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

The purpose of this study was to present a retrospective analysis of the frequency of nontuberculous mycobacteria (NTM)-related pulmonary infections among the AFB-positive and/or culture-positive patients in the Warsaw region who were suspected of tuberculosis (TB) and hospitalized in the university hospital between 1999 and 2005. All the AFB-positive pulmonary samples were examined with a molecular method using the Amplicor MTB test (Roche) for detection of Mycobacterium tuberculosis complex, and all mycobacterial isolates were speciated by high performance liquid chromatography (HPLC) analysis of mycolic acids. Patients who met clinical, radiological, and bacteriological criteria of mycobacteriosis were classified according to the American Thoracic Society (ATS) guidelines for diagnosis of NTM related disease. Among the 445 smear-positive or/and culture-positive patients, 142 subjects (31.9%) were found to be infected with M. tuberculosis. Among 303 non-TB patients, mycobacteriosis was found in 27 (8.9%) subjects. The frequency of NTM-related lung disease as compared to the bacteriologically-confirmed lung TB was estimated at 1:5. The rapid, precise methods of NTM speciation are necessary for progress in diagnostics of NTM related diseases.

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Introduction: The GenoType Mycobacterium CM and the GenoType Mycobacterium AS (HAIN Lifescience, Germany) were evaluated for the ability to differentiate mycobacterial species of clinical isolates. Serial use of the both assays is aimed to identify 38 different molecular patterns, of which 24 patterns can be assigned to single species, 10 patterns are allocated to two or more Mycobacterium species, and 4 patterns correspond to Mycobacterium species and gram-positive bacteria with a high G + C content. The analysis of mycolic acids by high pressure liquid chromatography (HPLC) was the reference method. Materials and methods: A set of 127 nontuberculous mycobacterial isolates on Loewenstein-Jensen media, derived from different patients between 1999 and 2007, was analyzed. The strains were primary classified by HPLC following the diagnostic procedure, and retyped by GenoType Mycobacterium CM/AS. Results: In total, results obtained by both methods were interpretable for 113 strains. Concordant results were obtained for 105 (93%) mycobacterial strains. One out of 8 incorcondantly classified strains, which was classified as M. abscessus/M. chelonae by HPLC, displayed a pattern of M. tuberculosis complex by a molecular method. Eleven clinical strains were differentiated only by one of used methods, either by HPLC (6 strains) or by GenoType CM/AS (5 strains). Three strains were not classified at all. Conclusions: Our results show that the GenoType Mycobacterium CM/AS system represents a useful tool to identify mycobacterial clinical isolates. The molecular system is as rapid and reliable as the HPLC, but much easier to perform and more friendly for the environment.

Introduction: The GenoType system (HAIN Lifescience, Germany) offers new perspectives of detecting the tuberculous and non-tuberculous mycobacteria at the molecular level. The system compromises five independent tests that could be performed either on direct specimens or isolated strains, to identify the strains and test the resistance against rifampin and isoniazid. Up to now, non GenoType test was applied in Poland. The aim of the study was an evaluation the accuracy of GenoType MTBC test in speciation of the clinical isolates, previously classified as M. tuberculosis complex by HPLC analyze of mycolic acids. Material and methods: 161 clinical isolates, derived from the TB patients hospitalized in the Warsaw Medical University Hospital between 1999 and 2007 were assayed. Results: On the basis of the hybridization patterns, all 161 studied strains were identified as M. tuberculosis/M. canettii. Conclusions: 1. The GenoType MTBC test (HAIN Lifescience, Germany) precisely recognizes M. tuberculosis complex. The 100% accordance in speciation of M. tuberculosis by the GenoType MTBC test as compared to HPLC method was demonstrated. The GenoType MTBC test can replace HPLC in detection of tuberculous mycobacteria in clinical isolates. 2. As the GenoType MTBC test performs well, the other tests of GenoType system may be considered to be verified indiagnostic procedure of mycobacterial infection.

Mycobacterium celatum is nontuberculous mycobacterium which is rarely pathogenic in human. We describe the case of M. celatum infection in immunocompetent patient. Caucasian 64-years old female patient was referred to the outpatient service because of pulmonary hemorrhage, cough and fever up to 39oC. She reported previous pulmonary tuberculosis (TB) 3 years ago. At presentation the physical examination was unremarkable.  Chest computed tomographic scan showed multifocal bronchiectases and upper lobes consolidations suggestive. Empiric antibiotic therapy with amoxicillin was undertaken. At the same time the consecutive sputum samples were immediately analyzed. Smears of six from eight sputum specimens were positive for acid-fast bacilli by Ziehl-Neelsen staining. Culture from each specimen produced slow-growing mycobacterium, identified as M. celatum by mycolic acid analysis with high performance liquid chromatography. The treatment with clarithromycin and ciprofloxacin was continued for 18 months with clinical improvement. Conclusion- we present the accuracy of microbiological diagnosis of M. celatum in patient with normal immune status, which allowed successful treatment. M. celatum infection was reported in HIV infected patients or with the history of pulmonary TB prior to mycobacterial infection. Also in our patient the TB in anamnesis may indicate “hidden immunodeficiency”.

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The polymorphism of the short fragment of the heat shock protein 65 encoding gene was evaluated by the PCR–RFLP technique described by Telenti and further developed by Devallois for identification of mycobac-terial species in routine laboratory work. We analysed 58 strains representing 25 different mycobacterial species (24 reference strains and 34 clinical isolates). The results obtained by PCR-RFLP and HPLC identification techniques were highly concordant The results were compatible for 87.5% (21/24) reference strains and for 97.1% (33/34) clinical isolates. The PCR–RFLP method allowed for accurate identification mycobacterial species, especially pathogenic strains. Restriction patterns obtained for 25 species of Mycobacteriaceae genus could help in constructing the data base and algorithms used in routine laboratory practice.