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J Clin Microbiol. 2013 January; 51(1): 383.
PMCID: PMC3536205

Reply to “Role of rpsA Gene Sequencing in Diagnosis of Pyrazinamide Resistance”


We appreciate the comments made by Simons and colleagues regarding the value of rpsA gene sequencing in the diagnosis of pyrazinamide resistance. The diversity of mutations associated with drug resistance in Mycobacterium tuberculosis has been a challenge to the sensitivity and predictive value of molecular diagnostic tools. For most clinical laboratories, full-gene sequencing of all resistance-associated loci is not a feasible approach (1). Until rapid, cost-effective methods for identifying every mutation in every strain are developed, operational demands dictate that routine diagnostic algorithms focus on the most common and informative targets. For example, most molecular assays target only the short “rifampin resistance-determining region” of rpoB, even though mutations outside this region can confer resistance to rifampin (2, 3).

Consistently, studies have identified pncA mutations in >80% of pyrazinamide-resistant Mycobacterium tuberculosis isolates (4). The paper by Shi and colleagues raised the possibility that rpsA mutations may explain resistance in wild-type pncA (pncAWT) strains (5). However, in our clinical collection of pyrazinamide-resistant isolates, no nonsynonymous mutations were observed (6). Although our colleagues report that 1 of the 5 pncAWT strains they examined contained a rpsA mutation, they do not indicate if they confirmed the phenotypic impact of the A778→C/Val260→Ile change (e.g., by cloning the mutant rpsA gene into a pyrazinamide-sensitive strain and measuring a decrease in susceptibility). To be clear, we do not discount a role for rpsA, but the current data indicate that rpsA mutations account for resistance in only a small subset of strains. We also concede that a two-step approach, where only pncAWT isolates are subjected to rpsA sequencing, may be useful. However, our initial intent was routine analysis of both genes, just as common algorithms for investigating isoniazid resistance target both katG and inhA.

Irrespective of the possible value of rpsA sequencing, it is evident that additional determinants of pyrazinamide resistance remain to be characterized. Notably, two of the isolates described by Simons and colleagues exhibited resistance to both pyrazinamide and isoniazid. We also observed this resistance pattern among pncAWT and rpsAWT isolates. Considering that pyrazinamide and isoniazid have somewhat similar chemical structures and that both are administered as prodrugs, we have wondered if some shared mechanism may mediate resistance to both agents. Thorough analysis of such strains may uncover novel determinants of resistance to pyrazinamide and other antimycobacterial agents and provide useful information for the effective treatment of tuberculosis.


This is a response to a letter by Simons et al. (doi:10.1128/JCM.02739-12).


1. Abebe G, Paasch F, Apers L, Rigouts L, Colebunders R. 2011. Tuberculosis drug resistance testing by molecular methods: opportunities and challenges in resource limited settings. J. Microbiol. Methods 84: 155–160 [PubMed]
2. Tan Y, Hu A, Zhao Y, Cai X, Luo C, Zou C, Liu X. 2012. The beginning of the rpoB gene in addition to the rifampin resistance determination region might be needed for identifying rifampin/rifabutin cross-resistance in multidrug-resistant Mycobacterium tuberculosis isolates from southern China. J. Clin. Microbiol. 50: 81–85 [PMC free article] [PubMed]
3. Telenti A, Imboden P, Marchesi F, Lowrie D, Cole S, Colston MJ, Matter L, Schopfer K, Bodmer T. 1993. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341: 647–650 [PubMed]
4. Chang KC, Yew WW, Zhang Y. 2011. Pyrazinamide susceptibility testing in Mycobacterium tuberculosis: a systematic review with meta-analyses. Antimicrob. Agents Chemother. 55: 4499–4505 [PMC free article] [PubMed]
5. Shi W, Zhang X, Jiang X, Yuan H, Lee JS, Barry CE, III, Wang H, Zhang W, Zhang Y. 2011. Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis. Science 333: 1630–1632 [PMC free article] [PubMed]
6. Alexander DC, Ma JH, Guthrie JL, Blair J, Chedore P, Jamieson FB. 2012. Gene sequencing for routine verification of pyrazinamide resistance in Mycobacterium tuberculosis: a role for pncA but not rpsA. J. Clin. Microbiol. 50: 3726–3728 [PMC free article] [PubMed]

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