Genotypic Characterization of pncA Gene in Patients with Multidrug-resistant Tuberculosis from South Africa
Pyrazinamide (PZA) is an essential component of empiric first- and second-line TB treatment regimens. However, PZA drug susceptibility testing (DST) is difficult to perform phenotypically and not routinely available. Genetic mutations in the pncA gene of M.tuberculosis commonly confer PZA resistance, but limited data on the prevalence and diversity of these mutations are available from clinical populations of TB patients. We utilized pncA sequencing to estimate PZA resistance among MDR and XDR TB patients in KwaZulu-Natal province, South Africa.
We prospectively enrolled 206 MDR TB and 289 XDR TB patients from September 2011 to April 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.
To date, targeted sequencing has been completed for 266 unique isolates from 80 MDR TB (16 resistant to H&R only, 64 resistant to H,R,S) and 186 XDR TB participants (all resistant to all five drugs [H, R, S, Km, Ofx]. A mutation in the pncA gene was observed in 55 (69%) of MDR TB and 178 (96%) of XDR TB subjects. We found 31 distinct pncA mutations (Figure); 20 different mutations were seen among MDR TB subjects, of which, only 7 were seen in more than one subject. Among XDR TB subjects, one mutation (insertion of cytosine after the 456 locus) was present in 133 (72%), consistent with the known genotypic clonality of the XDR cases in KwaZulu-Natal province.
Nearly 70% of MDR and nearly all XDR TB subjects 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 subjects, 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 in the future.
J. Brust, None
T. Mthiyane, None
K. Mlisana, None
P. Moodley, None
S. Shah, None
N. Gandhi, None