First Evaluation of an Improved Assay for Molecular Genetic Detection of Tuberculosis as Well as Rifampin and Isoniazid Resistances

Abstract

bThe commercially available line probe assay MTBDRplus 2.0 (Hain Lifescience, Nehren, Germany) was evaluated for its ability to detect Mycobacterium tuberculosis complex (MTBC) and mutations conferring resistance to rifampin (RMP) and isoniazid (INH) directly in smear-negative and smear-positive pulmonary clinical specimens under routine laboratory conditions. A total of 348 samples originating from Moldova, a high-incidence country for tuberculosis (TB), were investigated. Two hundred fiftyseven (73.9%) were smear negative, 12 samples were excluded, and 81 (23.3%) were smear positive. Two DNA extraction methods were applied. The National TB Reference Laboratory (NRL) is located at the Phthisiopneumology Institute and functions as the coordinating body for microbiological services for TB diagnostics in Moldova. The NRL performs microscopy, the culture of M. tuberculosis, drug susceptibility testing (DST) for first-and second-line drugs, molecular resistance testing, the training of laboratory network staff, the standardization of laboratory methods for the country, the epidemiological survey of TB drug resistance, and external quality control for other laboratories. The laboratory annually performs 75,000 investigations for TB diagnostics. The laboratory participates in external quality assurance (EQA) (Supranational Reference Laboratory [SNRL], Borstel, Germany). The last results of external quality control (EQC) demonstrated 100% coincidence of DST for first-line drugs and 96% for second-line drugs.The rapid detection of MDR-TB ensures treatment with appropriate drugs, thereby reducing morbidity, mortality, economic costs, the further transmission of infection, and the emergence of XDR strains (3).Line probe assays (LPA) for the fast and reliable detection of M. tuberculosis complex (MTBC) and resistance to RMP and INH have recently been endorsed by the WHO (19,21). The commonly used MTBDRplus 1.0 (Hain Lifescience, Nehren, Germany), which detects RMP and INH resistance, was evaluated with culture samples and smear-positive specimens in a variety of settings (6, 9, 10). As the sensitivity for the detection of mycobacteria by microscopy is known to be low (16), a substantial number of bacteria are not detected, and culture methods have to be performed to detect mycobacteria sufficiently in smear-negative samples. To overcome this limitation, the MTBDRplus 2.0 test (Hain Lifescience, Nehren, Germany) was further improved to detect mycobacteria and their resistance status against RMP and INH in smear-positive, smear-negative, and culture-positive specimens.Here, we describe the evaluation of this new MTBDRplus 2.0 test for smear-positive and smear-negative pulmonary specimens in a high-TB-prevalence country.

Molecular detection of rifampicin resistance in multidrug-resistant Mycobacterium tuberculosis strains from North-Eastern Romania

“…However, this percentage is greater than those reported in Republic of Moldova (0.9%) and Hungary (6.9%) (29,28). Other studies also mention low frequencies for this type of mutation in countries like: Italy (6.5%), South Africa (8.6%), China (10%), Ukraine (12.5%), Germany (13.6%) and Turkey (17.1%) (10,21,22,24,26,27).…”

“…This is in accordance with reports from other regions, such as China and South Africa (63.2% and 70.5%, respectively) (21,22) and some European countries: Turkey -56.1%, Italy -60.4%, Bulgaria -65%, Ukraine -73%, Germany -75.7% (10, 24 -27). However, our study showed great differences from our neighboring countries, Hungary with a lower frequency (31%) and Republic of Moldova with a higher frequency (86.8%) (28,29).…”

“…Mutation in codon 516 occurred at a rate of 12.76%, higher than in neighboring countries: Republic of Moldova (4.4%) and Ukraine (8%), but much smaller than in Hungary (37.9%) (29,26,28). Other studies show that this mutation occurs with frequencies varying greatly from 2% in China and 2.9% in Germany (representing the rarest mutation among the three) to 9.6% in South Africa (21,22,27).…”

Multidrug-resistant and extensively drug-resistant tuberculosis: a review of current concepts and future challenges

“…An early evaluation study on smear-negative samples indicated 90.7% sensitivity and 96% specifi city for rifampicin resistance, and 93.5% sensitivity and 82.3% specifi city for isoniazid resistance. 16 The use of the assay is technically more demanding and has a longer turn-around time compared with Xpert ® MTB/Rif, but is a good alternative test for isoniazid and rifampicin resistance in reference laboratories.…”

Rapid Detection of Isoniazid Resistance in Mycobacterium tuberculosis Isolates by Use of Real-Time-PCR-Based Melting Curve Analysis

“…However, only a limited number of assays have so far entered clinical laboratories. One example is Genotype MDRTBplus (Hain, Germany), which could detect both RIF and INH resistance mutations in a single assay and has been extensively validated (3)(4)(5)(6)(7). However, this reverse hybridization-based assay requires multiple post-PCR manipulations, and the color readout is subject to visual estimation.…”