Diversity and uncommon HPV types in HIV seropositive and seronegative women attending an STI clinic

Given the causal relationship between specific types of HPV with cervical cancer and precursor lesions, it is important to identify the viral type involved. The aim of this study is to access the prevalence of HPV types in HIV seropositive and seronegative women. Accordingly, 77 HPV positive cervical samples were obtained from 284 women (seropositive (n=112) and seronegative (n=172) for HIV) who attended a Sexually Transmitted Infection clinic, in Vitoria, Southeastern Brazil. Viral DNA was amplified by PCR using MY09/MY11 degenerated primers and the genotyping was performed by Restriction Fragment Length Polymorphism. Seventy five out of the 77 HPV samples were genotyped: 6, 11, 13, 16, 18, 26, 31, 31b, 32, 33, 34, 35, 52, 53, 55, 56, 58, 59, 61, 62, 64, 66, 71, 81, 83, 84. The most prevalent type was HPV16 followed by HPV types 6, 11 and 53. Fifty five percent and 45% belonged to high and low risk types, respectively. High risk types corresponded to 59% and 54.5% of the HPV detected in HIV seronegative and seropositive women, respectively. The uncommon HPV 13 type in cervical samples was also observed in this study. The oncogenic types were more common in the HIV seronegative samples and the number of cases with multiple infections was similar for the two groups. HPV typing is not only important clinically for the establishment of monitoring and treatment of a patient, it also provides knowledge of the viral types circulating in a population, which is of interest in the development of prevention and treatment programs for this disease.

HCMV gB genotypes in cervical secretion and placenta tissues in the state of Espírito Santo, Southeastearn Brazil

Human cytomegalovirus (HCMV) displays genetic variability in several regions, supposed to be related with strain-specific tissue tropism and immunopathogenesis. Based on sequence variation in the UL55 gene that encodes gB glycoprotein, HCMV strains can be assigned to one of four genotypes. Previous studies have addressed gB genotyping mostly by investigating strains derived from immunosuppressed patients, sometimes without previous knowledge about genotype distribution in a geographic area. The present study verified the distribution of HCMV gB genotypes of strains obtained from immunocompetent women at Vitória City, Espírito Santo State, Southeastern, Brazil. The HCMV genome was extracted from their cervical secretion, fetal and maternal placenta tissues (chorionic villous and decidua) from abortion cases and from white blood cells (WBCs). HCMV genotyping was performed by restriction fragment length polymorphism analyses of amplified product from the high variability site of the UL55 gene. All four genotypes were observed in both cervical secretion and placenta, whereas in WBCs a single gB1 genotype was detected. HCMV gB1 and gB2 genotypes were detected, respectively, in nine and in six of the 23 studied samples, while gB3 and gB4 were each found in four separate samples of the total. The differences in genotype frequency were not considered statistically significant. No mixed genotype infection was observed. The results indicated that the four gB HCMV genotypes had no particular tropism for placenta tissues and that all genotypes circulated within immunocompetent women at the time and in the region of study.

O citomegalovírus humano (HCMV) apresenta variabilidade em diversas regiões do genoma, supostamente relacionada ao tropismo tecidual e imunopatogênese viral. Baseando-se na variação de seqüência do gene UL55 que codifica a glicoproteína gB, o HCMV pode ser classificado em um dos quatro genótipos. Estudos prévios têm investigado a associação destes genótipos a partir de cepas obtidas de pacientes imunossuprimidos. O presente estudo determinou os genótipos gB de cepas de HCMV obtidas de mulheres imunocompetentes em Vitória, Espírito Santo, Sudeste do Brasil. O genoma do HCMV foi extraído de secreção cervical, tecidos placentários fetais e maternos (vilosidade coriônica e decídua) obtidos de casos de aborto e de leucócitos do sangue periférico. A genotipagem foi realizada através da análise de polimorfismo de fragmentos de restrição do produto amplificado da região de alta variabilidade do gene UL55. Todos os quatro genótipos foram detectados na secreção cervical e na placenta, enquanto que somente o genótipo gB1 foi detectado em leucócitos. Genótipos gB1 e gB2 foram detectados em nove e seis das 23 cepas estudadas, respectivamente, enquanto gB3 e gB4 foram encontrados cada um em quatro casos. A diferença na freqüência de genótipos não foi estatisticamente significante. Infecção mista não foi detectada. Estes resultados indicam que os quarto genótipos de HCMV apresentam tropismo para os tecidos placentários e que todos eles circularam nas mulheres imunocompetentes no período e região geográfica do estudo.

False positive reaction due to endogenous biotin activity in glandular epithelium of decidua

Biotin-labeled probe was used in an in situ hybridisation assay to localize virus infection in formalin-fixed, paraffin embedded tissues taken from eleven abortion cases. Probes for human cytomegalovirus (HCMV), human Parvovirus B19 (B19) and human adenovirus type 2 (HAd2), were labeled with biotin-11-dUTP by nick-translation reaction. Streptavidin-alkaline-phosphatase (SAP) was used to detect biotin, followed by 4-nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) solution. Positive reaction was observed in nucleus of glandular ephitelium cells of decidua either in positive or in negative control at first and second gestational trimester. The reaction was not inhibited with blocking solution for alkaline phosphatase endogenous activity and it persisted even with probes omission. The use of adequate negative control permitted to reveal the presence of nuclear biotin in glandular epithelium of decidua, responsible for false positivity in detection systems involving streptavidin biotin system (StrepABC). The stained cells resembled to cytophatic effect due to herpesvirus, which could induce further misinterpretation. The results obtained in this study strongly recommend that DNA detection by in situ hybridisation reaction in gestational endometrium should be done without using StrepABC system.

Sondas marcadas com biotina foram utilizadas neste trabalho para detecção de infecção viral por hibridização in situ em tecidos fixados com formalina e embebidos em parafina de 11 casos obtidos de abortamento. Sondas para citomegalovírus humano (HCMV), parvovírus B19 humano (B19) e adenovírus humano tipo 2 (HAd2), foram marcadas com biotina-11-dUTP através da reação de nick-translation. Estreptavidina conjugada com fosfatase alcalina (SAP) seguida por solução de 4-nitro-azul de tetrazolio/5-bromo-4-cloro-3-indolil fosfato (NBT/BCIP) foram utilizadas para detecção da biotina após a reação de hibridização. Reação positiva foi observada no núcleo de células do epitélio glandular da decídua tanto no controle positivo quanto no negativo em tecidos de primeiro e segundo trimestre gestacional. Esta reação não foi inibida com solução bloqueadora da atividade endógena de fosfatase alcalina e persistiu mesmo com a omissão das sondas. O uso de controles negativos permitiu revelar atividade endógena de biotina nuclear em epitélio glandular da decídua, responsável por reações falso positivas em sistemas de detecção estreptavidina-biotina (StrepABC). Os resultados obtidos neste estudo fortemente recomendam que a detecção de ADN por hibridização in situ em endométrio gestacional seja feita com outro sistema de detecção que não o StrepABC.