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Evaluation of real-time polymerase chain reaction for direct identification of mycobacteria in clinical samples and liquid culture bottles

The direct identification of mycobacteria with polymerase chain reaction (PCR) is becoming a very interesting issue, especially regarding its efficacy and feasibility. Reduced identification rate when PCR is applied on acid-fast bacilli (AFB) smear-positive sputum samples of low positivity (AFB1+) was recently noticed. The aim of our study was to determine would it be reasonable to perform PCR only on smear-positive clinical samples of higher positivity (AFB 2+ and AFB 3+). Furthermore, we wanted to determine is it more convenient to use PCR on smear-positive liquid culture bottles because of larger amount of present mycobacteria than in a clinical samples. The study was conducted at the University Hospital Centre Zagreb, Zagreb, Croatia. We performed PCR for mycobacteria on smear-positive clinical samples and diagnostic yield was evaluated according to the AFB score (AFB 1+, AFB 2+, AFB 3+). PCR was also done on smear-positive Mycobacterium Growth Indicator Tube (MGIT) culture bottles. LightCycler real-time PCR assay (LightMix®, TIBMOLBIOL, Berlin, Germany) to discriminate between Mycobacterium tuberculosis complex (MTBC) and other Mycobacterium spp. was used. Conventional auramin stain and culture on solid Lowenstein-Jensen medium was also done. Fifty-two smear-positive clinical samples included 33 sputum samples, 12 bronchoalveolar lavage (BAL) samples, 6 bronchial aspirates and 1 pleural fluid. Among them 51 MTBC and 1 nontuberculous mycobacterium (NTM) was isolated by conventional culture. The PCR had 100% identification rate for AFB 2+ and AFB 3+ samples, but only 14 out of 27 (52%) AFB 1+ samples were identified by PCR (Table 1). Among the 28 smear-positive MGIT bottles, 21 MTBC and 7 NTM isolates including M. gordonae (n=6) and M. intracellulare (n=1) were identified by conventional culture. PCR had 100% identification rate for smear-positive MGIT bottles. PCR efficacy is related to the mycobacterial burden in tested clinical sample. It is excellent and rapid method for identification of mycobacteria grown in MGIT culture bottles. PCR used on clinical samples is not reliable enough to rule out the diagnosis of tuberculosis in paucibacillary pulmonary tuberculosis, but should be used on clinical samples with higher AFB positivity. With this approach financial and practical demands regarding the influence of PCR on diagnostic process and choice of treatment would be justified.

tuberculosis; real-time polymerase chain reaction; clinical samples; liquid culture bottles

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