Fingerprinting of Mycobacterium tuberculosis isolates by MIRU-VNTR genotyping and detection of isoniazid resistance by real-time PCR

Abstract

Introduction. Tuberculosis (TB) is a great public health problem in developing countries such as Egypt. Genotyping of Mycobacterium tuberculosis isolates has a prominent role in the field of TB prevention. Aim. This study aimed to evaluate real-time PCR using Minor Groove Binder (MGB) probes and to identify circulating lineages/sub-lineages of M. tuberculosis and their transmission patterns. Hypothesis. We hypothesize that MIRU-VNTR technique is efficient in identifying circulating M. tuberculosis lineages in Egypt. Methodology. Fifty sputum specimens positive for acid-fast bacilli were included. Isoniazid (INH) resistance was detected using the 1 % proportion method. Real-time PCR using MGB-probes was used for simultaneous detection of TB infection and INH resistance. Partial sequencing of the katG gene was used to confirm INH resistance results. A standard 15 Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (15-MIRU-VNTR) approach was used for genotyping through the MIRU-VNTRplus online platform. Results. Only seven specimens showed phenotypic resistance to INH. M. tuberculosis was detected in all samples, while a mutation in the katG gene codon 315 was detected only in five samples, which were also phenotypically INH-resistant. Sequencing of the katG gene showed codon 315 mutation genotypically and phenotypically in the five INH-resistant isolates. Molecular genotyping of M. tuberculosis isolates revealed that the majority of isolates (26/50, 52 %) belonged to the S family of lineage_4. A low clustering rate (2 %) was observed among our isolates. According to the Hunter-Gaston Discriminatory Index (HGDI), 11 MIRU-VNTR loci were highly or moderately discriminative, while four loci were less polymorphic. Conclusion. MIRU-VNTR genotyping revealed a low clustering rate with a low recent transmission rate of M. tuberculosis strains in Alexandria, Egypt.