Cost analysis of GenoType® MTBDRplus and GenoType® MTBDRsl at the State Laboratory of São Paulo, Brazil

ABSTRACT Background: We aimed to evaluate the costs of GenoType® MTBDRplus and MTBDRsl incurred during the diagnosis of first- and second-line drug-resistant tuberculosis (TB) in São Paulo, Brazil. Methods: Mean and activity-based costs of GenoType® were calculated in a referral laboratory for TB in Brazil. Results: The mean cost value and activity-based cost of GenoType® MTBDRplus were USD 19.78 and USD 35.80 and those of MTBDRsl were USD 54.25 and USD 41.85, respectively. Conclusions: The cost of GenoType® MTBDRplus was reduced owing to the high number of examinations performed and work optimization.

Clinical outcomes and molecular characterization of drug-resistant tuberculosis in pre- and extensively drug-resistant disease based on line probe assays

ABSTRACT Multidrug-resistant tuberculosis (MDR-TB) represents a significant impact in transmission, outcome, and health costs. The World Health Organization recommends implementation of rapid diagnostic methods for multidrug-resistance detection. This study was performed to evaluate the frequency of pre- and extensively drug resistant tuberculosis (pre-XDR-TB and XDR-TB) among MDR-TB patients, the pattern of resistance mutations for fluoroquinolones and the clinical outcome. Adult patients followed at a Brazilian regional reference center for TB, from January 2013 to June 2019 were included. Stored Mycobacterium tuberculosis (Mtb) cultures were recovered, the DNA was extracted, and the susceptibility test was performed using the line probe assay for second line antimycobacterial drugs, Genotype MTBDRsl version 2.0 (Hain Lifescience, CmbH, Germany). Among 33 MDR-TB included patients, we diagnosed XDR-TB or pre-XDR in five (15%) cases. Of these, mutations related to fluoroquinolones resistance were observed in four Mtb isolates, including one who had no phenotypic resistance profile. In two other patients with phenotypic resistance to ofloxacin, genotypic resistance was not found. Case fatality rate was 60% in pre/XDR-TB group, compared to 3.6% in the remaining of patients. This study observed few cases of pre-XDR and XDR-TB among a MDR-TB cohort. Phenotypic and genotypic assays presented good agreement. Clinical outcome was more favorable for patients with susceptibility to fluoroquinolones and injectable drugs.

Early detection of Pre-XDR TB with line probe assay in a high TB burden country

Background - Worldwide, tuberculosis (TB) is one of the top 10 causes of death. Drug resistant tuberculosis has lately become a major public health problem that threatens progress made in Tuberculosis (TB) care and control worldwide. The aim of this study was to determine the prevalence of Pre-extensive drug resistant TB among MDR TB in North Central of Nigeria. Methods - This study was conducted from October, 2018 to August, 2019 with 150 samples. In Nigeria, guidelines for DR-TB as recommended by WHO is followed. All the samples from the patients who gave their consent were transported to a zonal reference TB laboratory (ZRL). Results - Mean age was 38.6 ± 13.4 years with peak age at 35-44. Out of these 103 samples processed with LPA, 101(98%) were rifampicin resistant and 2 were rifampicin sensitive, 99(96%) were INH resistant and 4 (4%) were INH sensitive, 5(5%) were fluoroquinolone resistant, 98(95%) were fluoroquinolone sensitive, 12 (12%) were Aminoglycoside + Capreomycin resistant, 91(83%) were Aminoglycoside + Capreomycin sensitive. Conclusion - Multidrug resistant TB and its severe forms (Pre-extensive & extensively drug resistant TB) can be detected early with rapid tool- Line Probe Assay rapid and prevented timely by early initiation on treatment.

Evaluation of GenoType® MTBDRplus VER2.0 kit for detecting mycobacterium tuberculosis complex in sputum / 公共卫生与预防医学

Objective To evaluate the performance of GenoType®MTBDRplus VER2.0 kit for detecting Mycobacterium tuberculosis complex (MTBC) in sputum. Methods Sputum samples from 177 patients with suspected tuberculosis were collected, and tested by smear, MGIT liquid culture and GenoType® MTBDRplus VER2.0. When the liquid culture was positive, identification of strains was carried out. The results were statistically analyzed using SPSS 22.0, and the consistency of the count data was compared using the Kappa test. Good consistency was defined as K≥0.75. The sensitivity and specificity were applied to evaluate the performance of GenoType® MTBDRplus VER2.0 kit for detecting MTBC in sputum. Results Based on the MGIT liquid culture results,，the sensitivity, specificity, positive predictive value, negative predictive value and total coincidence rate of the GenoType® MTBDRplus VER2.0 kit was 91.67%, 95.58%, 91.67%, 95.58% and 94.22%，respectively．The Kappa test was performed on both methods, K=0.872 (P® MTBDRplus VER2.0 kit had high sensitivity and specificity for detecting MTBC in sputum, and it has good application value for early diagnosis of tuberculosis.

Role of line probe assay in diagnosis and detection of drug resistance in Mycobacterium tuberculosis

Tuberculosis caused by Mycobacterium tuberculosis has remained a major global health problem worldwide. TB requires prolonged period of time for isolation by conventional culture methods. The emergence and spread of multi drug resistant (MDR-TB) poses great threats and challenges in controlling the infection. MDR-TB is resistant to both first line drugs rifampicin and isoniazid. PCR tests are based on targeting the mutation in rpoB, katG and inhA genes which can detect resistance to these drugs. To compare microscopy, conventional culture and Line probe assay for the detection of M. tuberculosis & detect rifampicin and isoniazid resistance using Lineprobe assay in various clinical samples. A total of 347 suspected patients of tuberculosis were included in the study. Demographic details & clinical presentation was noted. Various samples were received & processed for ZN staining, culture on LJ media and Line probe assay. Out of 347 cases, majority of cases were in the age group of 51-60 years (18.4%). Majority of the population was males (65.1%). Among suspected tuberculosis patients, cough with expectoration (55.9%) was the commonest complaint. Microscopy was positive in 17.3%, conventional culture was positive in 16.1% and line probe assay was positive in 26.2%. Out of 347, 91 were diagnosed with MTB, out of which 85.7% were sensitive to both rifampicin and isoniazid whereas 14.3% showed resistance to either rifampicin / isoniazid or both. LPA & direct microscopy are a good screening method for early diagnosis and detection of drug resistance but are not a complete replacement of conventional culture which is still a gold standard.

Distribution of hepatitis C virus genotypes in Istanbul, Turkey

Purpose: The hepatitis C virus (HCV) has seven main genotypes and multiple subtypes. The distribution of HCV genotypes varies across geographical regions worldwide. Updated estimates of HCV genotype distributions have a critical importance for developing strategies to manage or eliminate HCV infection. The aim of this study was to determine the distribution of HCV genotypes in patients with HCV admitted to a university hospital in Istanbul, Turkey. Materials and Methods: A total of 412 HCV RNA positive patients with 46.6% of males and 53.4% of females between January 2013 and September 2016 were included in the study. Genotyping of HCV of the study population was performed by a commercial reverse hybridisation line probe-based assay. Results: Genotype 1 (82.5%) was dominant genotype, followed by genotype 3 (10.7%), genotype 2 (4.6%) and genotype 4 (2.2%). Among patients with genotype 1, subtype 1a, 1b and undetermined subtype were 6.3%, 38.8% and 37.4%, respectively. It was observed that genotype proportion was dependent on gender and age of the patients. Genotype 1 and genotype 2 were more prevalent in females, whereas genotypes 3 and 4 were more prevalent in males. Genotype 1 in the older patients and genotype 3 in the younger patients were more prevalent. Conclusion: The majority of patients with HCV infection had genotype 1 (82.5%), followed by genotype 3, 2 and 4. Monitoring the change in HCV genotype distribution is critical for the development of effective strategies for HCV elimination.

Diagnostic accuracy of line probe assays for drug-resistant tuberculosis: a Meta-analysis / 中华流行病学杂志

Objective To evaluate the diagnostic accuracy of line probe assays for drugresistant tuberculosis (TB) in China.Methods Chinese databases (CNKI,Wanfang,SinoMed,VIP Information) and English databases (PubMed,Embase,Cochrane Library) were used to retrieve the literatures regarding the accuracy of line probe assays in the diagnosis of drug-resistant tuberculosis in China between January 1,2000 and September 1,2017.Quality Assessment of Diagnostic Accuracy Studies-2 was used to evaluate the quality of the included studies.Sensitivity and specificity in different studies (using drug sensitivity test or gene sequencing as gold standard) were combined by Meta-analysis using bivariate or univariate model.In addition,subgroup analysis (GenoType MTBDRplus,GenoType MTBDRsl and Reverse dot blot hybridization) and sensitivity analysis were also carried out.Results A total of 24 literatures involving 82 studies were included in the final analysis.The sensitivity and specificity of line probe assays for rifampicin resistant TB were 0.91 (0.88-0.94) and 0.98 (0.97-0.99),respectively.The sensitivity and specificity of line probe assays for isoniazid resistant TB were 0.80 (0.77-0.83) and 0.98 (0.96-0.99),respectively.The sensitivity and specificity of line probe assays for multidrug-resistant TB were 0.81 (0.76-0.85) and 0.99 (0.99-1.00),respectively.The sensitivity and specificity of line probe assays for quinolone resistant TB were 0.92 (0.88-0.95) and 0.94 (0.91-0.97),respectively.The sensitivity and specificity of line probe assays for second-line injectable drug resistant TB (including kanamycin,Capreomycin,amikacin) were 0.79 (0.58-0.91) and 0.98 (0.90-1.00),respectively.The sensitivity and specificity of line probe assays for extensively drug-resistant TB were 0.46 (0.19-0.75) and 1.00 (0.98-1.00),respectively.Subgroup analysis showed that the overall diagnostic accuracy of GenoType MTBDRplus and GenoType MTBDRsl was higher than that of Reverse dot blot hybridization.According to the results of sensitivity analysis,the results of this study were robust.Conclusion The diagnostic accuracy of line probe assays for drug-resistant TB is high.

Diagnostic accuracy of line probe assays for drug-resistant tuberculosis: a Meta-analysis / 中华流行病学杂志

Objective To evaluate the diagnostic accuracy of line probe assays for drugresistant tuberculosis (TB) in China.Methods Chinese databases (CNKI,Wanfang,SinoMed,VIP Information) and English databases (PubMed,Embase,Cochrane Library) were used to retrieve the literatures regarding the accuracy of line probe assays in the diagnosis of drug-resistant tuberculosis in China between January 1,2000 and September 1,2017.Quality Assessment of Diagnostic Accuracy Studies-2 was used to evaluate the quality of the included studies.Sensitivity and specificity in different studies (using drug sensitivity test or gene sequencing as gold standard) were combined by Meta-analysis using bivariate or univariate model.In addition,subgroup analysis (GenoType MTBDRplus,GenoType MTBDRsl and Reverse dot blot hybridization) and sensitivity analysis were also carried out.Results A total of 24 literatures involving 82 studies were included in the final analysis.The sensitivity and specificity of line probe assays for rifampicin resistant TB were 0.91 (0.88-0.94) and 0.98 (0.97-0.99),respectively.The sensitivity and specificity of line probe assays for isoniazid resistant TB were 0.80 (0.77-0.83) and 0.98 (0.96-0.99),respectively.The sensitivity and specificity of line probe assays for multidrug-resistant TB were 0.81 (0.76-0.85) and 0.99 (0.99-1.00),respectively.The sensitivity and specificity of line probe assays for quinolone resistant TB were 0.92 (0.88-0.95) and 0.94 (0.91-0.97),respectively.The sensitivity and specificity of line probe assays for second-line injectable drug resistant TB (including kanamycin,Capreomycin,amikacin) were 0.79 (0.58-0.91) and 0.98 (0.90-1.00),respectively.The sensitivity and specificity of line probe assays for extensively drug-resistant TB were 0.46 (0.19-0.75) and 1.00 (0.98-1.00),respectively.Subgroup analysis showed that the overall diagnostic accuracy of GenoType MTBDRplus and GenoType MTBDRsl was higher than that of Reverse dot blot hybridization.According to the results of sensitivity analysis,the results of this study were robust.Conclusion The diagnostic accuracy of line probe assays for drug-resistant TB is high.

Isolation, Identification and Drug Resistance Testing of Mycobacterium tuberculosis by Recent Diagnostic Modalities at Teaching Hospital in Moradabad (UP).

Objective: Tuberculosis is the second leading cause of death in developing countries among all infectious diseases. Globally, drug resistance strain of Mycobacterium tuberculosis is a public threat. Due to diagnostic delay, inadequate infection control and poor drug supply there is a emergence of MDR- TB and XDR- TB. Our aim was to isolate and identify the drug resistant strain of M. tuberculosis by using newer diagnostic modalities. Methods: Sputum sample and BAL fluid from 70 suspected cases were collected and analysed for Mycobacterium by Ziehl – Neelsen staining and liquid culture with molecular detection of drug resistant strain of M. tuberculosis. Result: In our study, among the 70 patients 27 (38.5%) were positive for AFB by microscopy. On testing for Mycobacterium by BacT/Alert 3D system, 54 were found to be positive. On performing further identification and susceptibility of 54 isolates towards rifampicin and isoniazid by molecular method, 5 isolates (9.25%) were resistant to both rifampicin and isoniazid confirming as multidrug resistant. 5 isolates (9.25%) were sensitive to rifampicin and resistant to isoniazid and 2 isolates (3.70%) were resistant to rifampicin and sensitive to isoniazid. Whereas 5 isolates (9.25%) found to be negative for M. tuberculosis. Conclusion: Our investigation highlights the importance of newer diagnostic modalities for isolation and identification of drug resistant strain of M. tuberculosis. Which ensure early and accurate diagnosis of patients with prevention of further transmission of disease.

Detection of rifampicin resistance in tuberculosis by molecular methods: A report from Eastern Uttar Pradesh, India.

Diagnosis of drug resistance tuberculosis (TB) by the gold standard method is labour intensive and time consuming. Hence, there is an urgent need for introduction of rapid diagnostic techniques. Line probe assay (LPA) and cartridge‑based nucleic acid amplification test (CBNAAT) have been introduced in India under Revised National Tuberculosis Control Program. Spot and morning sputum samples of previously treated patients by anti‑TB drugs were subjected to LPA or CBNAAT. Total 682/1253 (54.4%) were diagnosed as rifampicin‑resistant. The patients could be diagnosed early by molecular methods and put on second line treatment.

Early detection of multi-drug resistance and common mutations in Mycobacterium tuberculosis isolates from Delhi using GenoType MTBDRplus assay.

Purpose: There is scarcity of prevalence data of multi-drug–resistant tuberculosis (MDR-TB) data and common mutations responsible in North India. This study aimed to detect MDR-TB among MDR-TB suspects from Delhi and mutation patterns using GenoType MTBDRplus assay. Materials and Methods: All MDR suspects in fi ve districts of New Delhi were referred to the laboratory from 1st October 2011 to 31st December 2012 as per criterion defi ned by Programmatic Management of Drug Resistant Tuberculosis (PMDT). GenoType MTBDRplus assay was performed on 2182 samples or cultures and mutations in the rpoB gene for rifampicin (RIF) and katG and inhA genes for isoniazid (INH) were analyzed. Results: A total of 366 (16.8%) MDR-TB cases were diagnosed. MDR rate was found to be 32%, 16.6% and 10.2% during criterion A, B and C respectively. The most common mutation detected for RIF was S531L (59.0%) and for INH was S315T1 (88.3%). Mutations S531L and S315T1 occurred signifi cantly higher in MDR strains as compared to RIF mono-resistant and INH mono-resistant strains, respectively. Average laboratory turn-around time (TAT) for dispatch of result to districts for test conducted on samples was 4.4 days. Conclusion: GenoType MTBDRplus is a useful assay for rapid detection of MDR-TB. The common mutations for RIF and INH were similar to those seen in other regions. However, mutations determining MDR strains and mono-resistant strains differed signifi cantly for both RIF and INH.

Detection of multi-drug resistance & characterization of mutations in Mycobacterium tuberculosis isolates from North- Eastern States of India using GenoType MTBDRplus assay.

Background & objectives: information on drug resistance tuberculosis is sparse from North-East (N-E) States of Iindia. We undertook this study to detect multi-drug resistant tuberculosis (MDR-TB) among MDR-TB suspects, and common mutations among MDR-TB cases using GenoType MTBDRplus. Methods: All MDR suspect patients deposited sputum samples to peripheral designated microscopy centres (DMC) in North-East States. The district TB officers (DTOs) facilitated the transport of samples collected during January 2012 to August 2012 to our laboratory. The line probe assay to detect common mutations in the rpoB gene for rifampicin (RIiF) and katG and inhA genes for isoniazid (IiNH), respectively was performed on 339 samples or cultures. Results: A total of 553 sputum samples from MDR suspects were received of which, 181 (32.7%) isolates were found to be multi-drug resistant. Missing WT8 along with mutation in codon S531L was commonest pattern for rifampicin resistant isolates (65.1%) and missing WT along with mutations in codon S315T1 of katG gene was commonest pattern for isoniazid resistant isolates (86.2%). Average turn-around time for dispatch of LPA result to these sStates from cultures and samples was 23.4 and 5.2 days, respectively. Interpretations & conclusions: The MDR-TB among MDR-TB suspects in North-Eastern States of Iindia was found to be 32.7 per cent. The common mutations obtained for RIiF and IiNH in the region were mostly similar to those reported earlier.

Comparison between the conventional method & molecular line probe assay for identification & drug sensitivity of mycobacteria tuberculosis from clinical specimens.

Rapid susceptibility testing of Mycobacterium tuberculosis strains is imperative for therapy selection but traditional drug susceptibility tests take weeks or are expensive. Classical drug susceptibility (DST) may take up to 2 to 4 months. The line probe assay is a commercially available line-probe assay that rapidly detects Mycobacterium tuberculosis (MTB) complex, as well as the most common mutations associated with rifampicin and isoniazid. In this study we assessed the sensitivity and specificity of the rapid molecular method in comparison with the conventional method.

Detection of drug resistant Mycobacterium tuberculosis among patients with and without HIV infection in a rural setting / Detección de Mycobacterium tubersulosis resistente a los antibióticos entre pacientes con y sin infección por VIH en un área rural

OBJECTIVE: To analyse the sensitivity of Mycobacterium tuberculosis by nitrate reductase assay (NRA) and the Hain molecular line probe assay (LPA) in sputa of tuberculosis (TB)/HIV co-infected patients in Guyana. DESIGN: Sputum samples were collected from known TB patients at Georgetown Chest Clinic and were analysed at the Reference Laboratory, Guyana, over the period April 2010 to April 2011. RESULTS: Both methods recorded greater sensitivity for rifampin (RIF) than of isoniazid (INH). Both methods detected four RIF resistant, two INH resistant and two multi-drug resistant (MDR) strains and they had greater negative agreement indices than positive agreement indices. CONCLUSION: It was established that the sensitivity of Mycobacterium tuberculosis by the NRA and Hain LPA in TB/HIV co-infected patients has acceptable correlation and that HIV infection does not affect drug susceptibility testing.

OBJETIVO: Analizar la sensibilidad de Mycobacterium tuberculosis por medio del ensayo de nitrato reductasa (NRA) y el ensayo de sonda lineal (LPA) molecular de Hain en esputos de pacientes co-infectados TB/VIH en Guyana. DISEÑO: Muestras de esputo de pacientes de la Clínica del Tórax en Georgetown diagnosticados con tuberculosis, fueron analizadas en el Laboratorio de Referencias, en Guyana, en el período de abril de 2010 a abril de 2011. RESULTADOS: Ambos métodos registraron una mayor sensibilidad a la rifampicina (RIF) que a la isoniacida (INH). Ambos métodos detectaron cuatro cepas resistentes a RIF, dos resistentes a INH, y dos resistentes a mútiples medicamentos (RMM). Asimismo, presentaban mayores índices de concordancia negativa que de concordancia positiva. CONCLUSIÓN: Se estableció que la sensibilidad de Mycobacterium tuberculosis por medio del ensayo de NRA y el LPA de Hain en pacientes co-infectados TB/VIH, guarda una correlación aceptable, y que la infección por VIH no afecta la prueba de susceptibilidad a los medicamentos.

Evaluación de una técnica de hibridación reversa para identificación rápida de micobacterias en Chile / Evaluation of a reverse hybridization assay for the identification of Mycobacterium in Chile

Objective: Identification for Mycobacterium assay based in the new technology of reverse hybridization DNA probe assay was evaluated (Line Probe Assays-LiPAs). Methods: 74 strains belonging to 23 mycobacterial species or complex classified previously by classical biochemical methods, genetic probes and PRA (patterns of restriction analysis), with and without specific pattern expected to be identified at specie level were analysed.The utilized test, GenoType CM (Hain Lifescience, Nehren, Alemania), is able of identifying 14 of the most common mycobacterial species after a multiplex PCR technique targeting a 23S rRNA gene region followed by reverse hybridization technology. Results: Sensitivity of 94.0 percent (95 percent CI: 84.4-98.0 percent) and specificity of 88.0 percent (95 percent CI:46.7-99.3 percent) were obtained with the assay. Conclusion: GenoType CM is an appropriated tool for the identification of Mycobacteria, rapid, sensitive, operational in the current working conditions of the National Reference Laboratory of Mycobacteria in Chile and it might constitute a real breakthrough for shortening the time delay in the procedure, providing a better opportunity to use treatment only in cases where it is required.

Objetivos: Se evalúa una técnica para la identificación de micobacterias basada en la nueva tecnología de hibridación en tiras con sondas (Line Probe Assays-LiPAs). Métodos: Se analizaron 74 cepas, correspondientes a 23 especies y/o complejos, preclasificadas mediante pruebas bioquímicas tradicionales, sondas genéticas y PRA (análisis de patrones de restricción), identificables y no identi-ficables a nivel de especie por el kit utilizado. El kit evaluado, GenoType CM (Hain Lifescience, Nehren, Alemania), permite la identificación genética molecular de 14 de las especies micobacterianas más comunes, mediante una PCR múltiple e hibridación reversa del producto en tiras con sondas de regiones genéticas de ARNr de 23S. Resultados: Con la utilización de este ensayo para identificación de Micobacterias se obtuvo 94,0 por ciento(CI95 por ciento 84,4-98,0) de sensibilidad y 88,0 por ciento (CI95 por ciento 46,7-99,3) de especificidad totales. Conclusiones: Se concluye que GenoType CM constituye una herramienta adecuada para la identificación de micobacterias, rápida, sensible, operativa en las actuales condiciones de trabajo del Laboratorio de Referencia Nacional de Micobacterias en Chile y que podría constituir un avance para el acortamiento en los tiempos que demora el proceso, lo que implica una mejor oportunidad de aplicación de tratamiento sólo en los casos en que éste sea requerido.

New Diagnostic Methods for Tuberculosis / 대한내과학회지

Tuberculosis (TB) is one of the most important public issues for its global burden of mortality and morbidity. Rapid diagnosis and treatment of active TB are crucial in effective TB control because of lacking truly effective preventive strategy. In recent decades, drug-resistant TB (DR-TB) has emerged as an expanding threat, prompt and reliable laboratory detection of of DR-TB are urgently needed to limit the spread of them. Recently, nonmolecular and molecular assays has been developed for early detection of active TB with or without detection of drug resistance. Broth-based culture systems are capable of producing positive results in 3 weeks or less and many automated system can be widely used for drug susceptibility test. Nucleic acid amplication (NAA) assays have high specificity and positive predictive value, high sensitivity in smear positive specimens. Line probe assays shows high sensitivity for detecting resistance to rifampin and variables sensitivity for detecting resistance to isoniazid. XpertMTB/RIF assay, an automated rapid PCR device, showed high sensitivity and specificity for TB and rifampin resistance. Although new diagnostic methods can decrease turn-around time with considerable diagnostic accuracy and convenience, they are not a replacement for culture and conventional drug susceptibility test for diagnostic limitation in special situations.

The characteristics of drug resistant relevant genes in multidrug-resistant and extensively drug-resistant tuberculosis by fast molecular assay / 中华微生物学和免疫学杂志

ObjectiveTo analyze the characterstics of phenotype and genotype of multidrug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) by molecular line probe assay and liquid culture with MGIT960.MethodsGenoType MTBDR Kits were used for identifying the types of the first-line and second-line antituberculosis drug resistant genes partly and BD MGIT960 was used for detecting the chug susceptibility.Results( 1 ) Out of 94 MDR-TB strains,the rate of drug resistant to EMB,AMK,OFX and MFX by BD MGIT960 assay were 36.2%,17.0%,54.3% and 55.3%,respectively.Among these isolates,13 were extensively drug resistant tuberculosis (XDR-TB).(2) Compared with MGIT960,the concordance rate of GenoType MTBDRplus was 86.2% and 95.7% respectively.Taking MGIT960 results as reference,the sensitivity of GenoType MTBDRsl detecting the susceptibility of EMB,AMK,OFX and MFX to 94 isolates were 47.1%,81.3%,94.1%,94.2%,respectively.The specificity were 75.0%,98.7%,90.7%,92.9%,respectively.(3) Among the rpoB mutation categories,S531L accounts for most.MTB resistant to IFN caused by the mutation of katG chiefly and the S315T1 was in the majority.The gyrA mutation sites located at the ninety-fourth codon most.Out of 94 strains,23 were mixed with 2 kindsof Mycobacterium tuberculosis at least and 7 were undetectable mutations.Conclusion Among the M/XDR-TB,the strains resistant to INH,RFP,AMK,OFX and MFX were caused most by the mutation of katG,rpoB,rrs and gyrA,respectively.The relationship between EMB and embB was not so clear relatively.As a fast detecting drug susceptibility test kit,GenoType MTBDR possess good sensitivity and specificity.So,it could be as an assistant method to guide the therapy on clinic.

Rapid detection of rpoB mutation in rifampin-resistant mycobacterium tuberculosis strains with Line Probe Assay / 中华传染病杂志

Objective To detect rpoB mutations in rifampin resistant mycobacterium tuberculosis strains isolated from Shanghai, and to evaluate the implication of applicating line probe assay (LiPA). Methods A fragment (213bp) of rpoB gene of 58 Mycobacterium tuberculosis isolates was amplified and sequenced, 18 rifampin resistant strains and 10 susceptible strains were selected to detect mutation by LiPA. Results Mutations of rpoB gene in 17 strains of the 18 rifampin resistant isolates were found by LiPA, and there were no mutations in any of the 10 susceptible strains. The sensitivity of LiPA was 94.4% and the concordance with drug susceptibility of Mycobacterium tuberculosis was 96.4%. Conclusions LiPA is a useful method for the rapid detection of mutations of rpoB gene in rifampin resistant Mycobacterium tuberculosis with high sensitivity.

Clinical Usefulness of the Line Probe Assay for Rapid Detection of Rifampicin-resistant Tuberculosis / 결핵

BACKGROUND: RpoB gene mutations have been found in about 96-98% of rifampicin (RMP)-resistant Mycobacterium tuberculosis. Recent reports confirm that the in laboratory settings a rpoB gene mutation can be used as a surrogate marker for multi-drug resistant tuberculosis. However, its usefulness in clinical applications has not been evaluated. This study was performed to confirm whether mutation analysis of the rpoB gene of M.tuberculosis is useful in clinical settings. METHODS: The medical records of 33 patients in whom rpoB gene analysis was conducted using an INNOLiPA Rif. TB assay (LiPA) from June, 1998, to July, 2000, at the Asan Medical Center were retrospectively reviewed in 33 patients. the clinical characteristics in addition to the drug susceptibility and LiPA results were analyzed. The drug susceptibility test was considered as a gold standard method for M/ tuberculosis susceptibility and these results were compared with those of the rpoB gene study and sequencing analysis. sequencing analysis of the rpoB gene was done in cases where there was a discrepancy between the results of the drug susceptibility and rpoB gene study. RESULTS: The mean age and sex ratio was 42±18, and 24:9(M:F), respectively. there were 19 RMP susceptible (58%) and 14 RNP-resistant cases (42%) according to the rpoB gene study. The mean time from the request to reporting the results of the rpoB gene study was 5.2±2.6 days. The mean gap from reporting the rpoB gene study to reporting the susceptibility was 56±35 days. Twenty-eight cases (85%) showed identical results compared with the drug susceptibility results, wheres five cases (15%) showed contradictory results. When compared with the sequencing analysis, of the five cases that showed contradictory results, two had LiPA analysis errors and the remaining three were identical to the sequencing results. The rpoB gene study was of assistance in choosing the appropriate drugs in 28 cases (85%). CONCLUSIONS: An rpoB gene study using an LiPA assay was useful in rapidly diagnosing RMP-resistant tuberculosis, which enabled a proper choice of the appropriate drugs in clinical practices. However, an LiPA assay always should be performed in conjunction with microscopy, culture, and susceptibility tests.

Early Diagnosis of Rifampin-Resistant Mycobacterium tuberculosis by Gene Analysis of RNA Polymerase B Subunit

PURPOSE: The control of tuberculosis is seriously threatened worldwide by the recently emerging multidrug-resistant Mycobacterium tuberculosis. As a result, early detection of drug resistant M.tuberculosis strain has become very important but conventional laboratory methods are time consuming and delayed results often affect patients adversely in controlling tuberculosis. The authors studied the usefulness of the line probe assay to determine the mutaion in rpoB gene of rifampin resistant M.tuberculosis and to find out if this method can substitute conventional methods in the detection of resistant strain. METHODS: This study employed 40 clinical samples of M.tuberculosis which had been determined by culture and drug sensitivity test. After amplification of rpoB-the gene for the B subunit of the RNA polymerase-by PCR, the amplified products were hybridized with specific oligonucleotide probes immobilized on nitrocellulose strip and direct DNA sequencing was also performed. The results were compared with those of the classical susceptibility test. RESULTS: Among the 40 samples, 10 were identified as drug resistant strain by classical drug susceptibility test. Three of the ten resistant samples were rifampin resistant strains, which were identified by either method. All mutations were clustered within the region of 69bp of rpoB and all were single nucleotide mutations. Two isolates had a TCG->TTG(serine->leucine) mutation in codon 522. One isolate had a CAC->CTC(histidine->leucine) mutation in codon 526. CONCLUSION: In contrast to culture and sensitivity tests, line probe assay is an easy and speedy method for detecting rifampin resistant M.tuberculosis in clinical samples as well as a helpful tool for choosing antituberculosis drug in children.