Presence of 20% or more clue cells: an accurate criterion for the diagnosis of bacterial vaginosis in Papanicolaou cervical smears

A study was carried out in 135 women of reproductive age to evaluate the role of the Papanicolaou smear performed in cervical and vaginal samples for the diagnosis of bacterial vaginosis (BV), to validate the method for this diagnosis and to evaluate the reproducibility of the criterion used. The cervical and vaginal smears were stained using the Papanicolaou method and evaluated by two separate observers. The finding of 20% or more clue cells was considered positive for the presence of BV. This diagnostic criterion presented a sensitivity of 87%, specificity of 94%, a positive predictive value of 81%, and a negative predictive value of 96%, both in cervical and vaginal samples. There was excellent agreement in the diagnosis of BV between the two observers (kappa, 0.87) and between cervical and vaginal samples (kappa, 0.92). Therefore, the presence of 20% or more clue cells in the Papanicolaou smear is an accurate and reproducible criterion for the diagnosis of BV and may be used in screening for this infection, eliminating the need for further vaginal sample collection.     

Increased secretion patterns of interleukin-10 and tumor necrosis factor-alpha in cervical squamous intraepithelial lesions

Cytokines are released in response to infection of the uterine cervix by high-risk HPV. By using enzyme-linked immunosorbent assay, we measured the levels of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma), interleukin-6 (IL-6), and interleukin-10 (IL-10) in the cervical secretions of 120 cytologically normal or equivocal and 91 abnormal Japanese women. HPV infection of the cervical cells was typed by the LCR-E7 PCR method. The HPV DNA-negative samples were classified as either normal or inflamed, and the HPV DNA-positive samples were classified as HPV positive(+) n-ormal and as low- or high-grade squamous intraepithelial lesions (SILs). Compared with the normal cervices, all of the cytokines tested were elevated in inflamed, HPV+ normal, low-grade SILs (LSIL), and high-grade SILs (HSIL). The level of IL-10 was statistically higher in LSIL, and the level of TNF-alpha was higher in HSIL, relative to the cytokine levels in the inflamed and HPV+ normal samples (P <0.05; Mann-Whitney test). Multivariate analyses confirmed that increased levels of IL-10 were associated with LSIL (relative risk [RR]=3.9, 95% confidence interval [CI]=1.7-8.8) and that increased levels of TNF-alpha (RR=4.6, 95% CI=1.4-15) and age older than 40 years (RR=8.5, 95% CI=1.3-56) were associated with HSIL. The levels of INF-gamma and TNF-alpha (Th1-cytokines) correlated negatively with those of IL-6 and IL-10 (Th2-cytokines) in HPV+ normal and LSIL subjects, whereas no such correlation was observed for HSIL. The up-regulated secretion of IL-10 may inhibit immune responses against HPV infection in early cervical lesions, whereas up-regulated TNF-alpha and uncoordinated cytokine secretion (elevated both Th1 and Th2 cytokines) may reflect impaired or invalid responses in advanced stage lesions. The detection of IL-10 and TNF-alpha in cervical secretions may be a useful indicator of local immune responses and of the stage of the cervical lesions induced by HPV infection.     

Inflammatory events as detected in cervical smears and squamous intraepithelial lesions

The Dutch cytological coding system, KOPAC, enables to code for eight inflammatory events, that is koilocytosis (related to human papillomavirus (HPV)), Trichomonas, dysbacteriosis [related to bacterial vaginosis (BV)], Candida, Gardnerella, Actinomyces, Chlamydia, and non‐specific inflammation (leucocytosis). This study presents an analysis of 1,008,879 smears. Of each smear, the age of the woman and the reason for smear taking (screening or indication) was available. The cytoscores (per mille) for these codes were calculated. For the screening smears, the cytoscores were for koilocytosis (HPV) 2.6, for Trichomonas vaginalis 1.9, for dysbacteriosis 31.4, for Candida albicans 9.8, for Gardnerella vaginalis 0.7, for Actinomyces 6.9, for Chlamydia 0.8, and for non‐specific inflammatory changes 66.4. For the calculation of the Odds Ratio (OR), normal smears were used as a reference. The cytoscores for Chlamydia and Gardnerella covaried with high grade SIL (HSIL), with an OR of 7 and 12, respectively. In addition, the OR for Trichomonas vaginalis, for dysbacteriosis, and for leucocytosis proved to be significantly high in the indication smears. This study provides an oversight of HSIL and the full range of cervical infections as detected by cytology, proving that this infectious byproduct of screening can be very valuable. Diagn. Cytopathol. 2010. © 2009 Wiley‐Liss, Inc.     

MMP‐9 expression increases according to the grade of squamous intraepithelial lesion in cervical smears

Studies about cervical carcinogenesis have demonstrated the increased expression of matrix‐metalloproteinase (MMP) according to the grade of cervical intraepithelial lesions. Considering the importance of innovative techniques to introduce noninvasive and rapid diagnoses for patients, this study aimed to perform MMP‐9 immunocytochemistry in cervical smears according to the cytopathological diagnoses, in order to monitor MMP activity in cervical smears. This cross‐sectional study investigated the expression of MMP‐9 in normal cervical smears, inflammatory cervical smears, squamous intraepithelial lesions, and cervical carcinoma. Cervical smears from 630 women were collected for cytopathological diagnoses and immunocytochemistry. Women with squamous intraepithelial lesions showed an increase in MMP‐9 expression, with moderate to intense staining occurring with increasing cervical lesion grade. The prevalence of moderate to intense MMP‐9 staining was 9% in normal cervical smears, 12% in cervical inflammation, 24% in low‐grade squamous intraepithelial lesion (LSIL), 92% in high‐grade squamous intraepithelial lesions (HSIL) and 100% in cervical carcinoma cases. In the specific case of LSIL, we found that association with MMP‐9 is more evident when there is the simultaneous presence of an infectious agent. Thus, the expression of MMP‐9 in cervical smears increases according to the grade of cervical lesion and LSIL in the presence of infectious agents showed higher MMP‐9 expression than women with LSIL without infectious agents. Diagn. Cytopathol. 2014;42:827–833. © 2014 Wiley Periodicals, Inc.     

Bacterial vaginosis and inflammatory response showed association with severity of cervical neoplasia in HPV‐positive women

Vaginal infections may affect susceptibility to and clearance of human papillomavirus (HPV) infection and chronic inflammation has been linked to carcinogenesis. This study aimed to evaluate the association between bacterial vaginosis (BV) and inflammatory response (IR) with the severity of cervical neoplasia in HPV‐infected women. HPV DNA was amplified using PGMY09/11 primers and genotyping was performed using a reverse line blot hybridization assay in 211 cervical samples from women submitted to excision of the transformation zone. The bacterial flora was assessed in Papanicolaou stained smears, and positivity for BV was defined as ≥20% of clue cells. Present inflammatory response was defined as ≥30 neutrophils per field at 1000× magnification. Age higher than 29 years (OR:1.91 95% CI 1.06–3.45), infections by the types 16 and/or 18 (OR:1.92 95% CI 1.06–3.47), single or multiple infections associated with types 16 and/or 18 (OR: 1.92 CI 95% 1.06–3.47), BV (OR: 3.54 95% CI 1.62–7.73) and IR (OR: 6.33 95% CI 3.06–13.07) were associated with severity of cervical neoplasia (CIN 2 or worse diagnoses), while not smoking showed a protective effect (OR: 0.51 95% CI 0.26–0.98). After controlling for confounding factors, BV(OR: 3.90 95% CI 1.64–9.29) and IR (OR: 6.43 95% CI 2.92–14.15) maintained their association with the severity of cervical neoplasia. Bacterial vaginosis and inflammatory response were independently associated with severity of cervical neoplasia in HPV‐positive women, which seems to suggest that the microenvironment would relate to the natural history of cervical neoplasia. Diagn. Cytopathol. 2016;44:80–86. © 2015 Wiley Periodicals, Inc.     

DNA image cytometry and human papillomavirus (HPV) detection help to select smears at high risk of high-grade cervical lesions

Three samples were submitted from women undergoing routine screening (n=910): two smears (one for routine cytology and one for DNA image cytometry) and a scrape for human papillomavirus (HPV) testing. DNA histograms were classified as suspect in cases of aneuploidy, polyploidy, and/or diploidy with a high proliferation rate. Follow-up was available in 239 cases. The primary end-point was the presence of a high-grade squamous intraepithelial lesion (HGSIL) at biopsy. Seventy women (7.7%) had a high-risk (HR) HPV infection and a suspect DNA profile. In 77 women with cytological abnormalities, 28 HGSILs were detected: four with a prior diagnosis of ASCUS (all HR-HPV infected including three with a suspect DNA profile), three with smears evocative of LGSIL (all with HR-HPV infection and a suspect DNA profile), and 21 with smears evocative of HGSIL (all with HR-HPV infection and 20 with a suspect DNA profile). During the follow-up period, out of 239 women with a cytologically normal smear at first entry, five developed a HGSIL; all were HR-HPV-positive and four had a suspect DNA profile at the first smear. HR-HPV detection alone gives a sensitivity of 100% for the detection of HGSIL, with a specificity of 84.3%, whereas DNA measurement associated with HPV testing significantly enhances the specificity to 95.4%. Thus, the combination of HPV testing and DNA measurement provides a highly sensitive and specific evaluation of the risk of HGSIL on cervical smears.     

HPVDNA永生化人宫颈上皮细胞的生长方式与宫颈上皮内肿瘤的比较

目的：研究人乳头瘤病毒（HPV）DNA永生化人宫颈上皮细胞在器官培养的生长特点及将其与体内宫颈上皮内肿瘤（CIN）进行比较。方法：先用HPV16和18型DNA转染人宫颈上皮细胞,建立永生化的人宫颈上皮细胞株,再用胶原筏培养方法分析永生化人宫颈上皮细胞在器官培养的生长特点,将其与体内CIN的形态进行了比较。结果：人宫颈上皮细胞经HPV16和18DNA转染后,可以使其变成一种永生化细胞。细胞生物学研究显示永生化细胞是一种前恶性细胞。胶原筏培养显示水生化人宫颈上皮细胞的生长行为类似体内CIN。结论：HPV感染主要影响宫颈癌的发生的早期阶段。     

Prevalence of human papillomavirus genotypes and associated cervical squamous intraepithelial lesions in HIV-infected women in Botswana

Human papillomaviruses (HPV) constitute one of the most prevalent sexually transmitted infections and are the etiological agents for invasive cervical cancer, the predominant cancer among women in Botswana. However, the prevalence of HPV genotypes in Botswana has yet to be reported. One hundred thirty‐nine endocervical swabs were taken at baseline from HIV‐1 infected, HSV‐2 seropositive women enrolled in a longitudinal cohort study designed to assess the influence of herpes simplex virus‐2 (HSV‐2) infection on genital tract shedding of HIV‐1. Extracted DNA was evaluated for the presence of low‐risk and high‐risk HPV using the Roche Linear Array. Genotyping identified HPV in 95 of 139 women of which 61/95 were infected with high‐risk HPV and 56/95 with low‐risk HPV. The median number of genotypes was 2 (IQR: 1–4). The most prevalent HPV genotype in HIV‐infected women was HPV 58. Abnormal cervical cytology was detected in 87/127 women and was associated with contemporaneous HPV infection (RR = 1.43, 95% CI: 1.05–1.93; P = 0.02). HPV prevalence was high among HIV‐infected women with infection by multiple genotypes being widespread. The associations attributed to specific oncogenic HPV subtypes and cervical squamous intraepithelial lesions presented here provide critical information to inform future vaccine policy within Botswana. J. Med. Virol. 83:1689–1695, 2011. © 2011 Wiley‐Liss, Inc.     

Squamous intraepithelial lesions in cervical smears of human immunodeficiency virus-seropositive adolescents

Various authors have reported a high rate of human papillomavirus (HPV) infection and HPV-related neoplasias in human immunodeficiency virus (HIV)-seropositive women. On the other hand, young women are most susceptible to cervical infection because of immaturity of the cervix, as it appears that HPV has more access to the basal cells of the differentiating epithelium. The purpose of the present work was to study cervical smears of 82 adolescent HIV-seropositive women (13–21 years of age) to search for cytological evidence of cervical intraepithelial neoplasias. Twenty-one cases showed characteristic features of HPV infection and squamous intraepithelial lesions (SIL; 25.6%). Sixteen cases aged from 17 to 21 years (mean age 19.5 years) had low-grade SIL (LSIL; 19.5%) and five cases aged from 18 to 21 years (mean age 20.2 years) had high-grade SIL (HSIL; 6.1%). There was no significant difference between the mean age of patients with LSIL and HSIL. Two cases had atypical squamous cells of undetermined significance (ASCUS). In the present work it was found that HIV-seropositive adolescents have a high risk for preneoplastic lesions of the cervix (25.6%) as well as a high incidence of more aggressive lesions (6.1% of HSIL) when compared to the general population of adolescents. As it can be assumed that, if the age of acquisition of the infection in both groups (in the general population and HIV-seropositive women) is the same, it is probable that HIV infection in adolescents not only increases the frequency of HPV infections but also facilitates the evolution to more aggressive preneoplastic lesions of the cervix due to HPV. Diagn. Cytopathol. 1998;18:91–92. © 1998 Wiley-Liss, Inc.     

DLG1 polarity protein expression associates with the disease progress of low-grade cervical intraepithelial lesions

Human Discs large tumour suppressor (DLG1) participates in regulating cell polarity and proliferation, suggesting an important connection between epithelial organization and cellular growth control. However, it was demonstrated that DLG1 could acquire oncogenic attributes in some specific contexts. In this work, we evaluated the expression of DLG1 and its contribution to the progress of cervical lesions in order to investigate a potential role of this polarity protein in human oncogenic processes.We analyzed cervical biopsies from women with low-grade squamous intraepithelial lesion (LSIL) diagnosis (n = 30), for DLG1 expression by immunohistochemistry. These results were correlated with the clinical monitoring of the patients during a 24-month follow-up period. Our data indicate that while all LSIL patients with a DLG1 staining pattern similar to normal tissues are significantly more likely to regress (n = 23, Pattern I), all LSIL biopsy specimens showing a diffuse and intense DLG1 staining likely progress to high-grade lesions (n = 4, Pattern II). Finally, all persistent LSIL analyzed showed an undetermined DLG1 staining, with a diffuse distribution without a strong intensity (n = 3, Pattern III). We found a significant association between the expression pattern of DLG1 and the evolution of the lesion (p < 0.00001).This work contributes to the knowledge of DLG1 biological functions, suggesting that its expression may have an important role in the progression of early dysplastic cervical lesions, giving prognostic information.     

E‐cadherin expression in male urethral smears and correlation with PCR‐based detection of human papillomavirus infection

We performed cytological examination of urethral brushing to study aberrant E‐Cadherin expression as a possible marker for papillomavirus in cytological samples. A total of 30 cytobrush male urethral smears were examined E‐cadherin expression and human papillomavirus (hpv) was confirmed using PCR‐DNA. The age range was 19–36 years (mean: 27 years), 17 (56.7%) cases corresponded to low‐risk HPV and 13 (43.3%) cases were high‐risk HPV. The mean age ranges were 25.77 ± 5.90 years for high‐risk HPV and 26.77 ± 4.31years for low‐risk HPV. Pap smears showed dyskeratosis in 23 (76.7%) cases, koilocytes in 13 (43.3%) cases, infection or bacterial background in 14 (43.7%) cases, suggestive changes of Gardnerella infection in 7 (23.3%) cases, and Chlamydia in 3 (10%) cases. Immunohistochemistry was positive for membrane E‐Cadherin; there was weak expression in 13 (43.3%) cases, moderate expression in 11 (36.7%) cases (P = 0.109) (Figs. 3 a and b), and strong expression in 6 (20%) cases. There were a statistically significant correlations between E‐Cadherin expression and koilocytes (P = 0.007), individual cell dyskeratosis (P = 0.041), and HPV risk (P = 0.000). We concluded that the loss of E‐Cadherin membrane expression was greater in high‐risk HPV cases and was associated with individual cell dyskeratosis features and koilocytes. There were statistically significant correlations between E‐Cadherin expression and dyskeratosis (P = 0.043), koilocytes (P = 0.007), and type of HPV (P = 0.000). Using male urethral smears to test for the loss of E‐Cadherin membrane expression is simple, rapid, specific and more sensitive than conventional morphologic observations. We concluded that E‐Cadherin can be used to discriminate between high‐ and low‐risk papillomavirus in urethral cytologic specimens. Diagn. Cytopathol. 2010;38:583–589. 2009 Wiley‐Liss, Inc.     

Role of HPV DNA testing in predicting cervical intraepithelial lesions: comparison of HC HPV and ISH HPV

Human papillomavirus (HPV) is widely accepted as the primary agent involved in the development of squamous intraepithelial neoplasia and cervical carcinoma. Several commercial tests are available for detecting HPV DNA. This study compares the efficacy of INFORM HPV (in situ hybridization [ISH] HPV) and HCII (HC HPV) in predicting cervical lesions. A total of 762 sequential Papanicolaou (Pap) smears determined by cytologic examination to be either atypical squamous cells of undetermined significance (ASC-US) or low-grade squamous intraepithelial lesions (LSIL) were tested by both Hybrid Capture (HC) HPV and ISH HPV; 250 follow-up biopsies were reviewed as the reference standard for presence or absence of a lesion. ISH HPV and HC HPV differed significantly in accurately predicting biopsy findings from ASC-US and LSIL cases. The overall sensitivity and specificity of ISH HPV were 97% (28/29) and 86% (191/221); and HC HPV was 79% (23/29) and 56% (123/221). The positive predictive value (PPV) of ISH HPV was 48% (28/58) vs HC HPV value of 19% (23/121). Negative predictive value (NPV) was also better with ISH HPV at 99% (191/192) and HC HPV at 95% (123/129). Of equal importance, ISH HPV demonstrated a lower false-positive rate compared to HC HPV, 12% (30/250) vs 39% (98/250), as well as having a slightly lower false-negative rate 0.4% (1/250) vs 2.4% (6/250). ISH HPV is more predictive of biopsy histopathology in patients with detectable cervical lesions than is HC HPV. Effective triage of patients by HPV analysis using ISH HPV as compared to HC HPV has the potential of significant public health impact by reducing unnecessary colposcopies, as well as adverse medical, social, and psychological patient consequences.     

Comparison of cervical and blood T-cell responses to human papillomavirus-16 in women with human papillomavirus-associated cervical intraepithelial neoplasia

Human papillomaviruses (HPVs) are obligate epithelial pathogens and typically cause localized mucosal infections. We therefore hypothesized that T-cell responses to HPV antigens would be greater at sites of pathology than in the blood. Focusing on HPV-16 because of its association with cervical cancer, the magnitude of HPV-specific T-cell responses at the cervix was compared with those in the peripheral blood by intracellular cytokine staining following direct ex vivo stimulation with both virus-like particles assembled from the major capsid protein L1, and the major HPV oncoprotein, E7. We show that both CD4(+) and CD8(+) T cells from the cervix responded to the HPV-16 antigens and that interferon-gamma (IFN-gamma) production was HPV type-specific. Comparing HPV-specific T-cell IFN-gamma responses at the cervix with those in the blood, we found that while CD4(+) and CD8(+) T-cell responses to L1 were significantly correlated between compartments (P = 0.02 and P = 0.05, respectively), IFN-gamma responses in both T-cell subsets were significantly greater in magnitude at the cervix than in peripheral blood (P = 0.02 and P = 0.003, respectively). In contrast, both CD4(+) and CD8(+) T-cell IFN-gamma responses to E7 were of similar magnitude in both compartments and CD8(+) responses were significantly correlated between these distinct immunological compartments (P = 0.04). We therefore show that inflammatory T-cell responses against L1 (but not E7) demonstrate clear compartmental bias and the magnitude of these responses do reflect local viral replication but that correlation of HPV-specific responses between compartments indicates their linkage.     

Human papillomavirus DNA detection in the management of women with twice mildly abnormal cytological smears

This study was undertaken to investigate the value of HPV testing in women referred with two abnormal smears that were graded as mild dyskaryosis or less who attended for at least three follow-up visits. One hundred forty-nine women were included in the study and a total of 39 high-grade lesions including one cancer were detected. All of these were found to be associated with the persistent presence of one or more of 13 high-risk human papillomavirus types (HPV) as detected by a multiplex type specific PCR technique. Two high-grade lesions were initially missed by cytology. In contrast, no cytological or histological evidence of high-grade lesions was found during a follow-up period of up to 8 years in 62 women with no high-risk HPV infection or in 38 women with only transient high-risk HPV infection. The utility of high-risk HPV detection in the management of women presenting with mild cytological abnormalities is discussed.     

宫颈上皮内瘤变及其治疗对妊娠的影响

宫颈鳞状上皮内瘤变是一种和HPV感染有关的宫颈癌前病变。根据病变程度分为低度和高度鳞状上皮内病变,其中低度病变包括HPV感染和C IN1,高度病变包括C IN2~3。HPV感染和宫颈上皮内瘤变是一种生育年龄妇女常见的妇科疾病,HPV感染、宫颈病变本身及其治疗对妊娠的影响倍受关注。HPV感染在妊娠期间可能增加,经阴道分娩者新生儿暴露于HPV的机会增多,尚不能下结论对所有HPV感染者均采用剖宫产的分娩方式。妊娠期间发现C IN1,可以观察并产后随访。如果为C IN2~3,妊娠可能不会加重病变程度,但对阴道镜检查不满意者或高度怀疑浸润癌者应在孕期明确诊断,可于妊娠中期行宫颈锥切术。妊娠中期行宫颈锥切术可能使剖宫产率增加。C IN保守治疗对于患者受孕能力无显著的影响;宫颈冷刀锥切及LEEP锥切可能增加早产率,早产率的增加可能和胎膜早破的发生有关。     

吕梁地区宫颈病变患者人乳头状瘤病毒基因型的检测分析

目的调查吕梁地区21种人乳头状瘤病毒基因型的检测分析。方法收集568例女性宫颈病变患者宫颈分泌物中的脱落细胞,应用人乳头状瘤病毒导流杂交快速基因分型技术检测21种人乳头状瘤病毒亚型,包括13种高危亚型(16,18,31,33,35,39,45,51,52,56,58,59和68型)、5种低危亚型(6,11,42,43和44型)和3种中国人群常见亚型(53,66和CP8304型);分析21种基因型的流行病学特征。结果人乳头状瘤病毒感染率41.9%,单一感染率58.4%,混合性感染率39.9%。21种基因型中,高危型以16,53型为主,其次是52,58型,低危型以6,11型为主;人乳头状瘤病毒16型的感染率居首位。结论本地区21种人乳头状瘤病毒基因型的检测分析资料对人乳头状瘤病毒疫苗研究、应用及其感染的防治有重要意义。