Use of Multiplex Allele-Specific Polymerase Chain Reaction (MAS-PCR) to Detect Multidrug-Resistant Tuberculosis in Panama

Abstract

The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB) is phenotypic drug susceptibility testing (DST), which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allele-specific polymerase chain reaction (MAS-PCR) as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB . MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.

Multidrug-Resistant Tuberculosis in Panama Is Driven by Clonal Expansion of a Multidrug-Resistant Mycobacterium tuberculosis Strain Related to the KZN Extensively Drug-Resistant M. tuberculosis Strain from South Africa

“…As previously reported (34), all but one of the resistant strains had mutations in the RIF resistance-determining region of RpoB, which is commonly seen in other RIF-resistant strains (37). Most of the streptomycin-resistant strains had the nucleotide substitu- tion C517T (30 isolates) or A514C (1 isolate) in rrs (the 16S rRNA), 5 isolates had K43R or K88R mutations in RpsL, and 11 isolates had nonsynonymous or frameshift mutations in GidB (38-40) (see Table S1 in the supplemental material).…”

“…As noted previously (34), mutations at alternative (non-S315) residues in KatG appear to explain most of the remaining INH-resistant strains (8 of 10 strains) (see Table S2 in the supplemental material), as almost no mutations were observed in other genes associated with INH resistance (35,36). The inhA promoter mutation was associated primarily with non-LAM MDR strains (8 of 14 isolates), whereas 50 of 52 LAM MDR strains had some nonsynonymous mutations in KatG (and only 3 had the inhA CϪ15T mutation).…”

“…October 2013 Volume 51 Number 10 jcm.asm.org 3279 namide coresistance) (34). As noted previously (34), mutations at alternative (non-S315) residues in KatG appear to explain most of the remaining INH-resistant strains (8 of 10 strains) (see Table S2 in the supplemental material), as almost no mutations were observed in other genes associated with INH resistance (35,36).…”

“…Drug resistance patterns among the MDR strains are consistent with mutations in individual genes associated with resistance to each drug ( Table 2). The overall frequency of INH and RIF resistance mutations in this sample was described previously (34). The two most prevalent mutations for INH resistance observed among the 66 MDR isolates were the S315T mutation in KatG (70%, conferring high-level resistance) and the CϪ15T mutation in the inhA promoter (20%, conferring lower-level resistance but also ethio- …”

First Evaluation of GenoType MTBDR plus 2.0 Performed Directly on Respiratory Specimens in Central America

“…Although it is not a commonly recognized mutation (29), Y337C has been reported to confer INH resistance among nonclustered isolates (30). Our previous studies revealed that approximately 16% of clustered MDR isolates identified in Panama over the past decade lacked mutations in the katG and inhA loci (31). The second case with discordant results involved isolate 044-04, which contained a CϪ15T mutation detected with the LPA, although an indeterminate reaction was noted with the NRA.…”