Bacterias relacionadas con vaginosis bacteriana y su asociación a la infección por virus del papiloma humano / Vaginosis-associated bacteria and its association with HPV infection

Antecedentes y objetivo: En México y en el mundo cada vez son más frecuentes los casos de cáncer de cérvix y aunque se sabe que existen diversos factores de riesgo para padecer este tipo de cáncer, se ha sugerido que las alteraciones en el microbioma cervical pueden desempeñar un papel importante en el establecimiento y progresión del cáncer, específicamente hablando de la presencia de aquellas bacterias asociadas a vaginosis bacteriana como lo son Atopobium vaginae y Gardnerella vaginalis. Material y métodos: Se estudió el ADN de un total de 177 muestras de raspado de cérvix, de las cuales 104 pertenecían a mujeres sin alteraciones citológicas o colposcópicas y 73 muestras de lesiones precursoras con antecedente de infección por virus del papiloma humano (VPH). Todas las muestras fueron sometidas a detección de secuencias de Atopobium vaginae, Gardnerella vaginalis y VPH por medio de PCR. Resultados: El VPH fue encontrado en mayor proporción en las muestras precursoras, y poco más del 30% de las muestras sin lesión fueron positivas para VPH. La mayoría de las muestras contienen secuencias de ambas bacterias sin asociación con la presencia viral, lo que podría indicar que estos microorganismos serían parte del microbioma cervical en población mexicana. Conclusiones: Los resultados obtenidos indican que las bacterias analizadas potencialmente forman parte del bioma normal en las mujeres mexicanas por lo que es de especial importancia reconsiderar el papel patógeno de estos microorganismos

Background and objective: Cervical cancer is an important health problem in our country. It is known that there are several risk factors for this neoplasm, and it has been suggested that cervical microbiome alterations could play a role in the development and progress of cancer. Bacterial vaginosis associated bacteria such as Atopobium vaginae and Gardnerella vaginalis has been suggested as potential risk factor for cervical lesions and cervical cancer. Material and methods: DNA from 177 cervical scraping samples was studied: 104 belonged to women without cytological or colposcopic alterations and 73 samples from precursor lesions with previous human papillomavirus (HPV) infection history. All samples were screened for Atopobium vaginae, Gardnerella vaginalis and HPV by PCR. Results: High HPV prevalence was found in precursor samples, and 30% of samples without lesions were positive for HPV. Virtually all samples contained sequences of both bacteria, and interestingly, there was not HPV association observed; these results could suggest that these microorganisms could be part of the cervical microbiome in Mexican population. Conclusions: The results obtained indicate that the bacteria analysed could be part of normal biome in Mexican women, suggesting a potential reconsideration of the pathogen role of these microorganisms

Desempenho da citologia em meio líquido na identificação de agentes microbiológicos cérvico-vaginais / Liquid medium cytology performance in identification of cervico-vaginal microbiological agentes

Verificar o desempenho da citologia em meio líquido na identificação de agentes microbiológicos cérvico-vaginais em relação à citologia convencional. Métodos: Estudo analítico e transversal realizado no Laboratório de Citopatologia de um centro universitário. Foram realizadas coletas citopatológicas pelas duas metodologias: convencional e em meio líquido (Liqui-PREP®), além de coleta microbiológica. Resultados: Foram avaliadas 67 amostras cérvico-vaginais pelas duas técnicas citopatológicas, verificando-se que a citologia convencional identificou 26 esfregaços com microrganismos de interesse clínico, enquanto que a citologia em meio líquido identificou 20 casos, sendo observada boa concordância entre as técnicas na identificação de Gardnerella vaginalis e Candida spp. A citologia convencional também evidenciou mais casos de esfregaços com Trichomonas vaginalis e ocorreu boa concordância entre as técnicas citopatológicas e o exame microbiológico na identificação de Gardnerella vaginalis. Conclusão: Observou-se boa concordância entre as técnicas citopatológicas na identificação de Candida spp. e Gardnerella vaginalis, assim como entre as duas técnicas frente ao exame microbiológico na evidência de Gardnerella vaginalis.

More Easily Cultivated Than Identified: Classical Isolation With Molecular Identification of Vaginal Bacteria.

BACKGROUND: Women with bacterial vaginosis (BV) have complex communities of anaerobic bacteria. There are no cultivated isolates of several bacteria identified using molecular methods and associated with BV. It is unclear whether this is due to the inability to adequately propagate these bacteria or to correctly identify them in culture. METHODS: Vaginal fluid from 15 women was plated on 6 different media using classical cultivation approaches. Individual isolates were identified by 16S ribosomal RNA (rRNA) gene sequencing and compared with validly described species. Bacterial community profiles in vaginal samples were determined using broad-range 16S rRNA gene polymerase chain reaction and pyrosequencing. RESULTS: We isolated and identified 101 distinct bacterial strains spanning 6 phyla including (1) novel strains with <98% 16S rRNA sequence identity to validly described species, (2) closely related species within a genus, (3) bacteria previously isolated from body sites other than the vagina, and (4) known bacteria formerly isolated from the vagina. Pyrosequencing showed that novel strains Peptoniphilaceae DNF01163 and Prevotellaceae DNF00733 were prevalent in women with BV. CONCLUSIONS: We isolated a diverse set of novel and clinically significant anaerobes from the human vagina using conventional approaches with systematic molecular identification. Several previously "uncultivated" bacteria are amenable to conventional cultivation.

[ABOUT UNIFICATION OF LABORATORY CRITERIA OF DIFFERENTIATION OF BACTERIAL VAGINOSIS].

The article presents analysis of laboratory criteria and classifcations used to interpret results of laboratory analysis by technique of microscopy on bacterial vaginosis or dysbacteriosis of vagina. Their advantages and restrictions are demonstrated The unified criteria of evaluation are proposed concerning results of microscopy of mucosal discharge of vagina and corresponding classification. Thereafter, three degrees of bacterial vaginosis (dysbacteriosis of vagina) are differentiated: first degree--compensated dysbacteriosis of vagina, second degree--sub compensated dysbacteriosis of vagina and third degree--decompensated dysbacteriosis of vagina. The corresponding laboratory report of physician is formulated. The proposals are presented concerning development of common unified requirements to stages (pre-analytical, analytical, post-analytical) of laboratory diagnostic of bacterial vaginosis (dysbacteriosis of vagina) with purpose of their unambiguous understanding by clinicians and hence their decision making concerning necessity and tactics of management of patient.

More than meets the eye: associations of vaginal bacteria with gram stain morphotypes using molecular phylogenetic analysis.

Bacterial vaginosis (BV) is a highly prevalent condition associated with adverse health outcomes. Gram stain analysis of vaginal fluid is the standard for confirming the diagnosis of BV, wherein abundances of key bacterial morphotypes are assessed. These Lactobacillus, Gardnerella, Bacteroides, and Mobiluncus morphotypes were originally linked to particular bacterial species through cultivation studies, but no studies have systematically investigated associations between uncultivated bacteria detected by molecular methods and Gram stain findings. In this study, 16S-rRNA PCR/pyrosequencing was used to examine associations between vaginal bacteria and bacterial morphotypes in 220 women with and without BV. Species-specific quantitative PCR (qPCR) and fluorescence in Situ hybridization (FISH) methods were used to document concentrations of two bacteria with curved rod morphologies: Mobiluncus and the fastidious BV-associated bacterium-1 (BVAB1). Rank abundance of vaginal bacteria in samples with evidence of curved gram-negative rods showed that BVAB1 was dominant (26.1%), while Mobiluncus was rare (0.2% of sequence reads). BVAB1 sequence reads were associated with Mobiluncus morphotypes (p<0.001). Among women with curved rods, mean concentration of BVAB1 DNA was 2 log units greater than Mobiluncus (p<0.001) using species-specific quantitative PCR. FISH analyses revealed that mean number of BVAB1 cells was 2 log units greater than Mobiluncus cells in women with highest Nugent score (p<0.001). Prevotella and Porphyromonas spp. were significantly associated with the "Bacteroides morphotype," whereas Bacteroides species were rare. Gram-negative rods designated Mobiluncus morphotypes on Gram stain are more likely BVAB1. These findings provide a clearer picture of the bacteria associated with morphotypes on vaginal Gram stain.

Comparison of Affirm VPIII and Papanicolaou tests in the detection of infectious vaginitis.

To compare the Affirm VPIII molecular test (Becton Dickinson, Burlington, NC) with morphologic identification used in routine Papanicolaou (Pap) test screening in the detection and identification of Candida species, Trichomonas vaginalis, and Gardnerella vaginalis, we identified 431 cases with a concomitant Pap test and Affirm VPIII assay performed from the archives of a large academic institution. The study population consisted of women ranging in age from 17 to 79 years (mean and median ages, 33 and 31 years, respectively). With a routine Pap test, 60 patients (13.9%) were found to have bacterial vaginosis, 60 (13.9%) candidiasis, and 3 (0.7%) Trichomonas infection. With the Affirm VPIII assay, 183 (42.5%) patients tested positive for G vaginalis, 70 (16.2%) positive for Candida species, and 10 (2.3%) positive for T vaginalis. The differences were statistically significant. The results demonstrate that our patient population had a high incidence of bacterial vaginosis/Candida vaginitis; however, the Affirm VPIII was a more sensitive diagnostic test for the detection and identification of all 3 organisms compared with the Pap test.

Incidencia de infecciones vaginales diagnósticadas por citolog¡a cervicovaginal: en mujeres mayores de 40 años: Hospital Miliar Dr. Carlos Arvelo / Incidence of vaginal infections diagnosed by cervicovaginal cytology in women older than 40 years: Hospital Militar Dr. Carlos Arvelo

Las infecciones son probablemente, la patología más frecuente de consulta en ginecología. La abundante y molesta sintomatología que suele acompañar a estos procesos lleva a la mujer a consultar con su médico. Las infecciones vaginales o vaginitis se puede ocasionar por virus, bacterias, hongos o parásitos, estos gérmenes pueden estar en la vagina solos o asociados. Clínicamente pueden distinguirse según los síntomas y signos, pero el diagnóstico definitivo se establece por citología y/o microbiología. Los cambios hormonales durante la pubertad, en la adolescencia, el ambarazo y la menopausia, predisponen a la mujer a contraer vaginitis, al igual que la falta de higiene adecuada, sensibilidad a ciertos jabones, utilización de anticonceptivos, entre otros. En este tarbajo se presenta la casuística de esta patología en las pacientes examinadas entre enero y diciembre de 2004 en el Departamento de Ginecología y Obstetricia de nuestro hospital.

Light microscopy observation of lytic enzymatic activity of the organisms associated with bacterial vaginosis.

In this study, cervico-vaginal smears taken from 500 patients were examined cytologically using the Papanicolaou technique. Seventeen of the 500 were classified as having bacterial vaginosis. Lytic enzymatic activity of the organisms on clue cells were determined at light microscopic level. The integrity of the cell and the smoothness of the cell membrane were disrupted. Small cavities on the cell membrane and hollows in the cytoplasm were observed. Due to the loss of cytoplasm, very narrow and thin tracks around the nucleus and in the cytoplasm resembling a cobweb were seen. It is suggested that these lytic cellular changes might be formed by the organisms on clue cells.

Prevalência de vaginites específicas e inespecíficas em mulheres na pós-menopausa / Prevalence of non-specific and specific vaginitis in postmenopause women

O climatério é o período da vida feminina caracterizada pelo esgotamento dos folículos ovarianos, tendo como consequência à diminuiçäo dos níveis de estrogênio e progesterona, resultando em alteraçöes fisiológicas sobre a pele, mucosas, ossos e metabolismo lipoprotéico, sendo a menopausa um episódio dentro do climatério que representa a última menstruaçäo da vida da mulher. Com deficiência estrogênica, o epitélio vaginal torna-se atrófico e de pequena espessura, levando consequentemente a uma diminuiçäo da produçäo de glicogênio e baixo teor de acidez, favorecendo o surgimento de vaginites, as quais poderäo ser de origem específica ou inespecífica. Este trabalho teve como objetivo analisar a prevalência de vaginites específicas e inespecíficas em mulheres na pós-menopausa. Foram analisadas 533 mulheres de faixa etária compreendida entre 40 e 65 anos de idade, cujas amostras foram obtidas através de colheita tríplice (ectocérvice, endocérvice e fundo de saco vaginal), com posterior coloraçäo pela técnica de Papanicolaou. Os resultados demonstraram que das 533 mulheres examinadas, houve predomínio de vaginites inespecíficas determinadas por flora mista, em 44,5 porcento das pacientes, seguida por infecçöes ocasionadas pela Gardnerella vaginalis em 10 porcento das pacientes. As vaginites específicas por sua vez tiveram como predomínio às infecçöes provocadas pela Candida sp em 156 casos (27,3 porcento) seguidas pelo Trichomonas vaginalis observada em 72 pacientes (12,6 porcento), tendo sido observada uma maior incidência destes agentes em mulheres com faixa etária variando entre 40 a 49 anos, seguidas por aquela com idade variando entre 50 a 59 anos de idade. Estes dados chamam a atençäo para uma análise cuidadosa de esfregaço vaginal em mulheres na pós-menopausa em especial naquelas com sinais e sintomas de vaginites

Infección por Gardnerella vaginalis en parejas heterosexuales: estudio ultraestructural en células de descamación del epitelio estratificado / Gardnerella vaginalis infection in heterosexual couples: ultrastructural al study in descamation cells of stratified epithelium

La vaginosis bacteriana es una de las infecciones más frecuentes en la edad reproductiva de la mujer. La flora normal de lactobacilos es sustituída por concentraciones relativamente elevadas de Gardnerella vaginalis (GV), bacteroides anaerobios, Mobiluncus y Mycoplasma. El objetivo de este trabajo es hacer un análisis morfológico de los posibles mecanismos de adhesión y penetración de GV en la pareja heterosexual tanto en el epitelio escamoso de la pared vaginal como en el líquido seminal. Para cumplir con nuestro objetivo se estudiaron 10 parejas con cultivo de GV que tenían de 3 a 4 días de abstenencia sexual. Las mujeres presentaron al menos 3 de los 4 criterios de Amsel. Se obtuvieron muestras de las paredes laterales de la vagina y de líquido seminal, éstas se dividieron en dos partes: 1) para realizar cultivos para GV, Ureaplasma urealyticum y Mycoplasma hominis, y 2)para un análisis ultraestructural. Las muestras fueron procesadas con las técnicas habituales para microscopía electrónica. En las células vaginales se encontraron bacterias semejantes a GV en forma libre, adherencias a la membrana plasmática y dentro del citoplasma celular. En el líquido seminal se encontraron numerosas células ureterales de descamación que presentaron, al igual que en la mujer, bacterias en forma libre, adherida a la membrana plasmática y dentro del citoplasma. En 4 casos se encontraron bacterias semejantes a mycoplasma y un caso con partículas que sugieren citomegalovirus. En este trabajo se concluye que: 1) el varón presenta células uretrales semejantes a células guía" de la vagina de la mujer 2) la GV coloniza el epitelio ureteral del varón, 3) el varón es capaz de infectar y/o reinfectar a la mujer

Importância da detecçåo do Mobiluncus sp. nas gardnereloses resistentes ao tratamento com metronidazol / The importance of Mobiluncus sp. detection in vaginal infections caused by Gardnerella sp. which are results to metronidazole

O objetivo principal do trabalho é ressaltar o papel do citopatologista na detecçåo do bacilo curvo, móvel, anaeróbico, Gram-negativo e Gram-variável (Mobiluncus sp.) nas vaginoses bacterianas associadas principalmente à gardnerelose resistente. De um total de 506 exames citológicos rotineiros, vistos em um mês, foram detectados 77 esfregaços com flora bacteriana sugestiva de Gardnerella vaginalis. Utilizando-se lente de imersåo (o que rotineiramente nåo é feito pelo citopatologista), foi possível verificar a associaçåo de Mobiluncus sp. em 25 destas pacientes, quatro delas com resistência ao tratamento com metronidazol

Comparison of microscopic and cultural findings in the diagnosis of Gardnerella vaginalis infection.

The diagnosis of Gardnerella vaginalis infection on the basis of microscopic and cultural findings was compared. A total of 340 specimens of vaginal secretion were Gram stained and plated on a medium selective for Gardnerella vaginalis. Positive culture was obtained in 165 cases. Microscopy was unequivocally positive in 95, doubtful in 58 and negative in 187. Positive microscopy was confirmed by culture in 99%. On the other hand, 21% of the negative microscopy results gave a false negative diagnosis. Specimens for which microscopy was doubtful were culture positive in 53% of the cases, including 12% with heavy growth. Thus, positive microscopy proved to be sufficient for a reliable diagnosis of Gardnerella vaginalis infection. However, in specimens with negative or doubtful microscopic findings, additional culture is recommended.

Rapid presumptive identification of Gardnerella vaginalis (Haemophilus vaginalis) from human blood agar media.

Presumptive identification of Gardnerella vaginalis from 48-h human blood agar cultures by using a Gram stain, hemolysis, and colonial morphology was highly accurate.