BACKGROUND:To date, women are most often diagnosed with bacterial vaginosis (BV) using microscopy based Nugent scoring or Amsel criteria. However, the accuracy is less than optimal. The aim of the present study was to confirm the identity of known BV-associated composition profiles and evaluate indicators for BV using three molecular methods.METHODS:Evaluation of indicators for BV was carried out by 16S rRNA amplicon sequencing of the V5-V7 region, a tailor-made 16S rRNA oligonucleotide-based microarray, and a PCR-based profiling technique termed IS-profiling, which is based on fragment variability of the 16S-23S rRNA intergenic spacer region. An inventory of vaginal bacterial species was obtained from 40 females attending a Dutch sexually transmitted infection outpatient clinic, of which 20 diagnosed with BV (Nugent score 7-10), and 20 BV negative (Nugent score 0-3).RESULTS:Analysis of the bacterial communities by 16S rRNA amplicon sequencing revealed two clusters in the BV negative women, dominated by either Lactobacillus iners or Lactobacillus crispatus and three distinct clusters in the BV positive women. In the former, there was a virtually complete, negative correlation between L. crispatus and L. iners. BV positive subjects showed cluster profiles that were relatively high in bacterial species diversity and dominated by anaerobic species, including Gardnerella vaginalis, and those belonging to the Families of Lachnospiraceae and Leptotrichiaceae. Accordingly, the Gini-Simpson index of species diversity, and the relative abundance Lactobacillus species appeared consistent indicators for BV. Under the conditions used, only the 16S rRNA amplicon sequencing method was suitable to assess species diversity, while all three molecular composition profiling methods were able to indicate Lactobacillus abundance in the vaginal microbiota.CONCLUSION:An affordable and simple molecular test showing a depletion of the genus Lactobacillus in combination with an increased species diversity of vaginal microbiota could serve as an alternative and practical diagnostic method for the assessment of BV.

Persistent cervical high-risk human papillomavirus (HR-HPV) infection results in high-grade cervical intraepithelial neoplasia (CIN2/3) and cervical carcinoma. The susceptibility of the cervix to HPV carcinogenesis and the importance of HPV18 in cervical carcinoma despite relative infrequency in CIN2/3 could be linked to HR-HPV infection of immature metaplasia (IM) at the squamocolumnar junction. Atypical IM (AIM) is an equivocal category used to describe changes in IM suggestive of high-grade neoplasia, which causes diagnostic and management problems. We used laser capture microscopy combined with polymerase chain reaction in 24 women with HPV18, HPV16, or other HPV infections on cytologic analysis and a cervical loop electrosurgical excision procedure to locate HR-HPV in cervical tissue. HPV18-positive AIM and CIN2/3 were present in 7/12 cases with HPV18 on cytologic analysis. In 2 cases with HPV18 and other HPV types, HPV18 was only present in AIM and not in CIN2/3. HPV16-positive AIM was present in 3/7 and HPV16-positive CIN2/3 in 5/7 cases with HPV16. No cases had HPV16 AIM without CIN2/3. Other HR-HPV-positive AIM and CIN2/3 cases were present, respectively, in 1/6 and 5/6 cases positive for HR-HPV types other than HPV16/18. In a subset, 94% HPV18 AIM regions showed CK17 and p16 positivity, and 41% were CK7 positive. CIN2/3 and AIM with other HR-HPVs showed similar patterns. AIM was a particular feature of HPV18 infection in women with CIN2/3. HR-HPV infection of CK7/17-positive AIM expressing p16 was particularly seen for HPV18 with and without classical CIN2/3 and should be regarded as a high-grade precancer.

Cervical scrapes to diagnose cervical neoplasia, collected by the clinician with brushes, are sent to the Leiden Cytology and Pathology Laboratory (LCPL) in vials containing BoonFix, a noncross-linking coagulant fixative. Because the residual material left in the vials contains tissue fragments with important diagnostic information, we stored the residual material in our archives. The tissue fragments can be mummified and archived in commercially available histology cassettes. We can produce paraffin sections thereof. Immunostaining is beautiful on serial paraffin sections cut from these blocks. We experienced that it is important to leave the brush in the vial such that all tissue fragments can be used for histologic diagnosis. In order to optimize the system, tissue fragments left in the endocervical part of the brush are removed in a paint shaker. We illustrate this principle of recovering mummified tissue fragments in a false negative case with cytology containing many undiagnosable collapsed tissue fragments. This case shows clearly the efficacy of using the tissue fragments present in cervical scrapes for the histologic diagnosis of cervical neoplasia.

The Cellient Automated Cell Block System (Hologic) can be used to process cervical scrapes to paraffin sections. For the first study on this subject, cervical scrapes were fixed in the formalin-free fixative BoonFix. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS) and high-grade squamous lesion (HSIL) as diagnosed in the ThinPrep slide. The Cellient paraffin sections were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. Multiple HPV genotypes were encountered in 79% of the scrapes. This study showed that the Cellient system for paraffin sections can be combined with HPV testing thanks to the formalin-free BoonFix. In two additional studies it was shown that such samples can also be used for morphotyping the vaginal microbiome and preparing cytologic ThinPrep slides.

Forensic analysis of biological traces generally encompasses the investigation of both the person who contributed to the trace and the body site(s) from which the trace originates. For instance, for sexual assault cases, it can be beneficial to distinguish vaginal samples from skin or saliva samples. In this study, we explored the use of microbial flora to indicate vaginal origin. First, we explored the vaginal microbiome for a large set of clinical vaginal samples (n = 240) by next generation sequencing (n = 338,184 sequence reads) and found 1,619 different sequences. Next, we selected 389 candidate probes targeting genera or species and designed a microarray, with which we analysed a diverse set of samples; 43 DNA extracts from vaginal samples and 25 DNA extracts from samples from other body sites, including sites in close proximity of or in contact with the vagina. Finally, we used the microarray results and next generation sequencing dataset to assess the potential for a future approach that uses microbial markers to indicate vaginal origin. Since no candidate genera/species were found to positively identify all vaginal DNA extracts on their own, while excluding all non-vaginal DNA extracts, we deduce that a reliable statement about the cellular origin of a biological trace should be based on the detection of multiple species within various genera. Microarray analysis of a sample will then render a microbial flora pattern that is probably best analysed in a probabilistic approach.

Metaplastic cells with nebular cytoplasmic changes in the cervical smear are classified in the Dutch coding system for cervical screening as KOPAC O8 cells. Since these nebulated cells are already documented by Papanicolaou, we refer to these cells as Papanicolaou's nebular cells. We examined the simultaneous presence of these characteristic metaplastic cells and high-grade squamous intraepithelial lesion (HSIL) in a population-based data base from January 1991 and December 1996. The odds ratio (OR) of nebular cells concurring with HSIL increases with age. For the age cohort 30 years, the OR was 7.8 with a 95% confidence interval (CI) of 4.4-13.9. For the age cohort 60 years, the OR was 35.3 with a 95% CI of 7.8-159.2. Aiming to determine the nature of these nebular metaplastic cells, we performed Chlamydia and HPV PCR on 587 and 1,483 smears, respectively. With an OR of 0.9 [0.3-2.4] it is unlikely that Chlamydia plays a role in the appearance of these nebular cells in the smear. This study shows that with an OR of 5.9 [ 1.7-21.3] HPV is not only related to large koilocytosis but also to a nebular change of small metaplastic cells. This study reports that nebular changes of small metaplastic cells are related to cervical cancer and to HPV infection. Diagn. Cytopathol. 2012; 40: 197-200. (C) 2010 Wiley Periodicals, Inc.

Background. There are limited data on high-risk human papillomavirus (hr-HPV) genotypes among HIV-positive women in Africa, and little is known about their relationship with cervical cytology in these populations. Methods. We conducted a cross-sectional study among 194 HIV-positive women (143 from Tanzania, and 51 from South Africa) to evaluate HPV genotypes among HIV-positive women with normal and abnormal cytology. Cervical samples were genotyped for HPV types, and slides were evaluated for atypical squamous cell changes according to the Bethesda classification system. Results. Prevalence of high grade squamous intraepithelial dysplasia (HSIL) was 9%. Overall, more than half (56%) of women were infected with an hr-HPV type; 94% of women with HSIL (n = 16), 90% of women with LSIL (n = 35), and 42% of women within normal limits (WNL) (n = 58) tested positive for hr-HPV. Overall, the most prevalent hr-HPV subtypes were HPV16 (26%) and HPV52 (30%). Regional differences in the prevalence of HPV18 and HPV35 were found. Conclusion. Regional differences in HPV genotypes among African women warrant the need to consider different monitoring programmes for cervical preneoplasia. HPV-based screening tests for cervical preneoplasia would be highly inefficient unless coupled with cytology screening of the HPV-positive sample, especially in HIV-positive women.

The Cellient™ Automated Cell Block System (Hologic) can be used to analyze cells of HPV-positive cervical scrapes staining positive with the biomarker p16. For this study fourteen cervical scrapes of Tanzanian women infected with HIV testing positive for HPV were selected. The paraffin Cellient sections were stained with the Papanicolaou method, with hematoxylin eosin (HE), and with the biomarker p16. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS) and high-grade squamous lesion (HSIL) as diagnosed in the ThinPrep slide. The Cellient paraffin sections (cut from paraffin blocks prepared from the residual cervical sample) were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. Multiple HPV genotypes were encountered in 79% of the scrapes. HPV16 was found in six scrapes and HPV52 in four. In the Papanicolaou sections, it was easy to detect dyskeratotic cells. Eleven of the 14 cases were p16 positive and five contained p16 positive dyskeratocytes. Of the 10 ASCUS scrapes, two contained p16 positive CIN 1 epithelial fragments. All four HSIL cases contained p16 positive CIN 3epithelial fragments. In HIV-positive HPV-positive women, the Cellient system resulted in high quality histology sections with perfect p16 images of dyskeratocytes.

Objective: To report the prevalence of Gardnerella, Trichomonas and Candida in the cervical smears of 9 immigrant groups participating in the Dutch national cervical screening program. Study Design: Cervical smears were taken from 58,904 immigrant participants and 498,405 Dutch participants. As part of the routine screening process, all smears were screened for the overgrowth of Gardnerella (i.e. smears with an abundance of clue cells) and for the presence of Trichomonas and Candida. The smears were screened by 6 laboratories, all of which use the Dutch KOPAC coding system. The odds ratio and confidence interval were calculated for the 9 immigrant groups and compared to Dutch participants. Results: Immigrants from Suriname, Turkey and the Dutch Antilles have a 2–5 times higher prevalence of Gardnerella and Trichomonas when compared to native Dutch women. Interestingly, the prevalence of Trichomonas in cervical smears of Moroccan immigrants is twice as high, yet the prevalence of Gardnerella is 3 times lower than in native Dutch women. Conclusions: Immigrants with a high prevalence of Gardnerella also have a high prevalence of Trichomonas. In the context of the increased risk of squamous abnormalities in smears with Gardnerella, such slides should be screened with extra care.

Vaginal lactobacilli assessed by PCR-based microarray and PCR-based genotyping of HPV in South African women at risk for HIV and BV. Vaginal lactobacilli can be defined by microarray techniques in fixed cervical samples of South African women. Cervical brush samples suspended in the coagulant fixative BoonFix of one hundred women attending a health centre for HIV testing in South Africa were available for this study. In the Ndlovu Medical Centre in Elandsdoorn, South Africa, identification of 18 hr-HPV genotypes was done using the INNO-LiPA method. An inventory of lactobacilli organisms was performed using microarray technology. On the basis of the Lactobacillus and Lactobacillus biofilm scoring, the cases were identified as Leiden bacterial vaginosis (BV) negative (BV-; n = 41), Leiden BV intermediate (BV±; n = 25), and Leiden BV positive (BV+; n = 34). Fifty-one women were HIV positive and 49 HIV negative. Out of the 51 HIV positive women, 35 were HPV infected. These 51 HIV positive women were frequently infected with HPV16 and HPV18. In addition, HPV35, HPV52, HPV33, and HPV66 were often detected in these samples. Lactobacillus salivarius and Lactobacillus iners were the most prevalent lactobacilli as established by the microarray technique. In women with HPV infection, the prevalence of Lactobacillus crispatus was significantly reduced. In both HIV and HPV infection, a similar (but not identical) shift in the composition of the lactobacillus flora was observed. We conclude that there is a shift in the composition of vaginal lactobacilli in HIV-infected women. Because of the prominence of HPV35, HPV52, HPV33, and HPV66, vaccination for exclusively HPV16 and HPV18 might be insufficient in South African HIV+ women.

Objective: The large set of ThinPrep slides prepared in the Leiden Cytology and Pathology Laboratory is exploited for calculating the impact of the transition from PAPNET neural network scanning to the Imager technology. Study Design: All cervical samples were suspended and fixed in the coagulant fixative BoonFix. We compared 57,541 ThinPrep slides which were scanned by PAPNET and 64,273 ThinPrep slides processed with the Imager: 99,157 cases originated from the Dutch population screening program of asymptomatic women (screenees) and the remaining 22,657 samples were of symptomatic women. In the PAPNET series, 23% were diagnosed by additional light microscopy; in the Imager method, all slides were studied light microscopically. The cytoscores (positive cytology per 1,000 samples) were calculated for normal, atypical squamous cells of undetermined significance (ASC-US), cervical intraepithelial neoplasia (CIN) grades I–II, and for CIN III+. The odds ratios (ORs) for the positive cytoscores were assessed for both the screenees and the symptomatic women. Results: The cytoscores, per 1,000 cases, for ASC-US varied from 17.77 to 40.59, for CIN I–II from 7.17 to 33.35, and for CIN III+ from 2.81 to 8.8. These 6 cytoscores were higher for symptomatic women than for screenees. We observe significantly elevated ORs for the Imager for ASC-US (1.26 and 1.23), CIN I–II (1.45) and for CIN III+ (1.58 and 1.45). These 3 ORs are higher for screenees than for symptomatic women. Conclusion: The Imager technology is more efficacious, particularly for handling screenee slides.

Periodic episodes of increased sunspot activity (solar electromagnetic storms) occur with 10-11 and 5-6 year periodicities and may be associated with measurable biological events. We investigated whether this sunspot periodicity characterized the incidence of Pap smear-determined cervical epithelial histopathologies and human physiologic functions. From January 1983 through December 2003, monthly averages were obtained for solar flux and sunspot numbers; six infectious, premalignant and malignant changes in the cervical epithelium from 1,182,421 consecutive, serially independent, screening Pap smears (59°9″N, 4°29″E); and six human physiologic functions of a healthy man (oral temperature, pulse, systolic and diastolic blood pressure, respiration, and peak expiratory flow), which were measured ∼5 times daily during ∼34,500 self-measurement sessions (44°56″N, 93°8″W). After determining that sunspot numbers and solar flux, which were not annually rhythmic, occurred with a prominent 10-year and a less-prominent 5.75-year periodicity during this 21-year study span, each biological data set was analyzed with the same curve-fitting procedures. All six annually rhythmic Pap smear-detected infectious, premalignant and malignant cervical epithelial pathologies showed strong 10-year and weaker 5.75-year cycles, as did all six self-measured, annually rhythmic, physiologic functions. The phases (maxima) for the six histopathologic findings and five of six physiologic measurements were very near, or within, the first two quarters following the 10-year solar maxima. These findings add to the growing evidence that solar magnetic storm periodicities are mirrored by cyclic phase-locked rhythms of similar period length or lengths in human physiology and pathophysiology.

OBJECTIVE: The objective was to examine the use of a tailor-made DNA microarray containing probes representing the vaginal microbiota to examine bacterial vaginosis.STUDY DESIGN: One hundred one women attending a health center for HIV testing in South Africa were enrolled. Stained, liquid-based cytology slides were scored for bacterial vaginosis. An inventory of organisms was obtained using microarray technology, probing genera associated with bacterial vaginosis in more detail, namely Gardnerella, Atopobium, Dialister, Leptotrichia, Megasphaera, Mobiluncus, Peptostreptococcus, Prevotella, and Sneathia.RESULTS: Of 101 women, 34 were diagnosed positive for bacterial vaginosis. This condition was associated with an increased microbial diversity. It is no longer useful to base the diagnosis of bacterial vaginosis on Gardnerella alone. Rather, its presence with Leptotrichia and Prevotella species, and especially Atopobium was more indicative of an aberrant state of the vaginal flora.CONCLUSION: To understand the vaginal microbiota in more detail, microarray-based identification can be used after microscopic scoring.

OBJECTIVE:To exploit cervical thin layer brush samples through cytohistology in cases with invasive carcinoma with application of antibodies.STUDY DESIGN:Fourteen cases from women with carcinoma diagnosed in 2006 were selected out of 29 invasive carcinomas. From these 14 cases liquid-based cervical cytology material was available to prepare cytohistology. Eight women had squamous cell carcinoma, 4 endocervical adenocarcinoma, 1 endometrial adenocarcinoma and 1 ovarian adenocarcinoma. The residual material from the thin layer sample, collected by brushes by general practitioners, was used to prepare paraffin sections. These were stained with the Papanicolaou method and for the biomarkers Ki-67 and p16 and, if desired, for differentiation markers, including carcinoembryonic antigen, vimentin, cytokeratin 7 and cytokeratin 20 to establish the immunoprofile of the carcinoma.RESULTS:The morphologic details in the cancer nuclei in the paraffin sections were excellent, while in all cases the thin layer cytology slide contained thick epithelial fragments with blurred nuclei. In 5 of the 6 adenocarcinomas, the glandular architecture diagnostic of adenocarcinoma was visible in the cytohistology, which was highlighted in the biomarker stainings, particularly so in the Ki-67 sections. With the exception of endometrial adenocarcinoma, all p16(INK4a) stainings were positive, as they were in the ovarian adenocarcinoma case.CONCLUSION:Cytohistology is an adjunct to routine cervical cytologic examination of thin layer samples, allowing an unequivocal and refined diagnosis.

OBJECTIVE To describe recent trends in the prevalence of cytologic patterns of the vaginal flora (koilocytosis, Trichomonas, dys-bacteriosis, Candida, Gardnerella, Actinomyces, Chlamydia trachomatis) over the last decade. STUDY DESIGN From 1996 to 2005 > 500,000 cervical smears were screened in the context of the Dutch national screening program on a 5-year basis. Data from the first screening period were compared with those of the second screening period. RESULTS Prevalences differed from 34.8 for dysbacteriosis to 0.2 for C trachomatis. Bacterial imbalance (dysbacteriosis, unequivocal Gardnerella and Trichomonas) showed a decline in all age groups. Cases of human papillomavirus (HPV)-related koilocytosis have dramatically increased among young women (30 and 35 years). CONCLUSION Bacterial imbalance of the vaginal flora has significantly decreased during the past decade in all age cohorts. Campaigns on consciousness of vaginal hygiene might have contributed to this amazing effect. We ought to be concerned about the increase in HPV-related koilocytosis.

OBJECTIVE To analyze the impact of the 1995 revision of the Dutch cervical screening program guidelines (e.g., the introduction of more stringent criteria for cytologic diagnosis of atypical squamous cells of undetermined significance [ASCUS]) on the negative side effects of screening in Region West. STUDY DESIGN The data for this study were retrieved as two complementary datasets, both of which contained data on the invited screenees, including their cytology and histology follow-up. One dataset additionally included data on other smears. For invited screenees, we analyzed changes in cytoscores and histoscores between 1994, the last year before new screening guidelines were implemented, and 2003, the latest year for which follow-up to screening abnormalities was available in the retrieved data. Additionally, we analyzed changes in the total number of primary and repeat smears made. RESULTS Between 1994 and 2003, the cytoscore for ASCUS decreased from 19.4% to 1.3% of screenee smears. The total number of smears taken decreased by 30%. The cervical intraepithelial neoplasia 3+ histoscore remained the same (OR = 0.97, p = 0.91). CONCLUSION The reduction of equivocal ASCUS diagnoses resulted in a decrease of costly repeat smears, without measurably decreasing the effectiveness of the screening program.