Evaluation of Multidrug Resistant Loop-mediated Isothermal Amplification Assay for Detecting the Drug Resistance of / 生物医学与环境科学（英文）

Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of

Fusion expression with catalase improves the stability of multicopper oxidase and its efficiency in degrading biogenic amines / 生物工程学报

Some enzymes belonging to the multicopper oxidase (MCO) family can degrade the hazardous biogenic amine (BA) present in food. However, the oxidation of MCO in the process of degrading BAs may reduce its activity and stability, resulting in decreased catalytic efficiency. In this work, an MCO from Lactobacillus fermentum (MCOF) was fused with a Bacillus subtilis catalase (CAT) using different strategies and the fusion enzymes were respectively expressed in Escherichia coli BL21(DE3). The tolerance of eight fused MCOFs to H2O2 increased by 51%-68%, and the stability of CAT&MCOF increased by 17%, compared to the wild type MCOF. Using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a substrate, the substrate affinity (Km), the catalytic efficiency (kcat/Km) and the molar specific activity of CAT&MCOF increased by 1.0-fold, 1.7-fold and 1.2-fold than those of MCOF, respectively. The stability of CAT&MCOF under acidic conditions (pH 2.5-4.5) and moderate temperatures (35-55 °C) also improved. Moreover, the degradation rates of putrescine, cadaverine and histamine catalyzed by CAT&MCOF reached 31.7%, 36.0% and 57.8%, respectively, which increased by 132.5%, 45.7% and 38.9% compared to that of MCOF. The improvement on the stability and catalytic efficiency of MCOF by fusion expression with CAT provides a good example for improving the applicability of enzymes through molecular modifications.

miR-9-1 gene methylation and DNMT3B (rs2424913) polymorphism may contribute to periodontitis

Abstract Genetic and epigenetic changes have been associated with periodontitis in various genes; however, little is known about genes involved in epigenetic mechanisms and in oxidative stress. Objective: This study aims to investigate the association of polymorphisms C677T in MTHFR (rs1801133) and −149C→T in DNMT3B (rs2424913), as well as the methylation profiles of MTHFR, miR-9-1, miR-9-3, SOD1, and CAT with periodontitis. The association between polymorphisms and DNA methylation profiles was also analyzed. Methodology: The population studied was composed of 100 nonsmokers of both sexes, divided into healthy and periodontitis groups. Genomic DNA was extracted from the epithelial buccal cells, which were collected through a mouthwash. Polymorphism analysis was performed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), while methylation-specific PCR (MSP) or combined bisulfite restriction analysis techniques were applied for methylation analysis. Results: For DNMT3B, the T allele and the TT genotype were detected more frequently in the periodontitis group, as well as the methylated profile on the miR-9-1 promoter region. There was also a tendency towards promoter region methylation on the CAT sequence of individuals with periodontal disease. Conclusion: The polymorphism −149C→T in DNMT3B (rs2424913) and the methylated profile of the miR-9-1 promoter region are associated with periodontitis.

The association between genetic polymorphism of glutathione peroxidase 1 (rs1050450) and keratoconus in a Turkish population / A associação entre o polimorfismo genético da glutationa-peroxidase 1 (rs1050450) e pacientes com ceratocone em uma população turca

ABSTRACT Purpose: To investigate the potential associations between keratoconus and catalase rs1001179, superoxide dismutase 2 rs4880, and glutathione peroxidase 1 rs1050450 gene polymorphisms in a Turkish population. Methods: The study group included 121 unrelated keratoconus patients and 94 unrelated healthy controls. Blood samples (200 ml) were collected from all patients and controls to isolate genomic DNA. Genotyping was performed to identify rs1001179, rs4880, and rs1050450 using real-time polymerase chain reaction (PCR). Genotype and allele frequencies were calculated; their associations with keratoconus risk were assayed, and the association with keratoconus risk and demographic factors was examined. Results: Glutathione peroxidase 1 rs1050450 polymorphism was present in 41% cases compared with 29% controls (OR=1.66; 95% CI=1.11-2.50; p=0.014). No association was observed between catalase rs1001179 and SOD2 rs4880 polymorphisms and keratoconus (for all, p>0.05). Conclusions: This study evaluated possible relationships between rs1050450, rs1001179, and rs4880 polymorphisms and keratoconus susceptibility. We found a possible association between glutathione peroxidase 1 rs1050450 polymorphism and an increased risk of keratoconus. However, the genotype and allele frequencies were identical in the catalase rs1001179 and superoxide dismutase 2 rs4880 polymorphisms. Further studies are needed to analyze the effect of such variations in identifying keratoconus susceptibility.

RESUMO Objetivo: Investigar as possíveis associações entre o ceratocone e os polimorfismos rs1001179 da catalase, rs4880 da superóxido-dismutase 2 e rs1050450 da glutationa-peroxidase 1 rs1050450 em uma população turca. Métodos: O grupo de estudo incluiu 121 pacientes com ceratocone não relacionados e 94 controles saudáveis também sem pa rentesco. Amostra de sangue (200 mL) foram coletadas de todos os pacientes e controle para isolar o DNA genômico. A genotipagem foi realizada para identificar rs1001179, rs4880 e rs1050450 utilizando a reação em cadeia da polimerase (PCR) em tempo real. As frequências de genótipos e alelos foram calculadas, suas associações com o risco de ceratocone foram avaliadas, e a associação com risco de ceratocone e fatores demográficos foi examinada. Resultados: O polimorfismo da glutationa-peroxidase 1 rs1050450 estava presente em 41% dos casos, comparado com 29% dos controles (OR=1,66, IC 95%=1,11-2,50; p=0,014). Não foi observada associação entre o ceratocone e os polimorfismos rs1001179 e SOD2 rs4880 da catalase (para todos, p>0,05). Conclusões: Este estudo avaliou possíveis relações entre os polimorfismos rs1001179, rs4880 e suscetibilidade a cerato cone. Encontramos uma possível associação entre po limorfis mo da glutationa-peroxidase 1 rs1050450 e um risco aumentado de ceratocone. No entanto, o genótipo e as frequências alélicas foram idênticas nos polimorfismos rs1001179 da catalase e superóxido-dismutase 2 rs4880. Mais estudos são necessários para esclarecer o efeito dessas va riações na detecção da sus cetibilidade ao ceratocone.

Enhanced alkaline catalase production by Serratia marcescens FZSF01: enzyme purification, characterization, and recombinant expression

Background: Catalase (CAT) is an important enzyme that degrades H2O2 into H2O and O2. To obtain an efficient catalase, in this study, a new strain of high catalase-producing Serratia marcescens, named FZSF01, was screened and its catalase was purified and characterized. Results: After optimization of fermentation conditions, the yield of catalase produced by this strain was as high as 51,468 U/ml. This catalase was further purified using two steps: DEAE-fast flow and Sephedex-G150. The purified catalase showed a specific activity of 197,575 U/mg with a molecular mass of 58 kDa. This catalase exhibited high activity at 20­70°C and pH 5.0­11.0. Km of the catalase was approximately 68 mM, and Vmax was 1886.8 mol/min mg. This catalase was further identified by LC­MS/MS, and the encoding gene was cloned and expressed in Escherichia coli BL21 (DE3) with a production of 17,267 ± 2037 U/ml. Conclusions: To our knowledge, these results represent one of the highest fermentation levels reported among current catalase-producing strains. This FZSF01 catalase may be suitable for several industrial applications that comprise exposure to alkaline conditions and under a wide range of temperatures.

Detection of multidrug-resistant Mycobacterium tuberculosis strains isolated in Brazil using a multimarker genetic assay for katG and rpoB genes

Abstract Multidrug-resistant tuberculosis (MDRTB) is a serious world health problem that limits public actions to control tuberculosis, because the most used anti-tuberculosis first-line drugs fail to stop mycobacterium spread. Consequently, a quick detection through molecular diagnosis is essential to reduce morbidity and medical costs. Despite the availability of several molecular-based commercial-kits to diagnose multidrug-resistant tuberculosis, their diagnostic value might diverge worldwide since Mycobacterium tuberculosis genetic variability differs according to geographic location. Here, we studied the predictive value of four common mycobacterial mutations in strains isolated from endemic areas of Brazil. Mutations were found at the frequency of 41.9% for katG, 25.6% for inhA, and 69.8% for rpoB genes in multidrug-resistant strains. Multimarker analysis revealed that combination of only two mutations (“katG/S315T + rpoB/S531L”) was a better surrogate of multidrug-resistant tuberculosis than single-marker analysis (86% sensitivity vs. 62.8%). Prediction of multidrug-resistant tuberculosis was not improved by adding a third or fourth mutation in the model. Therefore, rather than using diagnostic kits detecting several mutations, we propose a simple dual-marker panel to detect multidrug-resistant tuberculosis, with 86% sensitivity and 100% specificity. In conclusion, this approach (previous genetic study + analysis of only prevalent markers) would considerably decrease the processing costs while retaining diagnostic accuracy.

Novel nonsense mutation in the katA gene of a catalase-negative Staphylococcus aureus strain

Abstract We report the first description of a rare catalase-negative strain of Staphylococcus aureus in Chile. This new variant was isolated from blood and synovial tissue samples of a pediatric patient. Sequencing analysis revealed that this catalase-negative strain is related to ST10 strain, which has earlier been described in relation to S. aureus carriers. Interestingly, sequence analysis of the catalase gene katA revealed presence of a novel nonsense mutation that causes premature translational truncation of the C-terminus of the enzyme leading to a loss of 222 amino acids. Our study suggests that loss of catalase activity in this rare catalase-negative Chilean strain is due to this novel nonsense mutation in the katA gene, which truncates the enzyme to just 283 amino acids.

GenoType MTBDR plus 1.0 ® para la detección de resistencia cruzada entre isoniacida y etionamida en aislamientos de Mycobacterium tuberculosis multirresistentes / GenoType MTBDR plus 1.0 ® for the detection of cross-resistance between isoniazide and ethionamide in isolates of multidrug-resistant Mycobacterium tuberculosis

Introducción. Una parte de los aislamientos de Mycobacterium tuberculosis multirresistente también presenta resistencia a la etionamida. Es importante determinar si la resistencia a la isoniacida es independiente o se cruza con la resistencia a la etionamida, ya que si sucede lo segundo habría que reevaluar el tratamiento antituberculoso de segunda línea. La prueba molecular GenoType MTBDR plus ® detecta las mutaciones asociadas con la resistencia a isoniacida y podría detectar la resistencia cruzada a la etionamida. Objetivo. Evaluar la prueba GenoType MTBDR plus ® y comparar su desempeño con el de la secuenciación, en la detección de mutaciones en el gen katG y en el promotor inhA en aislamientos clínicos de M. tuberculosis multirresistente. Materiales y métodos. Se utilizaron el estuche comercial GenoType MTBDR plus 1.0 ® y la secuenciación para evaluar mutaciones en el gen katG y en el promotor inhA en 30 aislamientos de M. tuberculosis multirresistente con resistencia a la etionamida. La cepa de laboratorio H37Rv y tres aislamientos sensibles a los medicamentos de primera línea, sirvieron de control. Resultados. Al comparar los resultados de la secuenciación y de la prueba GenoType MTBDR plus ® , el índice kappa fue de 1. Todos los aislamientos resistentes a la isoniacida y la etionamida tenían las mutaciones detectadas con la prueba GenoTypeMTBDR plus ® en el gen katG, y 40 % de ellos, las detectadas en el promotor inhA. Mediante secuenciación se encontraron, además, mutaciones en katG en posiciones diferentes a las detectadas por la prueba GenoType MTBDR plus ® . Conclusión. La prueba GenoTypeMTBDR plus ® tiene la capacidad de detectar rápidamente la resistencia a isoniacida. Además, los resultados del estudio sugieren que también podría utilizarse como prueba de tamización para detectar la resistencia cruzada a etionamida.

Introduction: A variable proportion of isolates of multidrug-resistant Mycobacterium tuberculosis also presents resistance to ethionamide. It is important to determine whether resistance to isoniazid is independent or crossed with resistance to ethionamide, given that this could lead to the re-evaluation of second-line anti-tuberculosis treatment. The GenoType MTBDR plus ® molecular test is used for the detection of MDR-MTB, as it identifies mutations associated with resistance to isoniazide and could detect cross-resistance with ethionamide. Objective: To evaluate the performance of GenoType MTBDR plus ® in comparison with sequencing in the detection of mutations in gene katG and promotor inhA in clinical isolates of multidrug-resistant M. tuberculosis . Materials and methods: The GenoType MTBDR plus 1.0 ® commercial kit and sequencing were used to evaluate mutations in gene katG and promotor inhA in 30 multidrug-resistant M. tuberculosis isolates that were resistant to ethionamide. The laboratory strain H37Rv and three pan-sensitive isolates acted as controls. Results: The kappa index for the comparison between the results of sequencing and GenoType MTBDR plus ® was 1. All the isolates resistant to isoniazid and ethionamide had the mutations detected by GenoTypeMTBDR plus ® in the katG gene and 40% of them in promotor inhA. Sequencing also revealed katG mutations in positions different to those detected by GenoType MTBDR plus ® . Conclusion: GenoType MTBDR plus ® is able to detect resistance to isoniazid rapidly. Our results suggest that it could also be used to screen for cross-resistance with ethionamide.

Cobertura de la estrategia preventiva de cáncer de vesícula biliar en Chile: Resultados de la Encuesta Nacional de Salud 2009-2010 / Coverage of the gallbladder cancer prevention strategy in Chile: results from the 2009-2010 National Health Survey

Background: In Chile, gallbladder cancer (GBC) is one of the most important causes of death and gallstone disease (GSD) is its main risk factor. Abdominal ultrasonography (AU) is used for the diagnosis of GSD and cholecystectomy is used to prevent it. Aim: To estimate GSD prevalence in the general population and to assess the diagnostic and therapeutic coverage of GSD as a preventive strategy for GBC in Chile. Material and Methods: A standardized digestive symptoms questionnaire of the 2009-2010 Chilean National Health Survey was answered by 5412 adults over 15 years old. Self-reports of AU, GBD and cholecystectomies were recorded. Results: The prevalence of biliary-type pain was 7.1%. During the last five years, the prevalence of AU was 16%. GSD was reported in 20% of these tests and 84% of them were asymptomatic. The prevalence of AU was significantly lower in Araucanía region and among people with less than 12 years of education. Life cholecystectomy prevalence was 11% and reached 40% in people aged over 60 years. Women accounted for 75% of total cholecystectomies. Twenty-one percent of individuals who referred biliary-type pain, were studied with an AU. Only 60% of people with GSD confirmed by AU underwent a cholecystectomy. Conclusions: GSD affects at least 27% of the Chilean adult population. Important deficits and inequities in GSD diagnostic and therapeutic coverage were identified.

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

A resposta oxidativa em corações de camundongos é modulada por background genético / The oxidative response of mouse hearts is modulated by genetic background

FUNDAMENTO: o tabagismo apresenta importante papel sobre as doenças cardiovasculares, entretanto permanecem pouco compreendidos os motivos pelos quais alguns seres humanos as desenvolvem e outros não. OBJETIVO: nosso objetivo foi analisar o perfil redox do coração de diferentes linhagens de camundongos após exposição à fumaça de cigarro. MÉTODOS: camundongos machos suíços (n = 10), C3H (n = 10), BALB/c (n = 10) e C57BL/6 (n = 10) foram expostos à fumaça de cigarro (12 cigarros/dia), enquanto os respectivos controles (n = 10) ao ar ambiente por 60 dias. Após sacrifício, o coração foi retirado para análises bioquímicas. RESULTADOS: embora o conteúdo de malondialdeído não tenha aumentado em nenhum grupo, a atividade da catalase diminuiu no grupo suíço (p < 0,05), BALB/c (p < 0,05) quando comparados aos respectivos grupos-controle, enquanto a mieloperoxidase diminuiu no grupo C3H (p < 0,05) e C57BL/6 (p < 0,001) quando comparados aos respectivos grupos controle. O conteúdo de glutationa reduzida diminuiu nos grupos suíço, C3H, C57BL/6 (p < 0,05) e no grupo BALB/c (p < 0,001) quando comparados com os respectivos controles. Observamos aumento do conteúdo da glutationa oxidada no grupo Suíço (p < 0,05) e diminuição nos grupos C3H (p < 0,05) e BALB/c (p < 0,001) quando comparados aos respectivos grupos-controle. A razão glutationa reduzida/ glutationa oxidada apresentou redução nos grupos suíço e C57BL/6 (p < 0.05) quando comparados aos grupos controle. CONCLUSÃO: o background genético nos camundongos pode influenciar na resposta antioxidante após a exposição à fumaça de cigarro e parece ser um fator determinante para o desequilíbrio redox no suíço e C57BL/6. Compreender as respostas antioxidantes e do background genético C3H e BALB/c podem fornecer importantes informações quanto à resistência cardíaca a fumaça de cigarro.

BACKGROUND: Smoking plays an important role in cardiovascular diseases. However, the reasons why some individuals develop those diseases and others do not remain to be explained. OBJECTIVE: This study aimed at assessing the redox profile of the heart of different mouse strains after exposure to cigarette smoke. METHODS: Male mice of the Swiss (n = 10), C3H (n = 10), BALB/c (n = 10) and C57BL/6 (n = 10) strains were exposed to cigarette smoke (12 cigarettes/day), while their respective controls (n = 10) were exposed to ambient air for 60 days. After being euthanized, their heart was removed for biochemical analyses. RESULTS: Although the malondialdehyde content did not increase in any of the groups, catalase activity decreased in the Swiss (p < 0.05) and BALB/c (p < 0.05) strain mice as compared with their respective control groups, while myeloperoxidase decreased in the C3H (p < 0.05) and C57BL/6 (p < 0.001) strain mice as compared with their respective control groups. The reduced glutathione content decreased in the Swiss, C3H, C57BL/6 (p < 0.05) and BALB/c (p < 0,001) strain mice as compared with their respective control groups. Regarding reduced glutathione content, an increase was observed in the Swiss strain mice (p < 0.05), while a decrease was observed in the C3H (p < 0.05) and BALB/c (p < 0.001) strain mice as compared with their respective control groups. The reduced glutathione/reduced glutathione ratio showed a reduction in the Swiss and C57BL/6 (p < 0.05) strain mice as compared with their respective control groups. CONCLUSIONS: The genetic background of mice can influence the antioxidant response after exposure to cigarette smoke and seems to be a determinant factor for redox imbalance in Swiss and C57BL/6 strain mice. Understanding antioxidant responses and genetic background of C3H and BALB/c strain mice might provide important information regarding cardiac resistance to cigarette smoke.

Detección de la mutación S315T en el gen katg como estrategia para identificación de Mycobacterium tuberculosis resistente a isoniacida en un laboratorio de referencia / Detection of S315T mutation in the katg gene as a strategy for identification of isoniazid-resistant Mycobacterium tuberculosis in a reference laboratory

Introduction: The emergence of strains of Mycobacterium tuberculosis resistant to drugs is a public health problem. Aim: To characterize the resistance to isoniazid (INH) in M. tuberculosis. Methods: Phenotypic and genotypic methods were used to determine the contribution of mutations at 315 codon of katG gene to the phe-notypic expression of resistance. The analysis of susceptibility to antibiotics was performed by the proportional method of Canetti and nitrate reductase method.Genotypic analysis of INH resistance was performed by PCR-RFLP. Results: 193 strains of M. tuberculosis from patients with respiratory symptoms were analyzed. Nineteen (9.8%) strains resistant to INH were identified, of which 12 (63.2%) showed resistance to other drugs. Genotypic analysis allowed to detect the mutation S315T in the katG gene in 15 of 17 strains phenotypically resistant to INH, showing a sensitivity of 88.24%, 100% specificity, 100% positive predictive value, 92% negative predictive value and high concordance with phenotypic methods (kappa = 0.85 (p < 0.01). Conclusion: The S315T mutation in the katG gene is the predominant mechanism of INH resistance in our circulating strains. This feature could be used as a rapid diagnostic tool with potential to detect at least 88% of isoniazid resistant strains, with great impact on the therapeutic management of patients.

Introducción: La emergencia de cepas de Mycobacterium tuberculosis multi-resistentes a antimicrobianos constituye un problema de salud pública. Objetivo: Caracterizar la resistencia a isoniacida (HIN) en cepas de M. tuberculosis. Métodos: Se aplicaron métodos fenotí-picos y genotípicos para determinar la contribución de mutaciones en el codón 315 del gen katG a la expresión fenotípica de resistencia. El análisis fenotípico de susceptibilidad a antimicrobianos se realizó por métodos de las proporciones de Canetti y nitrato reductasa. El análisis genotípico se realizó por RPC-PLFR. Resultados: Se analizaron 193 cepas de M. tuberculosis aisladas de pacientes sintomáticos respiratorios. Se identificaron 19 (9,8%) cepas resistentes a HIN, de las cuales, 12 (63,2%) exhibieron resistencia a otros antimicrobianos. El análisis genotípico permitió detectar la mutación katG S315T en 15/17 cepas fenotípicamente resistentes a HIN, exhibiendo una sensibilidad de 88,24%, especificidad de 100%, valor predictor positivo de 100%, valor predictor negativo de 92% y alta concordancia al comparar con los métodos fenotípicos (kappa = 0,85 (p < 0,01). Conclusión: La mutación S315T en el gen katG representa un mecanismo de desarrollo de resistencia a HIN predominante en las cepas circulantes en nuestro medio, característica que podría ser utilizada como herramienta diagnóstica rápida para detectar al menos 88% de las cepas resistentes a isoniacida, con gran impacto en el manejo terapéutico de los pacientes.

Fast test for assessing the susceptibility of Mycobacterium tuberculosis to isoniazid and rifampin by real-time PCR

Mycobacterium tuberculosis is the bacterium that causes tuberculosis (TB), a leading cause of death from infectious disease worldwide. Rapid diagnosis of resistant strains is important for the control of TB. Real-time polymerase chain reaction (RT-PCR) assays may detect all of the mutations that occur in the M. tuberculosis 81-bp core region of the rpoB gene, which is responsible for resistance to rifampin (RIF) and codon 315 of the katG gene and the inhA ribosomal binding site, which are responsible for isoniazid (INH). The goal of this study was to assess the performance of RT-PCR compared to traditional culture-based methods for determining the drug susceptibility of M. tuberculosis. BACTEC TM MGIT TM 960 was used as the gold standard method for phenotypic drug susceptibility testing. Susceptibilities to INH and RIF were also determined by genotyping of katG, inhA and rpoB genes. RT-PCR based on molecular beacons probes was used to detect specific point mutations associated with resistance. The sensitivities of RT-PCR in detecting INH resistance using katG and inhA targets individually were 55% and 25%, respectively and 73% when combined. The sensitivity of the RT-PCR assay in detecting RIF resistance was 99%. The median time to complete the RT-PCR assay was three-four hours. The specificities for tests were both 100%. Our results confirm that RT-PCR can detect INH and RIF resistance in less than four hours with high sensitivity.

Detection of genomic mutations in katG, inhA and rpoB genes of Mycobacterium tuberculosis isolates using polymerase chain reaction and multiplex allele-specific polymerase chain reaction

OBJECTIVE: Isoniazid (INH) and rifampin (RIF) are the most effective first line antibiotics against Mycobacterium tuberculosis. Mutations in several genes determine resistance of M. tuberculosis to INH, with the most common gene target of katG, and resistance to RIF is due to mutation in rpoB gene. The aim of present study was to assess the mutations in the regions related to RIF and INH resistance. METHODS: We characterized 80 clinical isolates of confirmed M. tuberculosis to analyze the most commonly observed INH and RIF mutations. PCR analysis and sequencing were used to detect mutations related to RIF and INH resistance. The multiplex allele-specific-PCR (MAS-PCR) was performed as a comparative assay and for evaluation of this method. RESULTS: The sequencing of the 250-bp region of katG codon 315, revealed point mutations at 5 different codons in 13.7 percent of the M. tuberculosis isolates. The sequencing of the 270-bp central region of the rpoB gene revealed point mutations at 7 different codons in 12 (15 percent) of the M. tuberculosis isolates. The results obtained with MAS-PCR are in accordance with PCR-sequencing with high sensitivity and specificity for katG315, inhA15, and rpoB (531, 516, 526). CONCLUSION: The results of this study suggested that molecular techniques can be used as a rapid tool for the identification of drug resistance in clinical isolates of M. tuberculosis. Both DNA sequencing and MAS-PCR yielded high sensitivity for the detection of RIF and INH mutations and detecting multi-drug resistant tuberculosis cases.

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.

Association of the Superoxide Dismutase (V16A) and Catalase (C262T) Genetic Polymorphisms with the Clinical Outcome of Patients with Acute Paraquat Intoxication

BACKGROUND/AIMS: Many patients with acute paraquat (PQ) intoxication die even at low PQ concentrations, whereas others with similar concentrations recover. Therefore, it is possible that individual differences in antioxidant capacity are responsible for the variable clinical outcome in patients with acute PQ intoxication. METHODS: We investigated whether there was a relationship between the genetic polymorphisms of SOD (V16A), catalase (C262T), and GPX1 (C593T) in 62 patients with acute PQ intoxication and the clinical outcomes of these patients. RESULTS: The frequency of the Mn-SOD V/V, V/A, and A/A genotypes were 56.3, 43.5, and 0% in survivors and 86.9, 13.1, and 0% in non-survivors (p > 0.05). The GPX1 C/C, C/T, and T/T genotypes were present in 100, 0, and 0% of all subjects. The catalase C/C, C/T, and T/T genotypes were present in 100, 0, and 0% of survivors, and in 82.6, 17.4, and 0% of non-survivors. Neither erythrocyte SOD activity nor catalase activity were significantly different between survivors and non-survivors. CONCLUSIONS: No association was found between clinical outcome of acute PQ intoxication and the genetic polymorphism of GPX1 (C593T) or the genetic polymorphisms or enzyme activity of superoxide dismutase (V16A) or catalase (C262T).

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.

Resistência do Mycobacterium tuberculosis à isoniazida por mutações em duas regiões diferentes do gene katG / Isoniazid-resistant Mycobacterium tuberculosis strains arising from mutations in two different regions of the katG gene

OBJETIVO: Analisar e comparar as mutações em duas regiões diferentes do gene katG, responsáveis pela resistência à isoniazida (INH). MÉTODOS: As análises foram feitas em 97 cepas de Mycobacterium tuberculosis multirresistentes isoladas de culturas de escarro provenientes do Centro de Referência Professor Hélio Fraga. Outras 6 cepas, sensíveis à INH, não apresentaram mutações e foram incluídas como controle. Duas regiões do gene katG (GenBank nº de acesso U06258) - região 1, do códon 1 até o códon 119, e região 2, do códon 267 até o códon 504 - foram amplificadas por PCR e sequenciadas para a identificação das mutações. RESULTADOS: Sete cepas eram resistentes à INH e não mostraram mutação nas duas regiões. Trinta cepas apresentaram mutações na região 1, que se caracterizou por um grande número de deleções, especialmente no códon 4 (24 cepas). A região 2 mostrou 83 mutações pontuais, principalmente no códon 315, com 73 casos de troca de serina (AGC) para treonina (ACC). A análise da região 2 permitiu o diagnóstico de resistência à INH em 81,4 por cento das cepas. Nove cepas tiveram mutações somente na região 1, e isso permitiu o aumento de identificação de cepas resistentes à INH para 90,6 por cento. CONCLUSÕES: O número de mutações do códon 315 foi elevado, compatível com os casos descritos no Brasil e em outros países, e a análise da região 1 aumentou a detecção de mutações em mais 9,2 por cento.

OBJECTIVE: To analyze and compare the mutations in two different regions of the katG gene, which is responsible for isoniazid (INH) resistance. METHODS: We analyzed 97 multidrug-resistant Mycobacterium tuberculosis strains isolated in cultures of sputum samples obtained from the Professor Hélio Fraga Referral Center, in Brasília, Brazil. Another 6 INH-sensitive strains did not present mutations and were included as controls. We used PCR to amplify two regions of the katG gene (GenBank accession no. U06258)-region 1, (from codon 1 to codon 119) and region 2 (from codon 267 to codon 504)-which were then sequenced in order to identify mutations. RESULTS: Seven strains were resistant to INH and did not contain mutations in either region. Thirty strains carried mutations in region 1, which was characterized by a high number of deletions, especially at codon 4 (24 strains). Region 2 carried 83 point mutations, especially at codon 315, and there was a serine-to-threonine (AGC-to-ACC) substitution in 73 of those cases. The analysis of region 2 allowed INH resistance to be diagnosed in 81.4 percent of the strains. Nine strains had mutations exclusively in region 1, which allowed the proportion of INH-resistant strains identified to be increased to 90.6 percent. CONCLUSIONS: The number of mutations at codon 315 was high, which is consistent with cases described in Brazil and in other countries, and the analysis of region 1 resulted in a 9.2 percent increase in the rate at which mutations were identified.

Mutations in rpoB and katG genes in Mycobacterium isolates from the Southeast of Mexico

The most frequent mutations associated with rifampin and isoniazid resistance in Mycobacterium are the substitutions at codons 531 and 315 in the rpoB and katG genes, respectively. Hence, the aim of this study was to characterize these mutations in Mycobacterium isolates from patients suspected to be infected with drug-resistant (DR) pulmonary tuberculosis (TB) in Veracruz, Mexico. Drug susceptibility testing of 25 clinical isolates revealed that five were susceptible while 20 (80 percent) were DR (15 percent of the annual prevalence for Veracruz). Of the DR isolates, 15 (75 percent) were resistant to rifampin, 17 (85 percent) to isoniazid and 15 (75 percent) were resistant to both drugs (MDR). Sequencing analysis performed in the isolates showed that 14 (93 percent) had mutations in the rpoB gene; seven of these (47 percent) exhibited a mutation at 531 (S[L). Ten (58 percent) of the 20 resistant isolates showed mutations in katG; nine (52 percent) of these 10 exhibited a mutation at 315 (S[T). In conclusion, the DR profile of the isolates suggests a significant number of different DR-TB strains with a low frequency of mutation at codons 531 and 315 in rpoB and katG, respectively. This result leads us to consider different regions of the same genes, as well as other genes for further analysis, which is important if a genetic-based diagnosis of DR-TB is to be developed for this region.

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.