A significant knowledge gap exists concerning the geographical distribution of nontuberculous mycobacteria (NTM) isolation worldwide.To provide a snapshot of NTM species distribution, global partners in the NTM-Network European Trials Group (NET) framework (www.ntm-net.org), a branch of the Tuberculosis Network European Trials Group (TB-NET), provided identification results of the total number of patients in 2008 in whom NTM were isolated from pulmonary samples. From these data, we visualised the relative distribution of the different NTM found per continent and per country.We received species identification data for 20 182 patients, from 62 laboratories in 30 countries across six continents. 91 different NTM species were isolated. Mycobacterium avium complex (MAC) bacteria predominated in most countries, followed by M. gordonae and M. xenopi. Important differences in geographical distribution of MAC species as well as M. xenopi, M. kansasii and rapid-growing mycobacteria were observed.This snapshot demonstrates that the species distribution among NTM isolates from pulmonary specimens in the year 2008 differed by continent and differed by country within these continents. These differences in species distribution may partly determine the frequency and manifestations of pulmonary NTM disease in each geographical location. @ERSpublications Species distribution among nontuberculous mycobacteria isolates from pulmonary specimens is geographically diverse

Twenty (3.8%) of the 521 specimens gave no interpretable result. Whereas the sensitivity of the Xpert assay with tissue specimens was 69.0% (20 out of 29 culture-positive cases detected), 100% sensitivity was found with the urine and stool specimens. The combined sensitivity and specificity of the Xpert assay were calculated to be 77.3% and 98.2%, respectively.With an estimated 9 million new cases and 2 million deaths every year, tuberculosis (TB) remains a leading public health problem worldwide (6). In the majority of cases, the disease affects the lungs, but there are also not negligible numbers of cases (about 15%) with extrapulmonary involvement in lowincidence countries. There are even higher rates in high-incidence countries. HIV-coinfected TB patients often develop extrapulmonary involvement and may progress rapidly unless the infection is diagnosed and they are treated appropriately (8).Extrapulmonary infection with members of the Mycobacterium tuberculosis complex (MTBC) remains a diagnosis that is often difficult to establish, since the number of bacteria in extrapulmonary specimens is often lower than the number in pulmonary specimens. Furthermore, collection of extrapulmonary material often requires invasive procedures, and it is not easy to obtain additional samples. In recent times, attention has been devoted to new nucleic acid amplification diagnostic technologies, owing to their rapidity, sensitivity, and specificity.One of the latest systems, the GeneXpert MTB/RIF (Xpert) assay, was evaluated only recently in a large study with pulmonary specimens. The Xpert assay uses heminested real-time PCR to amplify an M. tuberculosis-specific sequence of the rpoB gene. To determine rifampin (RMP) resistance, the rifampin resistance-determining region of the rpoB gene is probed with molecular beacons (7). The assay can be carried out in a nearly fully automated manner, including bacterial lysis, nucleic acid extraction and amplification, and amplicon detection. The test runs on the GeneXpert platform (Cepheid, Sunnyvale, CA) using a disposable plastic cartridge with all required reagents (16).It could be shown that the Xpert assay detected pulmonary TB in all TB patients, including over 90% of smear-negative patients, with a high sensitivity of over 97% (2). The purpose of this study was to test the efficiency and reliability of the Xpert system for the detection of M. tuberculosis bacteria in extrapulmonary specimens and to compare it to conventional culture methods. MATERIALS AND METHODSSpecimens. All nonrespiratory specimens that were submitted to the German National Reference Laboratory for Mycobacteria (NRL) from May 2009 to August 2010 were included in the study. The specimens originated from patients with suspected M. tuberculosis or nontuberculous mycobacterial (NTM) infection on the basis of clinical criteria or to rule out these infections. Consecutive specimens were used, and specimens were not selected by the use of special criteria. In total, 521 specimens were tested. These comprised 91 urin...

The new GenoType MTBDRplus assay (Hain LifescienceGmbHThe worldwide increase in the rates of multidrug-resistant (MDR) tuberculosis (resistance to at least rifampin [RMP] and isoniazid [INH]) has made the timely identification of resistant Mycobacterium tuberculosis complex (MTBC) strains to achieve effective disease management and to prevent their spread extremely important. INH and RMP are the most important first-line antituberculosis drugs, and resistance to these drugs often results in treatment failures and fatal clinical outcomes (6, 7).Recently, nonradiometric fully automated systems that are used to screen for resistance and that have technical and safety advantages have been introduced (23). However, the time for resistance testing still is about 7 to 10 days, beginning from the time that a positive culture is obtained (23). The most rapid results could be achieved by direct testing of patient specimens by fast molecular methods (11, 25). These methods are based on the knowledge that resistance to RMP and INH in M. tuberculosis is most often attributed to mutations in the rpoB, katG, and inhA genes. By targeting mutations in the 81-bp "core region" of the rpoB gene, more than 95% of all RMPresistant strains can be detected (28). On the contrary, the mutations that cause INH resistance are located in several genes and regions. Between 50% and 95% of INH-resistant strains have been found to contain mutations in codon 315 of the katG gene (18,20,28), between 20 and 35% of INHresistant strains have been found to contain mutations in the inhA regulatory region (20,22,28), and an additional 10 to 15% of INH-resistant strains had mutations in the ahpC-oxyR intergenic region (13,22,28), often in conjunction with katG mutations outside of codon 315 (26). In a recent study, the strong statistical association between specific mutations in the katG, inhA, and ahpC genes and INH resistance could be confirmed (8). The authors estimated that a simple test for five molecular markers is able to detect 74% of INH-resistant (INH r ) isolates; 0 to 5% of the INH r M. tuberculosis isolates had mutations in the inhA open reading frame and 8 to 20% had mutations in the inhA promoter region (8,20,31).DNA strip assays targeting rpoB (INNO-LiPA Rif; Innogenetics N.V., Ghent, Belgium) or rpoB plus katG (GenoType MTBDR; Hain Lifescience GmbH, Nehren, Germany) were developed and evaluated for use with M. tuberculosis cultures and smear-positive specimens (1,11,16,17,29). The DNA strip assays are based on a multiplex PCR in combination with reverse hybridization. Either the omission of a wild-type band or the appearance of bands of DNA signals representing specific mutations indicates the existence of a resistant strain.In order to enlarge the capacity for the detection of drug resistance, the new GenoType MTBDRplus assay was developed. The assay has the ability to detect a broader variety of rpoB gene mutations and inhA gene mutations. By covering mutations in the regulatory region of inhA, it can be expected that additional INH-...

The new GenoTypeThe worldwide emergence of extensively drug-resistant tuberculosis (XDR TB, resistant at least to rifampin and isoniazid, a fluoroquinolone [FLQ], and one of the three injectable second-line drugs amikacin [AM], kanamycin [KM], and capreomycin [CM]) is a serious global health problem (20,25). In the World Health Organization fourth global report on drug resistance (25), it was documented that more than 45 countries have reported XDR cases. The actual incidence could be underestimated, because second-line drug susceptibility testing (DST) is not available in many countries. To avoid a progressive development similar to that observed in multidrugresistant TB (resistant at least to rifampin and isoniazid) worldwide, now having the highest rate ever at 5.3%, timely identification of resistant Mycobacterium tuberculosis complex (MTBC) strains is mandatory.Conventional DST for XDR strains is performed sequentially in a two-step procedure beginning with a culture and first-line drug testing, proceeding to further drug testing in the case of multidrug resistance. The time needed for testing, even with the most rapid liquid methods, is still around 1 week per test, constrained by the relatively slow growth of M. tuberculosis (15, 18). The required time can be shortened by fast molecular methods to 1 day per test (3,8,21). Since recently broad-based knowledge about mutations that cause resistance to ethambutol (EMB) and some second-line drugs is available. Resistance to FLQs, AM-CM, and EMB in M. tuberculosis is most frequently attributed to mutations in the gyrA, rrs, and embB genes, respectively. First investigations have shown that by targeting mutations in codons 90, 91, and 94 in the gyrA gene, approximately 70 to 90% of all FLQ-resistant strains can be correctly detected (2, 13, 24). Previous reports have linked mutations A1401G, C1402T, and G1484T in the rrs gene to AM, CM, and KAN resistance (1, 11, 12), each of them being responsible for a specific resistance pattern. Mutations G1484T and A1401G were found to cause high-level resistance to all drugs, whereas C1402T causes resistance to only CM and KAN.Furthermore, mutations at embB codon 306 are found in 30 to 68% of EMB-resistant clinical strains (16,17,26).PCR-based techniques provide new possibilities for the rapid diagnosis of first-and second-line drug resistance; however, not all mycobacterial laboratories have access to DNAsequencing facilities. As an alternative, DNA strip assays for the detection of rifampin (INNO-LiPA Rif. TB; Innogenetics, Ghent, Belgium) or rifampin and isoniazid resistance of M. tuberculosis in a single assay (GenoType MTBDR; Hain Lifescience, Nehren, Germany) are now commercially available. These assays have been evaluated for M. tuberculosis cultures and specimens (3,7,8,10,21). The DNA strip assays are based on PCR or multiplex PCR in combination with reverse hybridization. The existence of a resistant strain is signaled either by the omission of a wild-type band or the appearance of bands representing specific mut...

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) substantially challenges TB control, especially in the European Region of the World Health Organization, where the highest prevalence of MDR/XDR cases is reported. The current management of patients with MDR/XDR-TB is extremely complex for medical, social and public health systems. The treatment with currently available anti-TB therapies to achieve relapse-free cure is long and undermined by a high frequency of adverse drug events, suboptimal treatment adherence, high costs and low treatment success rates. Availability of optimal management for patients with MDR/XDR-TB is limited even in the European Region. In the absence of a preventive vaccine, more effective diagnostic tools and novel therapeutic interventions the control of MDR/XDR-TB will be extremely difficult. Despite recent scientific advances in MDR/XDR-TB care, decisions for the management of patients with MDR/XDR-TB and their contacts often rely on expert opinions, rather than on clinical evidence.This document summarises the current knowledge on the prevention, diagnosis and treatment of adults and children with MDR/XDR-TB and their contacts, and provides expert consensus recommendations on questions where scientific evidence is still lacking.