Annie J. Vereecken

Definition of a type of abnormal vaginal flora that is distinct from bacterial vaginosis: aerobic vaginitis

Objective To define an entity of abnormal vaginal flora: aerobic vaginitis

Pathogenesis of abnormal vaginal bacterial flora

OBJECTIVE This study was undertaken to determine the relationships between microscopy findings on wet mounts, such as lactobacillary grade or vaginal leukocytosis, and results of vaginal culture, lactate and succinate content of the vagina, and levels of selected cytokines. STUDY DESIGN In a population of 631 unselected women seeking treatment at an obstetrics and gynecology outpatient clinic, vaginal fluid was obtained by wooden Ayre spatula for wet mounting and pH measurement, by high vaginal swab for culture, and by standardized vaginal rinsing with 2 mL 0.9% sodium chloride solution for measurements of lactate, succinate, interleukin 1beta, interleukin 8, leukemia inhibitory factor, and interleukin 1 receptor antagonist concentrations. Lactate and succinate levels were measured by gas-liquid chromatography and the cytokine concentrations were measured by specific immunoassays. Both univariate analysis (Student t test, Welch test, chi(2) test, and Fisher exact test) and multivariate regression analysis (Cox analysis) were used. RESULTS Increasing disturbance of the lactobacillary flora (lactobacillary grades I, IIa, IIb, and III) was highly correlated with the presence of Gardnerella vaginalis, Trichomonas vaginalis, enterococci, group B streptococci, and Escherichia coli. Vaginal pH and interleukin 8 and interleukin 1beta concentrations increased linearly with increasing lactobacillary grade, whereas lactate concentrations and the presence of epithelial cell lysis decreased. A similar pattern of associations with increasing leukocyte count was clear, but in addition there was an increase in leukemia inhibitory factor concentration. Multivariate analysis of vaginal leukocytosis, lactobacillary grades, and the presence of positive vaginal culture results showed that interleukin 1beta concentration was most closely related to the lactobacillary grade, leukemia inhibitory factor concentration was most closely related to the lactobacillary grade and positive culture results, interleukin 8 concentration was most closely related to positive culture results, and interleukin 1 receptor antagonist concentration was most closely related to vaginal leukocytosis and positive culture results. The concentration ratio of interleukin 1beta to interleukin 1 receptor antagonist remained stable, except when vaginal leukocytosis increased. In its most severe form, with >10 leukocytes per epithelial cell present, a decompensation of the vaginal flora with a collapse in interleukin 1beta and interleukin 1 receptor antagonist concentrations was seen, but there was a concurrent sharp increase in leukemia inhibitory factor concentration. This pattern was completely different from the course of the cytokine concentrations associated with a lactobacillary grade increase. CONCLUSION Both disturbed lactobacillary grade and the presence of increasing vaginal leukocytosis were correlated with lactobacillary substrate (lactate) concentration, pH, and the concentrations of a variety of cytokines. There was a remarkably linear increase in these cytokines as either leukocytosis or lactobacillary grade became more severe. In circumstances in which leukocytosis was extreme, however, interleukin 1beta was no longer produced but leukemia inhibitory factor concentrations increased. We speculate that in extreme inflammation the body tries to limit the damage that can be done by exaggerated cytokine production.

Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types

The causal relationship between persistent infection with high‐risk HPV and cervical cancer has resulted in the development of HPV DNA detection systems. The widely used MY09/11 consensus PCR targets a 450bp conserved sequence in the HPV L1 gene, and can therefore amplify a broad spectrum of HPV types. However, limitations of these consensus primers are evident, particularly in regard to the variability in detection sensitivity among different HPV types. This study compared MY09/11 PCR with type‐specific PCRs in the detection of oncogenic HPV types. The study population comprised 15, 774 patients. Consensus PCR failed to detect 522 (10.9%) HPV infections indicated by type‐specific PCRs. A significant correlation between failure of consensus PCR and HPV type was found. HPV types 51, 68 and 45 were missed most frequently. The clinical relevance of the HPV infections missed by MY09/11 PCR was reflected in the fraction of cases with cytological abnormalities and in follow‐up, showing 104 (25.4%) CIN2+ cases. The MY09/11 false negativity could be the result of poor sensitivity, mismatch of MY09/11 primers or disruption of L1 target by HPV integration or DNA degradation. Furthermore, MY09/11 PCR lacked specificity for oncogenic HPVs. Diagnostic accuracy of the PCR systems, in terms of sensitivity (MY09/11 PCR: 87.9%; type‐specific PCRs: 98.3%) and specificity (MY09/11 PCR: 38.7%; type‐specific PCRs: 76.14%), and predictive values for histologically confirmed CIN2+, suggest that type‐specific PCRs could be used in a clinical setting as a reliable screening tool.

P16INK4a as an adjunct marker in liquid‐based cervical cytology

Cytological screening for cervical cancer is hampered by high false negative rates. Inter‐observer reproducibility needs optimizing. The potential of p16INK4a as a biomarker for cervical lesions was examined in a study of liquid‐based cytology (LBC), HPV DNA testing by MY09/MY11 consensus PCR and type‐specific PCRs and p16INK4a immunocytochemistry on a series of 291 patients selected from routine screening. Comparison of the number of p16INK4a immunoreactive cells/1,000 cells exhibited a significantly higher mean count in HSIL (8.80 ± 1.13) than other cytological groups. The mean count of LSIL (1.09 ± 0.18) was significantly higher than that of the negative group (0.82 ± 0.40). ASC‐H and HSIL combined showed a significantly higher mean count (6.46 ± 1.17) than negative, ASC, ASC‐US and LSIL. The mean count of immunoreactive cells/1,000 cells was significantly higher in HPV16 positive samples (3.22 ± 0.72) than in samples containing infections with types of unknown malignant potential (0.83 ± 0.26) or HPV negative samples (1.17 ± 0.41). The mean count in infections with other high‐risk HPV types (2.55 ± 0.52) was significantly higher than that in HPV negative samples. Receiver‐operating characteristic curves yielded a test accuracy (area under curve) of 0.76, 0.79, 0.88 and 0.95 for ASCUS, LSIL, ASC‐H/HSIL and HSIL, respectively. Thresholds for 95% sensitivity were at 0.005, 0.007, 0.098 and 0.445 immunopositive cells/1,000 cells for ASCUS, LSIL, ASC‐H/HSIL and HSIL, respectively. The 95% specificity threshold for the detection of HSIL was at 1.87 immunopositive cells/1,000 cells. P16INK4a immunocytochemistry can be used as an adjunct to LBC in cervical screening, because it has a good diagnostic accuracy to discriminate HSIL and ASC‐H from other lesions. It could be used as a surrogate marker of high‐risk HPV infections.

Individualized decreasing-dose maintenance fluconazole regimen for recurrent vulvovaginal candidiasis (ReCiDiF trial)

OBJECTIVE Although many women with recurrent vulvovaginal candidiasis initially benefit from prophylactic intermittent treatment with antimycotics, most of them experience relapse after cessation of therapy, and often they return to the pretreatment recurrence rate. The purpose of this study was to demonstrate the efficacy and safety of an individualized, degressive, prophylactic regimen in 136 women with recurrent vulvovaginal candidiasis. STUDY DESIGN After an induction dose of 600 mg fluconazole during the first week, 117 women started maintenance therapy: 200 mg fluconazole weekly for 2 months, followed by 200 mg biweekly for 4 months, and 200 mg monthly for 6 months, according to their individual response to therapy. All women were tested for recurrences monthly with wet mount microscopy and vaginal culture during the first 6 months and bimonthly during the next 6 months. Patients were allowed to move on to the next level of maintenance therapy only if they were symptom free and microscopy and culture negative. RESULTS Of the women who were cured successfully after the induction phase, 101 women (90%) were disease-free after 6 months of maintenance therapy with this degressive regimen, and 80 women (77%) were disease-free after 1 year. The weekly incidence of the first clinical relapse was 0.5% during any period of the maintenance phase, and the rate of all new relapses, which included evidence of mycologic or microscopic colonization, was 1% per week. Women who experienced several relapses (poor responders) had experienced more relapses before entering the study compared with the optimal responders (odds ratio, 4.9; 95% CI,1.8-13.7; P = .002), experienced the disease for a longer period of time (6.5 vs 3.7 years; P = .06), and harbored significantly more Candida non-albicans during maintenance therapy (P = .001). No serious side-effects were noted. CONCLUSION Individualized, degressive, prophylactic maintenance therapy with oral fluconazole is an efficient treatment regimen to prevent clinical relapses in women with recurrent vulvovaginal candidiasis.

Vaginal cytokines in normal pregnancy

OBJECTIVE The purpose of this study was to determine whether the vaginal cytokine concentration varies during the course of uncomplicated pregnancy. STUDY DESIGN Prenatal visits of healthy women to University Hospital Gasthuisberg, Leuven, Belgium were considered. Cytokine levels in vaginal washings from 30 unselected healthy women with uncomplicated pregnancies were monitored during pregnancy and compared with those from 62 nonpregnant healthy control subjects. Exclusion criteria included bacterial vaginosis, moderate or severe aerobic vaginitis, Trichomonas vaginalis, Candida vaginitis (wet mount or culture), gonorrhea, and Chlamydia. Interleukin-6, interleukin-8, interleukin-1beta, interleukin-1-receptor antagonist, leukemia inhibitory factor, and tumor necrosis factor were measured. Nonparametric Kruskal-Wallis and Welch tests were used for univariate analysis, and the Spearman rank test was used for multivariate analysis. RESULTS Compared with concentrations in nonpregnant women, interleukin-1beta concentrations were similar, but interleukin-1-receptor antagonist production was depressed throughout pregnancy. Vaginal interleukin-6 and interleukin-8 were less often discovered during pregnancy than outside pregnancy and dipped significantly in the middle trimester, to rise again to prepregnancy levels in the third trimester. Leukemia inhibitory factor was lower during the beginning of pregnancy (P=.038) but otherwise did not differ from nonpregnant values throughout pregnancy nor did tumor necrosis factor. Sexual activity could not explain these findings. CONCLUSION Vaginal cytokine levels, especially interleukin-1 receptor antagonist, from pregnant women may differ from nonpregnant values; some levels, such as interleukin-6 and interleukin-8, may fluctuate during normal pregnancy. These spontaneous variations during pregnancy must be taken into account when mucosal immunologic responses to infection of the lower genital tract are being studied.

Immunocytochemistry in liquid-based cervical cytology: analysis of clinical use following a cross-sectional study

Cytological screening for cervical cancer is hampered by imperfect sensitivity and low inter‐observer reproducibility. Human papillomavirus (HPV) testing lacks specificity as a primary screening method. Studies indicate that immunocytochemical detection of alterations caused by HPV in the host cells can optimise screening. Here, the potential of p16INK4a (cyclin‐dependent kinase inhibitor p16) and MIB‐1 (Ki‐67 proliferation marker) as adjunct molecular markers for cervical lesions was investigated in a prospective, cross‐sectional study of 500 samples in the framework of opportunistic screening in Flanders, Belgium. A consecutive series of 200 samples and 100 samples from the cytological categories ASC, LSIL and HSIL were investigated. Surepath samples were interpreted according to the Bethesda 2001 reporting system. HPV testing was done with MY09/MY11 consensus PCR. Immunocytochemistry for p16INK4a and MIB‐1 was performed with an automated staining protocol. The number of immunoreactive cells/1,000 cervical cells was assessed. There was a higher mean number of p16INK4A and MIB‐1 immunoreactive cells/1,000 cells in HSIL (4.06 ± 1.93 and 11.13 ± 2.83, respectively) compared to other cytological categories. Both markers showed a large spread in counts, for all categories. In cases of HSIL without immunoreactive cells for either marker, low cellularity and long‐term storage in water were often the cause of false negativity. This study confirms that positive staining for p16INK4a and MIB‐1 is highly correlated with presence of high‐grade lesions. These markers could be used as adjuncts to increase the sensitivity of cytological screening as well as the specificity of the HPV test. However, clear methodological standards are needed for optimal performance of immunocytochemistry in a clinical setting.

Improved endocervical sampling and HPV viral load detection by Cervex-Brush® Combi

Objective:  Liquid‐based cytology (LBC) for cervical screening is becoming increasingly used. Together with SurePath® LBC, various collecting devices can be utilized, among which the Cervex‐Brush® is the most widely used. The new Rovers® Cervex‐Brush® Combi combines the advantages of the Cervex‐Brush® with the EndoCervex‐Brush® increasing sampling of the endocervical canal. The objective of this study was to analyse and to compare the Cervex‐Brush® Combi with the Cervex‐Brush® for the collection of squamous and endocervical cells, human papillomavirus (HPV) typing/quantification and disease detection in SurePath® LBC.

Wet mount microscopy reflects functional vaginal lactobacillary flora better than Gram stain

Aim—The status of vaginal lactobacillary flora, an indicator of possible genital infection and pregnancy complications, can be assessed on wet mount or Gram stained specimens. The former is quick, the latter more routine. The accuracy of the two preparative techniques to detect normal vaginal lactobacillary microflora was compared for 646 patients. The effect of delay in transport medium before Gram staining was also investigated. Methods—Patients presented with infectious vaginitis or for a routine prenatal visit. After placement of a speculum, duplicate smears were taken from the upper vaginal vault and examined fresh or after Gram staining. Lactobacillary grades from both methods were compared with lactate concentration in vaginal rinses. In a subgroup of 238 patients, Gram staining was performed both on fresh smears and those that had been transported in Stuarts growth medium. Results—Higher lactobacillary grades (more disrupted flora) were diagnosed 2.9 times more frequently on Gram stained specimens than on wet mounts (p < 0.0001), a difference even more pronounced after transport in Stuarts medium (relative risk, 4.2; p < 0.0001). Lactobacillary grades assessed on wet mounts correlated better with vaginal lactate concentration than those assessed on Gram stains. Conclusions—Easier recognition of lactobacillary morphotypes on wet mounts than on Gram stains might result from the loss of lactobacilli by the process of fixation or Gram staining. Wet mount microscopy of vaginal smears for assessment of lactobacillary grades, rather than Gram staining, is strongly recommended.

Thin-layer liquid-based cervical cytology and PCR for detecting and typing human papillomavirus DNA in Flemish women

The objective of this study was to document the occurrence and to correlate the prevalence of different human papillomavirus (HPV) types with the cytological results on simultaneously performed thin-layer preparations in a large population of Flemish women. During 1 year, 69 290 thin-layer preparations were interpreted using the Bethesda classification system. Using an algorithm for HPV testing based on consensus primers and type-specific PCRs in combination with liquid-based cytology, we determined the occurrence and distribution of 14 different oncogenic HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). Reflex HPV testing was performed on cytologically abnormal samples and on an age matched randomly selected control group with normal cervical cytology (n=1351). Correlation between cytology, age and prevalence for the 14 different high-risk HPV types is given. There is a significant increase in predominance of high-risk HPV types, with increasing abnormal cytology. Coinfection with multiple HPV types also increased with cytological abnormalities, and was highest in HSIL (16.7%). In Flanders, HSIL was most often associated with HPV types 16, 33, 35, 31, 18 and 51. Using thin-layer liquid-based cytology and PCR to detect HPV, it is feasible to screen large numbers of women.