ABSTRACT
BACKGROUND: Rapid and accurate techniques are always welcomed for the detection of resistant strains of Mycobacterium tuberculosis MTB. OBJECTIVES: The objective of this study is to evaluate the pyrosequencing technology for the detection of MTB resistance to Rifampicin (RIF) and Isoniazid (INH) in Syrian and Lebanese clinical strains; 66 strains resistant to INH, among them 56 resistant also to RIF, were tested. METHODS: Four pyrosequencing assays were optimized and applied to the following loci: rpoBrpoB RIF resistance-determining region, katG, the promoter regions of inhA and ahpC-oxyR intergenic region. RESULTS: The prevalence of mutations on codon 315 of the katG gene, inhA and ahpc-oxyR were 42.4%, 21.2% and 9.0%, respectively, which make an overall sensitivity of 72.6% for INH resistance. All RIF-resistant strains contained at least one non-synonymous codon change in the sequenced rpoB region (507-533) relative to the ATCC reference strain. The RIF drug resistance region (RRDR) sequencing identified 96 modified codons representing 34 different mutations. CONCLUSIONS: The high sensitivity and the short turnaround time combined with multilocus sequencing of several isolates in parallel make pyrosequencing an attractive method for drug resistance screening for MTB.
Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Bacterial/drug effects , Drug Resistance, Bacterial/genetics , Isoniazid/pharmacology , Mycobacterium tuberculosis/drug effects , Rifampin/pharmacology , Tuberculosis, Multidrug-Resistant/microbiology , Codon , DNA Mutational Analysis , Genes, Bacterial , Humans , Lebanon/epidemiology , Microbial Sensitivity Tests , Mutation, Missense , Mycobacterium tuberculosis/genetics , Public Health , Refugees , Sensitivity and Specificity , Syria/epidemiology , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/epidemiologyABSTRACT
Genotyping Mycobacterium tuberculosis in Lebanon on the national level may be beneficial for assessing patients and monitoring the therapeutic response to DOTS. This study aimed to characterize the spoligotypes of clinical isolates of M. tuberculosis patients collected between April 2004 and October 2005 from all Lebanese provinces. Isolates (n = 60) were cultured and identified by their biochemical characteristics. DNA extracts of these samples were amplified by PCR and genotyped by spoligotyping. Thirteen (13) patterns of M. tuberculosis complex family strains were identified: 41.6% of the strains belonged to the T 1 family, 25.0% to LAM 9, 10.0% to Haarlem 3, 3.3% to each of CAS, LAM 8, BCG and Family 36 and 1.7% to each of Haarlem 1, LAM 10, S, M. africanum, X 1 and T 3 families. The noticeable absence of Beijing and East African Indian families was not consistent with the patterns reported in neighbouring countries. A more inclusive study of the Lebanese population is necessary to accurately identify most of the prevailing families in the country.
Subject(s)
Bacterial Typing Techniques/methods , Genotype , Mycobacterium tuberculosis/classification , Tuberculosis, Pulmonary/microbiology , Female , Humans , Lebanon/epidemiology , Male , Mycobacterium tuberculosis/isolation & purification , Polymerase Chain Reaction/methods , Retrospective Studies , Sputum/microbiology , Tuberculosis, Pulmonary/epidemiologyABSTRACT
Genotyping Mycobacterium tuberculosis in Lebanon on the national level may be beneficial for assessing patients and monitoring the therapeutic response to DOTS. This study aimed to characterize the spoligotypes of clinical isolates of M tuberculosis patients collected between April 2004 and October 2005 from all Lebanese provinces. Isolates [n = 60] were cultured and identified by their biochemical characteristics. DNA extracts of these samples were amplified by PCR and genotyped by spoligotyping. Thirteen [13] patterns of M tuberculosis complex family strains were identified: 41.6% of the strains belonged to the T 1 family, 25.0% to LAM 9,10.0% to Haarlem 3, 3.3% to each of CAS, LAM 8, BCG and Family 36 and17% to each of Haarlem 1, LAM 10, S, M. africanum, X 1 and T 3 families. The noticeable absence of Beijing and East African Indian families was not,consistent with the patterns reported in neighbouring countries. A more inclusive study of the Lebanese population Is necessary to accurately identify most of the prevailing families in the country
