Comparison of the Tellgenplex HPV DNA test with the PCR-reverse dot blot assay for human papillomavirus genotyping

Objective: To access the performance of the Tellgenplex human papillomavirus (HPV) DNA test compared to the polymerase chain reaction-reverse dot blot (PCR-RDB) assay for the HPV genotyping. Methods: Sixty cervical swab samples were genotyped by the Tellgenplex HPV DNA test and the PCR-RDB assay. The Tellgenplex HPV DNA test and the PCR-RDB assay can detect 26 and 23 HPV genotypes, respectively. Each sample showed discrepancy was genotyped using sequencing. Results: The percent agreement between the two tests ranged from 83.3% to 100.0% according to different genotype. This showed perfect agreement (>0.81) for high-risk HPV genotypes (35, 39, 45, 53, 56, 59, 66, 68, and 82), substantial agreement (>0.65) for high-risk HPV genotypes (16, 18, 33, 52, and 58) and low-risk HPV genotype 43 between the two assays by the kappa analysis. The positive rates of the two assays for frequent HPV genotypes (16, 35, 39, 45, 52, 53, 58, 59, 66, and 82) were not statistically different, but the PCR-RDB assay showed higher positive rates than the Tellgenplex HPV DNA test for HPV genotypes 81 (P<0.05). As for more than 10 positive results by the Tellgenplex HPV DNA test and/or the PCR-RDB assay, the PCR-RDB assay showed higher relative sensitivity and specificity than the Tellgenplex HPV DNA test for the three HPV genotypes (16, 52, and 81). All HPV genotypes that can be detected by only the Tellgenplex HPV DNA test (HPV genotypes 44 and 55) were confirmed by sequencing. Conclusions: In conclusion, our results demonstrated that the PCR-RDB assay which can detect more multiple HPV genotypes in each specimen shows higher relative sensitivity and specificity than the Tellgenplex HPV DNA test, which makes it a better option for routine clinical use.

Comparison of the Tellgenplex HPV DNA test with the PCR-reverse dot blot assay for human papillomavirus genotyping

Objective:To access the performance of the Tellgenplex human papillomavirus (HPV) DNA test compared to the polymerase chain reaction-reverse dot blot (PCR-RDB) assay for the HPV genotyping.Methods:Sixty cervical swab samples were genotyped by the Tellgenplex HPV DNA test and the PCR-RDB assay. The Tellgenplex HPV DNA test and the PCR-RDB assay can detect 26 and 23 HPV genotypes, respectively. Each sample showed discrepancy was genotyped using sequencing.Results:The percent agreement between the two tests ranged from 83.3% to 100.0% according to different genotype. This showed perfect agreement (>0.81) for high-risk HPV genotypes (35, 39, 45, 53, 56, 59, 66, 68, and 82), substantial agreement (>0.65) for high-risk HPV genotypes (16, 18, 33, 52, and 58) and low-risk HPV genotype 43 between the two assays by the kappa analysis. The positive rates of the two assays for frequent HPV genotypes (16, 35, 39, 45, 52, 53, 58, 59, 66, and 82) were not statistically different, but the PCR-RDB assay showed higher positive rates than the Tellgenplex HPV DNA test for HPV genotypes 81 (P<0.05). As for more than 10 positive results by the Tellgenplex HPV DNA test and/or the PCR-RDB assay, the PCR-RDB assay showed higher relative sensitivity and specificity than the Tellgenplex HPV DNA test for the three HPV genotypes (16, 52, and 81). All HPV genotypes that can be detected by only the Tellgenplex HPV DNA test (HPV genotypes 44 and 55) were confirmed by sequencing.Conclusions:In conclusion, our results demonstrated that the PCR-RDB assay which can detect more multiple HPV genotypes in each specimen shows higher relative sensitivity and specificity than the Tellgenplex HPV DNA test, which makes it a better option for routine clinical use.

Study on relationship between hepatitis B virus genotypes and drug resistance mutation / 国际检验医学杂志

Objective To explore correlations between hepatitis B virus (HBV)genotyping,other clinical information and drug resistance mutations.Methods 358 cases of patients with chronic hepatitis B (CHB)were selected as subjects,and the resistance loci and genotypes were detected by using the polymerase chain reaction(PCR)-reverse dot blot technique.Clinical data,such as ser-um HBV DNA loads,serum levels of alanine aminotransferase(ALT)and hepatitis B virus e antigen(HBeAg),gender,age and length of nucleoside analogues use were collected.Results All samples were successfully amplified positive band.Type B(267 ca-ses)was the main HBV genotype,followed by type C(81 cases)and type D(10 cases).In the 31 1 cases of patients taking nucleoside analogues,269 cases were completely wild type.No drug resistance mutation was found in 47 cases of patients not taking medicine. The drug resistance mutations mainly occured in 204 and 180/204 site.There was no significant correlation between resistance mu-tations and gender,age,serum HBV DNA loads,genotype,serum levels of ALT and HBeAg(P >0.05).While the medication time was longer,the incidence of resistant mutants was greater(P <0.05).The 180 mutation had a certain correlation with 204 site mu-tation(P <0.05).Conclusion PCR-reverse dot blot technology can effectively detect the HBV genotype and mutations,which could effectively guide the clinical medication.

Assessment of messenger RNA (mRNA) of Mycobacterium tuberculosis as a marker of cure in patients with pulmonary tuberculosis.

AIMS: To analyse the performance of RT-qPCR using 85B mRNA in the diagnosis of Mycobacterium tuberculosis infection and in the assessment of the response to treatment for pulmonary tuberculosis (TB). METHODS AND RESULTS: Ninety-eight patients with signs of pulmonary TB were selected: 56 were considered infected with Myco. tuberculosis and they had positive cultures or evident clinical response to anti-TB treatment. Patients with pulmonary tuberculosis were evaluated by culture and RT-qPCR for a 30-day specific treatment. It was found that both tests demonstrated a decline in viable bacilli at 15 and 30 days after the beginning of the therapy in most of the patients. The quantification of the 85B mRNA target was performed in 52 patients who had initially shown positive results by RT-qPCR and who were followed on the days 15 and 30 after the specific treatment. Thus 85B mRNA was detectable in sputum samples in 52 patients with a confirmed diagnosis of pulmonary tuberculosis on day 0. During the specific treatment the 85B mRNA was detectable in 13 patients on day 15 and in only three patients on day 30. CONCLUSIONS: Mycobacterium tuberculosis mRNA in the sputum is a useful prognostic marker and its quantification, an early and reliable indicator for monitoring response to treatment, drug resistance, re-infection and relapse. SIGNIFICANCE AND IMPACT OF THE STUDY: RT-qPCR is a tool that can be used in clinical and therapeutic monitoring as an indicator of bacterial resistance and indicator of the period of transmissibility of Myco. tuberculosis in patients with pulmonary TB undergoing treatment.

Comparison of real time PCR and PCRˉreverse dot blot hybridization for detection of Human papillomavirus / 国际检验医学杂志

Objective To compare real time PCR with PCR-reverse dot blot hybridization (PCR-RDB)for detecting human pap-illomavirus (HPV)infection in women.Methods A total of 109 genital specimens from women were collected in the study.All specimens were tested HPV by using real time PCR and PCR-RDB,discrepant samples were tested again by PCR-xMAP.Results The concordant rate was 83.5%(91/109)between real time PCR and PCR-RDB (kappa=0.671),the other 18 discrepant samples were retested by PCR-xMAP,7 of those were identical with real time PCR and 11 with PCR-RDB.No differences of PCR-RDB pos-itive rates were found between the high and low viral load groups (χ2 =1.476,P =0.224).Conclusion It demonstrated moderate consistency between real time PCR and PCR-RDB.The HPV positive rates of PCR-RDB were stable when the viral loads were 103-108 .

MammaGene: ensaio quantitativo de 21 genes para prognóstico do câncer de mama receptor de estrogênio positivo / MammaGene®: a quantitative 21-gene assay for prognostic of breast cancer estrogen receptor-positive

Objetivos: Padronizar em nosso meio um ensaio que analisa, por RT-PCR, 21 genes e descrever a experiência inicial com 95 casos consecutivos de carcinoma inicial de mama receptor de estrogênio positivo. Métodos: O teste foi desenvolvido a partir dos relatos publicados por Cronin et al. (2004) e Paik et al. (2004) para a avaliação da expressão de genes em tecido fixado em formalina e incluído em parafina. O teste foi aplicado em uma coorte consecutiva de 95 amostras de câncer de mama receptor de estrogênio positivo e os escores finais foram comparados com a idade da paciente, O tamanho do tumor, o tipo e o grau histológico, expressão imunoistoquímica do receptor de estrogênio, índice de Ki67 e subtipo molecular. Resultados: Os escores finais variaram de 3 a 90 e as categorias de risco de recorrência em dez anos foram: baixa (34 casos), intermediária (38 casos) e alta (23 casos). Não houve associação das categorias de risco com idade, comprometimento linfonodal e tipo histológico. A media do tamanho dos tumores foi maior no grupo de alto escore (2,0 versus 1,2 cm). Observou-se associação entre o escore obtido pelo teste e grau histológico, Ki-67, nível de expressão de receptor de estrogênio e subtipo molecular. Conclusão: A realização do teste de 21 genes foi factível em nosso meio. Alem disso, os dados preliminares, aliados aos dados da literatura, sugerem que tal teste pode ser uma ferramenta útil na avaliação do risco de recorrência a distância em câncer de mama receptor de estrogênio positivo. No entanto, estudos adicionais são necessários para comparar os resultados deste trabalho com séries amplas publicadas na literatura.

Objectives: To standardize a homemade RT-PCR-based 21-gene assay and to describe the preliminary experience with 95 early positive estrogen receptor breast cancer consecutive cases. Methods: The test was developed using the reports described by Cronin et al. (2004) and Paik et al. (2004) for the evaluation of gene expression in fixed, paraffin-embedded tumor tissue. The test was performed in a consecutive cohort of 95 positive estrogen receptor breast carcinomas, and the final scores were compared with the patient's age, tumor size, histological type, histological degree, estrogen receptor immunohistochemical expression, Ki-67 expression, and molecular luminal subtype. Results: Final scores ranged from 3 to 90 and risk categories of recurrence in ten years were: low (34 cases), intermediate (38 cases), and high (23 cases). There was no association between score categorical distribution and age, lymph node status, or histological type. Mean tumor size was higher in the high score group (2.0 versus 1.2 cm). We have observed an overall concordance between the score obtained by the test, and the histological degree, Ki-67, estrogen receptor level, and molecular subtype. Conclusion: The developed 21-gene assay is a feasible test to be performed in a homemade setting. Besides, the preliminary data from this study suggest, in comparison with data from the literature, that this test has the potential to be a useful tool to evaluate the risk of breast cancer distant recurrence. However, further data are necessary in order to compare this paper's results with larger series published in the literature.

Detección molecular de patógenos emergentes de importancia médica y veterinaria en garrapatas capturadas sobre caballos domésticos / Molecular detection of emerging pathogens of medical and veterinary importance in ticks found in domestic horses

En la actualidad son varias las especies de patógenos emergentes de importancia médica y veterinaria transmitidos por garrapatas. Los estudios sobre estos agentes y sus enfermedades han sido escasos en Cuba. Conocer la presencia de algunos de estos patógenos en garrapatas cubanas que afectan el ganado equino. Se procesaron 95 garrapatas colectadas de caballos domésticos, conservadas en alcohol e identificadas taxonómicamente según claves convencionales. A cada una se le realizó extracción de ADN y posteriormente diferentes reacciones en cadena de la polimerasa utilizando cebadores específicos para los grupos microbianos Borrelia burgdorferi sensu lato, Anaplasma-Ehrlichia, y Babesia-Theileria. Cada uno de los productos de las reacciones en cadena de la polimerasa fue sometido a hibridaciones en línea reversa utilizando sondas para cada grupo en cuestión, así como específicas para las principales especies de estos. Las garrapatas estudiadas pertenecían a las especies Dermacentor (Anocentor) nitens (60 por ciento), Amblyomma cajennense (38 por ciento) y Rhipicephalus (Boophilus) microplus (2 por ciento). Se detectaron 7 garrapatas Dermacentor (Anocentor) nitens infectadas con bacterias del grupo Anaplasma/Ehrlichia, y no se pudo identificar la especie en cuestión con las sondas utilizadas. Una de estas garrapatas estaba además coinfectada con Babesia bovis. Se sugiere la circulación de una nueva especie de Anaplasma o Ehrlichia no reportada antes en Cuba, por lo que se necesita estudiar un número mayor de garrapatas, así como la incorporación de nuevas sondas en la hibridación en línea reversa u otras metodologías que permitan conocer con exactitud las especies que pudiesen afectar hoy día los caballos domésticos.

At present, there are several tick-borne emerging pathogen species of medical and veterinary importance. Few studies on these agents and its diseases have been made in Cuba. To determine the presence of some of these pathogens in Cuban ticks existing in the equine cattle. Ninety five ticks collected from domestic use horses were processed, preserved in alcohol and taxonomically identified according to the set classifications. Their DNA was extracted and subjected to several polymerase chain reactions with specific primers for microbial groups Borrelia burgdorferi sensu lato, Anaplasma-Ehrlichia, y Babesia-Theileria. Each of the products from polymerase chain reactions underwent reverse line blot hybridation using probes for each group as well as specific probes for the main species included in these groups. The studied ticks belonged to Dermacentor (Anocentor) nitens (60 percent), Amblyomma cajennense (38 percent) y Rhipicephalus (Boophilus) microplus (2 percent). Seven Dermacentor (Anocentor) nitens ticks infested with Anaplasma/Ehrlichia bacteria were detected but the species in question could not be detected by the used probes. One of these ticks was also co-infested with Babesia bovis. It is suggested that a new species of Anaplasma o Ehrlichia, not reported in Cuba before now, is circulating, so studying a higher number of ticks is needed and new probes in reverse line blot hybridation or other methodologies must be incorporated to allow exactly determining the species that may affect the Cuban domestic horses at present.