Altered Vaginal Microbiota Composition Correlates With Human Papillomavirus and Mucosal Immune Responses in Women With Symptomatic Cervical Ectopy.

Cervical ectopy is a benign condition of the lower genital tract that is frequently detected in women of reproductive age. Although cervical ectopy is regarded as a physiological condition, some women experience symptoms such as leucorrhoea, persistent bleeding and recurrent vaginal infections that require medical intervention. Cervical ectopy has not been linked to cervical cancer, but it is thought to facilitate the acquisition of sexually transmitted diseases (STDs), like Human Papillomavirus (HPV) infection, as it provides a favorable microenvironment for virus infection and dissemination. We and others have described the presence of oncogenic HPV types in women with symptomatic cervical ectopy. The relevance of this finding and the impact of symptomatic cervical ectopy on the cervicovaginal microenvironment (vaginal microbiota, immune and inflammatory responses) are currently unknown. To shed some light into the interplay between HPV, the vaginal microbiota and mucosal immune and inflammatory responses in the context of this condition, we enrolled 156 women with symptomatic cervical ectopy and determined the presence of HPV using a type-specific multiplex genotyping assay. Overall, HPV was detected in 54.48% women, oncogenic HPV types were found in more than 90% of HPV-positive cases. The most prevalent HPV types were HPV16 (29.4%), HPV31 (21.17%) and HPV18 (15.29%). Next, we evaluated the vaginal microbial composition and diversity by 16S rDNA sequencing, and quantified levels of cytokines and chemokines by flow cytometry using bead-based multiplex assays in a sub-cohort of 63 women. IL-21 and CXCL9 were significantly upregulated in HPV-positive women (p=0.0002 and p=0.013, respectively). Women with symptomatic cervical ectopy and HPV infection had increased diversity (p<0.001), and their vaginal microbiota was enriched in bacterial vaginosis-associated anaerobes (Sneathia, Shuttleworthia, Prevotella, and Atopobium) and depleted in Lactobacillus spp. Furthermore, the vaginal microbiota of women with symptomatic cervical ectopy and HPV infection correlated with vaginal inflammation (IL-1ß, rho=0.56, p=0.0004) and increased mucosal homeostatic response (IL-22, rho=0.60, p=0.0001). Taken together, our results suggest that HPV infection and dysbiotic vaginal communities could favor a vaginal microenvironment that might delay the recovery of the cervical epithelium in women with symptomatic cervical ectopy and favor STDs acquisition.

Peripheral blood lymphocytes from low-grade squamous intraepithelial lesions patients recognize vaccine antigens in the presence of activated dendritic cells, and produced high levels of CD8 + IFNγ + T cells and low levels of IL-2 when induced to proliferate.

BACKGROUND: Most infections with human papillomavirus (HPV) are resolved without clinical intervention, but a minority evolves into chronic lesions of distinct grades, including cervical-uterine cancer. It is known that in most cases the immune system mediates elimination of HPV infection. However, the mechanism of immune evasion leading to HPV persistence and development of early cervical lesions is not fully understood. The aim of the present work was to evaluate the potential of peripheral blood leukocytes (PBL) from low-grade squamous intraepithelial lesions (LSIL) patients to be activated ex-vivo by vaccine antigens, the participation of cytotoxic lymphocytes and regulatory T cells, and to determine the secretion of Th1 and Th2 cytokines mediated by stimulation of T cell receptors. RESULTS: We found that PBL from LSIL patients showed a significantly lower proliferation rate to vaccine antigens as compared to that of healthy donors, even though there was not a difference in the presence of antibodies to those antigens in sera from both groups. We did not find differences in either the frequency of CD4 + CD25 + FoxP3+ in PBL, or the levels of IL-4, IL-5 and IL-10 in plasma or conditioned media from PBL incubated with TcR agonists in vitro, between the two groups. However, we detected a lower production of IL-2 and a higher proportion of CD8 + IFNγ + cells in PBL from LSIL patients as compared with PBL from normal donors. We also observed that PBL from patients infected by HPV-16 and -18 were not able to proliferate in the presence of soluble HPV antigens added to the culture; however, a high level of proliferation was attained when these antigens were presented by activated dendritic cells. CONCLUSIONS: Our results suggest that the immunodeficiency reported in LSIL patients could be due to the inability of specific cytotoxic T lymphocytes that for some unknown reason are present but unable to mount a response when challenged with their antigens, probably related to an in situ IL-2 production deficiency.

The Amerindian mtDNA haplogroup B2 enhances the risk of HPV for cervical cancer: de-regulation of mitochondrial genes may be involved.

Although human papillomavirus (HPV) infection is the main causal factor for cervical cancer (CC), there are data suggesting that genetic factors could modulate the risk for CC. Sibling studies suggest that maternally inherited factors could be involved in CC. To assess whether mitochondrial DNA (mtDNA) polymorphisms are associated to CC, HPV infection and HPV types, a case-control study was performed in the Mexican population. Polymorphism of mtDNA D-loop was investigated in 187 CC patients and 270 healthy controls. HPV was detected and typed in cervical scrapes. The expression of 29 mitochondrial genes was analyzed in a subset of 45 tumor biopsies using the expression microarray ST1.0. The Amerindian haplogroup B2 increased the risk for CC (odds ratio (OR)=1.6; 95% confidence interval (CI): 1.05-2.58) and enhanced 36% (OR=208; 95% CI: 25.2-1735.5) the risk conferred by the HPV alone (OR=152.9; 95% CI: 65.4-357.5). In cases, the distribution of HPV types was similar in all haplogroups but one (D1), in which is remarkable the absence of HPV18, a very low frequency of HPV16 and high frequencies of HPV45, HPV31 and other HPV types. Two mtDNA genes (mitochondrial aspartic acid tRNA (MT-TD), mitochondrial lysine tRNA (MT-TK)) could be involved in the increased risk conferred by the haplogroup B2, as they were upregulated exclusively in B2 tumors (P<0.01, t-test). Although the association of mtDNA with CC and HPV infection is clear, other studies with higher sample size will be needed to elucidate the role of mtDNA in cervical carcinogenesis.

Prevalence of human papillomavirus genotypes, and mucosal IgA anti-viral responses in women with cervical ectopy.

BACKGROUND: Data on the prevalence of different human papillomavirus (HPV) genotypes and the associated mucosal immune response in women with cervical ectopy are scarce. OBJECTIVE: To assess the prevalence of different HPV genotypes and the mucosal anti-viral immune response in cervical ectopy. STUDY DESIGN: Detection and typing of HPV DNA was determined in 141 women with cervical ectopy, 272 cytologically normal controls and 98 low-grade squamous intraepithelial lesions (LSIL) by PCR and direct sequencing. Mucosal IgA antibodies to HPV16 and HPV18 were evaluated in cervical mucus by ELISA. RESULTS: The prevalence of HPV in cervical ectopy was higher (73.7%) than that observed in control samples (30.5% in endocervix, and 1.8% in exocervix), but similar to the prevalence in LSIL (62.2%). Typing showed that the overall distribution frequency concerned 14 different genotypes, with HPV18 being the most prevalent in cervical ectopy (53.9%), whereas HPV16 predominated in LSIL (38.7%). High-risk HPV genotypes were 2.2 times more frequent in cervical ectopy than in the normal endocervix (p<0.0001). HPV infection in cervical ectopy patients was accompanied by a mucosal IgA-antibody response. Antibody reactivity to HPV18 was significantly higher than the response to HPV16. CONCLUSION: Cervical ectopy is a risk factor for infection with high-risk HPV genotypes, in particular HPV18. Our results emphasize the need of further studies to clarify the oncogenic potential of this virus in cervical ectopy.

A great diversity of Amerindian mitochondrial DNA ancestry is present in the Mexican mestizo population.

There are limited data on mitochondrial DNA (mtDNA) variation in the Mexican mestizo population. To examine the genetic diversity and matrilineal ancestry, the full mtDNA hypervariable regions I and II were sequenced in 270 unrelated mestizos from different regions of Mexico. A total of 202 different haplotypes were identified and the haplotype diversity was 0.9945. Amerindian haplotypes predominated in the sample with a proportion of 93.3%, followed by European (6.0%) and African haplotypes (0.7%). The frequency of the Amerindian haplogroups A2, B2, C1 and D1 was 51.1, 17.8, 18.5 and 5.9%, respectively. The frequency of Amerindian haplogroups was higher in the central region than in Mexico City, whereas it was the contrary for European haplogroups. This difference was accounted principally by the high frequency of B2 haplotypes in the central region. The minimum spanning network, the mismatch distribution and Tajima's D neutrality test suggest a population expansion for each Amerindian haplogroup, which could be initiated more recently for haplogroups A2 and D1. The present knowledge combined with other nuclear genetic markers will be essential in future association studies to correct for genetic substructure in mestizo populations.

Binding of human papillomavirus type 16 to heparan sulfate is inhibited by mucosal antibodies from patients with low-grade squamous intraepithelial lesions but not from cervical cancer patients.

Mucosal antibodies against human papillomavirus type 16 (HPV16) capsids have been detected in infected women. To determine whether these antibodies recognize and block the receptor site mediating attachment of HPV16 to heparan sulfate, mucus samples from 126 HPV16-associated low-grade squamous intraepithelial lesion (LSIL) and 85 cervical cancer patients, previously found to react to HPV16 virus-like particles (VLP), and 101 normal controls were tested in an inhibition assay, using HPV16 VLP and heparan sulfate proteoglycan-coated plates. Inhibition levels of 9.3-67.2% were mediated by type-specific antibodies in 94.4% of LSIL patients. Cervical cancer cases showed significantly lower levels of inhibition than LSIL samples (P < 0.0001). The potential of antibodies to inhibit infection was explored in a pseudoinfection system using HPV16 pseudovirions. Inhibition of pseudoinfection by LSIL samples was significantly higher than that observed in the controls (P < 0.001) and cervical cancer cases (P < 0.005). These results indicate that mucosal antibodies inhibiting binding of VLP to heparan sulfate are developed in most LSIL patients, but are hardly present in cervical cancer patients.

Detection of autoantibodies to survivin in cervical mucus from patients with human papillomavirus-associated cervical cancer and precursor lesions.

OBJECTIVES: To investigate the prevalence of mucosal autoantibodies to survivin in patients with human papillomavirus (HPV)-associated cervical cancer and precursor lesions. METHODS: Cervical mucus from 117 HPV-associated cervical cancer, 102 high-grade squamous intraepithelial lesion (HSIL), 107 low-grade SIL (LSIL), and 80 normal controls were tested by ELISA using either full length recombinant survivin or survivin-derived peptides. Survivin expression in cervical tissue biopsies was studied by Western Blotting. RESULTS: Cervical mucus from 33 cervical cancer (28.2%), 17 HSIL (16.6%), and 8 LSIL (7.4%) patients reacted with recombinant survivin. The IgA-class antibody response was significantly higher than that observed in the normal controls. The level of mucosal anti-survivin response was associated to the level and intensity of survivin expression in the different lesions. Finally, reactivity against a survivin Nt-derived peptide was found more frequently than reactivity against a Ct-derived peptide. CONCLUSIONS: IgA-class autoantibodies against survivin are present in a substantial proportion of cervical mucus from patients with HPV-associated cervical cancer, and precursor lesions. Mucosal anti-survivin response is positively associated with the level of survivin expression and the grade of cervical lesion.

Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions.

Trichomonosis, a chronic sexually transmitted disease, remains a public health problem affecting yearly over 170 million people worldwide. This disease is caused by Trichomonas vaginalis, a protozoan flagellate rich in cysteine proteinases (CPs). Although CPs are involved in trichomonal cytopathogenicity, only few of them have been defined as virulence factors. In this study, we characterize a T. vaginalis 39-kDa proteinase (CP39) found in vaginal secretions from patients with trichomonosis. The CP39 proteinase bound to HeLa epithelial cells, vaginal epithelial cells (VECs), and human prostatic cancer cells (DU-145). CP39 did not bind to a human colon cancer (CaCo) cell line, suggesting tissue-specific binding. CP39 was found in six fresh trichomonad isolates tested. In two-dimensional gels, CP39 appeared as a single spot with a pI 4.5. CP39 is inhibited by E-64, stable at 50 degrees C, and active in a wide pH range (3.6-9.0), with an optimum pH at 7.0. In addition, CP39 degraded collagens I, III, IV, and V, human fibronectin, human hemoglobin, and human immunoglobulins A and G. Indirect immunofluorescence detected CP39 on the parasite surface with specific polyclonal antibody to purified CP39. Finally, CP39 was found to be immunogenic, as evidenced by detection on immunoblots with serum of patients with trichomonosis, but not control individuals. These data suggest that CP39 may play a role during trichomonal infection.

Detection of antibodies against a human papillomavirus (HPV) type 16 peptide that differentiate high-risk from low-risk HPV-associated low-grade squamous intraepithelial lesions.

A nonapeptide (16L1) was derived from the human papillomavirus type 16 (HPV-16) major capsid protein and tested for detection of potential cross-reactive serum IgG and cervical IgA antibodies in low- and high-risk HPV-associated low-grade squamous intraepithelial lesions (LSIL) and cervical cancer patients by ELISA. The IgG response was similar in women with low-risk HPV-associated LSIL and controls (P=0.1). In contrast, more than 90 % of patients with high-risk HPV-associated LSIL were seropositive. Although tumours from cancer patients were all positive for the presence of high-risk HPV DNA, the level of seropositivity decreased significantly in this group (P<0.0001). Cervical IgA antibodies were also detected in a significantly high proportion of women with high-risk HPV-associated LSIL compared with controls. However, the proportion of IgA-positive patients was lower than the proportion of IgG seropositives. In conclusion, the 16L1 peptide appears to be a high-risk type-common epitope that induces cross-reactive antibodies in high-risk, but not low-risk, HPV-associated LSIL patients, allowing differentiation of high- and low-risk infected women at this stage of infection.

[High-risk human papilloma virus and cervical intraepithelial neoplasia in women at 2 hospitals in Mexico City]. / Virus de papiloma humano de alto riesgo (VPH-AR) y neoplasia intraepitelial cervical (NIC) en mujeres de dos hospitales de la Ciudad de México.

OBJECTIVE: To determine the high risk HPV (HR-HPV) association with Cervical Intraepithelial Neoplasia (CIN) in women of two Dysplasia Clinics in Mexico City. MATERIAL AND METHODS: Prolective case-control study was done. Women with and without security affiliation attended in Instituto Mexicano del Seguro Social (Hospital 1) and Hospital General de México (Hospital 2) were included in the study. Cases were women with histopathologic diagnosis of CIN and controls were women with negative dysplasia in cytologic study (Pap). Information was obtained by direct interview. HR-HPV was determined by Hybrid Capture II assay, in cervical samples. Bivariate and logistic regression analysis was done. RESULTS: One hundred and two cases and 192 controls from Hospital 1 and 89 cases and 66 controls from Hospital 2 were included. 83.3% and 77.3% of women from Hospital 1 and 2 respectively were positive to HR-HPV. The association HR-HPV and CIN in Hospital 1 was ORa = 40.6, C.I. 95% = 17-96.8; while in Hospital 2 there was not association. Age was an effect modifier in the HR-HVP and CIN association, in Hospital 1. It was observed a correlation between viral load and CIN degree. CONCLUSIONS: The HR-HPV infection frequency in controls and CIN I was higher than the reported in other studies. Age was a modifier in the HR-HPV association and CIN. In dysplasia clinics without medical referral system of patients is possible to observe similar risk factors to cervical cancer.

[Study on Candida no-albicans species and its relation to recurrent vulvovaginal candidiasis]. / Estudio de especies de Candida no albicans y su relación con candidiasis vulvovaginal recurrente.

Genital candidiasis is a frequent pathology among women. It has a 5% incidence rate of presenting itself in a recurrent way, which leads to a longer treatment. Candida gender has various species. The ones, which are the most usual and the cause of vaginal, cervicovaginal, and vulvovaginal candidiasis are: Candida albicans, C. glabrata and C. krusei. Their presence is related to further appearances. A case study was made to identify the species of Candida gender. It was based on the diagnostics made in three Mexico City hospitals on female genital candidiasis cases. The identified and isolated Candida species obtained were: albicans with a 71.91%, C. glabrata with a 11.80%, and C. tropicalis with a 7.87%.

Virus de papiloma humano de alto riesgo (VPH-AR) y neoplasia intraepitelial cervical (NIC) en mujeres de dos hospitales de la Ciudad de México / High-risk human papilloma virus and cervical intraepithelial neoplasia in women at 2 hospitals in Mexico City

OBJECTIVE: To determine the high risk HPV (HR-HPV) association with Cervical Intraepithelial Neoplasia (CIN) in women of two Dysplasia Clinics in Mexico City. MATERIAL AND METHODS: Prolective case-control study was done. Women with and without security affiliation attended in Instituto Mexicano del Seguro Social (Hospital 1) and Hospital General de MÚxico (Hospital 2) were included in the study. Cases were women with histopathologic diagnosis of CIN and controls were women with negative dysplasia in cytologic study (Pap). Information was obtained by direct interview. HR-HPV was determined by Hybrid Capture II assay, in cervical samples. Bivariate and logistic regression analysis was done. RESULTS: One hundred and two cases and 192 controls from Hospital 1 and 89 cases and 66 controls from Hospital 2 were included. 83.3 and 77.3 of women from Hospital 1 and 2 respectively were positive to HR-HPV. The association HR-HPV and CIN in Hospital 1 was ORa = 40.6, C.I. 95 = 17-96.8; while in Hospital 2 there was not association. Age was an effect modifier in the HR-HVP and CIN association, in Hospital 1. It was observed a correlation between viral load and CIN degree. CONCLUSIONS: The HR-HPV infection frequency in controls and CIN I was higher than the reported in other studies. Age was a modifier in the HR-HPV association and CIN. In dysplasia clinics without medical referral system of patients is possible to observe similar risk factors to cervical cancer.

Molecular evaluation of the prevalence of oncogenic human papillomavirus genotypes in cervical acetowhite lesions.

The actual prevalence of cancer-related human papillomavirus (HPV) genotypes in cervical acetowhite lesions has not been established. In this work, the presence of oncogenic types of HPV in cervical acetowhite tissue was evaluated by molecular means. The presence of HPV DNA was determined in a group of women with and without cervical acetowhite lesions by a polymerase chain reaction (PCR) using the MY09/MY11 primers. The presence of 13 oncogenic HPV types was evaluated using the Hybrid Capture II test, and the prevalence of HPV type 16 (HPV16) was studied using an HPV16-specific PCR. HPV DNA was detected in 85.9% patients with acetowhite lesions; oncogenic HPV types were found in 83.7% of them; HPV16 was identified in 51.1% of the cases. HPV DNA was detected in 87.3% of the patients without acetowhite changes. Interestingly only 16.8% were infected by oncogenic genotypes and 2.5% were positive for the presence of HPV16. In conclusion, subclinical infection by oncogenic HPV genotypes is associated with the presence of acetowhite cervical tissue (p < 0.0005). Therefore, women showing acetowhite lesions might be at risk of developing cervical cancer.

Eficacia y seguridad de ciclopirox olamina en crema vaginal al 1 por ciento contra terconazol en crema vaginal al 0.8 por ciento en el tratamiento de candidiasis genital / Ciclopirox olamine 1 percent cream versus terconazole 0.8 percent cream effectiveness and safety in genital candidiasis treatment

Se efectuó un estudio multicéntrico comparativo, aleatorio, seleccionando 170 pacientes de 18 años o más, con diagnóstico clínico de candidiasis genital y con cultivo positivo a Candida, los cuales se asignaron aleatoriamente al tratamiento con ciclopirox olamina en crema vaginal al 1 por ciento (85 pacientes) o con terconazol en crema vaginal al 0.8 por ciento (85 pacientes) durante seis días, con el objeto de comparar la eficacia clínica, la eficacia fungicida y la seguridad de ambos tratamientos. Los resultados de la eficacia mixta (clínica y micológica) para ciclopirox olamina fue de cura al final del tratamiento - día siete - en 48 pacientes (62.3 por ciento) y al seguimiento - día 21 - de 42 (55.30 por ciento) y la mejoría al final en 25 pacientes (32.5 por ciento) y en el seguimiento 21 (27.6 por ciento) y para terconazol fue de cura al final en 45 pacientes (61.6 por ciento) y al seguimiento en 39 (57.4 por ciento) y de mejoría al final en 23 (31.5 por ciento) y en el seguimiento, 22 (32.4 por ciento). Se concluye que ambos medicamentos son eficaces y seguros para el tratamiento de candidiasis genital.

Factores pronósticos de la histerectomía radial por cáncer cérvico-uterino. Un análisis de 201 pacientes / Pronostic factores for patients undergoing radial histerectomy for cervical cancer

Se presenta un análisis retrospectivo, de los factores que influyeron en el pronóstico de 201 pacientes con cáncer invasor del cérvix, tratadas mediante histerectomía con linfadenectomía pélvica, (histerectomía radical) en el Servicio de Ginecología de la Unidad de Oncología, Hospital General de México, S.S. Se señala que la edad media del grupo fue 43 años y que 20 de 143 en estado Ib, (13.9%) y 10 de 51 en estadio IIa, (19.6%) desarrollaron metástasis ganglionares. P<0.05. En cuanto al pronóstico, 20 pacientes presentaron recurrencias tumorales, las que en el 89.5%se desarrollaron durante los 2 primeros años consecutivos a la cirugía. Se obtuvo una media de seguimiento de 2 años sin enfermedad, en 92 de 101, (91.0%) pacientes en estadio Ib, en 24 de 32, (75%) en estadio IIa, (P<0.05) y en 3 de 7, (42.8%) recurrencias a radiación. Las pacientes con mejor pronóstico fueron aquellas que tuvieron lesiones de menos de 2 cms (69/73, 94.5%) y las que mostraron ausencia de metástasis ganglionares, (111/125, 88.8%). El pronóstico menos favorable se obtuvo en enfermas con primario mayor de 4 cms, (12/18, 66%); en pacientes con carcinomas poco diferenciados, (13/20, 65.8%) y en las que se reportó la presencia de metástasis ganglionares, (8/15, 53.3%) .

Cáncer ovárico avanzado (etapa clínica III): valor pronóstico de un tratamiento quirúrgico óptimo / Advanced ovarian cancer (stage III): pronoctic value of an optimal surgical treatment

Se presenta un análisis retrospectivo de los factores que influyeron en el pronóstico de 115 enfermas con cáncer de ovario etapa III, con especial enfoque al papel desempeñado por la cirugía como parte del manejo integral de estas pacientes. Se señala que evolucionaron dos o más años sin evidencia de enfemedad, independientemente del tratamiento complementario administrado, 15 de 44 enfermas (30.4%) tratadas con panhisterectomía más omentectomía; 3 de 51 (5.8%) de las manejadas con cirugías insuficentes, (p < 0.05) y 4 de 17, (23.5%) de las que se sometieron a procedimientos quirúrgicos aún más radicales que la pnahisterectomía más omentectomía. Mientras que sólo 2 de 41 pacientes (4.8%), tratadas únicamente con cirugía lograron control del padecimiento por el lapso mencionado, el pronóstico de 74 enfermas manejadas en forma multidisciplinaria, varió de acuerdo con los siguientes factores: mejores resultados en pacientes reexploradas quirúrgicamente en el servicio, en relacion al grupo restante (11/23, 47.8% vs. 9/51, 17.6% p < 0.05); en enfermas tratadas con cirugías completas seguidas de quimioterapia, fundamentalmente a base de ciclofosfamida o bien adriamicina más ciclofosfamida, en relación a los resultados logrados con cirugías completas más radioterapia, (11/20, 55% vs. 6/24%, p < 0.05); y mejores resultados en el grupo de pacientes cuya edad promedio correspondió a 38 años