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1.
Rev. Soc. Bras. Med. Trop ; 54: e0728-2020, 2021. tab, graf
Article in English | LILACS | ID: biblio-1155535

ABSTRACT

Abstract INTRODUCTION: Mycobacterium tuberculosis (MTB) is a causative agent of tuberculosis (TB) that causes death worldwide. METHODS: MTB was subjected to phenotypic drug-susceptibility tests (DST), and drug-resistant genes were sequenced. RESULTS: Previously treated patients were more likely to have positive smear results and exhibit drug resistance. New patients were more likely to be mono SM-resistant and less likely to be INH- and RIF-resistant. The most common mutations were katG (S315T), rpoB (S450L), rpsL (K43R), and embB (M306V). CONCLUSIONS: The proportion of mono-SM-resistant TB among new patients was higher.


Subject(s)
Humans , Pharmaceutical Preparations , Tuberculosis, Multidrug-Resistant , Mycobacterium tuberculosis/genetics , Bacterial Proteins/genetics , Microbial Sensitivity Tests , China , Mutation , Antitubercular Agents/pharmacology
2.
Mem. Inst. Oswaldo Cruz ; 115: e200055, 2020. tab, graf
Article in English | LILACS, SES-SP | ID: biblio-1135234

ABSTRACT

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide. OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution. METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB). FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression. MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Young Adult , Tuberculosis, Multidrug-Resistant/microbiology , Drug Resistance, Multiple, Bacterial/genetics , Mutation/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Antitubercular Agents/pharmacology , Socioeconomic Factors , Brazil , Microbial Sensitivity Tests , Extensively Drug-Resistant Tuberculosis/microbiology , Middle Aged , Mycobacterium tuberculosis/isolation & purification
3.
Mem. Inst. Oswaldo Cruz ; 115: e200215, 2020. tab, graf
Article in English | LILACS, SES-SP | ID: biblio-1135232

ABSTRACT

The human-adapted strains of the Mycobacterium tuberculosis complex (MTBC) comprise seven phylogenetic lineages originally associated with their geographical distribution. Here, we report the genomes of three drug-resistant clinical isolates of the Latin American-Mediterranean (LAM) family collected in Kazakhstan. We utilised whole-genome sequencing to study the distribution and drug resistance of these isolates. Phylogenetic analysis grouped the genomes described in this study with the sequences from Russia, Uzbekistan, and Kazakhstan belonging to the LAM family. One isolate has acquired extensive drug resistance to seven antituberculosis drugs. Our results suggest at least two multi-drug resistant (MDR)/extensively drug-resistant (XDR)-associated genotypes of the LAM family circulate in Kazakhstan.


Subject(s)
Humans , Tuberculosis, Multidrug-Resistant/drug therapy , Drug Resistance, Multiple, Bacterial/drug effects , Mycobacterium tuberculosis/genetics , Antitubercular Agents/pharmacology , Phylogeny , Kazakhstan , Tuberculosis, Multidrug-Resistant/genetics , Genomics , Genotype , Latin America
4.
Mem. Inst. Oswaldo Cruz ; 115: e190407, 2020. tab
Article in English | LILACS | ID: biblio-1101275

ABSTRACT

BACKGROUND Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.


Subject(s)
Humans , Genetic Markers/genetics , Drug Resistance, Bacterial/genetics , Isoniazid/pharmacology , Mutation/genetics , Mycobacterium tuberculosis/genetics , Antitubercular Agents/pharmacology , DNA, Bacterial/genetics , Microbial Sensitivity Tests , Sensitivity and Specificity , Real-Time Polymerase Chain Reaction , Genotype , Mycobacterium tuberculosis/drug effects
5.
Mem. Inst. Oswaldo Cruz ; 115: e200520, 2020. tab, graf
Article in English | LILACS | ID: biblio-1154871

ABSTRACT

BACKGROUND The evaluation of procedures for drug susceptibility prediction of Mycobacterium tuberculosis based on genomic data against the conventional reference method test based on culture is realistic considering the scenario of growing number of tools proposals based on whole-genome sequences (WGS). OBJECTIVES This study aimed to evaluate drug susceptibility testing (DST) outcome based on WGS tools and the phenotypic methods performed on isolates of M. tuberculosis Lineage 1 from the state of Pará, Brazil, generally associated with low levels of drug resistance. METHODOLOGY Culture based DST was performed using the Proportion Method in Löwenstein-Jensen medium on 71 isolates that had been submitted to WGS. We analysed the seven main genome sequence-based tools for resistance and lineage prediction applied to M. tuberculosis and for comparison evaluation we have used the Kappa concordance test. FINDINGS When comparing the WGS-based tools against the DST, we observed the highest level of agreement using TB-profiler. Among the tools, TB-profiler, KvarQ and Mykrobe were those which identified the largest number of TB-MDR cases. Comparing the four most sensitive tools regarding resistance prediction, agreement was observed for 43 genomes. MAIN CONCLUSIONS Drug resistance profiling using next-generation sequencing offers rapid assessment of resistance-associated mutations, therefore facilitating rapid access to effective treatment.


Subject(s)
Humans , Tuberculosis, Multidrug-Resistant/genetics , Drug Resistance, Multiple, Bacterial/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Antitubercular Agents/pharmacology , Brazil , Pharmaceutical Preparations , Microbial Sensitivity Tests , Tuberculosis, Multidrug-Resistant/drug therapy , Drug Resistance, Multiple, Bacterial/drug effects , Whole Genome Sequencing , Mycobacterium tuberculosis/isolation & purification , Antitubercular Agents/therapeutic use
6.
Electron. j. biotechnol ; 41: 81-87, sept. 2019. tab, graf, ilus
Article in English | LILACS | ID: biblio-1087242

ABSTRACT

Background: The search for innovative anti-tubercular agents has received increasing attention in tuberculosis chemotherapy because Mycobacterium tuberculosis infection has steadily increased over the years. This underlines the necessity for new methods of preparation for polymer-drug adducts to treat this important infectious disease. The use of poly(ethylene glycol)(PEG) is an alternative producing anti-tubercular derivatives. However, it is not yet known whether PEGylated isonicotinylhydrazide conjugates obtained by direct links with PEG are useful for therapeutic applications. Results: Here, we synthesized a PEGylated isoniazid (PEG-g-INH or PEG­INH) by gamma radiation-induced polymerization, for the first time. The new prodrugs were characterized using Raman and UV/Vis spectrometry. The mechanism of PEGylated INH synthesis was proposed. The in vitro evaluation of a PEGylated isonicotinylhydrazide macromolecular prodrug was also carried out. The results indicated that PEG­INH inhibited the bacterial growth above 95% as compared with INH, which showed a lower value (80%) at a concentration of 0.25 µM. Similar trends are observed for 0.1, 1, and 5 µM. Conclusions: In summary, the research suggests that it is possible to covalently attach the PEG onto INH by the proposed method and to obtain a slow-acting isoniazid derivative with little toxicity in vitro and higher antimycobacterial potency than the neat drug.


Subject(s)
Polyethylene Glycols/chemistry , Isoniazid/chemistry , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/chemistry , Polyethylene Glycols/pharmacology , Polymers , Spectrum Analysis, Raman , In Vitro Techniques , Prodrugs , Polymerization , Gamma Rays , Isoniazid/pharmacology , Antitubercular Agents/pharmacology
7.
Ciênc. Saúde Colet ; 24(7): 2379-2386, jul. 2019. tab, graf
Article in Portuguese | LILACS | ID: biblio-1011840

ABSTRACT

Resumo A tuberculose tem estreita relação com as condições de vida das pessoas. O presente estudo classifica os municípios brasileiros de acordo com a presença ou ausência de casos de tuberculose (TB) e sua forma drogarresistente (TB-DR), e os descreve quanto às condições de saúde da população, os indicadores de controle da tuberculose, perfil demográfico e socioeconômico. Em 2014, 327 municípios registraram casos de TB-DR. Regiões integradas de desenvolvimento ou regiões metropolitanas concentraram 80,1% dos casos de TB-DR do País. Os municípios com casos de TB-DR tiveram piores indicadores de desfecho da TB, mas maiores percentuais de realização de exame de cultura, além de mais habitantes e melhores indicadores socioeconômicos. Os 3.644 municípios com casos de TB, mas sem casos de TB-DR, tiveram os piores indicadores socioeconômicos entre os três grupos. Os 1.594 municípios sem casos de TB tiveram as menores taxas de desemprego e de detecção de AIDS, e maior cobertura de atenção básica. Os diferentes perfis encontrados no estudo podem dar suporte à lapidação de estratégias nacionais de controle da doença e sua forma drogarresistente no País.


Abstract Tuberculosis is closely related to living conditions. This study classifies Brazilian municipalities according to the occurrence of tuberculosis (TB) and drug-resistant TB (DR-TB) cases and describes them with regard to the population's health conditions, tuberculosis control indicators, demographic and socioeconomic profile. In 2014, 327 municipalities reported DR-TB cases. Integrated regions of development or metropolitan regions accounted for 80.1% of national DR-TB cases. Municipalities with DR-TB cases had worse TB outcome indicators, but higher culture test percentages, and more inhabitants and better socioeconomic indicators. The 3,644 municipalities with TB cases, but without DR-TB cases, had the worst socioeconomic indicators among the three groups. The 1,594 municipalities without TB cases had the lowest rates of unemployment and AIDS detection and greater coverage of primary healthcare. The different profiles found in the study can sustain improved national interventions for TB and drug-resistant TB control in Brazil.


Subject(s)
Humans , Tuberculosis/epidemiology , Unemployment/statistics & numerical data , Antitubercular Agents/pharmacology , Socioeconomic Factors , Tuberculosis/microbiology , Brazil/epidemiology , Acquired Immunodeficiency Syndrome/epidemiology , Cities , Tuberculosis, Multidrug-Resistant/epidemiology
8.
J. bras. pneumol ; 45(2): e20180128, 2019. tab, graf
Article in English | LILACS | ID: biblio-1002440

ABSTRACT

ABSTRACT Objective: To evaluate the rapid diagnosis of multidrug-resistant tuberculosis, by using a commercial line probe assay for rifampicin and isoniazid detection (LPA-plus), in the routine workflow of a tuberculosis reference laboratory. Methods: The LPA-plus was prospectively evaluated on 341 isolates concurrently submitted to the automated liquid drug susceptibility testing system. Results: Among 303 phenotypically valid results, none was genotypically rifampicin false-susceptible (13/13; 100% sensitivity). Two rifampicin-susceptible isolates harboured rpoB mutations (288/290; 99.3% specificity) which, however, were non-resistance-conferring mutations. LPA-plus missed three isoniazid-resistant isolates (23/26; 88.5% sensitivity) and detected all isoniazid-susceptible isolates (277/277; 100% specificity). Among the 38 (11%) invalid phenotypic results, LPA-plus identified 31 rifampicin- and isoniazid-susceptible isolates, one isoniazid-resistant and six as non-Mycobacterium tuberculosis complex. Conclusions: LPA-plus showed excellent agreement (≥91%) and accuracy (≥99%). Implementing LPA-plus in our setting can speed up the diagnosis of multidrug-resistant tuberculosis, yield a significantly higher number of valid results than phenotypic drug susceptibility testing and provide further information on the drug-resistance level.


RESUMO Objetivo: Avaliar o diagnóstico rápido de tuberculose multirresistente, utilizando um teste comercial de sondas em linha (LPA-plus), na rotina de um laboratório de referência de tuberculose. Métodos: O teste LPA-plus foi avaliado prospectivamente em 341 isolados simultaneamente submetidos ao teste de suscetibilidade aos antimicrobianos em meio líquido, pelo sistema automatizado. Resultados: Entre os 303 resultados fenotipicamente válidos, nenhum foi genotipicamente falso suscetível à rifampicina (13/13; 100% de sensibilidade). Dois isolados sensíveis à rifampicina apresentavam mutações no gene rpoB (288/290; especificidade de 99,3%), as quais, no entanto, não são associadas à resistência a rifampicina. O LPA-plus não identificou resistência à isoniazida em três isolados fenotipicamente resistentes (23/26; 88,5% de sensibilidade) e detectou todos os isolados sensíveis à isoniazida (277/277; especificidade de 100%). Entre os 38 (11%) resultados fenotípicos inválidos, o LPA-plus identificou 31 isolados sensíveis à rifampicina e à isoniazida, um resistente à isoniazida e seis como micobactérias não tuberculosas. Conclusões: O LPA-plus mostrou excelente concordância (≥91%) e acurácia (≥99%). Sua implementação pode acelerar o diagnóstico da tuberculose multirresistente, produzir número significativamente maior de resultados válidos do que o teste fenotípico de suscetibilidade aos antimicrobianos e fornecer informações adicionais sobre o nível de resistência aos fármacos.


Subject(s)
Humans , Male , Female , Infant , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Young Adult , Tuberculosis, Multidrug-Resistant/diagnosis , Molecular Diagnostic Techniques/methods , Phenotype , Rifampin/pharmacology , Time Factors , DNA, Bacterial , Microbial Sensitivity Tests , Prospective Studies , Reproducibility of Results , Sensitivity and Specificity , Tuberculosis, Multidrug-Resistant/microbiology , Nucleic Acid Amplification Techniques/methods , Early Diagnosis , Isoniazid/pharmacology , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/pharmacology
9.
Arch. argent. pediatr ; 116(3): 463-467, jun. 2018. ilus
Article in Spanish | LILACS, BINACIS | ID: biblio-950028

ABSTRACT

La tuberculosis, considerada desde 2003 por la Organización Mundial de la Salud una emergencia global de salud, provoca una mortalidad anual de alrededor de 2 millones de personas, fundamentalmente, en países en vías de desarrollo. En la población pediátrica española, la incidencia es de 5 casos/100 000 niños de entre 5 y 14 años y 13 casos/100 000 niños de entre 0 y 4 años. La infección se transmite por vía respiratoria por enfermos bacilíferos. Los niños eliminan escasos bacilos en secreciones respiratorias y no suelen transmitir la infección. En España, el porcentaje de resistencias a isoniazida en la población general es de 5% y es superior en la población inmigrante, lo cual es importante tener en cuenta para el tratamiento de los casos. Se presenta un caso de tuberculosis por Mycobacterium africanum multirresistente al tratamiento, con evolución satisfactoria posterior a la terapia múltiple.


Tuberculosis, considered since 2003 by the World Health Organization a global health emergency, causes annual mortality of approximately 2 million people, mainly in developing countries. In the Spanish pediatric population, the incidence is 5 cases/100 000 children between 5 and 14 years and 13 cases/100 000 children between 0 and 4 years. The infection is transmitted through the respiratory tract by baciliferous patients. Children eliminate few bacilli in respiratory secretions and do not usually transmit the infection. In Spain, the resistance to isoniazid in the general population is 5%, being higher in the immigrant population, which is important to take into account for the treatment of cases. A case of tuberculosis due to Mycobacterium africanum multiresistant to treatment is presented, with satisfactory evolution after multiple therapy.


Subject(s)
Humans , Female , Child, Preschool , Tuberculosis, Multidrug-Resistant/diagnosis , Mycobacterium/isolation & purification , Antitubercular Agents/administration & dosage , Treatment Outcome , Mycobacterium/drug effects , Antitubercular Agents/pharmacology
10.
Braz. j. microbiol ; 48(4): 785-790, Oct.-Dec. 2017. tab, graf
Article in English | LILACS | ID: biblio-889167

ABSTRACT

ABSTRACT Early diagnosis of tuberculosis is of major clinical importance. Among 4733 clinical specimens collected from 3363 patients and subjected to Ziehl-Neelsen microscopy, 4109 were inoculated onto Löwenstein-Jensen slants and 3139 in Bactec/9000MB. Polymerase Chain Reaction (PCR) was performed in 3139 specimens, whereas, a genotypic assay was directly applied in 93 Mycobacterium tuberculosis complex PCR-positive for isoniazid and rifampicin resistance detection specimens (GenoType MTBDRplus). Recovered M. tuberculosis isolates (64) as well as, 21 more sent from Regional Hospitals were tested for antimycobacterial resistance with a phenotypic (manual MGIT-SIRE) and a genotypic assay (GenoType MTBDRplus). PCR in the clinical specimens showed excellent specificity (97.4%) and accuracy (96.8%), good sensitivity (70.4%), but low positive predictive value (40.3%). MGIT-SIRE performed to M. tuberculosis did not confer a reliable result in 16 isolates. Of the remaining 69 isolates, 15 were resistant to streptomycin, seven to isoniazid, seven to ethambutol and five to rifampicin. GenoType MTBDRplus correctly detected isoniazid (seven) and rifampicin-resistant M. tuberculosis strains (five), showing an excellent performance overall (100%). Susceptibility results by the molecular assay applied directly to clinical specimens were identical to those obtained from recovered isolates of the corresponding patients. Combining molecular and conventional methods greatly contribute to early diagnosis and accurate susceptibility testing of tuberculosis.


Subject(s)
Humans , Culture Techniques/methods , Molecular Diagnostic Techniques/methods , Mycobacterium tuberculosis/isolation & purification , Tuberculosis, Multidrug-Resistant/diagnosis , Tuberculosis, Pulmonary/diagnosis , Antitubercular Agents/pharmacology , Culture Techniques/economics , Drug Resistance, Bacterial , Genotype , Microbial Sensitivity Tests , Molecular Diagnostic Techniques/economics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/microbiology , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/microbiology
11.
An. acad. bras. ciênc ; 89(1): 31-43, Jan,-Mar. 2017. graf
Article in English | LILACS | ID: biblio-886638

ABSTRACT

ABSTRACT In Mexican Traditional Medicine 187 plant species are used in the treatment of respiratory conditions that may be associated with tuberculosis. In this contribution, we review the ethnobotany, chemistry and pharmacology of 63 species whose extracts have been assayed for antimycobacterial activity in vitro. Among these, the most potent is Aristolochia brevipes (MIC= 12.5 µg/mL), followed by Aristolochia taliscana, Citrus sinensis, Chrysactinia mexicana, Persea americana, and Olea europaea (MIC<64 µg/mL). Other potent extracts (inhibition > 95%, 50 µg/mL) include: Amphipterygium adstringens, Larrea divaricata, and Phoradendron robinsoni. Several active compounds have been identified, the most potent are: Licarin A (isolated from A. taliscana), and 9-amino-9-methoxy-3,4-dihydro-2H-benzo[h]-chromen-2-one (transformation product of 9-methoxytariacuripyrone isolated from Aristolochia brevipes), both with MIC= 3.125 µg/mL, that is 8-fold less potent than the reference drug Rifampicin (MIC= 0.5 µg/mL). Any of the compounds or extracts here reviewed has been studied in clinical trials or with animal models; however, these should be accomplished since several are active against strains resistant to common drugs.


Subject(s)
Plants, Medicinal/chemistry , Plant Extracts/pharmacology , Plant Extracts/chemistry , Antitubercular Agents/pharmacology , Antitubercular Agents/chemistry , Tetrazolium Salts , Colony Count, Microbial , Microbial Sensitivity Tests , Reproducibility of Results , Ethnobotany , Formazans , Mexico , Mycobacterium tuberculosis/drug effects
12.
Mem. Inst. Oswaldo Cruz ; 111(7): 454-459, tab, graf
Article in English | LILACS | ID: lil-787556

ABSTRACT

In this study we evaluated the crystal violet decolorization assay (CVDA) for detection of minimum inhibitory concentration (MIC) of antituberculosis drugs. 53 isolates were tested in this study and 13 of them were multidrug resistant (MDR) isolates. The antibiotics concentrations were 2-0.06 mg/L for isoniazid (INH) and rifampicin (RIF) and were 16-0.25 mg/L for streptomycin (STM) and ethambutol (EMB). Crystal violet (CV-25 mg/L) was added into the microwells on the seventh day of incubation and incubation was continued until decolorization. Decolorization of CV was the predictor of bacterial growth. Overall agreements for four drugs were detected as 98.1%, and the average time was detected as 9.5 ± 0.89 day after inoculation. One isolate for INH and two isolates for STM were determined resistant in the reference method, but susceptible by the CVDA. One isolate was susceptible to EMB by the reference method, but resistant by the CVDA. All results were concordant for RIF. This study shows that CVDA is a rapid, reliable and suitable for determination of MIC values of Mycobacterium tuberculosis. And it can be used easily especially in countries with limited-sources.


Subject(s)
Humans , Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/administration & dosage , Biological Assay , Drug Resistance, Multiple, Bacterial/drug effects , Ethambutol/administration & dosage , Ethambutol/pharmacology , Gentian Violet/chemistry , Indicators and Reagents/chemistry , Isoniazid/administration & dosage , Isoniazid/pharmacology , Microbial Sensitivity Tests/methods , Mycobacterium tuberculosis/growth & development , Rifampin/administration & dosage , Rifampin/pharmacology , Streptomycin/administration & dosage , Streptomycin/pharmacology , Tuberculosis, Multidrug-Resistant/microbiology
13.
Braz. j. infect. dis ; 20(2): 166-172, Mar.-Apr. 2016. tab, graf
Article in English | LILACS | ID: lil-780813

ABSTRACT

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.


Subject(s)
Humans , Bacterial Proteins/genetics , DNA-Directed RNA Polymerases/genetics , Catalase/genetics , Drug Resistance, Multiple, Bacterial/genetics , Isoniazid/pharmacology , Antitubercular Agents/pharmacology , Rifampin/pharmacology , DNA, Bacterial , Microbial Sensitivity Tests , Genetic Markers , Polymerase Chain Reaction , Predictive Value of Tests , Sensitivity and Specificity , Tuberculosis, Multidrug-Resistant/microbiology , Genotype , Mutation/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics
14.
Braz. j. infect. dis ; 20(1): 33-40, Jan.-Feb. 2016. tab, graf
Article in English | LILACS | ID: lil-776457

ABSTRACT

Abstract The current study was aimed to evaluate the performance of direct 2,3,5-triphenyl tetrazolium chloride assay and direct microscopic observation drug susceptibility assay with indirect Löwenstein-Jensen proportion method directly on Ziehl-Neelsen smear positive sputum specimens. Methods Direct acid fast bacilli smear positive sputum specimens (n = 264) were subjected to isoniazid and rifampicin drug susceptibility testing by direct 2,3,5-triphenyl tetrazolium chloride assay, direct microscopic observation drug susceptibility assay, and the performances were compared with indirect Löwenstein-Jensen proportion method. Results The direct 2,3,5-triphenyl tetrazolium chloride assay demonstrated an overall sensitivity, specificity, positive predictive value, and negative predictive value of 99.2%, 82.4%, 99.2%, and 88.5%, respectively, for the detection of isoniazid and rifampicin resistant Mycobacterium tuberculosis isolates when compared to indirect Löwenstein-Jensen proportion method. Likewise, the overall sensitivity, specificity, positive predictive value and negative predictive value of direct microscopic observation drug susceptibility assay were 98.8%, 82.4%, 99.2%, and 78.2%, respectively. Conclusion The direct 2,3,5-triphenyl tetrazolium chloride assay was found to be an economical alternative method for the rapid and accurate detection of isoniazid and rifampicin resistance from direct acid fast bacilli smear positive sputum specimens.


Subject(s)
Adult , Female , Humans , Male , Antitubercular Agents/pharmacology , Isoniazid/pharmacology , Microbial Sensitivity Tests/methods , Rifampin/pharmacology , Mycobacterium tuberculosis/drug effects , Sensitivity and Specificity , Sputum/microbiology , Tetrazolium Salts , Tuberculosis, Pulmonary/microbiology
15.
Braz. j. infect. dis ; 20(1): 41-47, Jan.-Feb. 2016. tab, graf
Article in English | LILACS | ID: lil-776468

ABSTRACT

Abstract Background Fluoroquinolones are the backbone of multidrug resistant tuberculosis treatment regimens. Despite the high burden of multidrug resistant tuberculosis in the country, little is known about drug resistance patterns, prevalence, and predictors of fluoroquinolones resistance among multidrug resistant tuberculosis patients from Pakistan. Objective To evaluate drug resistance patterns, prevalence, and predictors of fluoroquinolones resistance in multidrug resistant tuberculosis patients. Methods This was a cross-sectional study conducted at a programmatic management unit of drug resistant tuberculosis, Lady Reading Hospital Peshawar, Pakistan. Two hundred and forty-three newly diagnosed multidrug resistant tuberculosis patients consecutively enrolled for treatment at study site from January 1, 2012 to July 28, 2013 were included in the study. A standardized data collection form was used to collect patients’ socio-demographic, microbiological, and clinical data. SPSS 16 was used for data analysis. Results High degree of drug resistance (median 5 drugs, range 2–8) was observed. High proportion of patients was resistant to all five first-line anti-tuberculosis drugs (62.6%), and more than half were resistant to second line drugs (55.1%). The majority of the patients were ofloxacin resistant (52.7%). Upon multivariate analysis previous tuberculosis treatment at private (OR = 1.953, p = 0.034) and public private mix (OR = 2.824, p = 0.046) sectors were predictors of ofloxacin resistance. Conclusion The high degree of drug resistance observed, particularly to fluoroquinolones, is alarming. We recommend the adoption of more restrictive policies to control non-prescription sale of fluoroquinolones, its rational use by physicians, and training doctors in both private and public–private mix sectors to prevent further increase in fluoroquinolones resistant Mycobacterium tuberculosis strains.


Subject(s)
Adolescent , Adult , Child , Female , Humans , Male , Middle Aged , Young Adult , Antitubercular Agents/pharmacology , Fluoroquinolones/pharmacology , Mycobacterium tuberculosis/drug effects , Tuberculosis, Multidrug-Resistant/microbiology , Cross-Sectional Studies , Microbial Sensitivity Tests , Pakistan , Prevalence
16.
Braz. j. microbiol ; 46(3): 641-647, July-Sept. 2015. tab, ilus
Article in English | LILACS | ID: lil-755830

ABSTRACT

Dormancy models for Mycobacterium tuberculosis play important roles in understanding various aspects of tuberculosis pathogenesis and in the testing of novel therapeutic regimens. By simulating the latent tuberculosis infection, in which the bacteria exist in a non-replicative state, the models demonstrate reduced susceptibility to antimycobacterial agents. This minireview outlines the models available for simulating latent tuberculosis both in vitro and in several animal species. Additionally, this minireview discusses the advantages and disadvantages of these models for investigating the bacterial subpopulations and susceptibilities to sterilization by various antituberculosis drugs.

.


Subject(s)
Animals , Guinea Pigs , Mice , Rabbits , Antitubercular Agents/pharmacology , Disease Models, Animal , Latent Tuberculosis/pathology , Mycobacterium tuberculosis/growth & development , Tuberculosis, Pulmonary/pathology , Drug Resistance, Bacterial , Indicators and Reagents/pharmacology , Latent Tuberculosis/drug therapy , Latent Tuberculosis/microbiology , Macaca fascicularis , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Oxazines/pharmacology , Tuberculosis, Pulmonary/drug therapy , Tuberculosis, Pulmonary/microbiology , Xanthenes/pharmacology , Zebrafish
17.
Mem. Inst. Oswaldo Cruz ; 110(5): 618-623, Aug. 2015. tab, ilus
Article in English | LILACS | ID: lil-755891

ABSTRACT

Drug resistance is a global threat and one of the main contributing factors to tuberculosis (TB) outbreaks. The goal of this study was to analyse the molecular profile of multidrug-resistant TB (MDR-TB) in the state of Santa Catarina in southern Brazil. Fifty-three MDR Mycobacterium tuberculosisclinical isolates were analysed by spoligotyping and a partial region of therpoB gene, which is associated with rifampicin resistance (RMP-R), was sequenced. Some isolates were also distinguished by their mycobacterial interspersed repetitive units (MIRU). S531L was the most prevalent mutation found within rpoBin RMP-R isolates (58.5%), followed by S531W (20.8%). Only two MDR isolates showed no mutations withinrpoB. Isolates of the Latin American Mediterranean (LAM) family were the most prevalent (45.3%) found by spoligotyping, followed by Haarlem (9.4%) and T (7.5%) families. SIT106 was found in 26.4% of isolates and all SIT106 isolates typed by MIRU-12 (5 out of 14) belong to MIT251. There was a high correlation between the S531W mutation and the LAM family mainly because all SIT2263 (LAM9) isolates carry this mutation. Among isolates with the S531W mutation in rpoB MIRU demonstrates a cluster formed by four isolates (SIT2263 and MIT163) and very similar profiles were observed between eight of the nine isolates. Better characterisation of TB isolates may lead to new ways in which to control and treat TB in this region of Brazil.

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Subject(s)
Adult , Female , Humans , Male , Antitubercular Agents/pharmacology , DNA, Bacterial , Drug Resistance, Multiple, Bacterial/genetics , Mutation/genetics , Mycobacterium tuberculosis/drug effects , Tuberculosis, Multidrug-Resistant/microbiology , Bacterial Typing Techniques , Brazil , Bacterial Proteins/genetics , Genotype , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNA
18.
Mem. Inst. Oswaldo Cruz ; 110(5): 649-654, Aug. 2015. tab, ilus
Article in English | LILACS | ID: lil-755892

ABSTRACT

The purpose of this study is to evaluate four rapid colourimetric methods, including the resazurin microtitre assay (REMA), malachite green decolourisation assay (MGDA), microplate nitrate reductase assay (MNRA) and crystal violet decolourisation assay (CVDA), for the rapid detection of multidrug-resistant (MDR) tuberculosis. Fifty Mycobacterium tuberculosisisolates were used in this study. Eighteen isolates were MDR, two isolates were only resistant to isoniazid (INH) and the remaining isolates were susceptible to both INH and rifampicin (RIF). INH and RIF were tested in 0.25 µg/mL and 0.5 µg/mL, respectively. The agar proportion method was used as a reference method. MNRA and REMA were performed with some modifications. MGDA and CVDA were performed as defined in the literature. The agreements of the MNRA for INH and RIF were 96% and 94%, respectively, while the agreement of the other assays for INH and RIF were 98%. In this study, while the specificities of the REMA, MGDA and CVDA were 100%, the specificity of the MNRA was lower than the others (93.3% for INH and 90.9% for RIF). In addition, while the sensitivity of the MNRA was 100%, the sensitivities of the others were lower than that of the MNRA (from 94.1-95%). The results were reported on the seventh-10th day of the incubation. All methods are reliable, easy to perform, inexpensive and easy to evaluate and do not require special equipment.

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Subject(s)
Humans , Antitubercular Agents/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Microbial Sensitivity Tests/methods , Mycobacterium tuberculosis/drug effects , Coloring Agents , Indicators and Reagents , Sensitivity and Specificity
19.
Rev. chil. infectol ; 32(4): 382-386, ago. 2015. tab
Article in Spanish | LILACS | ID: lil-762634

ABSTRACT

This publication presents the results of the Chilean initial study of resistance to first line anti-tuberculous drugs. The study was carried out between 2011 and 2012 by the National Reference Laboratory of the Institute of Public Health, as part of the Drug Surveillance Resistance in tuberculosis (TB) promoted by the World Health Organization. Methodology: Cross-sectional study performed using cluster sampling, representative of the entire country as recommended by the World Health Organization. Susceptibility testing to isoniazid, rifampicin, streptomycin, ethambutol and pyrazinamide was performed through the proportion method or Wayne's enzymatic method, as appropriate. Results: 594 susceptibility tests were performed, showing an overall level of TB drug resistance of 8.6% and a prevalence of multidrug resistance of 1.3%. Indeed, the study showed a decrease in streptomycin resistance and an increase of isoniazid resistance in both mono-resistance and accumulated resistance compared to previous studies. No cases of mono-resistance to rifampicin were detected. Conclusion: An increased resistance to anti-TB drugs in Chile is observed, which despite being still low, is no less worrisome. Since 2014 the monitoring of drug resistance to TB is universally performed to avoid sub - diagnosis and treat each case according to the susceptibility profile.


Esta publicación presenta los resultados del estudio de resistencia inicial a fármacos anti-tuberculosos de primera línea realizado entre los años 2011 y 2012 en Chile por el Laboratorio de Referencia Nacional del Instituto de Salud Pública, estudio que forma parte de la vigilancia de la fármaco-resistencia en tuberculosis (TBC) promovida por la Organización Mundial de la Salud. Metodología: Estudio transversal realizado mediante un muestreo por conglomerado, representativo de todo el país según recomendaciones de la Organización Mundial de la Salud. Se realizó prueba de susceptibilidad a isoniacida, rifampicina, estreptomicina, etambutol y pirazinamida a través del método de las proporciones o método enzimático de Wayne según corresponda. Resultados: Se realizó test de susceptibilidad a 594 casos de TBC, observándose una resistencia inicial global de 8,6% y una prevalencia de multi-resistencia de 1,3%. Además destaca la caída en la resistencia a estreptomicina y el aumento de la resistencia a isoniacida, tanto en mono-resistencia como en resistencia acumulada para ambos fármacos comparada con los estudios anteriores. No se observaron casos de mono-resistencia a rifampicina. Conclusión. Se observa un aumento de la resistencia a fármacos anti-tuberculosos en Chile la que, a pesar de ser aún baja, no deja de ser preocupante. Desde el año 2014 la vigilancia de fármaco-resistencia para TBC se hace en forma universal, de modo de evitar el sub-diagnóstico y realizar un tratamiento de acuerdo al perfil de susceptibilidad de cada caso.


Subject(s)
Humans , Antitubercular Agents/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Mycobacterium tuberculosis/drug effects , Tuberculosis, Multidrug-Resistant/microbiology , Chile , Cross-Sectional Studies , Microbial Sensitivity Tests , Mycobacterium tuberculosis/classification , Sputum/microbiology
20.
Braz. j. med. biol. res ; 48(8): 759-764, 08/2015. tab, graf
Article in English | LILACS | ID: lil-753059

ABSTRACT

Resistance to Mycobacterium tuberculosis is a reality worldwide, and its diagnosis continues to be difficult and time consuming. To face this challenge, the World Health Organization has recommended the use of rapid molecular tests. We evaluated the routine use (once a week) of a line probe assay (Genotype MTBDRplus) for early diagnosis of resistance and for assessment of the main related risk factors over 2 years. A total of 170 samples were tested: 15 (8.8%) were resistant, and multidrug resistance was detected in 10 (5.9%). The sensitivity profile took 3 weeks (2 weeks for culture and 1 week for rapid testing). Previous treatment for tuberculosis and the persistence of positive acid-fast smears after 4 months of supervised treatment were the major risk factors observed. The use of molecular tests enabled early diagnosis of drug-resistant bacilli and led to appropriate treatment of the disease. This information has the potential to interrupt the transmission chain of resistant M. tuberculosis.


Subject(s)
Humans , Male , Female , Adult , DNA, Bacterial/genetics , Genotyping Techniques/methods , Mycobacterium tuberculosis/genetics , Tuberculosis, Multidrug-Resistant/diagnosis , Antitubercular Agents/pharmacology , Bacteriological Techniques/methods , Brazil , Early Diagnosis , Isoniazid/pharmacology , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Rifampin/pharmacology , Risk Factors , Sensitivity and Specificity , Tuberculosis, Multidrug-Resistant/microbiology
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