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

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.

Profile of drug-resistant-conferring mutations among new and previously treated pulmonary tuberculosis cases from Aligarh region of Northern India.

Background: The prevalence of multidrug-resistant-tuberculosis (MDR-TB) among new and previously treated cases is increasing worldwide as well as in India. Rapid detection of MDR-TB allows the establishment of an effective treatment regimen; minimizes the risk of further resistance, and limits the spread of drug-resistant strains. Early diagnosis of MDR-TB is the need of the hour in high-TB burden countries like India, and GenotypeMTBDRplus is quite sensitive and specific in determining the molecular resistance in drugs such as rifampicin and isoniazid. Methods: The present study was done for molecular detection of rifampicin and isoniazid resistance and resistance patterns among MDR-TB suspects and comparison of resistance patterns among new and previously treated cases by GenoType® MTBDRplus Line Probe Assay. A total of 1268 sputum samples of MDR-TB suspects were subjected to fluorescent microscopy. Fluorescent microscopy positive samples were subjected to GenoType® MTBDRplus (HAIN Lifescience) assay. Results: MDR-TB was detected 11.02%, 20.03% in new and previously treated cases. Among MDR-TB patients S531 L was the most common mutation detected in rpoB gene; 71.43% in new, and 72.17% in previously treated cases. S315T1 was the most common mutation noted in katG gene; 100% in new and 81.74% in previously treated. While in hA gene, it was C15T (7.8%) among previously treated cases. Conclusion: MDR-TB has high prevalence in the western part of Uttar Pradesh, India. Previously treated cases have even more high rate of MDR-TB than new TB cases. The most dominant gene mutations associated with resistance to INH and RIF were observed in codon 315 of the katG gene and codon 531 of the rpoB gene. While comparing the mutation patterns by Genotype MTBDRplus assay, previously treated cases showed more diversity of mutations and had greater number of unknown mutations.

Analysis of discrepant results between the Genotype® MTBDRplus assay and an antimicrobial drug susceptibility test for isoniazid-resistant tuberculosis.

BACKGROUND: We investigated discrepant results determined using the Genotype®MTBDRplus assay and a conventional antimicrobial drug susceptibility test (ADST) for isoniazid (INH) resistance using sequencing analysis and analyzed the clinical course of patients with discrepant results. METHODS: Among 1373 MTBDRplus assays performed at our tertiary referral center in South Korea between August 2009 and December 2015, the results for 46 (3.4%) differed from those for ADST. KatG and inhA gene sequencing analysis results were available for 23 patients. ADSTs were carried out using the absolute concentration method with Löwenstein-Jensen media. RESULTS: Results from 11 patients indicated INH susceptibility by MTBDRplus assay and INH resistance by ADST. For 5 of these patients, sequencing revealed no evidence of mutations, whereas specific mutations were detected in the remaining 6 patients. These should have been detected using the MTBDRplus assay. The other 12 patients had isolates with the opposite discrepancy, that is INH resistance by MTBDRplus assay but INH susceptibility by ADST. For 7 of these cases, sequencing results were consistent with those of the MTBDRplus assay. However, sequencing analysis did not explain the discrepancies in the remaining 5 patients. All 23 patients with discrepant results received individualized treatment regimens determined by the attending physician according to their test results and susceptibility to other drugs, such as rifampin. Good outcomes were reported for the majority. CONCLUSION: Discrepancies between test results for INH resistance on the MTBDRplus assay and ADST appear to be infrequent. Gene sequencing analysis is useful for identifying the cause of the discrepancy.

The mutations of katG and inhA genes of isoniazid-resistant Mycobacterium tuberculosis isolates in Taiwan.

BACKGROUND/PURPOSE: The isoniazid (INH) resistance of Mycobacterium tuberculosis is caused by mutations in the katG and inhA genes encoding for catalase-peroxidase and inhA, respectively. Sequences of the katG and inhA gene of 70 isolates were analyzed to identify the mutations and to compare the mutations with their related susceptibilities. METHODS: Sequences of the katG and inhA genes and the resistance profiles were analyzed for the 70 M. tuberculosis isolates, collected from nine hospitals in Taiwan during the period from 1999 to 2011. RESULTS: Fifteen alleles were identified in the katG gene and two alleles were identified in the inhA gene. Among the 15 alleles identified in the katG gene, 14 alleles were found in isolates resistant to isoniazid, while only three alleles were found in isolates susceptible to isoniazid. The mutations of the katG gene and their frequencies of 41 INH-resistant isolates were Arg463Leu (51%), Ser315Thr (29%), Ser315Asn (9.8%), and other loci (22%). The sensitivity and specificity of the Ser315Thr mutation for the detection of INH-resistant isolates were 29% and 100%, respectively. The frequency of inhA gene mutation was low (2.44%) in the 41 INH-resistant isolates. CONCLUSION: The diverse alleles of the katG gene associated with INH resistance are present in the M. tuberculosis isolates in Taiwan. These data may be applied to develop new probes for various alleles associated with INH resistance in order to increase the sensitivity for the detection of genetically diverse M. tuberculosis isolates in different geographic areas. The diversity of mutations can also provide information for investigating the evolutional lineages of M. tuberculosis isolates.

Drug resistance-related mutations in multidrug-resistant Mycobacterium tuberculosis isolates from diverse geographical regions.

BACKGROUND: Drug resistance in Mycobacterium tuberculosis is associated with chromosomal mutations in selected genes. These mutations can be screened for an early warning system for drug-resistant tuberculosis. The prevalence of individual mutations differs geographically, which must be considered in developing globally applicable screening tests. METHODS: In order to analyse the geographical distribution and frequency of mutations conferring resistance to rifampicin, isoniazid and fluoroquinolones, the researchers investigated the presence of mutations in the rpoB gene, the katG gene, the mabA-inhA promoter region and the gyrA gene in clinical isolates of multidrug-resistant tuberculosis (MDR-TB) from Belarus, China, Iran/Iraq, Honduras, Romania and Uganda. For each study site, the researchers described the distribution of specific mutations in 20 clinical MDR-isolates. RESULTS: The distribution of resistance-related mutations varied significantly between the study sites. Settings with a high incidence of MDR-TB, such as Belarus, showed a narrower spectrum of mutations related to rifampicin and isoniazid resistance and also a higher prevalence of fluoroquinolone resistance than study sites with a lower MDR-TB prevalence. CONCLUSION: This study confirms that there are significant geographical differences in the distribution of resistance-related mutations and suggests that an increased understanding of such differences in the specific distribution of resistance conferring mutations is crucial for development of new, generally applicable, molecular tools for rapid diagnosis of drug-resistant TB. The fact that a narrower distribution of mutations in high MDR-TB prevalence settings was seen suggests that much of the problems in these settings can be a result of an ongoing transmission of certain MDR-TB strains.

Quick Detecting Mutation Site of 315 Codon in katG Gene of INH resistant MTB by Stem-ring Molecular Probe in Liquid / 中华医院感染学杂志

OBJECTIVE To detect 315 codon of mutation site in katG of isoniazid(INH)-resistant Mycobacterium tyberculosis(MTB) by stem-ring molecular probe quickly and detect out the fluorescence sign of hybridization between amplified products of katG 315 codon and probe in liquid by fluorescence spectrophotometer.The results were confirmed by sequencing.METHODS The software,Beacon designer,was used to design the katG 315 codon stem-ring molecular probe and the amplification system,and the relationship between the way and sequencing of the amplification products were compared.RESULTS The difference between PCR products from standard strain and INH-resistant one was significant in detecting the fluorescent light by use of fluorescence spectrophotometer.We detected fluorescent light signal between the 16 INH resistant strains and 10 H37RV standard strains.The resistant rate to INH detected was about 44%,and the rate of coincidence was about 97.5%.CONCLUSIONS The stem-ring molecular probe technology show high sensitivety in detecting mutation site of nucleic acid.The rate of coincidence is good between fluorescence spectrophotometer way and sequencing.

The Relationship between Isoniazid Resistance and 463 CodonMutation of katG Gne in Mycobacterium Tuberculosis / 결핵

BACKGROUND: The 463 codon mutation of katG gene has been reported as an useful marker for the detection of isoniazid(INH) resistant strains of M. tuberculosis. This study aimed to elucidate relationship between 463 mutation in katG gene and INH resistance in M. tuberculosis. METHOD: DNA was extracted from 28 INH susceptible strains(MIC > or = 0.2microg/ml on the Lowenstein Jensen media) and used for amplification of 189bp fragment containing 463 codon by PCR. Amplified fragments were digested by restriction enzyme Msp I, analyzed by single strand conformation polymorphism(SSCP) in the MDE gel and sequenced to prove mutation. RESULT: Only 7(25%) out of 28 were digestible by restriction enzyme Msp I. The SSCP pattern of 21 strains were distinctly different from that of M. tuberculosis H37Rv. Msp I undigestible PCR fragment was substituted at 463 codon from Arg(CGG) to Leu(CTG). CONCLUSION: This finding clearly indicate no relationship between 463 codon mutation of the katG gene and INH resistance.

Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis from Korea / 대한임상병리학회지

BACKGROUND: The genetic basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has been attributed to at least four genes. Mutations of the katG gene encoding catalase-eroxidase have been shown to cause resistance to INH. Among these mutations, the point mutation of codons 315 and 463 are frequently found. To determine whether simple screening method could potentially be useful for the detection of INH-esistant strains, we investigated the presence of mutation of codons 315 and 463 of katG gene in INH-esistant M. tuberculosis from Korea. METHODS: We used the polymerase chain reaction (PCR) and direct sequencing analysis to detect the point mutation of codons 315 and 463 of katG gene in 48 strains of INH-esistant and 10 strains of INH-usceptible M. tuberculosis. RESULTS: No amplification product was generated from 2 of 48 INH-esistant strains. Among the remaining 46 isolates, point mutations at codons 315 and 463 were identified in 19 isolates (41.3%) and 40 isolates (87.0%), respectively. Among the 10 INH-usceptible strains, the codon 463 point mutation was identified in 7 isolates, however, no point mutation of the codon 315 was found. CONCLUSIONS: These results suggest that the point mutation of codon 463 of katG gene of M. tuberculosis may be a polymorphism not related with INH resistance. Although the mutation of codon 315 of katG gene was limited to 41.3% of INH-esistant isolates, further investigation of the codon 315 of katG gene should lead to increased understanding of resistance genes and the deveolpment of rapid molecular detection methods.

Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis from Korea / 대한임상병리학회지

BACKGROUND: The genetic basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has been attributed to at least four genes. Mutations of the katG gene encoding catalase-eroxidase have been shown to cause resistance to INH. Among these mutations, the point mutation of codons 315 and 463 are frequently found. To determine whether simple screening method could potentially be useful for the detection of INH-esistant strains, we investigated the presence of mutation of codons 315 and 463 of katG gene in INH-esistant M. tuberculosis from Korea. METHODS: We used the polymerase chain reaction (PCR) and direct sequencing analysis to detect the point mutation of codons 315 and 463 of katG gene in 48 strains of INH-esistant and 10 strains of INH-usceptible M. tuberculosis. RESULTS: No amplification product was generated from 2 of 48 INH-esistant strains. Among the remaining 46 isolates, point mutations at codons 315 and 463 were identified in 19 isolates (41.3%) and 40 isolates (87.0%), respectively. Among the 10 INH-usceptible strains, the codon 463 point mutation was identified in 7 isolates, however, no point mutation of the codon 315 was found. CONCLUSIONS: These results suggest that the point mutation of codon 463 of katG gene of M. tuberculosis may be a polymorphism not related with INH resistance. Although the mutation of codon 315 of katG gene was limited to 41.3% of INH-esistant isolates, further investigation of the codon 315 of katG gene should lead to increased understanding of resistance genes and the deveolpment of rapid molecular detection methods.