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

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EVALUATION OF THE HIGH-THROUGHPUT REPETITIVE-SEQUENCE-BASED PCR
DIVERSILAB SYSTEM IN M. tuberculosis MOLECULAR EPIDEMIOLOGY STUDIES
Miotto Paolo, Baldan Rossella, Cirillo Daniela M.
Emerging Bacterial Pathogens Unit, San Raffaele Scientific Institute, Milan – ITALY
PCR-based methods have been developed to simplify and reduce the time required for genotyping M. tuberculosis by standard
approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-VNTR complemented
with spoligotyping has been proposed as an alternative. Repetitive-sequence-based PCR (rep-PCR) is useful for
generating DNA fingerprints of diverse bacterial species. Rep-PCR amplicon fingerprints represent genomic segments
lying between non-coding repetitive sequences. A commercial system (Diversilab, Biomerieux) that electrophoretically
separates rep-PCR amplicons on microfluidic chips, and provides computer-generated readouts of results has been
adapted for use with Mycobacterium species. The ability of this system to type M. tuberculosis was evaluated in comparison
with spoligotyping and MIRU-VNTR in two different panels.
First, we evaluated a 35 strains panel by MIRU-15 complemented with spoligotyping and Diversilab rep-PCR. Results
were analyzed with MIRU-VNTRplus database for spoligo-MIRU, and with Diversilab Software for rep-PCR using two
different algorithms (Pearson Correlation [PC] and Kullback-Leibler [KL]). Threshold for clusters was fixed at 98% of
similarity for rep-PCR. MIRU-15 showed a clustering rate of 11.4% whereas the rep-PCR reported a clustering rate of
28.6% (PC) and 17.1% (KL). The discriminatory power (Hunter-Gaston discriminatory index [HGDI]) for spoligo-MIRU-
15 resulted 0.983 whereas for rep-PCR was 0.978 (PC) and 0.983 (KL).
We also compared the two techniques on a panel composed by 8 closely related strains from a probable outbreak. In
this case the rep-PCR showed a discriminatory power of 0.571 (PC) and 0.679 (KL), compared to a HGDI of 0.643 for
spoligo-MIRU-15. Nevertheless, clustering rate for MIRU-15 was 50.0% whereas rep-PCR algorithms showed a clustering
rate of 100.0% (PC) and 62.5% (KL), respectively. To understand the meaning of the discrepancies still found between
spoligo-MIRU-15 and rep-PCR, analysis of epidemiological data for the clustered patients will be taken in consideration.
Preliminary data obtained by Diversilab suggest that the KL algorithm is more appropriate for M. tuberculosis typing analysis.
Nevertheless, even if rep-PCR results analyzed by KL algorithm showed a discriminatory power similar to MIRU-15,
clustering rate remains higher. This new technique could be useful for a routine use in clinical laboratories for real-time
genotyping and laboratory contaminations control.
European Society of Mycobacteriology | 30 th Annual Congress | July 2009 | Porto - Portugal
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