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.

The 1410 bp DNA region (gCnA) encoding glutamine synthetase I (GSI) from Sfrepfumyces viridochrumugenes was amplified by PCR, cloned and sequenced. The molecular mass of the deduced GSI protein (469 residues) was determined to be 50 kDa. The DNA region showed 90 % nucleotide identity with the Sfrepfomyces coeliculor A3 (2) glnA gene, but no significant nucleotide sequence similarity with the gfnZZ(GSI1) gene of S. vividochromugenes. The chromosomal g h A and ghZZ genes of S. viridochrumugenes were disrupted by site-specific mutagenesis. Neither glnA nor glnZZ single mutants required glutamine for growth and both were normal in their sporulation. Measurement of the GS activity in cultures grown with Merent nitrogen sources revealed that GSI (heat-stable) and GSII (heat-labile) were always expressed together, with GSI as the predominant activity. It could be proposed that GSI, but not GSII is inactivated by adenylylation under conditions of nitrogen excess. GSI and GSII activities are inhibited by amino acids and by nucleotides.

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