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1.
Braz. j. infect. dis ; 24(5): 398-404, Sept.-Oct. 2020. tab, graf
Article in English | LILACS-Express | LILACS, ColecionaSUS | ID: biblio-1142551

ABSTRACT

Abstract Tuberculosis (TB) is one of the infectious diseases with high mortality in the world. DNA amplification techniques have been used to overcome barriers to the diagnosis of this disease. However, the success of these methodologies is highly dependent on the DNA obtained from the sample. This study was carried out to verify whether the DNA extracted by sonication (in house method) could yield suitable DNA for amplification by real-time PCR (qPCR). Sixty sputum samples were submitted to DNA extraction using sonication compared to a commercial method (Detect-TB kit, Labtest/MG-Brazil). All DNA samples were amplified by qPCR for IS6110 region (IS6110-qPCR/SYBR Green assay). Out of 60 samples, 40 were positive for TB; of these, all had positive results when extracted by sonication (100%) and 80% when extracted by the commercial method. The limit of detection (LOD) of Mycobacterium tuberculosis (H37Rv strain) by qPCR was 14CFU/mL when the DNA was extracted by sonication, compared to countless colonies when extracted by commercial kit. In conclusion, the sonication protocol (without purification step) proved to be a simple, fast, and suitable method for obtaining DNA for use in qPCR from sputum samples.

2.
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
3.
DST j. bras. doenças sex. transm ; 29(3): 106-109, 20171111.
Article in Portuguese | LILACS | ID: biblio-879139

ABSTRACT

A sífilis gestacional é um problema global de saúde pública e é uma das mais comuns causas de efeitos adversos durante a gravidez devido à ausência ou inadequação do tratamento. Estabelecer um diagnóstico de sífilis durante o pré-natal, evita a transmissão de Treponema pallidum para a criança. Objetivo: o objetivo deste estudo foi avaliar o desempenho de OL Syphilis (OrangeLife, Brasil), um teste imunocromatográfico rápido, para o diagnóstico de sífilis gestacional. Métodos: Um total de 185 mulheres grávidas no pré-natal foram avaliadas por sífilis OL. Os resultados foram comparados com os métodos tradicionais: Laboratório de Pesquisa de Doenças Venéreas e Reaginas de Plasma Rápido (VDRL e RPR) como ensaios de seleção e FTA-ABS como teste confirmatório. Resultados: A prevalência de sífilis nessa população foi de 6,49% (IC 3,40 a 11,06%). A sensibilidade do teste rápido (TR) foi de 91,67% (IC95% 61,52 a 99,79%) e a especificidade foi de 100% (95%IC 97,89 a 100%). O PPV foi 100% (95%CI 71,51 a 100%) e o VPL foi de 99,43 (95%CI 96,84 a 100%). O acordo medido pelo coeficiente Kappa foi de 0,954 (IC95% 0,863 a 1,000). Conclusão: O teste OL Syphilis poderia ser usado no rastreio de mulheres grávidas, fornecendo diagnóstico rápido, aumentando a probabilidade de ter a doença diagnosticada e oportuna, evitando as consequências devastadoras da sífilis congênita.


Gestational syphilis is a global public health problem and one of the most common causes of adverse effects during pregnancy due to absence or inadequacy of treatment. Establishing a diagnosis of syphilis during prenatal care prevents the transmission of Treponema pallidum to the child. Objective: The objective of this study was to evaluate the performance of OL Syphilis (OrangeLife, Brazil), a rapid immunochromatographic test for gestational syphilis diagnosis. Methods: A total of 185 pregnant women in prenatal care were evaluated by OL Syphilis. The results were compared by traditional methods: Venereal Disease Research Laboratory and Rapid Plasma Reagin (VDRL and RPR) for screening and fluorescent treponemal antibody absorption test (FTA-Abs) for confirmation. Results: The prevalence of syphilis in this population was 6.49% (95%CI 3.40 to 11.06%). Rapid Test (RT) sensitivity was 91.67% (95%CI 61.52 to 99.79%) and specificity was 100% (95%CI 97.89 to 100%). Positive predictive value was 100% (95%CI 71.51 to 100%) and Negative predictive value was 99.43% (95%CI 96.84 to 100%). The agreement measured by Kappa coefficient was 0.954 (95%CI 0.863 to 1.000). Conclusion: The OL Syphilis test could be used for screening pregnant women, thus providing rapid diagnosis, increasing the probability of diagnosis and timely treatment, and preventing the devastating consequences of congenital syphilis.


Subject(s)
Humans , Female , Pregnancy , Pregnancy , Prenatal Care , Syphilis, Congenital , Syphilis/diagnosis , Treponema pallidum , Chromatography, Affinity/statistics & numerical data , Public Health , Reagins
4.
Mem. Inst. Oswaldo Cruz ; 112(4): 255-259, Apr. 2017. tab, graf
Article in English | LILACS | ID: biblio-841784

ABSTRACT

BACKGROUND Porto Alegre is the Brazilian state capital with second highest incidence of tuberculosis (TB) and the highest proportion of people infected with human immunodeficiency virus (HIV) among patients with TB. Hepatitis C virus (HCV) infection increases the risk of anti-TB drug-induced hepatotoxicity, which may result in discontinuation of the therapy. OBJECTIVES The aim of this study was (i) to estimate prevalence of HCV and HIV in a group of patients newly diagnosed with active TB in a public reference hospital in Porto Alegre and (ii) to compare demographic, behavioural, and clinical characteristics of patients in relation to their HCV infection status. METHODS One hundred and thirty-eight patients with TB were tested for anti-HCV antibody, HCV RNA, and anti-HIV1/2 antibody markers. HCV RNA from real-time polymerase chain reaction (PCR)-positive samples was submitted to reverse transcription and PCR amplification. The 5′ non-coding region of the HCV genome was sequenced, and genotypes of HCV isolates were determined. FINDINGS Anti-HCV antibody, HCV RNA, and anti-HIV antibodies were detected in 27 [20%; 95% confidence interval (CI), 13-26%], 17 (12%; 95% CI, 7-18%), and 34 (25%; 95% CI, 17-32%) patients, respectively. HCV isolates belonged to genotypes 1 (n = 12) and 3 (n = 4). Some characteristics were significantly more frequent in patients infected with HCV. Among them, non-white individuals, alcoholics, users of illicit drugs, imprisoned individuals, and those with history of previous TB episode were more commonly infected with HCV (p < 0.05). MAIN CONCLUSIONS HCV screening, including detection of anti-HCV antibody and HCV RNA, will be important to improving the management of co-infected patients, given their increased risk of developing TB treatment-related hepatotoxicity.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Middle Aged , Aged , Tuberculosis/diagnosis , Tuberculosis/epidemiology , HIV Antibodies/blood , HIV Infections/diagnosis , HIV Infections/epidemiology , Hepatitis C/diagnosis , Coinfection/diagnosis , Coinfection/epidemiology , Brazil/epidemiology , RNA, Viral/blood , Polymerase Chain Reaction
5.
Mem. Inst. Oswaldo Cruz ; 112(2): 94-99, Feb. 2017. tab
Article in English | LILACS | ID: biblio-841768

ABSTRACT

BACKGROUND In high tuberculosis (TB) burden countries, there are few data on the performance of new molecular commercialised assays developed locally. OBJECTIVE To evaluate the performance of a new molecular commercialised assay for TB diagnosis (Detect-TB) in three laboratories. METHODS A total of 302 sputum samples from an equal number of patients with presumptive diagnosis of pulmonary tuberculosis (PTB) were submitted for routine smear microscopy, culture, and Detect-TB assay at three different sites in Brazil (the cities of Caxias do Sul, São Paulo and Canoas). FINDINGS Seventy four (24.7%) TB cases were diagnosed (65 bacteriologically confirmed). When compared to smear microscopy/culture results, the overall sensitivity and specificity of Detect-TB assay was 84.6% (CI 95%; 73.7-91.6) and 93.1% (CI 95%; 89.1-95.8), respectively. When compared to bacteriological and clinical diagnostic criteria, the sensitivity and specificity of Detect-TB assay was 74.3% (CI 95%; 63.3-82.9) and 92.9% (CI 95%; 88.7-95.6), respectively. Among the three sites - Caxias do Sul, São Paulo and Canoas - the sensitivity and specificity were respectively 94.7% and 97.8%; 71.4% and 93.9%, 82.1% and 88.9%. MAIN CONCLUSIONS These findings suggest that the Detect-TB assay could be applied routinely in reference laboratories across different regions in Brazil.


Subject(s)
Humans , Sputum/microbiology , Tuberculosis, Pulmonary/diagnosis , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/genetics , Brazil , DNA, Bacterial , False Negative Reactions
6.
Comun. ciênc. saúde ; 28(1): 85-90, jan. 2017.
Article in Portuguese | LILACS | ID: biblio-972641

ABSTRACT

Além de identificar os doentes com tuberculose (TB), é importante monitorar os genótipos de M. tuberculosis circulantes. Mesmo após a implantação do Xpert® MTB/RIF, o diagnóstico é ainda realizado apenas pela baciloscopia, que apresenta baixa sensibilidade, na maioria dos laboratórios. OBJETIVO: Utilizar análises de DNA para o diagnóstico e identificação dos genótipos circulantes em uma população do Rio Grande do Sul, Brasil. METODOLOGIA: Amostras clínicas foram analisadas pelo Detect-TB (Labtest,MG), por PCR em tempo real (Xpert® MTB/RIF) e comparados a baciloscopia. A genotipagem foi realizada por spoligotyping. RESULTADOS: A acurácia do Detect-TB para a identificação da TB foi similarao Xpert® MTB/RIF, sendo que o Detect-TB foi mais custo-efetivo quando utilizado em conjunto com a baciloscopia. Os genótipos LAM5, RDRio e like-Pinni2, relacionados a resistência ao tratamento, estavam sendo transmitidos neste grupo, e a maioria dos resistentes a isoniazida (78,5%)e dos resistentes a rifampicina (92,1%) apresentavam as mutações mais conhecidas. CONCLUSÃO: A aplicação de tecnologias de DNA pode auxiliar no controle de TB, tanto no diagnóstico rápido quanto na identificação de perfis resistentes, viabilizando tratamento adequado aos pacientes.


In addition to detecting patients with tuberculosis (TB), it is important tomonitor circulating M. tuberculosis genotypes. Even after the implantation of Xpert® MTB/RIF, the diagnosis is still based only by bacilloscopy, which have a low sensitivity, in most laboratories. OBJECTIVE: To use DNA analysis for diagnosis and identification of circulating genotypes in a population of Rio Grande do Sul, Brazil. METHODOLOGY: Clinical samples were analyzed by Detect-TB (Labtest,MG), real-time PCR (Xpert® MTB/RIF) and compared to bacilloscopy. Genotyping was performed by spoligotyping. RESULTS: The accuracy of Detect-TB was similar to Xpert® MTB / RIF, butDetect-TB was more cost-effective when used with bacilloscopy. The genotypesLAM5, RDRio and like-Pinni2, related to treatment resistance, were being transmitted among this group, and the majority of the resistant to isoniazid (78.5%) and the resistant to rifampicin (92.1%) presented themost known mutations. CONCLUSION: The application of DNA technologies can help in the controlof TB, both in rapid diagnosis and in the identification of resistant profiles, allowing adequate treatment to the patients.


Subject(s)
Humans , Tuberculosis , Molecular Epidemiology , Diagnosis , Drug Resistance
7.
Mem. Inst. Oswaldo Cruz ; 109(3): 307-314, 06/2014. tab
Article in English | LILACS | ID: lil-711730

ABSTRACT

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.


Subject(s)
Bacterial Proteins/genetics , Catalase/genetics , Drug Resistance, Multiple, Bacterial/genetics , Mutation/genetics , Mycobacterium tuberculosis/genetics , Oxidoreductases/genetics , Colorimetry , DNA, Bacterial/genetics , Genotyping Techniques , Isoniazid/pharmacology , Multiplex Polymerase Chain Reaction , Mycobacterium tuberculosis/drug effects , Nucleic Acid Hybridization , Rifampin/pharmacology
8.
Mem. Inst. Oswaldo Cruz ; 109(3): 345-351, 06/2014. tab
Article in English | LILACS | ID: lil-711738

ABSTRACT

Certain host single nucleotide polymorphisms (SNPs) affect the likelihood of a sustained virological response (SVR) to treatment in subjects infected with hepatitis C virus (HCV). SNPs in the promoters of interleukin (IL)-10 (-1082 A/G, rs1800896), myxovirus resistance protein 1 (-123 C/A, rs17000900 and -88 G/T, rs2071430) and tumour necrosis factor (TNF) (-308 G/A, rs1800629 and -238 G/A, rs361525) genes and the outcome of PEGylated α-interferon plus ribavirin therapy were investigated. This analysis was performed in 114 Brazilian, HCV genotype 1-infected patients who had a SVR and in 85 non-responders and 64 relapsers. A significantly increased risk of having a null virological response was observed in patients carrying at least one A allele at positions -308 [odds ratios (OR) = 2.58, 95% confidence intervals (CI) = 1.44-4.63, p = 0.001] or -238 (OR = 7.33, 95% CI = 3.59-14.93, p < 0.001) in the TNF promoter. The risk of relapsing was also elevated (-308: OR = 2.87, 95% CI = 1.51-5.44, p = 0.001; -238: OR = 4.20, 95% CI = 1.93-9.10, p < 0.001). Multiple logistic regression of TNF diplotypes showed that patients with at least two copies of the A allele had an even higher risk of having a null virological response (OR = 16.43, 95% CI = 5.70-47.34, p < 0.001) or relapsing (OR = 6.71, 95% CI = 2.18-20.66, p = 0.001). No statistically significant association was found between the other SNPs under study and anti-HCV therapy response.


Subject(s)
Female , Humans , Male , Middle Aged , Antiviral Agents/administration & dosage , Hepacivirus/drug effects , Hepatitis C, Chronic/drug therapy , Interferon-alpha/administration & dosage , Promoter Regions, Genetic , Ribavirin/administration & dosage , Tumor Necrosis Factor-alpha/genetics , Drug Therapy, Combination , Genotype , Hepatitis C, Chronic/genetics , /genetics , Myxovirus Resistance Proteins/genetics , Polymorphism, Single Nucleotide , Treatment Failure , Viral Load
9.
Mem. Inst. Oswaldo Cruz ; 108(1): 48-53, Feb. 2013. graf, tab
Article in English | LILACS | ID: lil-666043

ABSTRACT

A single-nucleotide polymorphism (SNP) upstream of interleukin (IL)28B was recently identified as an important predictor of the outcome of chronic hepatitis C patients treated with pegylated interferon plus ribavirin (PEG-IFN/RBV). The aim of this study was to investigate the association between the IL28B gene polymorphism (rs12979860) and virological response in chronic hepatitis C patients. Brazilian patients (n = 263) who were infected with hepatitis C virus (HCV) genotype 1 and were receiving PEG-IFN/RBV were genotyped. Early virological response (EVR) (12 weeks), end-of-treatment response (EOTR) (48 weeks), sustained virological response (SVR) (72 weeks) and relapse were evaluated using conventional and quantitative polymerase chain reaction (PCR) assays. The frequency of the C allele in the population was 39%. Overall, 43% of patients experienced SVR. The IL28B CC genotype was significantly associated with higher treatment response rates and a lower relapse rate compared to the other genotypes [84% vs. 58% EVR, 92% vs. 63% EOTR, 76% vs. 38% SVR and 17% vs. 40% relapse rate in CC vs. other genotypes (CT and TT), respectively]. Thus, the IL28B genotype appears to be a strong predictor of SVR following PEG-IFN/RBV therapy in treatment-naïve Brazilian patients infected with HCV genotype 1. This study, together with similar research examining other SNPs, should help to define adequate protocols for the treatment of patients infected with HCV genotype 1, especially those with a poor prognosis.


Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Antiviral Agents/administration & dosage , Hepatitis C, Chronic/drug therapy , Interferon-alpha/administration & dosage , Interleukins/genetics , Polyethylene Glycols/administration & dosage , Polymorphism, Single Nucleotide/genetics , Ribavirin/administration & dosage , Alleles , Cohort Studies , Drug Therapy, Combination , Genotype , Hepatitis C, Chronic/genetics , Hepatitis C, Chronic/virology , Polymerase Chain Reaction , Prospective Studies , Recombinant Proteins/administration & dosage , Treatment Outcome
10.
Mem. Inst. Oswaldo Cruz ; 107(7): 909-915, Nov. 2012. tab
Article in English | LILACS | ID: lil-656048

ABSTRACT

The occurrence of tuberculosis (TB) in prisons has been described as an alarming public health problem in many countries, especially in developing nations. The objective of this study was to conduct a survey among prisoners with TB respiratory symptoms in order to estimate the incidence of the disease, to analyze the drug susceptibility profile and genotype the isolates of Mycobacterium tuberculosis in the city of Charqueadas, southern of Brazil. The TB incidence was 55/1,900 inhabitants in the prison; this corresponds to an incidence of 3,789/100,000 inhabitants, with a prevalence of 72/1,900 (4,960/100,000 inhabitants). Drug susceptibility test was performed and, among the analyzed isolates, 85% were susceptible to all drugs tested and 15% were resistant to at least one drug, of which 89% were resistant only to isoniazid (INH) or in combination with another drug. The genotype classification of spoligotyping analysis showed that 40% of the isolates belong to LAM family, 22% to T family, 17.5% to Haarlem family, 12.5% to U family and 3% to X family. The shared international spoligotypes most frequently found were 729 (27%), 50 (9.5%), 42 (8%), 53 (8%) and 863 (8%). In conclusion, it was observed that TB in this specific population had been caused, mostly, by strains that have been transmitted in the last few years, as demonstrated by the large level of genotype clustering. In addition, it was found specific large clusters, which were not often found in the general population from the same period and in the same region.


Subject(s)
Adult , Humans , Male , Middle Aged , Young Adult , DNA, Bacterial/analysis , Mycobacterium tuberculosis/genetics , Prisoners/statistics & numerical data , Tuberculosis, Pulmonary/epidemiology , Brazil/epidemiology , Genotype , Mycobacterium tuberculosis/drug effects , Prevalence , Tuberculosis, Pulmonary/diagnosis
11.
Cad. saúde colet., (Rio J.) ; 20(3)jul. 12. tab
Article in Portuguese | LILACS-Express | LILACS | ID: lil-684835

ABSTRACT

O objetivo do estudo foi descrever a prevalência do papilomavírus humano e da Chlamydia trachomatis entre mulheres assintomáticas na região Sul do Brasil. Estudo transversal, com 1.217 esfregaços cervicais testados para citologia, papilomavírus humano e Chlamydia trachomatis-DNA. Os resultados foram estimados por regressão logística múltipla. As prevalências de papilomavírus humano, Chlamydia trachomatis-DNA e coinfecção foram de 28,4, 12,6, e 6,5%, respectivamente. A infecção por papilomavírus humano foi associada com a raça não branca, estar empregada e ter parceiro sexual com história de condiloma genital. A infecção por Chlamydia trachomatis apresentou associação com o início da atividade sexual em idade ?20 anos e estar empregada. A coinfecção apresentou associação com ter ?3 parceiros sexuais. Anormalidades citológicas do colo versus papilomavírus humano e coinfecção apresentaram associação significativa (p>0,001). Elevadas prevalências de papilomavírus humano, Chlamydia trachomatis e coinfecção foram observadas em uma população de mulheres assintomáticas e os resultados indicam a importância de medidas de prevenção e promoção da saúde.

12.
Mem. Inst. Oswaldo Cruz ; 106(2): 194-199, Mar. 2011. tab
Article in English | LILACS | ID: lil-583945

ABSTRACT

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2 percent (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85 percent and 98 percent, and 94 percent and 100 percent, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.


Subject(s)
Humans , Mycobacterium tuberculosis , Nucleic Acid Hybridization/methods , Polymerase Chain Reaction/methods , Sputum , Tuberculosis, Pulmonary , Colorimetry , DNA, Bacterial , Mycobacterium tuberculosis , Oligonucleotide Probes , Reagent Kits, Diagnostic , Sensitivity and Specificity
13.
Mem. Inst. Oswaldo Cruz ; 104(5): 710-714, Aug. 2009. ilus
Article in English | LILACS | ID: lil-528078

ABSTRACT

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.


Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Catalase/genetics , Drug Resistance, Bacterial/genetics , Isoniazid/pharmacology , Mycobacterium tuberculosis , Mutation/genetics , Colorimetry/methods , DNA, Bacterial/analysis , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Nucleic Acid Hybridization , Polymerase Chain Reaction
14.
Rev. saúde pública ; 43(2): 283-290, abr. 2009. ilus, tab
Article in English | LILACS | ID: lil-507818

ABSTRACT

OBJECTIVE: To evaluate metabolic changes associated with highly active antiretroviral therapy (HAART) in HIV-positive patients, and to identify risk factors associated. METHODS: Retrospective study that included 110 HIV-positive patients who where on HAART in the city of Porto Alegre (Southern Brazil) between January 2003 and March 2004. Data on demographic variables, cigarette smoking, diabetes mellitus, cholesterol and triglyceride levels, stage of HIV infection, antiretroviral therapy and HCV coinfection were collected...


OBJETIVO: Avaliar as alterações metabólicas associadas à terapia anti-retroviral potente em pacientes HIV-positivos e identificar fatores de risco associados. MÉTODOS: Estudo retrospectivo com 110 pacientes HIV-positivos que estavam sob terapia anti-retroviral potente (HAART) na cidade de Porto Alegre (RS), entre janeiro de 2003 e março de 2004. Os dados coletados incluem variáveis demográficas, tabagismo, diabetes mellitus, níveis de colesterol e triglicerídeos, estágio da infecção viral, terapia anti-retroviral e co-infecção com hepatite C...


OBJETIVO: Evaluar las alteraciones metabólicas asociadas a la terapia anti-retroviral potente en pacientes HIV-positivos e identificar factores de riesgo asociados. MÉTODOS: Estudio retrospectivo con 110 pacientes HIV-positivos que estaban en terapia anti-retroviral potente (HAART) en la ciudad de Porto Alegre (Sur de Brasil), entre enero de 2003 y marzo de 2004. Los datos colectados incluyen variables demográficas, tabaquismo, diabetes mellitas, niveles de colesterol y triglicéridos, fase de la infección viral, terapia anti-retroviral y co-infección con hepatitis C...


Subject(s)
Adult , Female , Humans , Male , Anti-HIV Agents/therapeutic use , Antiretroviral Therapy, Highly Active/adverse effects , Dyslipidemias/chemically induced , Glucose/analysis , HIV Infections/blood , Hepatitis C/complications , Cohort Studies , Cholesterol/blood , Dyslipidemias/blood , HIV Infections/complications , HIV Infections/drug therapy , Hepatitis C/blood , Protease Inhibitors/therapeutic use , Retrospective Studies , Risk Factors , RNA, Viral , Triglycerides/blood
15.
Braz. arch. biol. technol ; 51(4): 485-491, June-Aug. 2008. ilus, tab
Article in English | LILACS-Express | LILACS | ID: lil-622655

ABSTRACT

The aim of this work was to construct and test a plasmidial Internal Control (IC) to detect the inhibition in the PCR test for M. tuberculosis and also its contribution for a Public Health Laboratory routine. The IC was a 600-bp of DNA linked to a plasmid with the same primer sites, allowing the amplification with the 245-bp diagnostic fragment. The amplification of the positive samples rendered the IC and the diagnostic fragment; instead negative samples only showed the IC. A total of 149 tuberculosis samples were studied and introduced the IC to monitor. Results showed 3.3% of the samples without amplification of the IC, suggesting the inhibition. These samples showed results in accordance with the clinical results. The objective of the IC was to identify the false negative results.


A PCR do elemento IS6110 para diagnóstico da Tuberculose (TB) é muito utilizada em laboratórios de pesquisa. As suas limitações incluem, a inibição da enzima Taq DNA Polimerase. A seguir descrevemos a construção de um Controle Interno (IC) e ensaios de detecção da inibição da PCR para M. tuberculosis. O IC é um fragmento de DNA de 600 pb com os mesmos sítios de anelamento do primer, permitindo a amplificação com o fragmento diagnóstico de 245 pb. As amostras positivas fornecem um padrão de bandas referentes ao IC (664 pb) e ao fragmento diagnóstico (245 pb), e as amostras negativas apresentam apenas o fragmento correspondente ao IC. 149 amostras com diagnóstico conhecido foram analisadas por PCR introduzindo o IC em todas elas. Os resultados mostraram 3.3% de amostras sem amplificação do IC sugerindo inibição. Estas amostras quando testadas novamente mostraram resultados concordantes com os resultados clínicos. O objetivo do IC e identificar os falsos resultados negativos.

16.
Mem. Inst. Oswaldo Cruz ; 102(7): 867-870, Nov. 2007. tab
Article in English | LILACS | ID: lil-470359

ABSTRACT

Hepatitis C virus (HCV) isolates have been divided into six genotypes (1 to 6). The duration of hepatitis C standard treatment is 48 weeks for patients infected with HCV genotype 1 vs 24 weeks for those infected with genotypes 2 and 3. A total of 1544 HCV isolates from chronic patients living in the southern Brazilian states of Rio Grande do Sul (RS, n = 627) and Santa Catarina (SC, n = 917) were genotyped by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products. In RS, 338 (53.9 percent; 95 percent CI 50.0 - 57.8 percent), 34 (5.4 percent; 95 percent CI 3.8 - 7.4 percent) and, 255 (40.7 percent; 95 percent CI 36.9 - 44.6 percent) samples were from genotypes 1, 2, and 3, respectively. In SC, 468 (51 percent; 95 percent CI 47.8 - 54.2 percent), 26 (2.9 percent; 95 percent CI 1.9 - 4.1 percent) and, 423 (46.1 percent; 95 percent CI 42.9 - 49.3 percent) samples were from genotypes 1, 2, and 3, respectively. Genotyping results were confirmed by direct nucleotide sequencing of PCR products derived from 68 samples, without any discrepancy between PCR-RFLP and nucleotide sequencing methods. In conclusion, almost half of the hepatitis C patients from South of Brazil are infected by genotypes 2 and 3 and, these results have important consequential therapeutic implications as they can be treated for only 24 weeks, not 48.


Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Hepacivirus/genetics , Hepatitis C, Chronic/virology , Polymorphism, Single Nucleotide , Brazil , Cohort Studies , Genotype , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Retrospective Studies , RNA, Viral/genetics
17.
J. bras. pneumol ; 30(4): 358-364, jul.-ago. 2004. ilus, tab
Article in Portuguese | LILACS | ID: lil-383146

ABSTRACT

INTRODUÇAO: A tuberculose é uma doença antiga que ainda se mantém como um dos maiores males da humanidade no século XXI. Nas últimas décadas, o advento de novas tecnologias utilizando os conhecimentos de biologia molecular tem levado a um aumento na investigação da etiologia, detecção e epidemiologia da tuberculose. OBJETIVO: Avaliar o grau de similaridade entre as cepas de Mycobacterium tuberculosis provenientes do setor de tisiologia do Centro de Saúde Navegantes, de Porto Alegre (RS). MÉTODO: Foi realizado um estudo retrospectivo utilizando 55 amostras de escarro de pacientes atendidos ambulatorialmente no Centro de Saúde Navegantes para realização da técnica de RFLP. Os resultados obtidos pela genotipagem foram correlacionados com os dados gerados a partir da epidemiologia convencional. RESULTADOS: Trinta e nove isolados (70,9 por cento) apresentaram padrão único, enquanto dezesseis isolados (29,1 por cento) apresentaram padrões agrupáveis e formaram 8 clusters, com 2 pacientes em cada. Foi encontrada relação epidemiológica em 6 (37,5 por cento) dos 16 pacientes em cluster. CONCLUSAO: A associação adequada entre epidemiologia convencional e genotipagem de M. tuberculosis contribui para um melhor entendimento da dinâmica de transmissão da tuberculose mesmo quando o estudo é realizado em um único local.


Subject(s)
Humans , Male , Female , Mycobacterium tuberculosis/genetics , Tuberculosis, Pulmonary/microbiology , Genotype , Mycobacterium tuberculosis/classification , Polymorphism, Restriction Fragment Length , Retrospective Studies , Risk Factors , Tuberculosis, Pulmonary/transmission
18.
J. bras. pneumol ; 30(4): 365-370, jul.-ago. 2004. tab
Article in Portuguese | LILACS | ID: lil-383147

ABSTRACT

INTRODUÇAO: As taxas de resistência aos fármacos constituem um dos pilares da avaliação dos programas de controle da tuberculose. A demora na obtenção dos resultados, conseqüência da metodologia convencional utilizada, faz com que haja a necessidade de avaliação de novos testes, mais rápidos e menos onerosos. OBJETIVO: Comparar técnicas fenotípicas rápidas para determinação do perfil de susceptibilidade de M. tuberculosis, utilizando indicadores de viabilidade celular, com o teste das proporções em Lõwenstein-Jensen, padrão-ouro. MÉTODO: Foram utilizadas 166 cepas de M. tuberculosis com o perfil de susceptibilidade conhecido. A concentração mínima inibitória de cada fármaco foi determinada, em microplaca, utilizando-se meio líquido e os indicadores de oxi-redução, Alamar Blue® e brometo de tetrazolium. O ponto de corte entre a cepa sensível e a resistente foi estabelecido como concentração mínima inibitória maior ou igual a 0,2 mg /mL para isoniazida e 1,0 mg /mL para rifampicina. RESULTADOS: Houve concordância total entre os dois métodos de determinação da concentração mínima inibitória. Comparando os resultados dos testes com o padrão-ouro, obteve-se uma concordância de 95 por cento, para isoniazida e rifampicina. O tempo para obtenção dos resultados foi de 7 dias, contrastando com os 28 dias pelo método convencional. CONCLUSAO: Os testes para determinação da concentração mínima inibitória, em meio líquido, utilizando indicadores de oxi-redução, são rápidos e podem se utilizados como alternativa rápida na determinação de susceptibilidade de cepas de M. tuberculosis.


Subject(s)
Humans , Antitubercular Agents/pharmacology , Isoniazid/pharmacology , Mycobacterium tuberculosis , Rifampin/pharmacology , Indicators and Reagents , Microbial Sensitivity Tests , Cell Survival , Time Factors
19.
Rev. saúde pública ; 36(4): 525-532, ago. 2002. ilus, tab
Article in Portuguese | LILACS | ID: lil-320451

ABSTRACT

O progresso na compreensäo dos mecanismos de resistência aos fármacos usados no tratamento da tuberculose tem permitido o desenvolvimento de novos métodos para a detecçäo da tuberculose resistente. A resistente aos fármacos representa uma ameaça para os programas de controle da tuberculose. Para tanto, é necessário conhecer o padräo de sensibilidade das linhagens para fornecer o tratamento adequado. Os estudos moleculares dos mecanismos de açäo dos fármacos antituberculose têm elucidado as bases genéticas da resistência aos fármacos em Mycobacterium tuberculosis. Os mecanismos de resistência aos fármacos na tuberculose säo causados por mutaçöes cromossomais em diferentes genes da bactéria. Durante a exposiçäo aos fármacos, há uma pressäo seletiva favorecendo o desenvolvimento de linhagens resistentes. A tuberculose multirresistente é um problema nacional e internacional que traz sérias dificuldades para o controle global da doença. Realizou-se uma revisäo sobre os mecanismos moleculares associados à resistência aos fármacos com ênfase nas novas perspectivas para detectar os isolados resistentes


Subject(s)
Antitubercular Agents , Tuberculosis, Multidrug-Resistant , Tuberculosis , Isoniazid , Mycobacterium tuberculosis , Drug Resistance, Multiple , Rifampin
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