In Bangkok, the GenoType® MTBDRplus detected INH resistance, RIF resistance, and MDR-TB with a high sensitivity and 100% specificity in both isolates and AFB-positive sputum specimens, with substantial reductions in turn-around time compared with MGIT AST.
This study provides assurance that TB patients in Thailand with MDR-TB detected with the GenoType® MTBDRplus assay should immediately commence treatment with second-line anti-TB drugs. The 100% specificity corresponds to a positive likelihood ratio of near infinity, suggesting that, regardless of the pre-test odds of MDR-TB, the post-test odds from a positive test are sufficiently high to warrant MDR-TB treatment. Ideally, the GenoType® MTBDRplus assay should be performed on AFB-positive sputum specimens, rather than isolates, to take advantage of the more than three week acceleration in turn-around-time. Use of the assay on AFB-positive sputum and rapid commencement of MDR-TB treatment for patients with genotypic resistance has the potential to improve patient outcomes, reduce TB transmission, and reduce amplification of resistance to first-line drugs (e.g., ethambutol, pyrazinamide, and streptomycin) to which the isolate may not yet be resistant.
One weakness of the GenoType®
is the lack of 100% sensitivity for MDR-TB, attributable to the test's failure to detect all mutations that confer INH resistance. Two AFB-positive sputum specimens were confirmed as INH resistant by MGIT AST and sensitive by the GenoType®
. We were able to obtain interpretable sequence data of the promoter region for inhA
for one specimen which confirmed the result of the GenoType®
. INH resistance in this strain may be due to mutations in regions other than codon 315 of katG
or nucleic acid positions -15, -16 and -8 in the inhA
promoter region; or mutations in genes not represented on the test strip, such as ahpC-oxyR
, and ndh
]. However, a recent evaluation of 160 INH resistant isolates in Thailand found that 92.5% (148) of the gene mutations were found in codon 315 of katG
and the inhA
promoter and coding regions (127 and 22, respectively) [23
]. These findings support the utility of the GenoType®
assay for detecting the majority of genetic mutations conferring INH resistance in Thailand. As we were not able to obtain quality sequencing results for our second specimen, the reason for the discordance remains unclear. Advances in understanding the molecular epidemiology of INH resistance may contribute to improved test performance.
Our validation study was conducted using stored sputum specimens and isolates from TB patients in Thailand. Therefore, our turn around time using the GenoType® MTBDRplus for identification of RIF and INH resistance in AFB positive sputum specimens is based on the time from confirmed AFB positive smear results to interpretation of the GenoType® MTBDRplus. The turn around time for isolates is based on the time required to subculture strains of MTB, obtain growth, perform and interpret results of the GenoType® MTBDRplus assay. On average, results were available using GenoType® MTBDRplus on AFB-positive sputum specimens in 3 days and in 16 days for isolates; compared with 25 days using MGIT AST. Our laboratory uses the manual method for the GenoType® MTBDRplus assay and performs DNA extraction and PCR on the first day and hybridization on the second day. Interpretation of the test strips required an additional 1-2 days. Laboratories with adequate staff may reduce their turn around time by performing DNA extraction, PCR, and hybridization in one day, and by using the automated system (GT-Blot 48; Hain LifeScience GmbH, Germany). Turn around time from specimen collection to susceptibility result will be determined during the implementation phase of our study.
This study evaluated a large number of sputum specimens at a routine public health facility in a high-burden TB setting. Most evaluations of the GenoType®
assay have involved a relatively small number of specimens (usually less than 50) and were conducted in primarily academic facilities located in low TB burden, high income countries [9
]. The only other evaluation that involved a large number of samples from a public health facility in a high-burden TB country was done in South Africa [16
]. Test performance in 536 sputum specimens was similar to our findings, although the test showed the additional ability to detect MTB and drug-resistant MTB in smear-negative, culture-positive specimens. The major limitation of our study is that it involved a relatively small number of sputum specimens compared with the number routinely processed in the laboratory, which may have been an insufficient sample to detect differences in test performance between isolates and AFB-positive sputum.
In conclusion, the GenoType® MTBDRplus assay has been validated as a rapid and reliable first-line diagnostic test on AFB-positive sputum or MTB isolates for INH resistance, RIF resistance, and MDR-TB in Bangkok, Thailand. Whether it should be implemented into routine public health programmatic use, however, is not yet clear. Further studies are needed to evaluate its impact on treatment outcome and the feasibility and cost associated with widespread implementation.