Optimización de unidades formadoras de colonias y detección del virus de papiloma humano en sangre de cordón umbilical / Colony-forming units optimization and human papillomavirus detection in umbilical cord blood

Resumen Introducción: Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos. Objetivo: Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU. Métodos: Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming. Resultados: La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p < 0.0001); así como la de SCU comparada con la de segmento (p < 0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+. Conclusiones: Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

Abstract Introduction: Analysis of several markers is required for adequate quality control in umbilical cord blood units (UCBU) when are used for therapeutic purposes. Objective: To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU. Methods: One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the SSP technique. Results: CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p < 0.0001), as well as that of UCB in comparison with that of the segment (p < 0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY11 and GP5/GP6+. Conclusions: Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.

High incidence of herpes simplex virus-1 in cord blood and placenta infection of women in southern Brazil / Alta incidência do vírus herpes simplex 1 em sangue de cordão e infecção na placenta de mulheres no sul do Brasil

Abstract Objective Estimate the prevalence of human herpesvirus type 1 HSV-1 DNA in placental samples, its incidence in umbilical cord blood of newborns and the associated risk factors. Methods Placental biopsies and umbilical cord blood were analyzed, totaling 480 samples, from asymptomatic parturients and their newborns at a University Hospital. Nested polymerase chain reaction (PCR) and gene sequencingwere used to identify the virus; odds ratio (OR) and relative risk (RR) were performed to compare risk factors associated with this condition. Results The prevalence of HSV-1 DNA in placental samples was 37.5%, and the incidence in cord blood was 27.5%. Hematogenous transplacental route was identified in 61.4% from HSV-1+ samples of umbilical cord blood paired with the placental tissue. No evidence of the virus was observed in the remaining 38.6% of placental tissues, suggesting an ascendant infection from the genital tract, without replication in the placental tissue, resulting in intra-amniotic infection and vertical transmission, seen by the virus in the cord blood. The lack of condom use increased the risk of finding HSV-1 in the placenta and umbilical cord blood. Conclusion The occurrence of HSV-1 DNA in the placenta and in cord blood found suggests vertical transmission from asymptomatic pregnant women to the fetus.

Resumo Objetivo Estimar a prevalência do DNA do vírus herpes humano 1 (HSV-1) em amostras de placenta, sua incidência no sangue do cordão umbilical de recém-nascidos e fatores de risco associados. Métodos Biópsias de placenta e de sangue de cordão umbilical foram analisadas, totalizando 480 amostras de parturientes assintomáticas e seus recém-nascidos emum hospital universitário. Reação de cadeia de polimerase (RCP) nested e sequenciamento gênico foram usados para identificar o vírus; odds ratio (OR) e risco relativo (RR) foram realizados para comparar os fatores de risco associados à essa condição. Resultados A prevalência do DNA do HSV-1 em amostras de placenta foi de 37,5%, e a incidência no sangue do cordão foi de 27,5%. A via transplacentária hematogênica foi identificada em 61,4% das amostras de HSV-1+do sangue do cordão umbilical, pareadas com o tecido placentário. Nenhuma evidência do vírus foi observada nos restantes 38,6% dos tecidos placentários, sugerindo uma infecção ascendente do trato genital. A falta de uso do preservativo aumentou o risco de encontrar o HSV-1 na placenta e no sangue do cordão umbilical. Conclusão A ocorrência de DNA do HSV-1 na placenta e no sangue do cordão umbilical sugere uma transmissão vertical de gestantes assintomáticas para o feto.

Frequência do Papilomavírus Humano na placenta, no colostro e no sangue do cordão umbilical / Frequency of Human Papillomavirus in the placenta, in the colostrum and in the umbilical cord blood

OBJETIVO: Determinar a frequência do Papilomavírus Humano (HPV) na placenta, no colostro e no sangue do cordão umbilical de parturientes e seus neonatos atendidos no Ambulatório de Ginecologia e Obstetrícia do Hospital Universitário de Rio Grande (RS), Brasil. MÉTODOS: Foram coletadas biópsias de 150 placentas do lado materno, 150 do lado fetal, 138 amostras do sangue do cordão umbilical e 118 amostras de colostro. As biópsias de placenta foram coletadas da porção central e periférica. O DNA foi extraído segundo protocolo do fabricante e conforme referência encontrada na literatura. O HPV foi detectado pela técnica da reação em cadeia da polimerase aninhada (PCR-Nested) com os primers MY09/11 e GP5/GP6. A genotipagem foi por sequenciamento direto. As participantes responderam a um questionário autoaplicado com dados demográficos e clínicos, a fim de caracterizar a amostra. RESULTADOS: O HPV foi detectado em 4% (6/150) do lado materno das placentas, 3,3% (5/150) do lado fetal; 2,2% (3/138) no sangue do cordão e 0,8% (1/118) no colostro. A taxa de transmissão vertical foi de 50%. O genótipo de baixo risco oncogênico encontrado foi o HPV-6 (60%) e de alto risco, os HPV-16 e HPV-18 (20% cada). CONCLUSÕES: Esses resultados sugerem que o HPV pode infectar a placenta, o colostro e o sangue do cordão umbilical. .

PURPOSE: To determine the frequency of Human Papillomavirus (HPV) in the placenta, in the colostrum and in the umbilical cord blood of parturient women and their newborns assisted at the Clinic of Gynecology and Obstetrics of the University Hospital of Rio Grande (RS), Brazil. METHODS: Biopsies were collected from 150 placentas on the maternal side, 150 on the fetal side, 138 samples of umbilical cord blood and 118 of the colostrum. The placenta biopsies were collected from the central and peripheral portions. DNA was extracted according to the manufacturer's protocol and to a reference found in the literature. HPV was detected by the nested polymerase chain reaction (PCR-Nested) using primers MY09/11 and GP5/GP6. Genotyping was performed by direct sequencing. The participants responded to a self-applied questionnaire with demographic and clinical data, in order to characterize the sample. RESULTS: HPV was detected in 4% (6/150) of cases on the mother's side of the placentas, in 3.3% (5/150) on the fetal side, in 2.2% (3/138) in umbilical cord blood and in 0.84% (1/118) in colostrum samples. The vertical transmission rate was 50%. HPV-6 was the low-risk genotype found (60%) and the high-risk genotypes were HPV-16 and HPV-18 (20% each). CONCLUSIONS: These results suggest that HPV can infect the placenta, the colostrum and the umbilical cord blood. .

Waterborne viruses associated with repeated abortion

In this study we tried to find the role of some waterborne viruses in repeated abortion of women. The study includes maternal blood serum and fetal tissue. The serum of full term delivered women was taken as a control. All collected samples were inoculated on BGM and Hep2G cells to detect entero and Hepatitis E viruses. Enzyme-linked immunosorbent assay was also carried out for IgM and IgG antibodies against HEV in all serum samples. HEV-Ag was determined by dot-ELISA, which used also for enterovirus typing. Reverse transcriptase polymerase chain reaction was used for detection of entero and HE virus RNAs in the collected serum samples. To follow up the source of virus transmission, the wastewater treatment plant which serves the area of samples population was studied at the intake and the final effluent for the presence of hepatitis E virus and enteroviruses with special reference to coxsackieviruses. Wastewater samples were collected for 1 year and for enterovirus concentration the adsorption-elution on nitrocellulose membrane was used and for HEV, two methods of virus concentration were used, urea arginine phosphate buffer [U-APB] and PEG 8000.The results of HEV investigation of aborted women sera was 22% for IgG, 3% for IgM, 20% HEV-Ag, and 16% of HEV-RNA by RT-PCR. For fetal tissue, HEV-Ag was detected in 5% of the collected samples. The detected enteroviruses were coxsackieviruses types 2, 3,4and 5 in all serum samples and wastewater samples. The results showed also, that virus concentration by U-APB is much better than PEG-8000 but not highly efficient

Prevalence of cytomegalovirus IgG and IgM antibodies in the sera of postpartum women and cord sera of newborn babies and their correlation with socioeconomic status

Using the ELISA technique, cytomegalovirus [CMV] IgG and IgM values were measured in the sera of 176 subjects who delivered healthy term infants and in the cord sera of the newborn babies. IgG was positive in 149 of 176 maternal sera and in 6 of 176 newborn cord sera. The subjects were classified into three socioeconomic levels: high, moderate and low. The distribution of IgG and IgM positivity among the three groups revealed significant differences [p <0.05]. The fetus and the placenta are affected during primary and reactive infections in pregnancy. The percentage of IgM-positive infants delivered by IgM-positive patients was found to be highly significant. CMV IgG is not always protective against reinfection. In our study, IgM was positive in 24 women in spite of IgG positivity. The distribution of CMV seropositivity was not significan is important risk factors in pregnancy as it is the most frequent congenital and early postnatal infectious agent