwent mycobacterial culture and DST for isoniazid (H), rifampin (R), streptomycin (S), kanamycin (Km) and ofloxocin (Ofx). We extracted DNA from pure Mtb isolates and performed targeted sequencing of the pncA gene, characterizing the proportion, frequency and structure of polymorphisms. Results: To date, targeted sequencing has been completed for 314 isolates from 80 MDR TB (16 resistant to HR 20 different mutations were seen among MDR TB subjects, of which, only 7 were seen in more than one participant. Among XDR TB participants, one mutation (insertion of cytosine after the 456 locus) was present in 166 (71%), consistent with the clonality of XDR TB patients in KwaZulu-Natal. Conclusion: Nearly 70% of MDR TB and nearly all XDR TB participants had pncA mutations, and likely PZA resistance, in our cohort. A wide diversity of mutations was seen. Most mutations were singlets, suggesting acquisition of mutations de novo, perhaps due to empiric use. One specific mutation was common among XDR TB participants, but it occurred in conjunction with a specific RFLP genotype associated with clonal expansion. Given the difficulty in determining PZA susceptibility, characterizing pncA mutations may provide important data for developing rapid genotypic PZA susceptibility assays. BACKGROUND • South Africa has one of the highest incidence rates of multidrug-resistant tuberculosis (MDR TB) and extensively drug-resistant tuberculosis (XDR TB) with high rates of HIV co-infection. • Pyrazinamide (PZA) is used empirically in drug-resistant TB treatment regimens. • Precise acidic conditions are required in vitro to conduct PZA drug susceptibility testing (DST). However, acidic environments inhibit the growth of Mycobacterium tuberculosis (Mtb) bacilli. • Therefore, conventional PZA DST is difficult to perform in vitro and not widely available. • Gene sequencing is an effective method to identify mutations associated with resistance for some firstand second-line anti-TB drugs. • Genetic mutations in the pncA gene of Mtb typically confer resistance to PZA. However data on the frequency and diversity of pncA mutations is limited. • Thus, the actual prevalence of PZA resistance in patients with TB and drug-resistant TB is unknown. Funding: NIH/NIAID R01AI087465 [PI Neel Gandhi] and NIH/NIAID R01AI089349 [PI Neel Gandhi] Contact: Salim Allana, MD, MPH; salim.allana@emory.edu SETTING • Study site: KwaZulu-Natal province, South Africa, which has a population of 10.2 million. • TB incidence rate is nearly 1,100 per 100,000 population, and more than 80% of TB cases are HIV co-infected. • In 2012, MDR TB incidence was 45 cases/100,000 population and XDR TB incidence was 3.1 cases/100,000 population. • The HIV prevalence among adults (15-49 years of age) is 16.8%. • MDR TB patients receive standardized second-line treatment which includes PZA, ethambutol, kanamycin, moxifloxacin, terizidone, and ethionamide. • XDR TB patients receive a similar regimen, except that capreomycin, moxifloxacin and PAS replace kanamycin. • DST testing to evaluate PZA susceptibility is not routinely performed in this setting. OBJECTIVE Among MDR and XDR TB subjects in KwaZulu-Natal province, South Africa: • To characterize the frequency and diversity of polymorphisms in the pncA gene. • To estimate the prevalence of pyrazinamide resistance.