Correlation between GenoType MTBDRplus Assay and Phenotypic Susceptibility Test for Prothionamide in Patients with Genotypic Isoniazid Resistance / 결핵

BACKGROUND: The purpose of this study was to analyze the relationship between the gene mutation patterns by the GenoType MTBDRplus (MTBDRplus) assay and the phenotypic drug susceptibility test (pDST) results of isoniazid (INH) and prothionamide (Pto). METHODS: A total of 206 patients whose MTBDRplus assay results revealed katG or inhA mutations were enrolled in the study. The pDST results were compared to mutation patterns on the MTBDRplus assay. RESULTS: The katG and inhA mutations were identified in 68.0% and 35.0% of patients, respectively. Among the 134 isolated katG mutations, three (2.2%), 127 (94.8%) and 11 (8.2%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Among the 66 isolated inhA mutations, 34 (51.5%), 18 (27.3%) and 21 (31.8%) were phenotypically resistant to low-level INH, high-level INH, and Pto, respectively. Of the 34 phenotypic Pto resistant isolates, 21 (61.8%), 11 (32.4%), and two (5.9%) had inhA, katG, and both gene mutations. CONCLUSION: It is noted that Pto may still be selected as one of the appropriate multidrug-resistant tuberculosis regimen, although inhA mutation is detected by the MTBDRplus assay until pDST confirms a Pto resistance. The reporting of detailed mutation patterns of the MTBDRplus assay may be important for clinical practice, rather than simply presenting resistance or susceptibility test results.

Gene mutation associated with drug resistance in M.tuberculosis strains isolated from national TB prevalence survey in Mongolia / Монголын Анагаах Ухаан

Background@#According to the First National Tuberculosis (TB) Prevalence Survey in Mongolia the prevalence of bacteriologically-confirmed pulmonary TB among adults was 559.6 (95% CI: 454.5–664.7) per 100000 population in 2014–2015. This was three times as high as previously estimated. Nationwide anti-tuberculosis (TB) drug resistance survey was conducted in 1999 and 2007 in Mongolia. Share of multidrug resistant TB (MDR-TB) cases among newly notified TB cases increased from 1.0% in 1999 to 1.4% in 2007. Accordingly, we aimed to perform drug susceptibility test on strains isolated from TB Prevalence Survey and to determine the prevalence of drug resistant TB.@*Material and Methods@#All 242 MTB strains isolated from the survey TB cases were tested GenoTypeMTBDRplus test and conventional 1st line DST on solid medium. @*Result@#Conventional DST and GenoTypeMTBDRplus tests done for 93.8% (227/242) of them and 6.2% (15/242) were tested by GenoTypeMTBDRplus only. A 61.6% (95%CI 55.3-67.4) of all cases were susceptible to first line anti-TB drugs, any drug resistance and MDR-TBdetected as 38.4% (95% CI 32.5-44.7)and 9.5% (95% CI 6.4-13.9), respectively. Prevalence of MDR-TB was7.8% (95% CI 4.9-12.4) among new and 17.9% (95% CI 9.0-32.7) among previously treated cases. The 64 strains were identified as a resistant to isoniazid, 32.8% (42/64) and 65.6% (21/64) were katG, and inhAmutation, respectively. One isolate (1.6%) was mutations in both the inhAand katGgenes.The predominant mutations detected in therpoB were S531L (91.3%) among rifampicin resistant isolates and the mutation in inhAwas C–15T (100%) and katG mutation was S315T1 (100%) among isoniazid-resistant isolates. @*Conclusion@#Prevalence of cases with DR-TB is high among prevalent TB cases, especially prevalence of MDR-TB among new cases. In comparison to previous studies, DR-TB cases seem to be increased. Rifampicin resistant strains have a mutation of the rpoBand resistance to isoniazid is predominantly associated with the inhA mutation.

Determinación de la mutación S315T del gen katG en aislados resistentes a Isoniacida de Mycobacterium tuberculosis mediante PCR-RFLP / Determination of S315T mutation within the katG gene in isoniazid-resistant Mycobacterium tuberculosis isolate by PCR-RFLP

Objetivo: determinar la mutación S315T del gen katG en aislados de Mycobacterium tuberculosis resistentes a isoniacida mediante la técnica PCR-RFLP. Materiales y métodos: A partir de 68 aislados de Mycobacterium tuberculosis se realizó el análisis de polimorfismo en productos de amplificación de 1054 y 630 pb que contenían la mutación S315T del gen katG mediante PCR-RFLP empleando las enzimas de restricción MspI y SatI. Mediante SPSS se determinó sensibilidad, especificidad, valores predictivo positivo y negativo, coeficientes de probabilidad positivo y negativo. Resultados: El 74,46% de aislados fenotípicamente resistentes y 4,76% fenotípicamente sensibles presentaron la mutación del gen katG S315T. La PCR-RFLP para S315T del gen katG presentó 85,4% de sensibilidad y 95,2% de especificidad con MspI y 85,4% de sensibilidad y 94,4% de especificidad con SatI. Discusión: La PCR-RFLP tiene una alta capacidad resolutiva que depende de la enzima que se emplee como se observó en estudios previos. La presencia de la mutación S315T en pacientes vírgenes al tratamiento sugiere la circulación de aislados resistentes a Isoniacida. Conclusión: La PCR-RFLP resultó una alternativa válida y rápida para el diagnóstico de la resistencia a isoniacida, mediante la detección de la mutación S315T del gen katG en comparación con el método convencional de las proporciones.

Objetive: To determine the S315T mutation of the katG gene in Mycobacterium tuberculosis isoniazid-resistant isolates by PCR-RFLP. Materials and Methods: Polymorphism analysis of 1054 and 630 bp products containing the S315T mutation of the katG gene was performed by PCR-RFLP using the MspI and SatI restriction enzymes from 68 Mycobacterium tuberculosis isolates. Sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratio were determined using SPSS. Results: 74.46% of isoniazid-resistant and 4.76% of isoniazid-sensitive isolates showed the S315T mutation in katG gene. The PCR-RFLP for S315T of the katG gene had 85.4% sensitivity and 95.2% specificity with MspI and 85.4% sensitivity and 94.4% specificity with SatI. Discussion: The PCR-RFLP has a high resolutive capacity that depends on the enzyme that is used as it was observed in previous studies. The presence of the S315T mutation in treatment-naive patients suggests the circulation of isolates resistant to isoniazid. Conclusion: PCR-RFLP is a valid and rapid alternative for the diagnosis of isoniazid resistance, by detection of S315T mutation in the katG gene compared to the conventional method of proportions.

Direct detection of rpoB and katG gene mutations of Mycobacterium tuberculosis in clinical samples

Objectives: To study the rpoB and katG gene mutation rate and its markers. Methods: Cross-sectional study methods were used to study Tuberculosis. A total of 45 sputum samples were collected from Annapurna Neurological Institute and Allied sci-ences. Then, acid fast bacilli staining were performed. Positive and negative samples were carried for conventional polymerase chain reaction identification and electrophoresis. Results: Out of 45 samples, 3 were acid fast bacilli positive and the rest were negative. Male participants were more as compare to female participants and the mutation in rpoB and katG gene was found similar i.e. 6.66％among the total samples. Conclusions: We can conclude that genetic mutation in Mycobacterium tuberculosis can be identified directly from the clinical samples. However, we have carried this study in less sample size and to validate research on large number of sample is recommended.

Direct detection of rpoB and katG gene mutations of Mycobacterium tuberculosis in clinical samples

Objectives To study the rpoB and katG gene mutation rate and its markers. Methods Cross-sectional study methods were used to study Tuberculosis. A total of 45 sputum samples were collected from Annapurna Neurological Institute and Allied sciences. Then, acid fast bacilli staining were performed. Positive and negative samples were carried for conventional polymerase chain reaction identification and electrophoresis. Results Out of 45 samples, 3 were acid fast bacilli positive and the rest were negative. Male participants were more as compare to female participants and the mutation in rpoB and katG gene was found similar i.e. 6.66% among the total samples. Conclusions We can conclude that genetic mutation in Mycobacterium tuberculosis can be identified directly from the clinical samples. However, we have carried this study in less sample size and to validate research on large number of sample is recommended.

Early detection of multidrug resistant (MDR) Mycobacterium tuberculosis in a single tube with in-house designed fluorescence resonance energy transfer (FRET) probes using real-time PCR.

Rapid and correct diagnosis is crucial for the management of multidrug resistance (MDR) in Mycobacterium tuberculosis (MTB). The present study aims at rapid diagnosis for identification of multidrug resistance tuberculosis (MDR-TB) using real-time PCR. FRET hybridization probes targeting most prominent four selected codons for rpoB526 and 531 and for katG314 and 315 genes were designed and evaluated on 143 clinical MTB isolates and paired sputa for rapid detection of MDR-TB. The results of real-time PCR were compared with gold standard L-J proportion method and further validated by DNA sequencing. Of the 143 MTB positive cultures, 85 and 58 isolates were found to be ‘MDR’ and ‘pan susceptible’, respectively by proportion L-J method. The sensitivity of real-time PCR for the detection of rifampicin (RIF) and isoniazid (INH) were 85.88 and 94.11%, respectively, and the specificity of method was found to be 98.27%. DNA sequencing of 31 MTB isolates having distinct melting temperature (Tm) as compared to the standard drug susceptible H37Rv strain showed 100% concordance with real-time PCR results. DNA sequencing revealed the mutations at Ser531Leu, His526Asp of rpoB gene and Ser315Thr, Thr314Pro of katG gene in RIF and INH resistance cases. This real-time PCR assay that targets limited number of loci in a selected range ensures direct and rapid detection of MDR-TB in Indian settings. However, future studies for revalidation as well as refinement are required to break the limitations of MDR-TB detection.

Genotypic Detection of rpoB and katG Gene Mutations Associated with Rifampicin and Isoniazid Resistance in Mycobacterium Tuberculosis Isolates: A Local Scenario (Kelantan)

Background: Drug resistant tuberculosis (DR-TB) remains a public health issue that is of major concern on a global scale. The characterisation of clinical isolates may provide key information regarding the underlying mechanisms of drug resistance, and helps to augment therapeutic options. This study aims to evaluate the frequency of gene mutations associated with Rifampicin (RIF) and Isoniazid (INH) resistance among nine clinical isolates. Methods: A total of nine drug resistant Mycobacterium tuberculosis clinical isolates were screened for genetic mutations in rpoB and katG using polymerase chain reaction (PCR) amplification and DNA sequencing. Genotypic analysis was performed to detect the mutations in the sequence of the target genes. Results: Our findings reveal that 80% of the isolates possess mutations at codon 119 (His119Tyr) and 135 (Arg135Trp and Ser135Leu) within the rpoB gene; and 70% possess mutations in the katG gene at codon 238 with amino acid change (Leu238Arg). Conclusion: Findings from this study provide an overview of the current situation of RIF and INH resistance in a hospital Universiti Sains Malaysia (HUSM) located in Kelantan, Malaysia, which could facilitate molecular-based detection methods of drug-resistant strains. Further information regarding the molecular mechanisms involved in resistance in RR-/MDR-TB should be addressed in the near future.

The characteristics of the genes mutations in rifampin and isoniazid resistant Mycobacterium tuberculosis clinical isolates from Baise district, Guangxi autonomous region / 实用医学杂志

Objective To analyze the characteristics of the rpoB, KatG and inhA genes mutations in rifampin and isoniazid resistant Mycobacterium tuberculosis (MTB) clinical isolates in Baise district, Guangxi autonomous region. Methods 128 MTB clinical strains were collected and isolated for drug susceptibility testing, and drug resistant strain DNA was subtracted for rpoB, KatG and inhA genes mutation analysis. Results 75%(27/36)isolates carried mutations in the rpoB gene,and 59.3%(16/27)isolates carried mutations in 531 sites. 44.1%(15/34) isolates carried mutations in KatG or inhA, and 66.7%(10/15) isolates appeared in KatG 315 site, with two new mutations found in KatG 279 and 427 site. In these mutation isolates, 13.3%(2/15) mutations appeared in inhA 5, 6.7%(1/15) in inhA 16, and 20%(3/15) in both katG and inhA. Conclusions The mutation of rpoB, katG and inhA genes in TB is highly correlated with its resistance to rifampin and isoniazid in Baise district, Guangxi autonomous region. The study will provide a basis for further understanding the anti-bacterium mechanism and quick diagnostic methods for drug-resistant tuberculosis.

Rapid screening of rpoB and katG mutations in Mycobacterium tuberculosis isolates by high-resolution melting curve analysis.

Background: Early detection of multidrug‑resistant tuberculosis (MDR‑TB) is essential to prevent its transmission in the community and initiate effective anti‑TB treatment regimen. Materials and Methods: High‑resolution melting curve (HRM) analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis clinical isolates including 20 rifampin resistant (RIF‑R), 21 isoniazid resistant (INH‑R) and 54 fully susceptible (S) isolates determined by proportion method of drug susceptibility testing. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. Results: HRM analysis of a 129‑bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF‑R and all RIF‑S isolates. All INH‑S isolates generated wild‑type HRM curves and 18 out of 21 INH‑R isolates harboured any mutation in 109‑bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF‑R and 3 INH‑R isolates were falsely identified as susceptible which were confirmed for having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF‑R isolates) and katG315 (85.7% of INH‑R isolates), respectively. Conclusion: HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource‑limited settings.

Rapid detection of MDR-Mycobacterium tuberculosis using modified PCR-SSCP from clinical Specimens

Objective: To design a rapid test to detect the rifampin (RIF) and isoniazid (INH) resistant mutant based on polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique that analyzes the katG, rpoB genes.Methods:tuberculosis. To determine the susceptibility of isolates to anti TB drugs, the proportional method was used. Mutations presented within the amplified products of the katG, rpoB genes were evaluated by SSCP.Results:Using proportional method, 12 (11.6%) and 9 (8.7%) isolates were resistant respectively Biochemical test as well as IS6110 targeting PCR revealed 103 clinical samples were to INH and RIF and 9 (8.7%) isolates showed resistance to both drug (multi-drug resistant tuberculosis). Three (2.9%) multi-drug resistant tuberculosis and two INH resistant isolates were detected by the PCR-SSCP and sequencing. The sensitivity and specificity of PCR-SSCP for multi-drug resistant isolates were 33% and 100%, respectively.Conclusions:Complete agreement between SSCP and sequencing can indicate that resistance-associated mutations have occurred in other genes except our considered genes.

Rapid detection of MDR-Mycobacterium tuberculosis using modified PCR-SSCP from clinical Specimens

<p><b>OBJECTIVE</b>To design a rapid test to detect the rifampin (RIF) and isoniazid (INH) resistant mutant based on polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique that analyzes the katG, rpoB genes.</p><p><b>METHODS</b>Biochemical test as well as IS6110 targeting PCR revealed 103 clinical samples were tuberculosis. To determine the susceptibility of isolates to anti TB drugs, the proportional method was used. Mutations presented within the amplified products of the katG, rpoB genes were evaluated by SSCP.</p><p><b>RESULTS</b>Using proportional method, 12 (11.6%) and 9 (8.7%) isolates were resistant respectively to INH and RIF and 9 (8.7%) isolates showed resistance to both drug (multi-drug resistant tuberculosis). Three (2.9%) multi-drug resistant tuberculosis and two INH resistant isolates were detected by the PCR-SSCP and sequencing. The sensitivity and specificity of PCR-SSCP for multi-drug resistant isolates were 33% and 100%, respectively.</p><p><b>CONCLUSIONS</b>Complete agreement between SSCP and sequencing can indicate that resistance-associated mutations have occurred in other genes except our considered genes.</p>

Mutation in katG315 is, possibly, a good prognostic marker for treatment with second-line drugs in multi-drug resistant tuberculosis: A preliminary study.

The aim of this study was to explore baseline data, laboratory and molecular analyses to determine if any could serve as potential prognostic marker(s) for treatment response to second line tuberculosis regimens. Of a total number of 50 multi-drug resistant tuberculosis (MDR-TB) patients starting second-line drug MDR-TB treatment in Iraq, only 21 showed treatment adherence and thus, included in this study. Response to treatment was monitored for 11 months by sputum microscopy and culture. We explored baseline data, laboratory and molecular analyses to determine if any could serve as potential prognostic marker(s) for treatment response. Highly signifi cant association (P = 0.019) was detected between mutations in katG315 codon and good response to second-line anti-TB drugs. Spoligotyping and mycobacterial interspersed repetitive unit variable number tandem repeat confi rmed that katG315-mutatnt isolates were genotypically unrelated. The katG315 mutation is a potential prognostic marker for treatment response to second-line anti-tuberculosis drugs. One possible explanation of our results is that the katG315-mutants are sensitive to bacterial killing by “oxidative killing.”

Research article Evaluation of Genotype MTBDRplus Assay for identifying Multidrug Resistant Mycobacterium tuberculosis isolates in Nepal

Background and Objectives: Multidrug-resistant (MDR) Mycobacterium tuberculosis strains are serious threats to the control of tuberculosis and comprise an increasing public health problem. Rapid detection of such strains is quite critical in timely management of such issues. The study was performed with an objective to compare Genotype MTBDRplus reverse hybridization probe assay (Hain Lifescince, GmBH, Nehern, Germany) with culture based proportion method for rapidly identifying MDR-TB strains from suspected multi drug resistant cases, referred to GENETUP Kathmandu, Nepal. Methodology: A commercially available new Genotype MTBDRplus assay was evaluated for its ability to detect mutations in Mycobacterial isolates conferring resistance to rifampicin (RMP) and isoniazid (INH). A total of 64 MDR isolates (i.e., at least resistant to RMP and INH), 5 fully susceptible strains and 1 RMP sensitive strains by conventional proportion method were analyzed using Genotype MTBDRplus assay. MTBDRplus assay is designed to detect the mutations in the hot spot region of rpoB gene, katG and regulatory region of inhA gene. Results: The MTBDRplus assay detected 59 of 61 RMP resistant strains (96.72%) with mutations on 81-bp hot spot region of rpoB gene and 60 of 63 INH resistant strains (95.23%) with mutation in codon 315 katG and regulatory region of inhA. The sensitivity and specificity for the detection of RMP resistance were 96.72% and 100% respectively. While, value of the same two variables for INH resistance were 95.23% and 100%, respectively. Conclusions: The new Genotype MTBDRplus assay represents a rapid, reliable, upgraded tool with high sensitivity and specificity for the detection of INH and RMP resistance strains that can readily be included in a routine laboratory work for the early diagnosis and control of MDR-TB.

Phenotypic and genotypic characteristics of drug resistance in Mycobacterium tuberculosis isolates from pediatric population of Chennai, India.

Purpose: Multidrug-resistant TB (MDR-TB) has been reported in almost all parts of the world. Childhood TB is accorded low priority by national TB control programs. Probable reasons include diagnostic difficulties, limited resources, misplaced faith in BCG and lack of data on treatment. Good data on the burden of all forms of TB among children in India are not available. Objective: To study the drug sensitivity pattern of tuberculosis in children aged from 3 months to 18 years and the outcome of drug-resistant tuberculosis by BACTEC culture system and PCR-based DNA sequencing technique. Materials and Methods: This is a retrospective study. One hundred and fifty-nine clinical specimens were processed for Ziehl-Neelsen stain, Mycobacterial culture by BACTEC method, phenotypic DST for first-line drugs for Mycobacterium tuberculosis (M. tuberculosis) isolates and PCR-based DNA sequencing was performed for the M. tuberculosis isolates targeting rpoB, katG, inhA, oxyR-ahpC, rpsL, rrs and pncA. Results and Conclusion: Out of the 159 Mycobacterial cultures performed during the study period, 17 clinical specimens (10.7%) were culture positive for M. tuberculosis. Among the 17 M. tuberculosis isolates, 2 were multidrug-resistant TB. PCR-based DNA sequencing revealed the presence of many novel mutations targeting katG, inhA, oxyR-ahpC and pncA and the most commonly reported mutation Ser531Leu in the rpoB gene. This study underlines the urgent need to take efforts to develop methods for rapid detection and drug susceptibility of tubercle bacilli in the pediatric population.

Detection and characterization of mutations of multidrug-resistant tuberculosis isolates of the Philippine General Hospital

BACKGROUND: Emergence of multidrug-resistant tuberculosis (MDR-TB) poses a major challenge to prevailing disease management. MDR-TB arises from mutations in several genes comprising the resistance determining regions, including rpoB, katG and gyrA. OBJECTIVE: To detect and characterize mutations in rpoB, katG and gyrA. METHODS: Thirty selected Mycobacterium tuberculosis isolates from the IDS-PGH were subjected to PCR amplification and sequencing. Sequences were compared to the wild type strain H37Rv. RESULTS: Mutations were detected in codons 512, 513, 516, 522, 526, 531 and 533 of rpoB, codons 280, 281, 315 and 333 of katG, and codons 90 and 94 of gyrA sequences. The most frequently mutating codons for rpoB, katG and gyrA were 531, 315 and 94, respectively. A clustering analysis of the sequences showed occurrence of seven, four and three clusters for the genes rpoB, katG and gyrA, respectively. The eight clusters obtained from the concatenated sequences of the three genes represent the eight potential genotypes of local strains. One cluster represents the wild type strain genotype, another cluster represents the XDR strain genotype, and six clusters represent the MDR strain genotypes. CONCLUSION: These findings indicate the utility of multiple RDR sequence analysis in both identifying specific drug resistance mutation and genotyping of various M. tuberculosis isolates.

Clinical data and molecular analysis of Mycobacterium tuberculosi isolates from drug-resistant tuberculosis patients in Goiás, Brazil

Drug resistance is one of the major concerns regarding tuberculosis (TB) infection worldwide because it hampers control of the disease. Understanding the underlying mechanisms responsible for drug resistance development is of the highest importance. To investigate clinical data from drug-resistant TB patients at the Tropical Diseases Hospital, Goiás (GO), Brazil and to evaluate the molecular basis of rifampin (R) and isoniazid (H) resistance in Mycobacterium tuberculosis. Drug susceptibility testing was performed on 124 isolates from 100 patients and 24 isolates displayed resistance to R and/or H. Molecular analysis of drug resistance was performed by partial sequencing of the rpoB and katGgenes and analysis of the inhA promoter region. Similarity analysis of isolates was performed by 15 loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. The molecular basis of drug resistance among the 24 isolates from 16 patients was confirmed in 18 isolates. Different susceptibility profiles among the isolates from the same individual were observed in five patients; using MIRU-VNTR, we have shown that those isolates were not genetically identical, with differences in one to three loci within the 15 analysed loci. Drug-resistant TB in GO is caused by M. tuberculosis strains with mutations in previously described sites of known genes and some patients harbour a mixed phenotype infection as a consequence of a single infective event; however, further and broader investigations are needed to support our findings.

Analysis of antimicrobial susceptibility and mutations of genes associated with rifampicin and isoniazid resistance in Mycobacteria tuberculosis isolates from 7 patients with cutaneous tuberculosis / 中华皮肤科杂志

Objective To assess the antimicrobial resistance and resistance-related mutations in rpoB and katG genes in Mycobacteria tuberculosis isolates from patients with cutaneous tuberculosis. Methods Seven strains of Mycobacteria were isolated from lesions or secretions of patients with cutaneous tuberculosis and identified as M. tuberculosis. Proportion method was used to test the susceptibility of M. tuberculosis isolates to rifampicin, isoniazid, streptomycin and ethambutol. PCR and sequencing were performed to analyze the mutations in rpoB and katG genes. Results Of the 7 isolates of M. tuberculosis, 1 was resistant to rifampicin,isoniazid and ethambutol simultaneously, and the other 6 were sensitive to rifampicin, isoniazid, streptomycin and ethambutol. All the 7 isolates were positive for the amplification of rpoB and katG genes by PCR. DNA sequencing revealed two mutations at codon 531 (TCG to TTG) and codon 315 (AGC to ACC) in the multi-drug resistant strain, which were absent in the other 6 strains. Conclusion Multi-drug resistance has emerged in M. tuberculosis isolates from patients with cutaneous tuberculosis, which is likely to be related to improper treatment.

In house colorimetric reverse hybridisation assay for detection of the mutation most frequently associated with resistance to isoniazid in Mycobacterium tuberculosis

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC→ACC (Ser→Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100 percent of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.

Detección de mutaciones en los Genes rpoß y katG asociados a resistencia en cepas de Mycobacterium tuberculosis de Bolivia / Detection of mutations in rpoß and katG genes associated to resistance in Mycobacterium tuberculosis strains from Bolivia

Objetivo: Evaluar la sensibilidad y especificidad de la genotipificación, como instrumento de diagnóstico rápido y confiable parala detección de mutaciones en los genes rpoß y katG asociados a resistencia,en cepas de Mycobacterium tuberculosis de Bolivia.Diseño: Test Diagnóstico Metodología: Las cepas analizadas fueron aisladas y enviadas por los diferentes Laboratorios de la Red Nacional de Diagnósticode Tuberculosis de Bolivia entre febrero y diciembre de 2007. La muestra para el presente estudio estuvo constituida por un totalde 65 aislamientos previamente caracterizados por métodos fenotípicos de cultivo y pruebas de sensibilidad a la RIF e INH, porel método de las proporciones Canetti-Rist. La genotipificación ha sido realizada utilizando el kit Genotype MTBDR, basado enla utilización de métodos de amplificación e hibridización, para detectar mutaciones a nivel de los marcadores de resistencia rpoßy katG.Resultados: Se procedió al cálculo de la sensibilidad y especificidad de la prueba de diagnóstico; además de los valores predictivospositivo y negativo. Dicho análisis muestra los siguientes resultados: sensibilidad 74...

Objective:To evaluate the sensitivity and specificity of genotyping as a tool for rapid and reliable detection of mutations in rpoß and katG genes associated with resistance in Mycobacterium tuberculosisstrains from Bolivia. Design:Diagnostic Test Methodology: The strains analyzed were isolated and submitted by different laboratories of the National Network for Diagnosisof Tuberculosis of Bolivia between February and December 2007. The sample for this study consisted of 65 isolates previousl y characterized by phenotypic methods of culture and sensitivity testing to RIF and INH by the Canetti-Rist proportion method. Genotyping of these samples has been done using the MTBDR Genotype kit, according to amplification and hybridization methodsto detect mutations at the rpoß and katG resistance markers.Results:The sensitivity and specificity of the diagnostic tests were calculated, as well as the positive and negative predictivevalues. This analysis shows the following results: sensitivity 74%, specificity 92%, positive predictive value 92%, and negative predictive value 73%. Conclusions: The genotyping test using Genotype MTBDR, meeting validation criteria for a diagnostic test study in our country,constitutes a quick, useful and reliable tool for use in diagnosis and routine determination of sensitivity and resistance in MTBCstrains.

Detection of Isoniazid and Rifampicin Resistance by Sequencing of katG, inhA, and rpoB Genes in Korea / 대한진단검사의학회지

BACKGROUND: In Korea, tuberculosis is resistant to isoniazid (INH) and/or rifampicin (RIF) in more than 10% of cases. To prevent the spread of resistant Mycobacterium tuberculosis strains, it is crucial to develop more rapid resistance detection methods. METHODS: To determine the feasibility of using direct sequencing for detecting INH- and RIF-resistant strains, the katG gene, the regulatory region of the inhA gene, and the 81-bp hot-spot region of the rpoB gene from 95 culture isolates and 46 respiratory specimens were sequenced. Total 141 culture isolates were classified by conventional drug susceptibility testing (DST) as INH(R)/RIF(R) (N=30), INH(R)/RIF(S) (N=23), INH(S)/RIF(R) (N=15), and INH(S)/RIF(S) (N=73). RESULTS: Compared with phenotypic DST, the overall sensitivity and specificity of sequencing were 83.0% (44/53) and 96.6% (85/88), respectively, for INH resistance, and 93.3% (42/45) and 100% (96/96), respectively, for RIF resistance. The rates were similar between culture isolates and respiratory specimens. Interestingly, three specimens with inhA -15C>T mutation were susceptible to INH by conventional DST. CONCLUSIONS: Detection of mutations in the katG codon 315, the inhA regulatory region, and the hot-spot region of rpoB would be useful for rapid detection of INH and RIF resistance in Korea.