European Society of Mycobacteriology - Instituto Nacional de Saúde ...

PP-76
THE UTILITY OF MOLECULAR TESTING IN
ROUTINE MYCOBACTERIOLOGY DIAGNOSIS
Fanourios Kontos, Loukia Zerva.
Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.
Objective
Molecular methods increasingly replace phenotypic tests in Mycobacteriology. This study describes a “molecular” strategy
that was developed for routine testing of clinical samples and isolates with the goal of shortening turnaround time
(TAT) and providing accurate results.
Methods
All samples submitted for mycobacterial cultures (from 12/2006 to 3/2009) were processed and cultured by standard
methodology. The first smear (+) specimen of any patient and every first (+) culture were tested by an in-house IS6110-
PCR in order to differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria
(NTM) (TAT 4 hours). IS6110-PCR (+) specimens or cultures were tested by Genotype MTBDRplus (Hain-LIFESCIENCE)
for MTBC species confirmation and detection of Isoniazid and Rifampicin resistance (TAT 2 days). Subsequent culture
(+) specimens from the same patient were tested by Accuprobe MTBC (Biomerieux) (TAT 2 hours). The susceptibility
testing of MTBC isolates was performed by MGIT960 (Becton Dickinson). If a (-) result was obtained by the IS6110-
PCR, identification of isolates proceeded using both Genotype Μycobacterium CM and AS (Hain-LIFESCIENCE) (TAT
two days). For NTM species confirmation a PCR-RFLP analysis of the hsp65 gene was applied (TAT 3 days); additionally,
sequencing of the 16S rRNA gene (TAT 5 days) represented the reference identification method for selected isolates.
Results
Out of 4.000 specimens, 160 were culture positive (4%) including 85 MTBC positives (63.5% acid fast stain [AFS] positive)
and 75 NTM positives (26.7% AFS positive). Five samples contained more than one NTM species, which were identified
only by molecular testing. There was complete agreement between all molecular identification methods; however
16S rRNA sequencing was more informative (examples: M. fortuitum and M. lentiflavum, M. celatum). All MTBC isolates
were Rifampicin susceptible, two were high- and one low-level Isoniazid resistant by MGIT960; only the latter was not
recognized by Genotype MTBDRplus.
Conclusions
Focusing on molecular methodology resulted in fast and accurate final reporting. The low incidence of MTBC positivity
among tested specimens (2,1%) justified the decision not to use indiscriminately a direct molecular test for tuberculosis
diagnosis. The frequent occurrence of NTM (48,5% of all culture positive samples) necessitates direct molecular testing
of all AFS (+) specimens.
148 ESM 2009