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New rapid molecular test detects MDR-TB mutations

A new rapid molecular test has shown the ability to detect, with reasonable certainty, mutations associated with multidrug-resistant tuberculosis (MDR-TB), an international team of researchers have reported.

In head-to-head comparisons against the gold standard tests for TB drug susceptibility, the cartridge-based, automated assay was able to identify mutations associated with resistance to isoniazid, fluoroquinolones, and aminoglycosides with relatively high sensitivity and specificity. Detection of resistance to fluoroquinolones and aminoglycosides is important because they are core components of the World Health Organisation (WHO)-endorsed shortened treatment regimen for uncomplicated MDR-TB.

Incorrect treatment of MDR-TB is associated with higher morbidity and mortality and ongoing transmission of the disease, the authors note, and tests that can rapidly identify patients who are eligible for the shortened treatment are needed, as current culture-based drug-susceptibility tests can take 2 to 6 weeks and require substantial laboratory equipment and training. The investigational assay tested in the study, which analyses sputum samples from TB patients for genetic mutations, is able to produce results in just over 2 hours.

The results of the study could be a significant development in global efforts to combat MDR-TB. Xpert MTB/RIF, a rapid molecular test currently used to simultaneously detect the presence of Mycobacterium tuberculosis and identify resistance to the first-line TB drug rifampin, has greatly enhanced the ability to detect drug-resistant TB cases, but it is not able to identify resistance to other TB drugs.

"I think it's an important study," said Dr David Dowdy, an infectious disease epidemiologist at Johns Hopkins Bloomberg School of Public Health who was not involved in the study. "It shows that we now have the ability to test for most second-line drugs with good accuracy, directly from sputum, and with results being available in just a couple of hours. It will be very helpful for patients with drug-resistant TB, to make sure that they are started on the right treatment from day one."

In the prospective study, the researchers tested the diagnostic accuracy of the assay in 308 patients from South Korea and China who had symptoms of pulmonary TB. The results of the molecular test were compared with the results of phenotypic culture-based drug-susceptibility testing and DNA sequencing, which also looks for mutations associated with drug resistance.

M tuberculosis resistance to isoniazid, fluoroquinolones (ofloxacin and moxifloxacin), and aminoglycosides (amikacin and kanamycin) is associated with 25 mutations in six genes and promoter regions, the authors note.

Phenotypic drug-susceptibility testing identified 194 of 308 patients (63%) as having infections resistant to one or more TB drugs, including 55 with MDR-TB (defined as TB that is resistant to at least rifampin and isoniazid), 54 with MDR-TB that was also resistant to fluoroquinolones or aminoglycosides, and 39 with extensively drug-resistant TB (defined as MDR-TB with additional resistance to any fluoroquinolone and at least one of the three second-line injectable drugs—amikacin, kanamycin, or capreomycin).

Compared with the culture-based test, the sensitivity and specificity of the investigational assay for detecting resistance were 83.3% and 99.2% for isoniazid, 88.4% and 96.6% for ofloxacin, 87.6% and 94.3% for moxifloxacin, 71.4% and 98.4% for kanamycin, and 70.7% and 99.6% for amikacin.

When compared with DNA sequencing, the sensitivity and specificity of the molecular test for the detection of resistance mutations were even higher: 98.1% and 100% for isoniazid, 95.8% and 100% for both fluoroquinolones, 92.7% and 99.6% for kanamycin, and 96.8% and 100% for amikacin.

This latter finding is important, the authors note, because the WHO has set a target for the diagnostic sensitivity and specificity of next-generation molecular drug-susceptibility tests of 95% and 98%, using DNA sequencing as the reference standard. When compared with DNA sequencing in this study, the investigational molecular assay met the sensitivity target for isoniazid, fluoroquinolones, and amikacin but missed the sensitivity target for kanamycin. The specificity target was met for all drugs.

Furthermore, the assay was able to identify 48 of 53 patients (90.6%) who had TB without resistance to fluoroquinolones or aminolgycosides and therefore would be microbiologically eligible for the shortened treatment regimen, and 81 of 92 patients (88%) with TB that was resistant to one or both of those drugs. That means those patients would be properly identified as inappropriate candidates for the shortened treatment regimen, potentially sparing antibiotic doses.

"We speculate that a positive test result could be used to triage patients away from the new shortened treatment regimen for multidrug-resistant tuberculosis and toward treatment centers with the capacity for comprehensive drug-susceptibility testing and with experience treating highly drug-resistant tuberculosis," the authors write.

Background: Fluoroquinolones and second-line injectable drugs are the backbone of treatment regimens for multidrug-resistant tuberculosis, and resistance to these drugs defines extensively drug-resistant tuberculosis. We assessed the accuracy of an automated, cartridge-based molecular assay for the detection, directly from sputum specimens, of Mycobacterium tuberculosis with resistance to fluoroquinolones, aminoglycosides, and isoniazid.
Methods: We conducted a prospective diagnostic accuracy study to compare the investigational assay against phenotypic drug-susceptibility testing and DNA sequencing among adults in China and South Korea who had symptoms of tuberculosis. The Xpert MTB/RIF assay and sputum culture were performed. M. tuberculosis isolates underwent phenotypic drug-susceptibility testing and DNA sequencing of the genes katG, gyrA, gyrB, and rrs and of the eis and inhA promoter regions.
Results: Among the 308 participants who were culture-positive for M. tuberculosis, when phenotypic drug-susceptibility testing was used as the reference standard, the sensitivities of the investigational assay for detecting resistance were 83.3% for isoniazid (95% confidence interval [CI], 77.1 to 88.5), 88.4% for ofloxacin (95% CI, 80.2 to 94.1), 87.6% for moxifloxacin at a critical concentration of 0.5 μg per milliliter (95% CI, 79.0 to 93.7), 96.2% for moxifloxacin at a critical concentration of 2.0 μg per milliliter (95% CI, 87.0 to 99.5), 71.4% for kanamycin (95% CI, 56.7 to 83.4), and 70.7% for amikacin (95% CI, 54.5 to 83.9). The specificity of the assay for the detection of phenotypic resistance was 94.3% or greater for all drugs except moxifloxacin at a critical concentration of 2.0 μg per milliliter (specificity, 84.0% [95% CI, 78.9 to 88.3]). When DNA sequencing was used as the reference standard, the sensitivities of the investigational assay for detecting mutations associated with resistance were 98.1% for isoniazid (95% CI, 94.4 to 99.6), 95.8% for fluoroquinolones (95% CI, 89.6 to 98.8), 92.7% for kanamycin (95% CI, 80.1 to 98.5), and 96.8% for amikacin (95% CI, 83.3 to 99.9), and the specificity for all drugs was 99.6% (95% CI, 97.9 to 100) or greater.
Conclusions: This investigational assay accurately detected M. tuberculosis mutations associated with resistance to isoniazid, fluoroquinolones, and aminoglycosides and holds promise as a rapid point-of-care test to guide therapeutic decisions for patients with tuberculosis.

Yingda L Xie, Soumitesh Chakravorty, Derek T Armstrong, Sandra L Hall, Laura E Via, Taeksun Song, Xing Yuan, Xiaoying Mo, Hong Zhu, Peng Xu, Qian Gao, Myungsun Lee, Jongseok Lee, Laura E Smith, Ray Y Chen, Joon Sung Joh, YoungSoo Cho, Xin Liu, Xianglin Ruan, Lili Liang, Nila Dharan, Sang-Nae Cho, Clifton E Barry III, Jerrold J Ellner, Susan E Dorman, David Alland

[link url=""]Centre for Infectious Disease Research and Policy/University of Minnesota material[/link]
[link url=""]New England Journal of Medicine abstract[/link]

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