Human papillomavirus and Chlamydia trachomatis in oral and genital mucosa of women with normal and abnormal cervical cytology.

BACKGROUND: HPV and C.trachomatis are the most prevalent, viral and bacterial STI worldwide. Both commonly have an asymptomatic development and can evolve into a persistent infection which, added to coinfections, may be important cofactors for the oncogenic transformation. OBJECTIVE: Evaluate the prevalence of oral and genital HPV and C.trachomatis infection in women with normal and abnormal cervical cytology. STUDY DESIGN: The cross-sectional study included 200 swabs, 100 oral and 100 cervical from 50 women with normal and 50 with abnormal cervical cytology. HPV and C.trachomatis infections were detected using PCR with specific primers. RESULTS: HPV DNA was detected in 27% (n = 27/100) of women with normal and abnormal cytology. Out of 100 genital samples we detected HPV DNA in 18% (n = 18/100) and 14% (n = 14/100) out of 100 oral samples. HPV genotypes detected were genotype 6 of low-risk and 16, 31, 52, 58 and 16-31 coinfection of high-risk. C.trachomatis DNA was detected in 49% (n = 49/100) of patients. Out of 100 genital samples we detected C.trachomatis in 35% (n = 35/100) and 31% (n = 31) out of 100 oral samples. There is statistically significant (p < 0.05) between cytology and HPV and C.trachomatis infection but there is no statistically significant between cytology and the other characteristics. CONCLUSIONS: Since the histology of oral mucosa resembles that of the uterine cervix, we can anticipate the presence of HPV and other STI which are detected in different lesions of genital areas and the oral mucosa. Therefore, is important C.trachomatis detection and specific treatment in asymptomatic women because this infection may increase the risk of HPV persistence and coinfection induces a pro-inflammatory environment that may promote the carcinogenesis.

Oral Human Papillomavirus: a multisite infection.

BACKGROUND: The Human Papillomavirus (HPV) has different strategies for persist in the cells. This characteristic has led us to consider the presence of the virus in tissues of the oral cavity that had no clinical signs of infection. The aim of this study was to detect the presence of DNA-HPV at multiple sites of the oral cavity. MATERIAL AND METHODS: A case-control study was designed: Oral Squamous Carcinoma Group (OSCG), healthy n=72 and Control Group (CG), n=72, healthy volunteers paired by sex and age with OSCG. Four samples were taken from OSCG: saliva, biopsy, brush scraping of lesion and contralateral healthy side. In CG a saliva sample and a scratch of the posterior border of tongue were collected. HPV was detected by PCR using Bioneer Accuprep genomic DNA Extraction kit, and consensus primers MY09 and MY11. Chi square test was applied. RESULTS: 432 samples were obtained from 144 individuals. DNA-HPV was detected in 30 (42%) of OSCG subjects and 3(4%) of CG. Two or more positive samples were obtained in 67% of the OSCG, 67% in saliva and 60% in biopsy; in CG 100% of the individuals were positive in the two samples. CONCLUSIONS: HPV is frequently present in oral cavity as a multifocal infection, even without the presence of clinical lesions.

[Prevalence and phylogenetic analysis of Chlamydia trachomatis in a population of women in Posadas, Misiones]. / Prevalencia y análisis filogenético de Chlamydia trachomatis en una población de mujeres de Posadas, Misiones.

OBJECTIVE: Chlamydia trachomatis is the most prevalent bacteria causing sexually transmitted infections. In women, this infection can cause cervicitis and urethritis, although it's usually asymptomatic. The aim of this study was to investigate the prevalence of C. trachomatis in women attending the lab Instituto de Previsión Social and detect the genotypes. METHODS: Endocervical samples from 505 symptomatic and asymptomatic women were assayed. It was determined the presence of C. trachomatis by PCR through amplification of a fragment of the cryptic plasmid. Positive samples were genotyped by the partial amplification of the ompA gene and analyzed phylogenetically. RESULTS: Forty-three positive samples were detected to infection with C. trachomatis, obtaining a prevalence of 8.5% (IC 95%: 6.4-11.3%). The prevalence of C. trachomatis was higher in women with vaginal symptoms [11.3% (30/265) vs. 5.4% (13/240)] (p = 0.018), as well as in women under 26 year-old [11.5% (28/244) vs. 6.2% (15/246)] (p = 0.021). Based on phylogenetic analysis, it was observed that 62% of the samples were genotype E, 15% genotype J, 15% genotype D, and 8% genotype F. CONCLUSIONS: This work is the first contribution on the molecular epidemiology of C. trachomatis in the Misiones province, Argentina, which shows the rate of prevalence of this bacterium and offers information on circulating genotypes.

HPV detection and genotyping in males from the city of Córdoba, Argentina.

A wide range of human papillomavirus (HPV) types can infect the anogenital region of males. Although there is a vast knowledge on HPV infections in women as well as on their association with cervical cancer, the study of HPV infections in males is scarce and controversial. The aim of the present work was to detect and typify HPV infections of the anogenital region in males and analyze the associated risk factors in the population studied. Anogenital samples from 37 patients (30 of whom were HIV carriers) attending the Infectology Service at the Hospital Nacional de Clinicas in C6rdoba, Argentina, were studied. Nine of these patients tested HPV-positive and five out of these nine were found to have mixed infections, being 18 and 61 the most frequent genotypes. There was a significant correlation between the HPV-positive patients and those having an HPV-compatible lesion or AIDS. The present work is the first study in the city of Cordoba which contributes relevant results to the knowledge of HPV infection and to the possible implementation of measures for its prevention.

Detection of rubella-virus-induced apoptosis in Vero cell cultures with hematoxylin and eosin staining.

In order to facilitate the detection of apoptotic cells (Apo C) in Rubella virus (RV) infected cultures in settings of low resources, we compared hematoxylin and eosin staining (H&E) with the conventional TUNEL technique, and confirmed our findings with DNA electrophoresis and transmission electron microscopy. H&E allowed to distinguish Apo C from non-apoptotic cells. The proportion of Apo C in infected cultures was proportional to the multiplicity of infection (MOI). At a MOI of 10, the percent of Apo C at 3, 4 and 5 days post infection (pi) were 26, 45 and 47%, respectively, which were significantly reduced when the caspase inhibitor z-VAD-fmk was present in the supernatant. By the TUNEL assay, the percent of Apo C in RV-infected cultures were lower (0.8, 1.2 and 1.2% at 3, 4 and 5 days pi, respectively). Our results have shown that H&E staining is an easy, rapid, economic and reproducible method to detect Apo C in RV infected Vero cells cultures. It is possible that H&E makes evident early stages of apoptosis, when an apoptotic cell shows chromatin condensation, nuclear and cytoplasmic contraction (but is still attached to the monolayer), while TUNEL detects later stages of apoptosis because it needs an extensive DNA fragmentation, when apoptotic cells are about to or have already detached from the substratum.

Detection of rubella-virus-induced apoptosis in Vero cell cultures with hematoxylin and eosin staining.

In order to facilitate the detection of apoptotic cells (Apo C) in Rubella virus (RV) infected cultures in settings of low resources, we compared hematoxylin and eosin staining (H&E) with the conventional TUNEL technique, and confirmed our findings with DNA electrophoresis and transmission electron microscopy. H&E allowed to distinguish Apo C from non-apoptotic cells. The proportion of Apo C in infected cultures was proportional to the multiplicity of infection (MOI). At a MOI of 10, the percent of Apo C at 3, 4 and 5 days post infection (pi) were 26, 45 and 47

, respectively, which were significantly reduced when the caspase inhibitor z-VAD-fmk was present in the supernatant. By the TUNEL assay, the percent of Apo C in RV-infected cultures were lower (0.8, 1.2 and 1.2

at 3, 4 and 5 days pi, respectively). Our results have shown that H&E staining is an easy, rapid, economic and reproducible method to detect Apo C in RV infected Vero cells cultures. It is possible that H&E makes evident early stages of apoptosis, when an apoptotic cell shows chromatin condensation, nuclear and cytoplasmic contraction (but is still attached to the monolayer), while TUNEL detects later stages of apoptosis because it needs an extensive DNA fragmentation, when apoptotic cells are about to or have already detached from the substratum.

Detection of rubella-virus-induced apoptosis in Vero cell cultures with hematoxylin and eosin staining

In order to facilitate the detection of apoptotic cells (Apo C) in Rubella virus (RV) infected cultures in settings of low resources, we compared hematoxylin and eosin staining (H&E) with the conventional TUNEL technique, and confirmed our findings with DNA electrophoresis and transmission electron microscopy. H&E allowed to distinguish Apo C from non-apoptotic cells. The proportion of Apo C in infected cultures was proportional to the multiplicity of infection (MOI). At a MOI of 10, the percent of Apo C at 3, 4 and 5 days post infection (pi) were 26, 45 and 47

, respectively, which were significantly reduced when the caspase inhibitor z-VAD-fmk was present in the supernatant. By the TUNEL assay, the percent of Apo C in RV-infected cultures were lower (0.8, 1.2 and 1.2

at 3, 4 and 5 days pi, respectively). Our results have shown that H&E staining is an easy, rapid, economic and reproducible method to detect Apo C in RV infected Vero cells cultures. It is possible that H&E makes evident early stages of apoptosis, when an apoptotic cell shows chromatin condensation, nuclear and cytoplasmic contraction (but is still attached to the monolayer), while TUNEL detects later stages of apoptosis because it needs an extensive DNA fragmentation, when apoptotic cells are about to or have already detached from the substratum.

[Production and characterization of monoclonal antibodies directed agains the major outer membrane protein and lipopolysaccharide of Chlamydia trachomatis]. / Producción y caracterización de anticuerpos monoclonales dirigidos contra la proteína mayor de membrana externa y contra el lipopolisacárido de Chlamydia trachomatis.

Two monoclonal antibodies (MnAb) directed against the lipopolysaccharide (LPS) and major outer membrane protein (MOMP) of Chlamydia trachomatis were produced for use in indirect immunofluorescence (IFI). The specificity of the antibodies was determined by Dot-blot, Immunoblotting (IB) and IFI onto culture cells infected with C. trachomatis and IFI onto commercial swabs (MRL). The MnAb 2D3 and 3C2 detected LPS and MOMP of C. trachomatis, respectively, by different methods. Neither MnAb showed cross-reactions when other gram-negative bacteria were used as antigens.

Producción y caracterización de anticuerpos monoclonales dirigidos contra la proteína mayor de membrana externa y contra el lipopolisacárido de Chlamydia trachomatis. / [Production and characterization of monoclonal antibodies directed agains the major outer membrane protein and lipopolysaccharide of Chlamydia trachomatis]

Two monoclonal antibodies (MnAb) directed against the lipopolysaccharide (LPS) and major outer membrane protein (MOMP) of Chlamydia trachomatis were produced for use in indirect immunofluorescence (IFI). The specificity of the antibodies was determined by Dot-blot, Immunoblotting (IB) and IFI onto culture cells infected with C. trachomatis and IFI onto commercial swabs (MRL). The MnAb 2D3 and 3C2 detected LPS and MOMP of C. trachomatis, respectively, by different methods. Neither MnAb showed cross-reactions when other gram-negative bacteria were used as antigens.

Producción y caracterización de anticuerpos monoclonales dirigidos contra la proteína mayor de membrana externa y contra el lipopolisacárido de Chlamydia trachomatis / [Production and characterization of monoclonal antibodies directed agains the major outer membrane protein and lipopolysaccharide of Chlamydia trachomatis]

Two monoclonal antibodies (MnAb) directed against the lipopolysaccharide (LPS) and major outer membrane protein (MOMP) of Chlamydia trachomatis were produced for use in indirect immunofluorescence (IFI). The specificity of the antibodies was determined by Dot-blot, Immunoblotting (IB) and IFI onto culture cells infected with C. trachomatis and IFI onto commercial swabs (MRL). The MnAb 2D3 and 3C2 detected LPS and MOMP of C. trachomatis, respectively, by different methods. Neither MnAb showed cross-reactions when other gram-negative bacteria were used as antigens.

Neutralizing monoclonal antibody to the E1 glycoprotein epitope of rubella virus mediates virus arrest in VERO cells.

The best-known mechanism of action of antibody-mediated virus neutralization is to impede the entrance of viruses to host cells, as determined by neutralization assays. Antibodies may also inhibit the exit of rubella virus (RV) from infected host cells; in this case, the interaction of the antibodies with their domains must occur on the plasma membrane, because antibodies cannot enter the cells. In the present study, we were able to block temporally the exit of virions from RV-infected cells by the binding of monoclonal antibody (mAb) H3 to their surface. The objective was accomplished in three steps: first, we determined the duration of the viral replication cycle; then we established the kinetics of the presence of the domains defined by our mAbs in the cytoplasm of RV-infected VERO cells; and, finally, we assessed the release of viral particles to the supernatant of infected VERO cells in the presence or absence of mAbs or positive and negative mice sera. RV-specific mice sera and mAb H3, which binds to the amino acid sequence 208-239 of the RV-E1 glycoprotein, were able to delay for 24 hours the release of virions from infected cultures, suggesting that the reaction of mAb H3 with its epitope may arrest any change necessary for the assembly and/or release of virions. In conclusion, the neutralizing domain recognized by mAb induces antibodies that can block the viral replication by several mechanisms of action, such as the obstruction of virus entry into cells and the delay of viral release. All of these mechanisms are intimately involved in the critical virus-host cell interactions that allow self-limitation of the infection.

Cell-fusion assay for the detection of rubella virus in Vero cells.

BACKGROUND AND OBJECTIVES: Rubella virus (RV) produces a subtle and slow-developing cytopathic effect in Vero cells that is difficult to recognize, especially at low multiplicities of infection. In order to facilitate the detection of RV in cell culture, we standardized a low-pH virus-mediated cell-fusion assay. STUDY DESIGN: The incubation periods, temperatures, pH and multiplicity of infection were established. The specificity of the method was tested by immunofluorescence assay and cell-fusion inhibition by specific sera. RESULTS: Six days post infection, Vero cells were treated for 5 min with fusion medium. After that, monolayers were incubated with medium at neutral pH for 16 h and then stained. Gigantic cells with multiple nuclei were observed. CONCLUSIONS: The method allowed the observation of unequivocal images that are easier to recognize than the cytopathic effect caused by RV in the same cell line. At the same time, the method is simple, accessible and shown to be specific to demonstrate the replication of several strains and isolates of RV in Vero cells.

Presence of a neutralizing domain in isolates of rubella virus in Cordoba, Argentina.

We studied the presence of a neutralizing epitope of rubella virus (RV) in locally circulating strains in Cordoba, Argentina, using binding by the monoclonal antibody (MAb) H3. This epitope is contained in a sequence of the E1 glycoprotein (E1208-239) represented by the synthetic peptide SP15. H3 MAb showed specific binding to SP15 by enzyme-linked immunosorbent assay (ELISA). One wild-type postnatal isolate, four clones derived from this isolate, and one congenital isolate were reactive with H3 by ELISA. These results suggest that the region of RV represented by SP15 is a domain present in locally circulating strains.

Detección de anticuerpos para rubéola en muestras de sangre recolectadas por filtro / Detection of rubella virus antibodies in whole blood collected on filter paper

El síndrome de rubéola congénita (CRS) puede ser fácilmente evitado si las mujeres en edad gestacional conocen su estado inmune y, en caso de ser seronegativas, reciben la vacunación previa al embarazo. Con el objetivo de determinar el estado inmune de grandes poblaciones se adoptó un método sencillo para la recolección y el almacenamiento de las muestras de sangre. A un total de 100 pacientes se les extrajo suero y sangre que fue absorbida en un círculo de papel de filtro, que capta aproximadamente 0,1 ml de suero. Fueron analizados los títulos de anticuerpos inhibidores de la hemaglutinación (TIH). En las muestras de papel, luego de ser almacenada en bolsas plásticas a 25 grados centígrados durante 7, 14, 21, 30, 60 y 100 días, con respecto a los sueros de cada paciente. De las muestras de sangre obtenidas en el papel de filtro a los 30 días de realizada la extracción, el 72 o/o tuvo el mismo TIH y el resto poseía una dilución de diferencia en relación con los sueros correspondientes. De las muestras ensayadas a los 60 días de extracción un 59 o/o tuvo el mismo TIH y el 41 o/o una dilución de diferencia. A los 100 días de extracción, el 51 o/o de las muestras tuvieron el mismo TIH, el 38 o/o una dilución de diferencia y el 11 o/o tuvieon 1 o más de una dilución de diferencia. En conclusión, los resultados de ese estudio mostraron que las muestras de sangre recolectadas sobre papel de filtro pueden ser usadas para la detección de anticuerpos antivirus rubéola determinados por IHA dentro de los 30 días post-obtención con una sensibilidad y especificidad del 100 o/o. Esta muestra en papel es estable a temperatura ambiente, sin ninguna restricción de aire o esterilización especial (AU)

Detección de anticuerpos para rubéola en muestras de sangre recolectadas por filtro / Detection of rubella virus antibodies in whole blood collected on filter paper

El síndrome de rubéola congénita (CRS) puede ser fácilmente evitado si las mujeres en edad gestacional conocen su estado inmune y, en caso de ser seronegativas, reciben la vacunación previa al embarazo. Con el objetivo de determinar el estado inmune de grandes poblaciones se adoptó un método sencillo para la recolección y el almacenamiento de las muestras de sangre. A un total de 100 pacientes se les extrajo suero y sangre que fue absorbida en un círculo de papel de filtro, que capta aproximadamente 0,1 ml de suero. Fueron analizados los títulos de anticuerpos inhibidores de la hemaglutinación (TIH). En las muestras de papel, luego de ser almacenada en bolsas plásticas a 25 grados centígrados durante 7, 14, 21, 30, 60 y 100 días, con respecto a los sueros de cada paciente. De las muestras de sangre obtenidas en el papel de filtro a los 30 días de realizada la extracción, el 72 o/o tuvo el mismo TIH y el resto poseía una dilución de diferencia en relación con los sueros correspondientes. De las muestras ensayadas a los 60 días de extracción un 59 o/o tuvo el mismo TIH y el 41 o/o una dilución de diferencia. A los 100 días de extracción, el 51 o/o de las muestras tuvieron el mismo TIH, el 38 o/o una dilución de diferencia y el 11 o/o tuvieon 1 o más de una dilución de diferencia. En conclusión, los resultados de ese estudio mostraron que las muestras de sangre recolectadas sobre papel de filtro pueden ser usadas para la detección de anticuerpos antivirus rubéola determinados por IHA dentro de los 30 días post-obtención con una sensibilidad y especificidad del 100 o/o. Esta muestra en papel es estable a temperatura ambiente, sin ninguna restricción de aire o esterilización especial

[Detection of rubella antibodies in blood samples collected on filter paper]. / Detección de anticuerpos para rubéola en muestras de sangre recolectadas en papel de filtro.

Congenital rubella syndrome (CRS) could be prevented if young women knew their immune status before pregnancy, contributing in this way to decrease the birth morbidity rate due to CRS among the children. Our objective was to optimize the detection of rubella virus-antibodies by HAI, using an easier and safer method to collect samples of big populations. One hundred specimens, obtained from patients in a pediatric hospital and pregnant women in an institute of Virology were used for this work. Venous blood was drawn and collected in a test tube, and few drops were spotted onto filter paper circles. These samples were kept in envelopes and stored at room temperature until analysis. Seventy two percent of dried blood samples had titers identical to those of the corresponding serum samples, and 28% dried blood samples showed 1 dilution of difference. Storage of dried blood at room temperature for 30 days did not affect the HAI titers. Up to 60 days post attainment, 59% dried blood samples had identical titers to those of the corresponding serum samples, and 41% dried blood samples showed one dilution of difference. At 100 days of storage 51% dried blood samples had identical titers to those of the corresponding serum samples, 38% dried blood samples showed 1 dilution of difference and 11% and more than 1 dilution of difference. In conclusion, dried blood on filter paper is an easier method to transport and store blood samples for the determination of rubella virus immunity, for as long as 30 days. It could be used for large-scale epidemiological studies. The sensitivity and specificity of HAI performed on dried blood samples was 100%. Only 0.25 ml of whole blood is needed and the samples are stable at room temperature, without air or sterile conditioning. The proposed methodology is a practical approach to collect, transport and store blood samples. Moreover the blood dried on paper spots can be placed in a plastic bag and mailed to a reference laboratory. This is an appropriate alternative method for serological screenings in developing countries.

Different affinity of monoclonal antibodies for conserved neutralizing epitopes on two strains of rubella virus.

There is, apparently, only one serological type of rubella virus (RV) in the population, although several isolates exist with different characteristics. Some authors failed to detect significant differences among RV strains by neutralization, hemagglutination inhibition, and enzyme immunoassay using polyclonal and monoclonal antibodies, but differences in growth, plaque morphology, and temperature sensitivity between vaccine and wild-type strains were shown by Chantler et al. (3) With the purpose of analyzing the possible differences among several strains of RV, we studied the affinity constant of two monoclonal antibodies (MAbs) for two conserved neutralizing epitopes. Wild-type Cordoba (regional isolation of a post-natal infection) and RA 27/3 (vaccine) strains of RV were tested. H3 and H14 MAbs were generated against wild-type Cordoba strain. They defined two epitopes with conserved neutralizing and hemagglutinating activity on both strains. The affinity of the MAbs (expressed as the affinity constant), was greater for Cordoba strain than for RA 27/3. Analyzing the results obtained, we conclude that the neutralizing epitopes defined by our MAbs on E1 glycoprotein are conserved in the two strains, but react with significative different affinities. This could be a way to characterize antigenically different viral strains of the same serotype.