| Abstract |
The present report evaluated all the samples referred to the Mycobacteriology laboratory of the University Hospital of Heraklion from January 2000 to December 2007. The purpose of the study was to determine methods for rapid and reliable diagnosis of mycobacterioses, especially tuberculosis, to evaluate the combined use of conventional and molecular techniques and to analyze demographic features of these infections. A total of 9542 consecutive specimens were stained with Ziehl-Neelsen stain, cultured on Lowenstein-Jensen (L-J) slants as well as in the BacT/Alert 3D system. Furthermore, three of the most recent commercial molecular assays (GenProbe, bio-Merieux, GenoType Mycobacterium Direct and Genotype MTBDRPlus, Hein-lifescience) were conducted and analyzed for direct detection of MTBC in respiratory and non-respiratory specimens. Their performance and usefulness, not only on smear-positive samples, but also on selected smear-negative specimens, for whom the degree of suspicion of TB was high, was emphasized. Mycobacterium tuberculosis and non-tuberculous mycobacteria (NTM) were identified by Accuprobe (bio-Merieux) and GenoType Mycobacterium CM, AS and MTBC (Hein-lifescience). For identification and confirmation of new and rare strains, the two “gold” standard methods: sequencing of 16SrRNA and analysis of the heat-shock 65 kd gene by PCR-Restriction Fragment Length Polymorphism (RFLP) were used. Resistance to first-line antituberculous drugs was
33
determined by the conventional proportional method on L-J medium and by the automated BacT/Alert 3Dsystem.Two new molecular assays, GenoType MTBDRPlus and GenoType MTBDRsl (Hein-lifescience), for rapid detection of genes involved in resistance in MTBC isolates were evaluated. Particularly, the GenoType MTBDRPlus detects mutations conferring resistance to rifampicine (rpoB) and isoniazid (katG and inhA), while the MTBDRsl permits the detection of resistance to fluoroquinolones, aminoglycosides and ethambutol by testing mutations in the genes gyrA, rrs and embB respectively, Drug sensitivity testing for non-tuberculous mycobacteria (NTM) was performed by the E-test method. A total of 320 mycobacterial strains were isolated including 179 MTBC and 141 NTM strains belonging to 19 mycobacterial species (51 rapid and 90 slow-growing). From the 179 MTBC only one was identified as M. bovis and 178 as M. tuberculosis. The analysis of the 51 rapid-growing mycobacteria revealed: 26 M. fortuitum, 9 M. peregrinum, 7 M. chelonae, 5 M, abscessus, 1 M. thermoresistible, 2 M. mucogenicum and 1 M.europaen. The first isolation of M. thermoresistible in Europe and seventh globally, is reported. The mycobacterium strain GN-10643, together with two similar strains isolated in Europe, establish a new strain named M. europaeum. The 90 slow-growing mycobacteria consisted of 24 M. avium, 4 M. intracellulare, 1 M. scrofulaceum, 4 M. kansasii, 1 M. marinum, 1 M. malmonse, 1 M. xenopi, 10 M. lentiflavum, 29 M. gordonae, 2 M. arupense, 12 M. GN01 (M. herakleense),
1M. GN-9680 (M. hellenicum). A new strain is reported and named M. herakleense sp. nov. because of the place of its first isolation and discovery. A second new one strain M. GN-9680 was isolated and studied. Furthermore, a third new, rapid-growing mycobacterium strain, discovered during the study period was named M. create (M. GN-
34
11124). Also, two rare, non-tuberculous mycobacteria M. arupense were isolated and identified (M. GN-9722and M. GN-10546). The sequences of the new strains and of the rare ones were deposited in the GenBank. The three new species were also deposited to International Public Collections for preservation and investigation. Drug susceptibility testing of all non-tuberculous mycobacteria was performed using E-test method (AB Biodisk, Solna, Sweden), according to the manifacturer’s instructions. A total of 132 (73,7%) positive samples were obtained from native Greeks and 47 (26,3%) from immigrants originated from 11 countries (Bulgaria 15, Romania 10, Albania 9. Georgia 4, Ukraine 2, India 2, and Finland, Kenya, Uganda and Nigeria one isolate each. One additional patient was of Kurdish origin).Drug susceptibility testing (DST) showed that 146 (81,6%) isolates were susceptible to all anti-tuberculosis agents, while 33 (18,4%) were resistant to one or more drugs, and only one (0,56%) was found MDR. Resistant to isoniazid (INH) increased significantly over the two study periods from 2,9% over the years 2000-2003 to 16% over the period 2004-2007 (p=0,002). No significant increase in the rate of resistance to rifampicin ( RMP), ethambutol (EMB), streptomycin (SM), piraziamid (PYR) or para-amino-salicylic acid (PAS) was seen. Resistance to ≥2 anti-tuberculous drugs rose over the two study periods from 2,9% to 12%, a significant increase (p=0,03). The overall INH-resistance rate of M. tuberculosis strains isolated from Greeks was 5,3% (7/132), while among immigrants was 17% (8/47, p=0,02). No significant difference in RMP and EMB was seen between Greeks and immigrants. On the other hand, SM resistance was significant higher in immigrants (9/47 versus 8/132, p=0,017), and this was the case with PAS (0/132 in Greeks versus 3/47 in immigrants, p=0,017). The concordance rates of DST and GenoType MTBDRPlus assay for INH and
35
RMP resistance were 93,3% (14/15) and 100% (1/1), respectively. Strengthened surveillance is necessary to control TB in our region, nevertheless the low level of MDR TB, because of the increasing rate of the general resistance.
|
Search
