Human papillomavirus infection situation and its genotype distribution among gynecological outpatients / 重庆医学

Objective To understand the status quo of human papillomavirus (HPV) infection and its genotypes distribution a-mong gynecological outpatients in Chengdu region .Methods The DNA microarray technique combined with PCR and DNA reverse hybridizatio technology was used to detect the genotypes of HPV infection .The data were analyzed by the SPSS 13 .0 software .Re-sults A total of 5 052 samples of cervical exfoliated cells among gynecological outpatients were detected ,and the total positive rate of HPV infection was 17 .52% .The differences of HPV infection among various age groups were statistically significant ,especially the positive rate of HPV infection in the 20-25 years age group was higher than that in the 26-30 years age group and the 31-35 years age group(P<0 .05) ,and which in the 36 -40 years age group was also higher than that in the 26 -30 years age group (P<0 .05) .The positive detection rate of high risk HPV subgenotypes was 18 .1% ,which was higher than 5 .5% of low risk sub-genotypes with statistical difference (P<0 .01) .HPV 52 was the most frequent subgenotypes in high risk subgenotypes ,accounting for 15 .03% ,followed by HPV 16 and HPV 58;HPV 81 was the most frequent subgenotypes in low risk subgenotypes ,accounting for 7 .98% .Conclusion The positive rate of HPV infection among gynecological outpatients is higher ,and the majority of geno-types are high risk .It is suggested that the routine examination of HPV subgenotypes detection focused on different age groups should be recommended .

Distribución de genotipos del virus de hepatitis C en la población Argentina / Distribution of hepatitis C virus genotypes in an Argentine population / Distribuição dos genótipos do vírus da hepatite C na população Argentina

Mundialmente se han identificado 6 genotipos (1 al 6) del virus de la hepa­titis C (HCV). Dichos genotipos se subdividen en diferentes subtipos (a, b, c y otros). La respuesta al tratamiento instaurado depende del genotipo del virus infectante. El objetivo del trabajo fue determinar la frecuencia de los diferentes genotipos del HCV en la población de Argentina. Se estudiaron 510 pacientes infectados con HCV; la genotipificación del virus se realizó utilizando el equipo Versant HCV genotype assay 2.0 (LiPA). Los resultados obtenidos indican que el genotipo predominante del HCV en Argentina es el 1 (67,6%), con una prevalencia similar de subtipos 1a y 1b (33,3% y 34,5%, respectivamente). Se observó también una frecuencia similar de los genotipos 2 y 3 (14,5% cada uno). Con este estudio se actualizan los datos de las frecuencias de los diferentes genotipos de HCV que circulan en Argentina utilizando la nueva versión del reactivo para diagnóstico, el cual permitió una correcta subtipificación de las muestras.

Six hepatitis C virus genotypes have been identified worldwide so far. The­se genotypes have been subclassified into different subtypes (a, b, c and others). It is known that the response to treatment is highly dependent on the genotype involved. The aim of this work was to assess the frequency of occurrence of the different HCV genotypes in the population of Argentina. To this end, 510 infected patients were subjected to HCV genotyping using the commercial kit Versant HCV genotype assay 2.0. Results indicated that the genotype 1 was the most frequent (67.6%), and that subtypes 1a and 1b showed a similar prevalence (33.3% and 34.5%, respectively). Genotypes 2 and 3 also displayed a similar frequency (14.5% and 14.5% respectively). This study provides an update regarding the frequency of all HCV genotypes circulating in Argentina. The results were obtained by the novel version of the genotyping kit, which enabled a correct subtyping of samples.

Globalmente foram identificados 6 genótipos (1 ao 6) do vírus da hepatite C (HCV). Estes genótipos são subdivididos em vários subtipos (a, b, c, etc.). A resposta ao tratamento depende do genótipo do vírus infectante. O objetivo do estudo foi determinar a freqüência dos diferentes genótipos do HCV na população Argentina. Foram estudados 510 pacientes infectados com o HCV, a genotipagem do vírus foi realizada utilizando o kit Versant HCV genotype assay 2.0 (LiPA). Os resultados obtidos indicam que o genótipo predominante na Argentina é o tipo 1 (67,6%), observando-se uma prevalência dos subtipos 1a e 1b (33,3% e 34,5% respectivamente). Observou-se também uma freqüência semelhante do genótipos 2 e 3 (14,5% e 14,5% respectivamente). Este estudo atualiza os dados das freqüências dos diferentes genótipos do HCV em circulação na Argentina usando a nova versão do kit, o que permitiu uma correta subtipagem das amostras.

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

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

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

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

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.

Hibridização reversa e sequenciamento na genotipagem do vírus da hepatite C / Reverse hybridization and sequencing for genotyping the hepatitis C virus

INTRODUÇÃO: Os métodos de genotipagem do vírus da hepatite C têm sido muito discutidos. O objetivo deste trabalho foi comparar as metodologias de hibridização reversa e sequenciamento direto para a genotipagem do vírus da hepatite C. MÉTODOS: Noventa e uma amostras de plasma de pacientes assistidos na Faculdade de Medicina de Botucatu da Universidade Estadual Paulista foram utilizadas. A genotipagem por hibridização reversa foi realizada utilizando o kit comercial INNO-LiPA® v.1.0. O sequenciamento direto foi efetuado em sequenciador automático utilizando protocolos in house. RESULTADOS: A genotipagem por sequenciamento direto mostrou-se eficiente na resolução dos resultados inconclusivos pelo kit comercial. O kit mostrou resultados errôneos em relação à subtipagem viral. Além disso, a genotipagem por sequenciamento direto revelou um erro do kit com relação à determinação genotípica questionando a eficiência do método também para a identificação do genótipo viral. CONCLUSÕES: A genotipagem realizada por meio de sequenciamento direto permite uma maior acurácia na classificação viral quando comparada à hibridização reversa.

INTRODUCTION: The methods for genotyping the hepatitis C virus have been much discussed. The aim of this study was to compare the methodologies of reverse hybridization and direct sequencing for genotyping the hepatitis C virus. METHODS: Ninety-one plasma samples from patients attended at the Botucatu Medical School, São Paulo State University, were used. Genotyping by reverse hybridization was performed using the INNO-LiPA® v.1.0 commercial kit. Direct sequencing was performed in an automated sequencer using in-house protocols. RESULTS: Genotyping by direct sequencing was shown to be efficient for resolving cases that had remained inconclusive after using the commercial kit. The kit showed erroneous results in relation to virus subtyping. Moreover, direct sequencing revealed an error of the kit regarding the genotypic determination, thereby raising doubts about the efficiency of reverse hybridization for identifying the virus genotype. CONCLUSIONS: Genotyping by direct sequencing allowed greater accuracy of virus classification than did reverse hybridization.

Frequency of the mdr-1 C>T gene polymorphism in patients with COPD

BACKGROUND AND AIM: The multi-drug resistant-1 (MDR-1) gene is located on human chromosome 7 and encodes a glycosylated membrane protein that is a member of the ATP-binding cassette transporters superfamily. The aim of the study was to reveal the role of the C3435T MDR-1 gene polymorphism in chronic obstructive pulmonary disease. METHOD: DNA samples from 41 patients with chronic obstructive pulmonary disease and 50 healthy control participants were used to compare MDR-1 gene profiles. Genotyping assays were performed using the StripAssay technique that is based on reverse-hybridization. RESULTS: The T allele polymorphism in the MDR-1 gene located at position 3435 in exon 26 was shown to correlate with chronic obstructive pulmonary disease. CONCLUSION: These preliminary results suggest that the T allele polymorphism of the MDR-1 gene is associated with chronic obstructive pulmonary disease.

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.

Detection of Clarithromycin-resistant Strains from Clinical Isolates of Mycobacterium abscessus / 결핵및호흡기질환

BACKGROUND: Mycobacterium abscessus is the most pathogenic and drug-resistant rapid-growing mycobacterium. Clarithromycin or azithromycin are the only regular oral antimycobacterial agents that have an effect on M. abscessus. We tried to detect the clarithromycin-resistant strains from the clinical isolates of M. abscessus. METHODS: We tried to isolate the clarithromycin-resistant strains from 220 clinical isolates of M. abscessus by performing using reverse hybridization assay (RHA) and the broth microdilution test (BMT). RESULTS: Seven resistant strains (3.2%) from all the tested clinical isolates were detected by BMT. Three of these resistant strains were also detected by RHA and it was confirmed that they had point mutants. CONCLUSION: These results showed that clarithromycin resistance in M. abscessus clinical isolates is related to a point mutation and other unknown mechanisms.

Determinación de las mutaciones más frecuentes en pacientes con fibrosis quística, Ecuador. Análisis de mutaciones en el gen CFTR por técnica de hibridación reversa in situ y heterodúplex: junio 1996–enero 2004 / The most frequent mutation in patients with Cystic Fybrosis in Ecuador. Using the techniques of reverse in situ hybridization and heteroduplex to analyze mutations of the CFTR gene: june 1996–january 2004

La fibrosis quística (FQ) es la enfermedad hereditaria más común y letal que existe; es de carácter autosómico recesivo, de afección multisistémica, causada por una mutación en el gen CFTR (cystic fibrosis transmembrane regulator) ubicado en el brazo largo del cromosoma 7 (7q31.2). Tipo de estudio: descriptivo. Justificativo: Ecuador es uno de los pocos países latinoamericanos en los que aún no se conoce la verdadera incidencia y frecuencia de las mutaciones que afectan a los pacientes que padecen de fibrosis quística. Objetivos: Establecer el primer registro molecular de las mutaciones más frecuentes de la población de FQ ecuatoriana. Establecer la incidencia estimada de la enfermedad en el Ecuador. Resultados: Se incluyeron 62 pacientes en el estudio desde 1996 al 2004. Se determinó una incidencia estimada de 1:11.252 nacidos vivos durante el año 2004, con un estimado de 25 nacidos vivos afectos de fibrosis quística durante el referido año. La frecuencia de las mutaciones halladas fue ∆F508 37.1%, G85E 8.9%, G542X 2.4%, N1303K 2.4%, G551D 1.6% y R334W 0.8%. La frecuencia de la mutación G85E (8,9%) encontrada en Ecuador es la más alta a nivel mundial, incluso mayor a la del sur de Grecia de donde se cree que es originaria dicha mutación. Conclusiones y recomendaciones: La sensibilidad de los métodos utilizados (heterodúplex e hibridación reversa in situ) en relación a la población ecuatoriana fue 53,22%, que representa el porcentaje de mutaciones que se pudieron encontrar. Aunque aceptable en relación a los resultados encontrados a nivel mundial, este porcentaje plantea la imprescindible necesidad de utilizar secuenciación para establecer ese gran porcentaje de mutaciones que permanecen como no conocidas (WT).

Cystic Fybrosis is a autosomal recessive disease that is very common. It affects multi-organs and caused by the mutation of CFTR gene ( cystic fibrosis transmembrane regulator) which is found on the long arm of chromosome 7. Type of study: descriptive. Justification: Ecuador is one of the countries in Latin America that we still don’t know the incidence and frequency of the mutations that affect patients with Cystic Fybrosis. Objectives: To establish which are the most common mutations in patients with Cystic Fybrosis in Ecuador. To establish the incidence of this disease in Ecuador. Results: In this study 62 patients were found during 1996 to 2004. An incidence of 1:11252 born alive a total of 25 newborns had cystic fybrosis during this year. The frequencies for the mutations found were: ∆F508:37.1%, G85E: 8.9%, G542X: 2.4%, N1303: 2.4%, G551D: 1.6%, R334W: 0.8%. The frequency of gene mutation G85E has been found to be the highest in Ecuador other to other countries including South of Greece where the mutation originated. Conclusion and Recommendations: The sensitivity of the techniques used was 53.22% which represents the percentage of mutations that were found. Even though this is acceptable in relation to the results found worldwide, this percentage shows us how important it is to use mutation screening to establish the percentage of mutations that are unknown.

Detection of embB Gene Mutation of Mycobacterium tuberculosis by Reverse Hybridization Assay / 결핵및호흡기질환

BACKGROUND: Ethambutol (EMB) is one of important first-line drug in the treatment of tuberculosis. Molecular techniques to detect embB gene mutations have been considered as an method to define the EMB resistance. We investigated the mutation rate within embB gene among EMB resistant strains using reverse hybridization techniques. METHODS: We made 11 probes that had wild or mutated sequences containing codons 306, 406, or 497 within embB gene respectively. These probes were reverse-hybridized with PCR products amplified from embB gene which were isolated from 149 ethambutol resistant strains and 50 pan-susceptible strains. RESULTS: Out of 149 ethambutol resistant strains, one hundred (67.1%) had mutation at least one base at codon 306, 406, or 497 in embB gene. Mutation at codon 306, 406, 497 were demonstrated in 75 (50.3%), 16 (10.7%), and 13 strains (8.7%) respectively. There were four strains that showed multi-mutation at codon 306 and codon 406 simultaneously. A high proportion (8.1%) had single mutation at codon 406. There was no mutation observed in embB gene among 50 pan-susceptible strains. CONCLUSION: Reverse hybridization will be useful technique for detection of gene mutation correlated to ethambutol resistance.

Early Detection of Mycobacterium Tuberculosis and Determination Rifampin-Resistance by PCR-Reverse Hybridization (Line Probe Assay) v / 감염

BACKGROUND: Recent worldwide increase of tuberculosis has been mainly caused by opportunistic infections in the patients with AIDS. Emergence of multidrug- resistant Mycobacterium tuberculosis has been also posing serious problems on the treatment of tuberculosis. The purpose of this study was to evaluate the usefulness of the PCR-reverse hybridization method (line probe assay, INNO-LIPA; Innogenetics, Belgium) for the identification of M. tuberculosis and detection of rifampicin- resistant M. tuberculosis. METHOD: Identification of 37 isolates was performed by both conventional method and line probe assay. Detection of rifampin resistance by line probe assay was based on the reaction of amplified gene products of isolates with either wild type probes (S1, S2, S3, S4, and S5) specific for rpoB gene or mutant type probes (R2, R4a, R4b, and R5) specific for the mutation sequence in the rpoB region, and the results were compared with those by the absolute concentration method. RESULTS: All of the 37 isolates were identified as M. tuberculosis by both line probe assay and conventional method. Eight isolates susceptible to rifampin by absolute concentration method showed no mutation by line probe assay. Twenty seven of 29 isolates resistant to rifampin by absolute concentration method showed the following mutations in rpoB gene: 11, S5-R5+(loss of S5 with R5); 7, S4-R4a; 1, S4-R4b+; 4, S2-R2+; 1, S1-S2+: 1; S2-S4-; 1, S4-; and 1, R5+. The sensitivity of rifampin resistance by line probe assay was 95%. CONCLUSION: Line probe assay is a useful tool for the early identification of M. tuberculosis as well as determination of its rifampin resistance, especially in cases of emergent need for rapid treatment for tuberculosis before getting the final of conventional antituberculous drug susceptibility test.