Application of GeneXpert MTB/RIF technology in rifampicin resistance gene mutation of Mycobacterium tuberculosis / 中国热带医学

@#Abstract: Objective To analyze the accuracy and feasibility of GeneXpert MTB/RIF (GeneXpert) detection in the detection of Mycobacterium tuberculosis and the characteristics of rifampicin-resistant rpoB gene mutations. Methods A total of 4 234 sputum samples from suspected tuberculosis patients diagnosed in Sanya tuberculosis designated hospitals from 2015 to 2021 were selected and subjected to sputum smear, solid culture, drug sensitivity test by solid proportion method and GeneXpert detection. Results The positive detection rates of sputum smear, solid culture and GeneXpert of 4 234 sputum samples were 29.24% (1 238/4 234), 32.17% (1 362/4 234) and 35.40% (1 499/4 234), respectively. The positive detection rate of GeneXpert was higher than that of sputum smear, and the difference was statistically significant (χ2=36.775, P<0.01). It was slightly higher than solid culture, and the difference was not statistically significant (χ2=9.908, P=0.02). Taking solid culture results as the gold standard, the sensitivity and specificity of GeneXpert for detecting MTB were 91.04% (1 240/1 362) and 90.98% (2 613/2 872), respectively. According to the proportional drug susceptibility test results as the gold standard, the sensitivity and specificity of GeneXpert in detecting rifampicin resistance were 96.96% (96/99) and 98.86% (1 128/1 141), respectively, with the consensus rate of 98.71%. The accuracy of rifampicin resistance in GeneXpert group without probe mutation was significantly lower than that in group with probe mutation. There was a statistical difference in probe mutation frequency between newly treated and retreated cases. The analysis of rpoB gene mutation frequency characteristics showed: Probe E (50.00%) > Probe A (22.12%) > Probe D (14.42%) > Probe B (6.73%) > combined probe (5.77%) > Probe C (0.96%). Conclusions GeneXpert detection can quickly and effectively diagnose rifampicin-resistant tuberculosis, which is helpful for early clinical diagnosis and treatment. In this region, the rpoB gene mutation probes of rifampicin-resistant tuberculosis mainly occurr in Probe E and Probe A, with the least mutations in Probe C.

Statistical analysis of 484 Mycobacterium tuberculosis genomes reveals an association between single nucleotide polymorphisms on ponA1 gene and LAM and Haarlem lineages / Análisis estadístico de 484 genomas de Mycobacterium tuberculosis revela una asociación entre los polimorfismos de un solo nucleótido en el gen ponA1 y los linajes LAM y Haarlem

Abstract Objectives: Evaluate the association between rifampicin resistance and the presence of at least one SNP in the rpoB and ponA1 genes and the spoligotype defined lineages. Material and Methods: This study analyzed two databases of 484 genomes of M. tuberculosis from strains isolated from patients in the cities of Lima and Callao, for which the odds ratio (OR) was calculated considering belonging to a certain spoligotype defined lineages as an exposure factor. Results: No statistically significant association (ρ value> 0.05) was found between the presence of at least one SNP in the rpoB gene and the lineages included in the study (LAM, Haarlem, T and Beijing). However, a statistically significant association was found between the presence of at least one SNP in the ponA1 gene and the LAM and Haarlem lineages (ρ value <0.05). An association was found between the P631S SNP in the ponA1 gene and the LAM and Haarlem lineages; and the A516T SNP, of this same gene, presented an association with the LAM lineage. Likewise, an association was found between rifampicin resistance and the LAM lineage. Conclusions: The presence of SNPs in the ponA1 gene is associated with the LAM and Haarlem lineages.

Resumen Objetivos: Evaluar la asociación entre la resistencia a rifampicina y la presencia de al menos un SNP en los genes rpoB y ponA1 y los linajes definidos por espoli gotipos. Material y Métodos: Este estudio analizó dos bases de datos de 484 genomas de M. tuberculosis de cepas aisladas de pacientes de las ciudades de Lima y Callao, para lo cual se calculó el odds ratio (OR) considerando la pertenencia a determinado linaje definido por espoligotipos como un factor de exposición. Resultados: No se encontró una asociación estadísticamente significativa (valor de ρ >0.05) entre la presencia de al menos un SNP en el gen rpoB y los linajes incluidos en el estudio (LAM, Haarlem, T y Beijing). No obstante, se halló una asociación estadísticamente significativa entre la presencia de al menos un SNP en el gen ponA1 y los linajes LAM y Haarlem (valor de ρ <0.05). Se encontró una asociación entre el SNP P631S del gen ponA1 y los linajes LAM y Haarlem; y el SNP A516T, de este mismo gen, presentó una asociación con el linaje LAM. Asimismo, se halló una asociación entre la resistencia a rifampicina y el linaje LAM. Conclusiones: La presencia de SNPs en el gen ponA1 está asociada con los linajes LAM y Haarlem.

Development of molecular markers of Mycobacterium tuberculosis rifampicin resistance gene rpoB by PARMS technology / 生物工程学报

The purpose of this study is to provide a simple and reliable genetic typing approach for molecular drug susceptibility test of Mycobacterium tuberculosis, through the developing of fluorescence molecular marker of rifampicin resistance gene rpoB. Eleven fluorescent molecular markers of the rpoB gene were established by using the sequence difference between the amino acid positions 531, 526, 516, 511 and 513 of rpoB gene of rifampicin-resistant strains and the alleles of rifampicin-sensitive strains, combined with the PARMS technique (Penta-primer amplification refractory mutation system). We used 104 clinical isolates of Mycobacterium tuberculosis to validate this marker and it was verified by sequencing as 100% correct. These samples were also tested with proportional drug sensitivity test. The coincidence rate was 94.23%. The molecular markers had high reliability for genotyping of rpoB gene. It can also detect low-concentration drug-resistant samples (511/533 unit point mutations) whose phenotypic susceptibility cannot be detected. The eleven sets of fluorescent molecular markers could cover 92%-96% of rpoB gene mutation types of rifampicin-resistant strains, and provide new idea for rapid detection of rifampin-resistant Mycobacterium tuberculosis.

Identifation methods of Brucella Melitensis / 中华地方病学杂志

Objective To establish genotyping methods for rapid identification of Brucella melitensis (B.melitensis) biovar 1,2 and 3 and to verify these method.Methods Single nucleotide polymorphism of RpoB gene and tandem repeat sequence (TRS) Bru42 of standard reference strain 16M were used to design primers,then the RpoB-PCR and TRS-PCR method were established for identification of B.melitensis standard reference strains,these two methods were used to identify clinical isolates of B.melitensis and compared with the conventional methods.Results The results of B.melitensis standard reference strains (biotype 1,2,3) identified by RpoB-PCR and TRS-PCR were consistent with those of the conventional identification methods.Totally 50 clinical isolates [including B.melitensis biovar 1 (17),2 (3) and 3 (30)] were identified as RpoB-2 genotype,only one B.melitensis biovar 1 strain was identified as RpoB-3 genotype.Genotype identification results of standard reference strains and clinical isolates with the same biotype were not exactly the same.Fothermore,TRS-PCR experiment displayed that 51 clinical isolates were all genotype 2 of B.melitensis (genotype TRS-2).Conclusions There is no clear relationship between biovars and genotypes within B.melitensis,and significant difference exists between B.melitensis standard reference strains and clinical isolates within RpoB gene.Bru42 can not be used for genotyping clinical isolates of B.melitensis.

Identifation methods of Brucella Melitensis / 中华地方病学杂志

Objective To establish genotyping methods for rapid identification of Brucella melitensis (B.melitensis) biovar 1,2 and 3 and to verify these method.Methods Single nucleotide polymorphism of RpoB gene and tandem repeat sequence (TRS) Bru42 of standard reference strain 16M were used to design primers,then the RpoB-PCR and TRS-PCR method were established for identification of B.melitensis standard reference strains,these two methods were used to identify clinical isolates of B.melitensis and compared with the conventional methods.Results The results of B.melitensis standard reference strains (biotype 1,2,3) identified by RpoB-PCR and TRS-PCR were consistent with those of the conventional identification methods.Totally 50 clinical isolates [including B.melitensis biovar 1 (17),2 (3) and 3 (30)] were identified as RpoB-2 genotype,only one B.melitensis biovar 1 strain was identified as RpoB-3 genotype.Genotype identification results of standard reference strains and clinical isolates with the same biotype were not exactly the same.Fothermore,TRS-PCR experiment displayed that 51 clinical isolates were all genotype 2 of B.melitensis (genotype TRS-2).Conclusions There is no clear relationship between biovars and genotypes within B.melitensis,and significant difference exists between B.melitensis standard reference strains and clinical isolates within RpoB gene.Bru42 can not be used for genotyping clinical isolates of B.melitensis.

Comparison phenotypic and genotypic identification of Staphylococcus species isolated from bovine mastitis / Comparação da identificação fenotípica e genotípica de espécies do gênero Staphylococcus isoladas de mastite bovina

In addition to Staphylococcus aureus nowadays other coagulase-positive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS), earlier considered of minor importance, are now accepted as relevant pathogens for humans and animals. The involvement of these microorganisms in bovine mastitis etiology and the possibility their transmission through milk to humans justify the requirement of developing reliable methods for identification of the most frequent species among them. The purpose of this study was to compare the phenotypic techniques with the genotypic method carried out by sequencing of the rpoB gene in identification of several species of the genus Staphylococcus isolated from bovine mastitis. A total of 300 staphylococci isolates of bovine mastitis cases from several Brazilian dairy herds were studied by phenotypic and genotypic techniques, respectively: 150 CoPS and 150 CoNS strains. A total of 18 CoNS different species and 4 CoPS species were identified. Among the CoNS the following species were recognized: 48 (32%) Staphylococcus warneri, 22(15%) S. epidermidis, 20(13%) S. hyicus, 10(7%) S. xylosus, 7(5%) S. haemolyticus, 6(4%) S. simulans, 6(4%) S. schleiferi subsp schleiferi, 6(4%) S. hominis, 5(3%) S. pasteuri, 4(2.7%) S. cohnii, 3(2%) S. saprophyticus subsp. saprophyticus 3(2%) S. chromogenes 3(2%) S. sciuri, 2(1%) S. saccharolyticus, 2(1%) S. lugdunensi, 1(0,7%) S. auricularis, 1(70%) S. saprophyticus subsp. bovis, 1(0.7%) S. capitis. And among the 150 CoPS were identified respectively: 105 (70%) S. aureus, 21(14%), S. hyicus, 19(13%) S. intermedius e 5(3%) S. schleiferi subsp coagulans. Considering the 150 CoNS isolates, the identifications performed by phenotypic and genotypic tests presented 96.7% of concordance, kappa coefficient of agreement = 0.933, SE (standard error) of kappa=0.021 (95% confidence interval: 0.893 to 0.974), Pearson's correlation coefficient (r) = 0.9977, (confidence interval 95%: 0.9938 a 0.9992) and in relation to 150 CPS isolates it was detected an agreement of 98.7%, kappa = 0.960, SE of kappa = 0.016, (95% confidence interval: 0.929 to 0.992) Pearson's correlation coefficient (r) = 0.9994 (95% confidence interval: 0.9681 to 1.0000). The verified agreement strength between the identification methods can be considered as excellent. These results assure that according to laboratory resources any of them will be suitable to perform the staphylococci identification.(AU)

Além de Staphylococcus aureus atualmente outros estafilococos coagulase positiva (SCP) e estafilococos coagulase-negativos (SCN), anteriormente considerados de menor relevância, são reconhecidos como importantes patógenos para humanos e animais. O envolvimento desses micro-organismos na etiologia da mastite bovina e a possibilidade da sua transmissão através do leite aos humanos justifica a utilização de métodos confiáveis para a identificação das espécies mais frequentes. O objetivo deste estudo foi comparar as técnicas fenotípicas com o método genotípico realizada por sequenciamento do gene rpoB na identificação de espécies do gênero Staphylococcus spp. isolados de mastite bovina. Um total de 300 estafilococos isolados de casos de mastite bovina em diferentes rebanhos leiteiros brasileiros foram estudados por técnicas fenotípicas e genotípicas, respectivamente: 150 linhagens de SCP e 150 linhagens de SCN. Foram identificados um total de 18 espécies de SCN e 4 espécies SCP. Entre os SCN as seguintes espécies identificadas: 48 (32%) Staphylococcus warneri, 22 (15%) S. epidermidis, 20 (13%) S. hyicus, 10 (7%) S. xylosus, 7 (5%) S. haemolyticus, 6 (4%) S. simulans, 6 (4%) S. schleiferi subsp schleiferi, 6 (4%) S. hominis, 5 (3%) S. pasteuri, 4 (2,7%) S. cohnii, 3 (2%) S. saprophyticus subsp. saprophyticus, 3 (2%) S. chromogenes, 3 (2%) S. sciuri, 2 (1%) S. saccharolyticus, 2 (1%) S. lugdunensi, 1 (0,7%) S. auricularis, 1 (70 %) S. saprophyticus subsp. bovis, 1 (0,7%) S. capitis. E entre as 150 SCP foram identificados, 105 (70%) S. aureus, 21 (14%), S. hyicus, 19 (13%) S. intermedius e 5 (3%) S. schleiferi subsp coagulans. Considerando-se os 150 SCN isolados, as identificações realizadas por testes fenotípicos e genotípicos apresentaram 96,7% de concordância, coeficiente de concordância kappa = 0,933, SE (erro padrão) de kappa = 0,021 (95% intervalo de confiança: 0,893-0,974), coeficiente de correlação de Pearson (r) = 0,9977, (intervalo de confiança de 95%: 0,9938 a 0,9992) e em relação a 150 SCP isolados foi observado uma concordância de 98,7%, kappa = 0,960, sE de kappa = 0,016, (95% de intervalo de confiança: 0,929 a 0,992) coeficiente de correlação de Pearson (r) = 0,9994 (95% intervalo de confiança: 0,9681-1,0000). A correlação entre os métodos de identificação pode ser considerada como excelente. Esses resultados demonstraram que de acordo com os recursos disponíveis no laboratório, poderia ser utilizada qualquer uma das metodologias.(AU)

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.

Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Evaluate “Rifampicin Resistance” as Surrogate Marker for Rapid Detection of MDR-TB Using Real-Time PCR Directly on FNAC Samples of Tuberculous Lymphadenitis.

Background: India has the dubious distinction of having second largest burden of MDR-TB cases in the world. According to WHO, MDR-TB is defined as resistance to isoniazid and rifampicin, the two most important drugs for treatment of TB. “Rifampicin resistance” is recommended as surrogate marker for MDR-TB by WHO, as at least, 90% of all rifampicin-resistant clinical isolates are also found resistant to isoniazid. Localization of genetic alterations in the 81-bp “Rifampicin Resistance-Determining Region” of rpoB gene in 96% of rifampicin resistant strains make it particularly amenable for early detection of MDR-TB by molecular techniques like Real-Time PCR. Aim: Evaluation of “rifamipicin resistance” as surrogate marker for rapid detection of MDR-TB using Real-Time PCR directly on FNAC samples of tuberculous lymphadenitis (TBLN). Materials and Methods: Eighty cases of TBLN undergoing anti-tubercular treatment (ATT) and 10 lymphadenitis cases of non-tuberculous origin (controls) were included in the study. To evaluate “rifamipicin resistance” as surrogate marker for rapid detection of MDR-TB, Real-Time PCR and conventional Drug Susceptible Testing (DST) were carried out. Results: Eighteen samples were identified as MDR-TB cases by DST. Real-Time PCR picked up mutated ropB gene in 17 cases out of these 18 MDR-TB cases. Conclusion: “Rifampicin resistance” is an efficient surrogate marker for timely detection of MDR-TB using rapid, accurate and sensitive molecular technique like Real-Time PCR.

Comparison of Rifampicin Resistance in Mycobacterium tuberculosis Isolates by Multiplex Allele Specific PCR (MAS-PCR) with Enzyme Linked Immunosorbent Assay (PCR-ELISA).

Aims: Detection of drug resistance M. tuberculosis isolates is one of the most important strategies to control the disease. Nowadays, with advances in molecular technology, various methods are available to detect drug resistant M. tuberculosis strains such as those based on capture specific probes. In this study, we aimed to investigate the frequency of mutation in the M. tuberculosis -rpoB gene by Polymerase Chain Reaction based on Enzyme Linked Immuno Sorbent Assay (PCR-ELISA) detection. Methodology: Thirty-three culture positive isolates were randomly selected for this study. All the isolates were subjected to a drug Susceptibility Test (DST) using the proportion method. Then the ability and the efficiency of Multiplex Allele Specific PCR (MAS-PCR) and PCR-ELISA to detect Rif resistant (Rifr) M. tuberculosis isolates was compared and evaluated. Results: Mutation of rpoB gene was detected in 19/33 isolates (57.6%) by PCR-ELISA. Hybridization with the specific mutant probes 516 and 526 codon occurred in 1/33 isolates each (3% respectively). Hybridization with the specific mutant probe 531 occurred in 13/33 isolates (39.4%). Three isolates (9.2%) showed simultaneous mutation in codons 516 and 531. The sensitivity and specificity of MAS-PCR in comparison to the Proportional Method was 100%. On the other hand, PCR-ELISA showed 75% sensitivity and 69.2% specificity. The positive predictive value for the PCR-ELISA method was 78.9% and the negative predictive value was 64.3%.The general efficacy of test was 72.7%. Conclusion: The study showed that the sensitivity and specificity of PCR-ELISA to detect mutations in the rpoB gene in Drug Resistant strains was low. Furthermore, this proved to be a complex, time consuming and expensive test. Therefore, this test is not recommended for determining Rifampicin resistance in M. tuberculosis strains.

rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis in Zhejiang Province / 中华临床感染病杂志

Objective To characterize rpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis (M.tuberculosis) in Zhejiang Province.Methods A total of 188 clinical isolates of M.tuberculosis from 188 patients with pulmonary tuberculosis in Tongde Hospital of Zhejiang Province and Integrated Chinese and Western Medicine Hospital of Zhejiang Province were collected.Conventional drug resistance analysis was performed and the mutation of rpoB gene was detected by PCR-based DNA sequencing.The association between gene mutations in rifampin-resistance determining region of M.tuberculosis and clinical resistance was analyzed.Results Fifty-seven out of 188 isolates (30.3％) were drug-resistant strains,including 18 isolates (9.6％) with single-resistance to rifampin,28 isolates (14.9％) with single-resistance to other anti-tuberculosis drugs (10 to isoniazid,12 to streptomycin and 6 to ethambutol),and 11 isolates (5.9％) with multi-drug-resistance (rifampin plus one or more drugs of isoniazid,streptomycin and ethambutol).Among 29 rifampin-resistant strains,rpoB gene mutation existed in 27 strains (93.1％),and the most frequently mutated sites were codons 526 (55.6％,16/27),513 (22.2％,5/27),531 (14.8 ％,4/27)) and 529 (7.4％,2/27).Among 28 strains which were resistant to other anti-tuberculosis drugs,rpoB mutations existed in 4 strains (14.3％),and the mutated sites were codons 526 (2 strains) and 513 (2 strains).All 13 sensitive isolates had no mutation in rpoB gene.Conclusion Rifampin resistance in M.tuberculosis is closely correlated with rpoB gene mutations in Zhejiang province,and the most frequent sites of mutation are at codons 526 and 513.

Comparison of the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide tube method with the conventional method and real-time polymerase chain reaction for the detection of rifampicin resistance in Mycobacterium tuberculosis.

Colorimetric methods are cheap, reproducible, and rapid methods of detecting drug resistance in Mycobacterium tuberculosis. The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) method is one such technique that has been established in our laboratory to detect rifampicin resistance. The present study compared the results of the MTT method with those of the proportion method and real-time polymerase chain reaction (RTPCR) in order to establish sensitivity and specificity of MTT. The mutations for rifampicin resistance occur in rpoB gene, and the commonest reported are in codons 526 and 531. Therefore, RTPCR was targeted at these two codons. The concordance of MTT with the proportion method and RTPCR was 94 and 72.77%, respectively, and that of RTPCR with the proportion method was 77.77%. While the study confirmed that the MTT method is a good method for detecting rifampicin resistance, it also brought out the fact that RTPCR when targeted for limited mutations is not a good tool. Either the genotypic method used should target the total 81-bp rpoB genome or methods such as DNA sequencing should be used. For resource-constraint laboratories, the MTT method can be considered as a better choice.

Chronic Pulmonary Disease Due to Mycobacterium monacense Infection: The First Case from Iran

We herein report a case in which the recently characterized species Mycobacterium monacense was isolated from the sputum of an Iranian patient. This case represents the first isolation of M. monacense from Iran. The isolate was identified by conventional and molecular techniques. Our findings show that M. monacense infection is not restricted to developed countries.

A Case of Mycobacterium Marinum Tenosynovitis Diagnosed by the PCR-restriction Fragment Length Polymorphism of the rpoB Gene / 대한내과학회지

Mycobacterium marinum is an uncommon cause of skin and soft-tissue infection. The diagnosis of M. marinum infection is often delayed when only a conventional tissue culture method is used. PCR-restriction fragment length polymorphism (RFLP) analysis using the novel region of the rpoB gene is now available for the rapid identification of Mycobacteria. We report a case of hand infection caused by M. marinum that was identified by PCR-RFLP analysis. The PCR-RFLP assay is a specific and rapid method for the identification of Mycobacteria that facilitates the early diagnosis of non-tuberculous Mycobacterium infection.

A Case of Mycobacterium Marinum Tenosynovitis Diagnosed by the PCR-restriction Fragment Length Polymorphism of the rpoB Gene / 대한내과학회지

Mycobacterium marinum is an uncommon cause of skin and soft-tissue infection. The diagnosis of M. marinum infection is often delayed when only a conventional tissue culture method is used. PCR-restriction fragment length polymorphism (RFLP) analysis using the novel region of the rpoB gene is now available for the rapid identification of Mycobacteria. We report a case of hand infection caused by M. marinum that was identified by PCR-RFLP analysis. The PCR-RFLP assay is a specific and rapid method for the identification of Mycobacteria that facilitates the early diagnosis of non-tuberculous Mycobacterium infection.

Susceptibilidad de M. tuberculosis a drogas antituberculosas, determinada por dos técnicas, en el estado Sucre, Venezuela / usceptibility of M. tuberculosis to antituberculosis drugs as determined by two methods, in Sucre state, Venezuela

El objetivo de este trabajo fue evaluar la resistencia a isoniacida (INH), rifampicina (RIF), estreptomicina (STR) y etambutol (EMB) de 59 cepas de Mycobacterium tuberculosis, aisladas en el período agosto 2005-diciembre 2006, en el estado Sucre, Venezuela, empleando el método de proporciones de Canetti y de nitrato reductasa. Se encontró 6,3% de resistencia primaria y 14,3% de adquirida. Una cepa fue considerada MDR, al presentar resistencia a RIF e INH. Se comparó la prueba de nitrato reductasa con el método de las proporciones, encontrándose 100% de concordancia entre los resultados de los dos métodos para INH, RIF y EMB, y 95,65% para STR. Además, la prueba nitrato reductasa produjo resultados en 10 a 14 días, comparado con 42 días para el método de proporciones, por lo que la primera se postula como una alternativa muy valiosa para acortar el tiempo de respuesta en la valoración de la susceptibilidad de M. tuberculosis. La secuencia del gen rpoB en la cepa resistente a RIF demostró la presencia de una mutación no descrita anteriormente en la región hipervariable de 81 pares de bases, donde se ha reportado el mayor número de mutaciones de cepas resistentes a RIF. Esta mutación produjo un cambio en el codón 456 de TCG > CAG. Al comparar nuestros resultados con los hallados en el último estudio de prevalencia de resistencia realizado en el estado, se demuestra una disminución en la circulación de cepas resistentes en la zona de estudio.

The objective of this study was to evaluate the resistance to isoniazid (INH), rifampicin (RIF), streptomycin (STR) and ethambutol (EMB), with the Canetti’s proportions method (PM) and the nitrate reductase assay (NRA) of 59 clinical strains of Mycobacterium tuberculosis, isolated in the period of august 2005 to december 2006, in Sucre state, Venezuela. Primary and acquired drug resistance was 6.3% and 14.3%, respectively. Only one strain was found to be multidrug resistant (MDR). The overall agreement between the NRA and PM was 100% for INH, RIF and EMB, and 96% for STR. The time to obtain results was 10 to 14 days for the NRA, compared to 42 days for the PM. The NRA was easy to perform and therefore represents a useful tool for rapid and accurate determination of drug-resistant M. tuberculosis. The sequence of the rpoB gene of the RIF resistant strain demonstrated a never described mutation (change in the codon 456; TCG > CAG) in the hypervariable region of 81 base pairs where most of the mutations of the RIF resistant strains have been reported. Comparison of our results with those of the last resistance prevalence study carried out in the years 1998-1999, shows a decrease in the studied area.

Sequencing analysis on rpoB gene mutation of rifampin-resistance in Mycobacterium tuberculosis / 基础医学与临床

Objective Genetic evaluation of relationship between rpoB gene mutation and rifampin-resistance of Mycobacterium tuberculosis (MTB).Methods This study was carded out with 81 clinical isolates and sensitivity test and genotypic analysis by PCR amplification and sequencing of rpoB gene were performed.Results Among these isolates,there were 27 rifampin-resistant and 54 sensitive strains.Of the 27 drug-resistant isolates,20 (74.1%) carried mutations on the amplified fragment of the rpoB gene with 10 mutation types at seven codons including 531 and 526,and two new mutation patterns were recognized.On the uther hand,one mutation (1.9%) appeared in 54 drug-sensitive strains.Conclusion The study shows geographical variation in the mutation types of rpoB gene in M.tuberculosis isolates from Guizhou,China.The result is valuable for the development of fast diagnostic methods.

rpoB Gene Mutations in Rifampicin - Resistant Mycobacterium Tuberculosis Strains in Vietnam

Introduction: Mycobacterium tuberculosis resists rifampicin (RIF) because of mutations in the rpoB (the p subunit of RNA polymerase) gene, mostly in the 81 bp region. \r\n', u'Objectives: Identify the frequency and characteristics relative to drug - resistant rpoB gene mutation in RIF - resistant M. tuberculosis strains. \r\n', u'Subjects and method: 40 M. tuberculosis strains including 11 RIF - sensitive strains and 29 RIF - resistant strains. Some bio molecular techniques were used such as extracting mycobacterial DNA, PCR, cloning, sequencing and analyzing mutation related RIF - resistance on rpoB gene. \r\n', u'Results: No mutation was found on the 81 bp region of rpoB gene of the RIF - sensitive M. tuberculosis strains. The rate of mutation on rpoB gene of 29 RIF - resistant M. tuberculosis strains is 96.6%. We found 12 mutation codon positions on the 81 bp region of the rpoB gene, and the mutation codon positions with high frequency were 531 (51.7%) and 526 (31%). The mutation position found in only one strain is codon 519 (3.4%) but not found in other reports. There are 15 types of drug resistant mutations in which TCG531 TCG is the most common with 50%. Multi - drug resistance was seen in mutable and none mutable cases, with all codon positions and mutable forms. \r\n', u'Conclusion: No mutation was found on the 81 bp region of the rpoB gene of RtF - sensitive M. tuberculosis strains. The rate of mutation on the rpoB gene of RIF - resistant M. tuberculosis strains is 96.6%. The new mutation position found is codon 519. The mutation on the rpoB gene does not determine the multi - drug resistance of M. tuberculosis. \r\n', u'

Mutation of rpoB gene in M leprae isolates from Korea / 나학회지

Global efforts to control leprosy by intensive chemotherapy, including rifampin, have led to a significant decrease in the number of registered patients. But emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases. Mutations in the rpoB gene, encoding the betasubunit of RNA polymerase, were reported to result in resistance to rifampin in several mycobacterial species, including M leprae. To prevent the emergence and transmission of drug-resistant leprosy and to identify and treat existing cases, it is necessary to establish rapid methods for detection of drug resistance in M leprae. However, M. leprae has not been cultivated on artificial media; therefore, to identify drug susceptibility patterns, bacteria must be tested using Shepard's mouse footpad assay. This in vivo method requires at least 6 months and relatively large numbers of bacteria. Recently, there have been advances in the elucidation of molecular events responsible for drug resistance in mycobacteria Sequences of the rpoB, genes were analyzed for 43 isolates of Mycobacterium leprae from leprosy patients in Korea. Three isolates(6.98%) showed representative mutations in the rpoB, genes, suggesting the emergence of rifampin-resistant M. leprae.

Mutation of DNA fragment of rpoB gene in different degrees of rifampin-resistance in Mycobacterium tuberculosis / 中国实用内科杂志

Objective To study the mutation of DNA fragment of rpoB gene in different degrees of rifampin-resistance in Mycobacterium tuberculosis.Methods DNA fragment of rpoB gene in Mycobacterium tuberculosis was sequenced,including 32 low-level (R50) rifampin-resistant strains (50?g/mL rifampin-resistant),22 high-level (R250) rifampin-resistant strains (250?g/mL rifampin-resistant),10 (R0)rifampin-sensative strains and 1 H 37 Rv strain.Results No mutation was detected in 10 rifampin-sensative strains and 1 H 37 Rv strain;25(78.1%)rifampin-resistant strains had mutations in R50 and 21(95.5%)rifampin-resistant strains had mutations in R250(P=0.170).The mutatione points were distributed disorderly in R50.The 531-Ser mutation(57.1%)and joint mutation(23.8%)were more in R250 than those in R50.Conclusion The frequency of mutation in the rpoB gene of rifampin-resistant strain is higher.The mutation points are distributed disorderly in R50.The 531-Ser mutation(57.1%)and joint mutation(23.8%)are major mutative characteristics in R250.