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Objective High‐risk human papillomavirus (HrHPV)‐positive women detected by self‐sampling require an extra visit at the general practitioner for additional cytology testing, but the loss to follow up within this triage is substantial. The aim of this study was to evaluate the clinical utility of reflex cytology on hrHPV‐positive self‐samples for immediate stratification of women who need referral for colposcopy. Design A prospective cohort study. Setting Two Dutch cervical cancer‐screening laboratories. Population 1014 screenees who tested hrHPV‐positive on self‐samples between 1 December 2018 and 1 August 2019. Methods Self‐samples were directly used for cytological analysis. Cytological and histological outcomes during follow up were obtained from the Dutch Pathology Registry (PALGA). Main outcome measures Test performance of reflex cytology on self‐samples was determined for different thresholds and compared with physician‐taken cytology and histological outcomes. Results Reflex cytology on self‐samples for detecting abnormal cytology showed a sensitivity of 26.4% (95% CI 21.8–31.3) and specificity of 90.5% (95% CI 87.7–92.8). Of all ≥CIN2 cases, 29.4% (95% CI 22.5–37.1) were detected with reflex cytology on self‐samples. The positive predictive value for detection of ≥CIN2 was higher with cytology on self‐collected samples than on physician‐collected samples. Of women who were lost to follow up, 12.9% were found to have abnormal cytology on their self‐sampled material. Conclusion Cytology testing is achievable on hrHPV‐positive self‐samples, could decrease the loss to follow up in screening and is easily implementable in the current clinical practice. Of all hrHPV‐positive women with abnormal cytology on additional physician‐collected samples, 26.4% could have been directly referred for colposcopy if triage with reflex cytology on self‐sampled material had been performed. Tweetable abstract Reflex cytology for triage of hrHPV+ self‐samples is of added value for direct referral of women for colposcopy., Tweetable abstract Reflex cytology for triage of hrHPV+ self‐samples is of added value for direct referral of women for colposcopy.

Primary high-risk human papillomavirus (hrHPV) DNA testing has been introduced in several countries worldwide, including The Netherlands. The objective of this study was to compare three automated workflow procedures for hrHPV testing of which the hrHPV detection assays meet the international guidelines for HPV testing. To mimic a realistic screening situation, we aimed to process 15 000 residual PreservCyt cervical samples in a period of 3 months. During a 3 months period, four technicians were involved in processing 5000 specimens per month on three automated platforms, (1) Qiagen Digene HC2 HPV DNA test (HC2, signal amplification); (2) Roche Cobas HPV test (DNA amplification), and (3) Hologic Aptima HPV test (RNA amplification). We measured and scored general aspects (time-to-results, hands-on-time (HOT)), maintenance, pre-run, run and post-run aspects, inventory (orders, storage), and number of errors on a scale from 1 to 10. As determined for one complete workflow each, maximum processing capacity and HOT were 296 samples and 2 h:55 m, 282 samples and 3 h:20 m, and 264 samples and 4 h:15 m for Aptima, Cobas, and HC2, respectively. The mean throughput time per run was 5 h:51 m for Cobas in which 94 samples could be processed. For Aptima, the mean throughput time per run was 6 h:30 m for 60 samples. Mean throughput time for HC2 is longer since results were provided on day 2. In this study, the fully automated Aptima workflow scores best with a 7.2, followed by Cobas with a score of 7.1 and HC2 with a score of 5.8. Although all HPV tests used in this comparison meet the international test guidelines, the performance (workflow) characteristics of the assays vary widely. A specific choice of a laboratory for high-throughput testing can be different based on the laboratory’s demands, but also hands-on-time, time-to-results/ # samples, maintenance, pre-run, run and post-run parameters, consumables, technical support, and number of errors are important operational factors for the selection of a fully automated workflow for hrHPV testing.

Objective Several studies have shown that there is an upward shift in the classification of cervical cytology when high-risk human papillomavirus (hrHPV) status is known to be positive. The Netherlands implemented primary hrHPV screening with reflex cytology as the primary screening test in 2017. Prior to implementation of the new programme, we investigated whether knowledge of hrHPV status influences cytology rating. Methods Using a set of 200 cytology slides that had been previously tested, two pairs of cytotechnicians rated 100 slides per pair twice: first without knowledge of hrHPV status and then, after a wash-out period of two months, with knowledge of hrHPV status. Results We found that hrHPV positive slides were more likely to be rated up over the referral threshold (i.e. from negative for intraepithelial lesion or malignancy to atypical squamous cells of undetermined significance+) than hrHPV negative slides at the second review when hrHPV status was known (relative risk = 3.2; 95% confidence interval: 1.3–7.9). Conclusions If the same upward shift in ratings were to be observed in the national programme, it may have implications for referrals of women with low-grade lesions.

Contains fulltext : 177357.pdf (Publisher’s version ) (Open Access) In 2017 the cervical cancer screening program in The Netherlands will be revised. Cervical smears will primarily be tested for the presence of high-risk human papillomavirus (hrHPV) instead of cytology, and vaginal self-sampling will be offered to non-responders. This includes a potential risk that part of the women who would otherwise opt for a cervical smear will wait for self-sampling. However, self-sampling for hrHPV in a responder population has never been studied yet. The aim of this study was to investigate the applicability and accuracy of self-sampling in detecting hrHPV in a screening responder population. A total of 2049 women, aged 30-60years, participating in the screening program in The Netherlands were included from April 2013 to May 2015. After they had their cervical smear taken, women self-collected a cervicovaginal sample with a brush-based device, the Evalyn Brush. Both the cervical smear and self-sample specimen were tested with the COBAS 4800 HPV platform. The hrHPV prevalence was 8.0% (95% CI 6.9-9.2) among the physician-taken samples, and 10.0% (95% CI 8.7-11.3) among the self-samples. There was 96.8% (95% CI 96.0-97.5) concordance of hrHPV prevalence between self-samples and physician-taken samples. Women in our study evaluated self-sampling as convenient (97.1%), user-friendly (98.5%), and 62.8% preferred self-sampling over a physician-taken sampling for the next screening round. In conclusion, self-sampling showed high concordance with physician-taken sampling for hrHPV detection in a responder screening population and highly acceptable to women. Implementation of HPV-self-sampling for the responder population as a primary screening tool may be considered.

Abstract Background In January 2017, the Dutch cervical cancer screening programme transitioned from cytomorphological to primary high-risk HPV (hrHPV) DNA screening, including the introduction of self-sampling, for women aged between 30 and 60 years. The Netherlands was the first country to switch to hrHPV screening at the national level. We investigated the health impact of this transition by comparing performance indicators from the new hrHPV-based programme with the previous cytology-based programme. Methods We obtained data from the Dutch nationwide network and registry of histo- and cytopathology (PALGA) for 454,573 women eligible for screening in 2017 who participated in the hrHPV-based programme between 1 January 2017 and 30 June 2018 (maximum follow-up of almost 21 months) and for 483,146 women eligible for screening in 2015 who participated in the cytology-based programme between 1 January 2015 and 31 March 2016 (maximum follow-up of 40 months). We compared indicators of participation (participation rate), referral (screen positivity; referral rate) and detection (cervical intraepithelial neoplasia (CIN) detection; number of referrals per detected CIN lesion). Results Participation in the hrHPV-based programme was significantly lower than that in the cytology-based programme (61% vs 64%). Screen positivity and direct referral rates were significantly higher in the hrHPV-based programme (positivity rate: 5% vs 9%; referral rate: 1% vs 3%). CIN2+ detection increased from 11 to 14 per 1000 women screened. Overall, approximately 2.2 times more clinical irrelevant findings (i.e. ≤CIN1) were found in the hrHPV-based programme, compared with approximately 1·3 times more clinically relevant findings (i.e. CIN2+); this difference was mostly due to a national policy change recommending colposcopy, rather than observation, of hrHPV-positive, ASC-US/LSIL results in the hrHPV-based programme. Conclusions This is the first time that comprehensive results of nationwide implementation of hrHPV-based screening have been reported using high-quality data with a long follow-up. We have shown that both benefits and potential harms are higher in one screening round of a well-implemented hrHPV-based screening programme than in an established cytology-based programme. Lower participation in the new hrHPV programme may be due to factors such as invitation policy changes and the phased roll-out of the new programme. Our findings add further to evidence from trials and modelling studies on the effectiveness of hrHPV-based screening.

Community-acquired pneumonia (CAP) is the most common form of pneumonia and is a leading infectious cause worldwide. Identification of patients that are at risk to develop severe disease has proven to be a major challenge. Soluble mannose receptor (sMR; sCD206) is a new serum marker for macrophage activation. Recent studies showed that sMR levels are increased in patients suffering from severe infections making it a potential biomarker for improved discrimination of disease severity. For measuring sMR, no standardized assay is available. Aim of this study is to develop an assay for standardized measurement of sMR. Next, this assay was used to assess sMR plasma levels for its ability to predict severe disease development in a patient cohort for community-acquired pneumonia.We developed a well-validated sandwich ELISA that enables standardized measurement of sMR in plasma and serum samples. Repeatability was tested by calculating the percentage coefficient of variation (%CV) within and between runs and within and between operators. sMR levels were assessed in a cohort of 100 patients with community-acquired pneumonia.All %CV values were10%, indicating low variation. Higher sMR levels were observed in patients with severe disease when compared to patients without severe disease development (p = 0.004). Patients with sMR levels between 100-430 ng/ml had 22.7% chance to develop severe disease whereas patients with levels between 430-1000 ng/ml had 33.3% chance to develop severe disease.We suggest that sMR has potential as a new biomarker for the prediction of disease severity in patients with community-acquired pneumonia.

The aim of this study was to examine sexual inactivity and occurrence of selected sexually transmitted infections in relation to body mass index. We used data from two large Danish population-based cross-sectional studies conducted in 1991-1995 (HPV study: 6869 women, aged 22-32 years) and in 2004-2005 (Liva study: 19,484 women, aged 18-45 years). Data were collected using a structured interview and measured weight, height, high-risk human papillomavirus DNA, Chlamydia DNA for the HPV study and a structured questionnaire for the Liva study. Overweight and obese women were more likely to have had no lifetime sexual partner or no sexual partner in the last year, e.g., obese women had a threefold (95 percent CI: 1.95-5.04) odds ratio of having had no sexual partner in the last year compared to normal weight women. Additionally, overweight and obese women had a lower likelihood of genital warts and high-risk human papillomavirus infection. A similar tendency was found for self-reported Chlamydia, but not with presence of Chlamydia DNA. If higher likelihood of no lifetime or recent sexual partners among overweight and obese women reflects unmet sexual needs, it could give rise to concern because quality of sexual life is associated with general quality of life.

Objectives Some studies suggest that Chlamydia trachomatis (CT) enhances cervical carcinogenesis; however, a possible confounding effect of persistent human papillomavirus (HPV) infection was not addressed. We examined the potential role of CT infection in the development of subsequent cervical intraepithelial neoplasia grade 3 or worse (CIN3+) in women with prevalent HPV infection and in a subgroup of women with persistent HPV infection. Methods Participants in this population-based cohort study underwent a structured interview, including history of CT infection, and subsequently cervical exfoliated cells were obtained for HPV DNA and CT DNA testing. Women with high-risk HPV DNA infection and no prevalent cervical disease constituted the overall study population (n=1390). A subgroup of women with persistent HPV infection (n=320) was also identified. All women were passively followed for development of cervical lesions in the national Pathology Data Bank. HRs and 95% CIs for CIN3+ during follow-up (up to 19 years) were estimated in an accelerated failure time model. Results Women who reported more than one CT infection had a statistically significantly increased risk of CIN3+ (high-risk HPV-positive, HR=2.51, 95% CI 1.44 to 4.37) (persistent HPV infection, HR=3.65, 95% CI 1.53 to 8.70). We found no association between CT DNA and subsequent risk of CIN3+ among women who were HPV-positive or had a persistent HPV infection at baseline. Conclusions Repeated CT infections increased the risk of CIN3+ among women with prevalent as well as persistent high-risk HPV infection.

Community-acquired pneumonia (CAP) is mostly caused by Streptococcus pneumoniae. Identification of the pathogen causing CAP can be achieved by conventional culture techniques of sputum and/or blood, antigen detection from urine or molecular analysis. However, it remains difficult to determine patients who are at risk of severe disease development (intensive care unit [ICU] admittance and/or death). In this retrospective study, 121 patients admitted to the emergency department with pneumonia symptoms were included. Several markers of infection (pneumococcal DNA load in blood (real-time LytA PCR), white blood cell (WBC) count, C-reactive protein (CRP), procalcitonin (PCT) and soluble urokinase plasminogen activator receptor (suPAR) levels) were assessed for their ability to predict severe disease development. Of 121 patients, 6 were excluded from the study because of an alternative diagnosis, whereas 8 were excluded from biomarker analysis because of the presence of co-morbidities. Of the 115 patients analysed by the LytA PCR, 23 were positive. PCR detected S. pneumoniae DNA in 82% of patients with positive blood culture for S. pneumoniae. PCR missed three samples from patients in which S. pneumoniae was recovered by blood cultures. However, eight additional LytA PCR-positive samples were detected from patients whose blood cultures remained negative. Pneumococcal DNA load was also monitored in time for 31 patients, of whom 11 had positive PCR results. For 10 out of 11 (91%) positive PCR patients, a clear increase in Ct-values was observed, indicating a lower pneumococcal DNA load in the blood as a result of antibiotic therapy. Biomarker analysis was performed in 107 patients, of whom 29 showed severe disease development. Pneumococcal DNA load (p = 0.026), PCT (p = 0.046) and suPAR (p = 0.001) levels most reliably predicted severe disease development. In conclusion, in patients with CAP, higher pneumococcal DNA load, PCT and suPAR values are associated with severe disease development (ICU admission and/or death). These biomarkers may be useful tools for triage of patients suspected of having CAP in the emergency department.

To investigate risk factors for incident and redetected Chlamydia trachomatis (CT) infection in women, including the role of high-risk human papillomavirus (HPV).In this population-based, prospective cohort study conducted in Copenhagen, Denmark, 10,729 women aged 20 to 29 years were tested for CT and HPV DNA and provided information on sexual and health behavior at baseline. Of these, 7998 (74.5%) participated in a follow-up visit 2 years later with identical data collection. We used logistic regression to investigate risk factors for incident and redetected CT infection at follow-up.Among CT DNA negative women at baseline (n = 7529), 106 (1.4%) were CT DNA positive at follow-up (incident infection). Increasing number of sexual partners during follow-up (odds ratio [OR], 1.07 per partner; 95% confidence interval (CI), 1.02-1.11), low educational level (OR, 1.69; 95% CI, 1.11-2.56; for basic education vs. high school or higher), and high-risk HPV positivity at baseline (OR, 1.66; 95% CI, 1.06-2.58) were risk factors for incident infection, whereas older age (OR, 0.86 per year increase; 95% CI, 0.80-0.93) and condom use (OR, 0.60; 95% CI, 0.38-0.94) were associated with reduced risk. Among CT DNA positive women at baseline (n = 469), 108 (23.0%) tested positive at follow-up (redetected infection). We found no statistically significant associations between age, educational level, sexual behavior, smoking, or high-risk HPV status and the risk for redetected CT.Young age, low educational level, high number of sexual partners, failure to use condoms, and high-risk HPV positivity are associated with increased risk for incident CT infection. These findings may guide the development of targeted CT prevention strategies, including screening and information campaigns.

Aim: Although the predictive and prognostic value of thymidylate synthase (TS) expression and gene polymorphism in colon cancer has been widely studied, the results are inconclusive probably because of methodological differences. With this study, we aimed to elucidate the role of TS gene polymorphisms genotyping in therapy response in stage III colon carcinoma patients treated with 5-FU adjuvant chemotherapy.Patients and Methods: 251 patients diagnosed with stage III colon carcinoma treated with surgery followed by 5-FU based adjuvant therapy were selected. The variable number of tandem repeats (VNTR) and the single nucleotide polymorphism (SNP) in the 5′-untranslated region of the TS gene were genotyped.Results: There was a positive association between tumor T stage and the VNTR genotypes (p=0.05). In both univariate and multivariate survival analysis no effects of the studied polymorphisms on survival were found. However, there was an association between both polymorphisms and age. Among patients younger than 60 years, the patients homozygous for 2R seemed to have a better overall survival, whereas among the patients older than 67 this longer survival was seen by the carriers of other genotypes.Conclusion: We conclude that the TS VNTR and SNP do not predict response to 5-FU therapy in patients with stage III colon carcinoma. However, age appears to modify the effects of TS polymorphisms on survival.

The prevalence of human papillomavirus (HPV) genotypes in relation to age was investigated by the polymerase chain reaction (PCR) method in cytologically normal smears from 4 different groups of women. Group A consisted of young women from a district population, aged 15-34 years, using oral contraceptives and visiting general practitioners for a check-up (n = 156); group B were asymptomatic women, aged 35-55, in a district population participating in a triennial screening program for cervical cancer (n = 1555); group C and D consisted of women, seen at the gynecological outpatient department for a wide spectrum of gynecological complaints or for control of their hormonal contraception, aged 15-34 years (n = 2320), and aged 35-55 years (n = 1826) respectively. An HPV (all types) prevalence of 14.1%, 4.1%, 13.9% and 6.6% and an HPV 16/18 prevalence of 3.8%, 0.9%, 3.3% and 1.5% were found in groups A, B, C and D respectively. Statistically significant differences (p value < 0.001) in HPV prevalence were found between women aged 15-34 years and women aged 35-55 years in the district population and in the hospital population. No statistically significant differences in HPV 16/18 were observed after age-matching between women in corresponding age-classes of both populations. In a 5-year interval analysis a strong age-dependent relationship was demonstrated, with a maximum between 20 and 24 years. After the age of 35 a constant level of 1-2% HPV 16/18 was observed. These results indicate that genital HPV infections are age-dependent and suggest that HPV infections at young age can be transient. The implications of these findings in the context of cervical cancer screening are discussed.

Item does not contain fulltext BACKGROUND: Neoadjuvant radiochemotherapy (RCT) is thought to result in a favorable oncological outcome in esophageal cancer patients. Unfortunately, it also implies that adjacent healthy tissue is preoperatively exposed to the potential damaging influence of RCT. Here, the impact of preoperative RCT on matrix metalloproteinase (MMP) expression in healthy esophageal tissue aligned with the tumor at the time of surgery is examined. PATIENTS AND METHODS: 23 patients participating in a clinical trial were randomized to either the control (n = 12) or the neoadjuvant RCT group (n = 11). In the latter group, surgery was performed 5 weeks after the last course of RCT. Full-thickness biopsies were taken from healthy esophageal tissue at the proximal border of the resection specimen and more distally next to the tumor. MMP-2 and MMP-9 activity in the samples was assessed by quantitative gelatin zymography and immunohistochemistry. RESULTS: In the proximal segment, the activities of the MMP-9-dimer (135 kDa) and proMMP-9 (92 kDa) were significantly increased in the RCT group as compared with the control group: 28.5 versus 3.0 (p = 0.025) and 87.7 versus 13.0 (p = 0.015) arbitrary units for 135 kDa and 92 kDa, respectively. In the distal part, RCT resulted in a significant increase of proMMP-2 (72 kDa: 35.8 versus 17.8, p = 0.005) and proMMP-9 (81.2 versus 23.3, p = 0.03). CONCLUSION: In esophageal cancer patients, neoadjuvant RCT results in increased MMP expression in healthy esophageal tissue as measured at the time of surgery. Since increased levels of MMPs are associated with severe postoperative complications including anastomotic leakage this finding necessitates further clinical research. 6 p.

Contains fulltext : 71387.pdf (Publisher’s version ) (Open Access) BACKGROUND: Not all patients with locally advanced rectal cancer (LARC) respond equally to neo-adjuvant radiochemotherapy (RCT). Patients with highly apoptotic less advanced rectal cancers do not benefit from short-term radiotherapy. This study investigates whether this is also the case in the setting of RCT for LARC. PATIENTS AND METHODS: Tissue microarrays were constructed of biopsy and resection specimens of 201 LARC patients. Apoptosis (M30) and several apoptosis-regulating proteins [p53, Bcl-2, Bax, cyclooxygenase-2 (Cox-2) and mamma serine protease inhibitor (maspin)] were studied with immunohistochemistry. Subsequently, predictive values for local recurrence (LR), overall survival (OS) and histological tumour regression were analysed. RESULTS: Apoptotic levels, quantified as the number of apoptotic cells/mm(2) tumour epithelium, were higher in posttherapy tissues compared with biopsies (P < 0.001). Biopsies from clinical T4 stage tumours demonstrated significantly higher levels of apoptosis than clinical T3 stage tumours (P = 0.020). Therapy-induced apoptosis was higher when the interval between the last day of irradiation and surgery increased (P < 0.001, correlation coefficient = 0.355). Pre- and posttherapy apoptosis, p53, Bcl-2, Bax and Cox-2 were not associated with LR, OS or tumour regression. Intense pretherapy cytoplasmatic staining of maspin indicated a higher risk on LR (P = 0.009) only. CONCLUSION: Combined RCT is also successful in highly apoptotic tumours and is therefore independent of intrinsic apoptosis.

Better information on the prevalence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infection is needed in many world areas.Cross-sectional study of population-based samples of nonpregnant women aged 15 to 44 years in Nigeria, Colombia, Argentina, Vietnam (2 areas), China, Thailand (2 areas), Korea, and Spain. 5,328 consenting women aged 15 to 44 years participated. Exfoliated cervical cells were collected and testing for CT and NG and human papillomavirus (HPV) was done using PCR-based assays.Age-standardized CT prevalence ranged between 0.2% (95% confidence interval, CI: 0.0-0.7%) in Spain and 5.6% (95% CI: 3.4-7.8%) in Nigeria. NG ranged between 0% (with broad CIs) in several areas and 2.6% (95% CI: 1.0-4.2%) in Nigeria. Prevalence of CT in all areas combined was greater in women aged 15 to 24 (4.5; 95% CI: 3.4-5.8%) than 25 to 44 (2.6; 95% CI: 2.1-3.1%), whereas NG prevalence was similar in the 2 age groups (0.3%). The only significant risk factors were NG infection (for CT), CT infection (for NG) and infection with high-risk HPV types (for both).The prevalence of CT and, most notably, NG was relatively low in a variety of countries. Our findings, however, do not apply to subsets of high-risk women who are likely to be underrepresented in our population-based samples.

Recently, we showed that Epstein-Barr virus (EBV)-positive gastric carcinoma (GC) forms a distinct clinicopathologic entity with a better prognosis due to lower incidence of lymph node metastases (LN+). Here we investigated whether in EBV-positive GC more pronounced activation of cellular immune responses is associated with absence of (micro)metastases. Twenty EBV-positive primary tumors (PT) (9 LN+) were matched with 28 EBV-negative GC (11 LN+) for T- and N-stage, gender, and age. The PT (n = 28) and its LNs were analyzed by EBER RNA in situ hybridization and by immunohistochemistry for MHC class I and II expression, for CD3, CD8, CD4, CD20, CD56, CD83, and Granzyme B (GzB) expression. In LN metastases of EBV-positive GC, the EBV genome is maintained, excluding tumor escape by virus deletion. All GC express MHC class I independently of EBV status. In comparison with EBV-negative GC, EBV-positive GC have higher expression of MHC class II on the tumor cells (P = 0.029) and a more extensive infiltrate (P < 0.0001) of activated GzB+ CD8+ T cells (P = 0.028), which is most abundant in those EBV-positive tumors that do not metastasize (P < 0.0001). In addition, in EBV-positive GC without metastases, the infiltrate contains higher numbers of mature dendritic cells (DC) (P = 0.018). At present, the antigenic target has to be determined. These data support the notion that local triggering of cellular immune responses in EBV-positive GC prevents lymph node metastasis formation.

Udgivelsesdato: 2006-null BACKGROUND: Infection with human papillomavirus is considered a necessary factor in developing high-grade squamous intraepithelial lesions of the cervix. However, most human papillomavirus positive women do not develop high-grade squamous intraepithelial lesions and other factors may be important for this transition. The objective of the present study was to examine if smoking and alcohol intake are associated with the risk of developing high-grade squamous intraepithelial lesions in women positive for high-risk human papillomavirus types. METHODS: We used baseline information on exposures on 548 high-risk human papillomavirus positive women with normal cytology, comparing 94 women who developed high-grade squamous intraepithelial lesions with 454 women who remained cytologically normal. Logistic regression was applied for statistical analysis. RESULTS: Compared with never smokers, the odds ratio for high-grade squamous intraepithelial lesions among current smokers was 1.99 (95% CI: 1.21-3.28). Among current smokers, number of cigarettes, years of smoking, and early age at smoking initiation were associated with increased risk. However, when modeled simultaneously, it seemed that smoking duration and age at smoking initiation were more associated with risk of high-grade squamous intraepithelial lesions than amount of smoking. Alcohol intake was not associated with risk of high-grade squamous intraepithelial lesions among these women. CONCLUSION: Smoking is associated with an increased risk of developing high-grade squamous intraepithelial lesions in women who are infected with oncogenic human papillomavirus.

Coinfection with multiple types of human papillomavirus (HPV) and its implications for the development of efficacious HPV vaccines is a subject of great interest. To describe the occurrence of concurrent infection with multiple HPV types and to determine whether genital HPV infection modifies the risk of acquiring a new HPV infection with another HPV type, 1610 subjects were monitored for an average of 4.1 years in Bogota, Colombia. Information on risk factors for HPV infection and cervical cells was collected for detection of HPV DNA of 36 types at study entry and at 6 consecutive 6-month follow-up visits. Clustering or the concurrent acquisition of multiple types occurred more often than would be expected by chance. Subjects with incident HPV-16 or -18 infection had 5-7 times higher odds of acquiring a subsequent HPV-58 infection than subjects not infected with HPV-16 or -18. This might affect the protection conferred by effective HPV vaccines.

BACKGROUND: Genital human papillomavirus (HPV) infections are generally accepted to be sexually transmitted, but studies of HPV infections in sex partners are limited. We investigated HPV type-specific concordance and viral load in 238 heterosexual couples. Women with cervical intraepithelial neoplasia were the index patients in these couples.METHODS: GP5+/6+ polymerase chain reaction (PCR), followed by reverse-line blot analysis, was used for the detection of 45 HPV types in cervical and penile scrape samples. Viral loads were subsequently determined in scrape samples positive for HPV types 16, 18, 31, and 33 by LightCycler-based real-time PCR assays.RESULTS: A total of 89.9% of the women and 72.9% of their male partners were HPV positive. Predominantly high-risk HPV types were found in persons of both sexes, but infections with multiple and non-high-risk HPV types were more common in men. Of the HPV-positive couples, 57.8% of the men had the same HPV type as their partners; this rate was significantly higher than that expected by chance (P < .001). Moreover, these HPV-concordant men had higher penile scrape viral loads than did the non-HPV-concordant men. For HPV type 16-positive women, higher cervical viral loads were predictive of presence of HPV type 16 in their sex partners.CONCLUSIONS: In sexually active couples, HPV type concordance was more prevalent than expected by chance and was associated with increased viral loads. These data provide biological support for HPV transmission between sex partners.

Human papillomavirus (HPV) infections and HPV-associated penile lesions are frequently found in male sexual partners of women with cervical intraepithelial neoplasia (CIN). To determine the significance of these findings, we studied the prevalence of HPV and HPV associated penile lesions in a male hospital population with non-STD complaints. Penoscopy was performed after application of acetic acid to identify flat lesions, papular lesions, condylomata acuminata and pearly penile papules (PPPs). Presence of HPV DNA in penile scrapes was tested by GP5+6+ PCR. In case of HPV 16 positivity, viral loads were quantified using a LightCycler based real-time PCR method. Comparing the non-STD male hospital population (n = 118) with the male sexual partners of women with CIN (n = 238), flat penile lesions were found in 14% vs. 60% and penile HPV in 25% vs. 59% of the men, respectively. We found that the presence of penile HPV and, in case of HPV 16 positivity, higher viral loads were associated with the presence of flat penile lesions. Amongst the HPV-positive men, flat penile lesions were more common and larger in size in male sexual partners of women with CIN than in the non-STD hospital population. HPV infections and HPV-associated flat penile lesions are commonly found in the non-STD male population. However, these lesions are less frequently present and smaller in size than in male sexual partners of women with CIN. Higher viral loads in penile scrapes of male sexual partners of women with CIN are reflected by a higher prevalence of flat penile lesions and a larger size of these lesions.

Background. Cost-effectiveness analyses of screening programmes for asymptomatic Chlamydia trachomatis infection suggest that screening at low prevalences in the population is cost-effective. However, the decision models in these studies are based on assumptions about the risk of complications, which are derived from the literature. Incorrect assumptions may lead to under- or overestimation of the effectiveness of screening. The first objective of this paper is to evaluate the assumptions about the probability of complications after an asymptomatic C. trachomatis infection. The second objective is to calculate alternative rates by using available data on the incidence of complications. Methods. We identified cost-effectiveness studies via Medline, and evaluated these for the evidence for the quoted probabilities. In addition, the probability of complications was calculated for Amsterdam from available registration data. Results. In the three studies that were identified, the assumptions for the rates of pelvic inflammatory disease (PID) (clinical and subclinical) after C. trachomatis infection varied from 15% to 80%, and for ectopic pregnancy, tubal factor infertility, and chronic pelvic pain after PID from 5-25%, 10-20%, and 18-30%, respectively. The assumptions were based on data from high-risk populations, case-control data, and data not accounting for misdiagnoses. Using data obtained from local registrations, we estimated the probability of a clinical PID (0.43%), ectopic pregnancy (0.07%), and tubal factor infertility (0.02%) for women with a current infection. These estimates were consistently lower than the estimates based on the literature. Conclusions. We argue that an overestimation of the current complication rates is likely. The effect of overestimation is potentially the greatest in populations with a low prevalence, since the currently assumed cost savings associated with screening may disappear when using more realistic estimates for complications. © International Epidemiological Association 2004; all rights reserved.

BACKGROUND The performance of two commercially available detection systems for high-risk HPV (hrHPV), Hybrid Capture 2 (HC2) and in situ hybridization (ISH), were compared on cervical scrapings. METHODS Using general primer (GP)-mediated GP5+/6+-polymerase chain reaction (PCR)-enzyme immunoassay and reverse line blot genotyping, 76 liquid-based cervical samples were identified with ≥ 1 of the 12 hrHPV types present in the probes of the HC2 and ISH assays. The positivity rate of the assays and the HC2 viral load were determined and related to cytologic findings (n = 76 samples) and histologic findings (n = 43 samples). RESULTS Overall, HC2 scored significantly more samples positive compared with ISH (P < 0.01). Seventy-four of 76 samples (97%) were positive according to HC2. Forty-six of 76 samples (61%) were positive according to ISH, including 80% and 70% of samples that were classified cytologically as moderate dysplasia and severe dysplasia, respectively. All women with underlying cervical intraepithelial neoplasia (CIN) lesions and 67% of women without CIN had positive HC2 samples. ISH scored 33%, 66%, 88%, and 73% of samples positive of women with no CIN, Grade 1 CIN (CIN 1), CIN 2, and CIN 3, respectively. The HC2 viral load was significantly higher in women who had a cytologic diagnosis of dysplasia (P < 0.01) and in women who had an underlying diagnosis of CIN (P < 0.01) compared with women who had neither. In addition, the viral load was significantly higher in ISH positive samples compared with ISH negative samples (P < 0.01). CONCLUSIONS An increased HC2 viral load was associated with an increased chance of underlying high-grade CIN disease in women who tested hrHPV GP5+/6+-PCR positive. Moreover, although positive ISH results were associated with an increased overall viral load in the sample, the analytic sensitivity of ISH was too low to detect all women with prevalent high-grade CIN. Cancer (Cancer Cytopathol) 2004;102:11–8. © 2003 American Cancer Society.

In this study we developed and evaluated a new PCR-based typing assay, directed to the VD2 region of the omp1 gene, for the detection and typing of urogenital Chlamydia trachomatis infections. A nested VD2 PCR-reverse line blot (RLB) assay was developed for the typing of nine different urogenital serovars of C. trachomatis . The assay developed was tested with reference strains of C. trachomatis serovars and cervical scrapes of 86 Colombian women previously found to be positive for C. trachomatis by using plasmid PCR. Two sets of primers directed to the VD2 region of the omp1 gene of C. trachomatis were designed, and fragments of 220 and 166 bp were generated in the primary and nested PCRs, respectively. In addition, an RLB assay was developed to identify nine different urogenital serovars of C. trachomatis (Ba, D, E, F, G, H, I, J, and K) and group controls, including group B (Ba, D, and E), group C (I, J, K, and H), and an intermediate group (F and G). Using this assay, we were able to type 81 of the 86 samples (94.2%). Of these samples, 91.3% were single C. trachomatis infections, and 8.7% were multiple infections. The most common serovars identified were serovars D (22.2%), F (18.5%), G (13.6%), and E (12.3%). Of the women with multiple C. trachomatis infections, >50% contained both serovars D and E. The nested VD2 PCR-RLB developed is a simple, fast, and specific method for the identification of individual urogenital C. trachomatis serovars previously detected by using plasmid PCR. Moreover, it is an appropriate method for studying multiple C. trachomatis infections and for use in large epidemiological studies.

Cytological cervical screening is rather inefficient because of relatively high proportions of false negative and false positive smears. To evaluate the efficiency of high-risk human papillomavirus (hrHPV) testing, by GP5+/6+ PCR-enzyme immunoassay (EIA), in conjunction with cytology (Intervention Group) to that of the classical cytology (Control Group), we initiated the Population Based Screening Study Amsterdam (POBASCAM). POBASCAM is a population-based randomized controlled trial for implementation of hrHPV testing in cervical screening. The outcome measure is the proportion of histologically confirmed ≥CIN3 lesions in each study arm up to and including the next screening round after 5 years. We present the design, methods and baseline data of POBASCAM. When, in the next 5 years, the follow-up will be completed, the data obtained will be used in model studies, including a cost-effectiveness study, to advise the Dutch Ministry of Public Health in deciding whether cervical screening should be based on combined hrHPV and cytology testing instead of cytology alone. Between January 1999 and September 2002, 44,102 women (mean age = 42.8 years; range = 29-61) that participated in the regular Dutch screening program were included in our study. In the Intervention Group the distribution of cytology and hrHPV by cytology class was as follows: normal cytology 96.6% (3. 6% hrHPV positive); borderline and mild dyskaryosis (BMD) 2.5% (34.6% hrHPV positive); and moderate dyskaryosis or worse (>BMD) 0.8% (88.3% hrHPV positive), i.e., 0.4% moderate dyskaryosis (82.9% hrHPV positive), 0.3% severe dyskaryosis (92.5% hrHPV positive), 0.1% carcinoma in situ (95.2% hrHPV positive), BMD, i.e., 0.4% moderate dyskaryosis, 0.3% severe dyskaryosis, 0.1% carcinoma in situ, BMD of 13.7% among women with a positive hrHPV test was not age-dependent. Our study indicates that large-scale hrHPV testing by GP5+/6+ PCR-EIA in the setting of population-based cervical screening is practically feasible, is accepted by both participating women and general practitioners and yields highly reproducible results.

Penile HPV-associated lesions are frequently seen in male sexual partners of women with CIN. The natural course and clinical significance of these lesions are unclear. Women with CIN and their male sexual partners were randomized for condom use (condom group n = 68, noncondom group n = 68). Males were screened for the presence of penile lesions, i.e., flat lesions, papular lesions and condylomata acuminata, and of HPV in their penile swabs by PCR testing. Median follow-up time was 13.1 months (range 2.9-57.4). The outcome of our study was clinical regression of penile lesions defined as disappearance of lesions at penoscopy. Potentially prognostic factors, i.e., HPV status, lesion type and age, were studied as well. Outcomes were assessed in 57 men of the condom group and in 43 men of the noncondom group. Condom use shortened the median time to regression of flat penile lesions (7.4 months condom group vs. 13.9 months noncondom group; HR = 2.1, 95% CI 1.2-3.7). This effect was not found for papular lesions (HR = 0.5, 95% CI 0.1-2.8). HPV-negative men showed a significantly shorter median time to regression of flat lesions (3.8 months) compared to men with either HPV-positive status (8.5 months; HR = 0.4, 95% CI 0.2-0.9) or inconsistent HPV status (13.1 months; HR = 0.2, 95% CI 0.1-0.6). Regression of flat penile lesions is HPV-dependent and accelerated by condom use. This effect is probably the result of blocking viral transmission between sexual partners.

Given the fact that infection with high-risk human papillomavirus (HPV) is causally involved in cervical cancer, addition of high-risk HPV testing to a cervical smear may improve the efficacy of cervical cancer screening programmes, the triage of women with equivocal or borderline Pap smears, and the monitoring of women who have been treated for cervical intraepithelial neoplasia grade 3 (CIN 3). Compared to a cervical smear HPV tests revealed a superior sensitivity (ie clinical sensitivity) for lesions >/= CIN 3, and a negative predictive value approaching 100%. However, a potential complication is the availability of several HPV testing methods, all displaying a different sensitivity and specificity to detect HPV-positive women (ie analytical sensitivity and specificity). There is now compelling evidence that the clinical sensitivity and specificity of HPV tests are not simply synonymous to their analytical sensitivity and specificity, respectively. In fact, a distinction between so-called clinically relevant and irrelevant high-risk HPV infections should be made when considering HPV tests for primary screening, triage policies, or post-treatment monitoring. Here, we discuss the potential importance of HPV load in the context of currently widely applied HPV detection methods, to distinguish clinically relevant from irrelevant HPV infections. From this it can be concluded that it is of utmost importance to define criteria, involving viral load threshold and the type of HPV detection method that should be fulfilled by an HPV test before implementation of such a test in clinical practice and population-based cervical cancer screening programmes.

Little is known about the factors that influence clearance of human papillomavirus (HPV), the primary cause of cervical carcinoma. A total of 227 women cytologically normal and HPV positive at baseline were identified from a population-based cohort of 1,995 Bogota, Colombia, women aged 13-85 years followed between 1993 and 2000 (mean follow-up, 5.3 years). HPV DNA detection and viral load determination were based on a GP5+/GP6+ polymerase chain reaction enzyme immunoassay. Rate ratio estimates for HPV clearance were calculated by using methods for interval-censored survival time data. Analyses were based on 316 type-specific HPV infections. HPV 16 had a significantly lower clearance rate than infections with low-risk types (rate ratio (RR) = 0.47, 95% confidence interval (CI): 0.32, 0.72), HPV types related to HPV 16 (types 31, 33, 35, 52, 58) had intermediate clearance rates (RR = 0.62, 95% CI: 0.47, 0.94), and other high-risk types did not show evidence of slower clearance compared with low-risk types. Infections with single and multiple HPV types had similar clearance rates. There was no evidence of a dose-response relation between clearance and viral load. Observed was slower clearance in parous women (RR = 0.64, 95% CI: 0.47, 0.89) and faster clearance in ever users of oral contraceptives (RR = 1.38, 95% CI: 1.07, 1.77).

Low grade squamous intra-epithelial lesions could be considered as a manifestation of human papillomavirus exposition, however the discrepancy between rates of infection with human papillomavirus and development of low grade squamous intra-epithelial lesions is notable. Here we report a cross-sectional three-armed case–control study in the Colombian population, to compare the risk factors of women with low grade squamous intra-epithelial lesions with that of human papillomavirus DNA-negative and positive women with normal cytology. British Journal of Cancer (2002) 87, 1417–1421. doi:10.1038/sj.bjc.6600650 www.bjcancer.com © 2002 Cancer Research UK

Human papillomavirus is the principal risk factor associated with cervical cancer, the most common malignancy among women in Colombia. We conducted a survey, aiming to report type specific prevalence and determinants of human papillomavirus infection in women with normal cytology. A total of 1859 women from Bogota, Colombia were interviewed and tested for human papillomavirus using a general primer GP5+/GP6+ mediated PCR–EIA. The overall HPV DNA prevalence was 14.8%; 9% of the women were infected by high risk types, 3.1% by low risk types, 2.3% by both high risk/low risk types and 0.4% by uncharacterized types (human papillomavirus X). Thirty-two different human papillomavirus types were detected, being human papillomavirus 16, 58, 56, 81(CP8304) and 18 the most common types. The human papillomavirus prevalence was 26.1% among women younger than 20 years, 2.3% in women aged 45–54 years, and 13.2% in women aged 55 years or more. For low risk types the highest peak of prevalence was observed in women aged 55 years or more. Compared to women aged 35–44 years, women aged less than 20 years had a 10-fold increased risk of having multiple infections. Besides age, there was a positive association between the risk of human papillomavirus infection and number of regular sexual partners and oral contraceptive use. In women aged below 25 years, high educational level and having had casual sexual partners predicted infection risk. In conclusion, there was a broad diversity of human papillomavirus infections with high risk types being the most common types detected. In this population multiplicity of sexual partners and, among young women, high educational level and casual sexual partners seem to determine risk. British Journal of Cancer (2002) 87, 324–333. doi:10.1038/sj.bjc.6600442 www.bjcancer.com © 2002 Cancer Research UK

We followed 353 women referred with abnormal cervical cytology in a non-intervention cohort study. In 91 pregnant women we compared high-risk human papilloma virus rates in the subsequent trimesters and postpartum in comparison to 262 non-pregnant women. High-risk human papilloma virus clearance was compared with 179 high-risk human papilloma virus positive non-pregnant women. Our main questions were: (1) do high-risk human papilloma virus rates change during pregnancy?; and (2) is there any difference between high-risk human papilloma virus clearance in pregnant and non-pregnant women? Women were monitored 3–4 monthly by cytology, colposcopy, and high-risk human papilloma virus testing. The median follow-up time was 33 months (range 3–74). Non-pregnant women showed prevalence rates of high-risk human papilloma virus of 64, 57, 53, and 50%, respectively, in four subsequent 3-months periods since the start of the study. These high-risk human papilloma virus rates were higher than in the three trimesters of pregnancy, and during the first 3 months postpartum, i.e. 50, 44, 45, and 31%, respectively. Postpartum only, this difference was statistically significant (P=0.004). Paired comparisons of high-risk human papilloma virus prevalence rates of the different trimesters with the postpartum rate showed (McNemar test) decreased rates: first trimester: 18% (P=0.02), second trimester: 13% (P=0.02) and third trimester: 23% (P

Objectives: Genital infection with certain types of human papillomavirus (HPV) is the most important risk factor for cervical cancer. The male sexual partner is supposed to be the vector of the infection. However, the knowledge of risk factors for genital HPV DNA in men is limited. The objective of this paper is to study the risk factors for HPV infection in men and to compare them with those found in women, including the study of whether there are different risk profiles for oncogenic and non-oncogenic HPV types. Methods: From a sexually transmitted diseases (STD) clinic in Denmark, 216 men were consecutively included. A personal interview was done and material for genital HPV DNA detection was obtained with swabs. HPV DNA was detected by polymerase chain reaction (PCR). Odds ratios (OR) for HPV as well as for oncogenic and non-oncogenic types separately were computed with a 95% confidence interval (CI) by means of unconditional multiple logistic regresssion. Results: The most important predictors of any HPV were lifetime number of sex partners (OR = 4.3; 95% CI 1.4 to 13.1 for 25‐39 v 1‐9 partners), young age, and being uncircumcised. The most important risk factor for oncogenic HPV types was lifetime number of partners, whereas number of partners in the past year and ever having genital warts were risk factors for the non-oncogenic HPV types. Young age predicted risk of both oncogenic and non-oncogenic HPV types. Conclusions: Most risk factors for HPV DNA detection in men resemble those found in women. As in women, the risk factor profile for the oncogenic HPV types was different from that of the non-oncogenic HPV types.

and 39%, respectively, for IgG and 7, 7, 3, and 7%, respectively, for IgA. The IgG seroprevalence rates for the PCR-positive women were two to three times higher than those for the PCR-negative women, i.e., 72 versus 29%, 72 versus 29%, 47 versus 26%, and 74 versus 25% for CT-EIA, SeroCT, CT pELISA, and the MIF assay, respectively. After discrepancy analysis, the sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the IgG assays; for CT-EIA they were 84.7, 98.6, 98.4, and 86.7%, respectively; for CT pELISA they were 71.4, 97.3, 96.2, and 78.3%, respectively; for SeroCT they were 84.7, 98.6, 98.4, and 86.3%, respectively; and for the MIF assay they were 79.2, 83.1, 98.3, and 83.1%, respectively. In conclusion, these peptide-based ELISA systems for the serological detection of C. trachomatis infection performed as well as the MIF assay. Since these tests are less time-consuming, less expensive, and easier to perform than the MIF assay, they might be useful in the serodiagnosis of chlamydial infection. Chlamydia trachomatis infection is the most prevalent sexually transmitted disease in Europe and the United States. In women, this infection can lead to severe sequelae like ectopic pregnancy and tubal infertility. C. trachomatis serology has been used for both diagnostic purposes and large epidemiological studies. However, widespread introduction of C. trachomatis-specific serology has not gained wide acceptance. The microimmunofluorescence (MIF) assay (10) is still regarded as the “gold standard” in the serological diagnosis of C. trachomatis infections. However, the MIF assay is not suited for use by routine laboratories since reading of the specific fluorescence requires a high degree of expertise. Also, current serological tests employ either group-specific lipopolysaccharide (LPS) or reticulate bodies as antigen and thus show crossreactivity with C. pneumoniae (4). Serological cross-reactivity leads to high rates of false-positive results, especially in a population with a low prevalence of C. trachomatis infection, since the rates of C. pneumoniae seroprevalence are up to 60%. In addition, there have been reports on serological cross-reactivity due to proteins from other bacteria, e.g., Acinetobacter (2). Several user-friendly enzyme immunoassays with increased specificity have been developed by using LPS-stripped C. trachomatis particles (8). Recently, three commercially available assays have been developed by using specific synthetic peptides based on the major outer membrane of C. trachomatis. These assays have the potential to be both specific and sensitive. Proper comparison of these serological assays to the MIF assay is strongly needed since these new tests are well standardized, less expensive, and less laborious than MIF. Therefore, we analyzed these new commercially available, peptide-based enzyme-linked immunosorbent assay (ELISA) systems for the detection of specific serum immunoglobulin G (IgG) and IgA antibodies to C. trachomatis in relation to the detection of C. trachomatis infections in the corresponding cervical scrapings by PCR. The performances of these new assays were compared to that of the MIF assay.

The relation between human papillomavirus type 16 (HPV 16) viral load in cervical scrapes and development of high-grade cervical intraepithelial neoplasia (CIN II or III) was studied in a nested case-control study of women with normal cytology (group A) and in a cohort of women with abnormal cytology (group B). HPV 16 DNA load was determined using a quantitative real-time PCR assay. In group A, case women (women with CIN II/III, n = 12) had a significantly higher viral load than control women (women with CIN ≤ I, n = 47). This resulted in an increased relative risk of women with the 50% highest viral load for development of CIN II/III (OR 7.7; CI 1.6–33). In group B, women with CIN II/III (n = 38) had a significantly higher viral load than women with CIN ≤ I (n = 25). Women with the 50% highest viral load had an increased relative risk of CIN II/III (OR 3.2; CI 1.1–9.3) and a decreased chance of both viral clearance and cytologic regression. Our data suggest that in women with normal cytology an increased HPV 16 load confers an increased risk of developing a CIN lesion. A sustained high viral load is subsequently informative for progression to a high-grade CIN lesion. © 2002 Wiley-Liss, Inc.

Background & Aims: Approximately 10% of gastric adenocarcinomas carry the human pathogenic Epstein-Barr virus (EBV). The role of EBV in the pathogenesis of these carcinomas remains to be established. Methods: To obtain a comprehensive overview of chromosomal aberrations in EBV-carrying and EBV-negative gastric carcinomas we performed comparative genomic hybridization (CGH) on 44 gastric carcinomas, 10 EBV-positive, and 34 EBV-negative. Additionally, DNA flow cytometry was done. Results: Loss of chromosome 4p (P < 0.001) and of 11p (P < 0.02) was exclusively restricted to EBV-carrying gastric carcinomas. In addition, loss of 18q (P < 0.02) was significantly more frequent in EBV-carrying gastric carcinomas. The latter involves loci, which have already been linked to gastric carcinogenesis such as the DCC and SMAD4 gene. In contrast, the losses on chromosome 4 and 11 do not yet harbor a gene related to gastric carcinogenesis. No significant correlation was found between DNA-ploidy and the EBV-status. A number of chromosomal aberrations were found at comparable frequencies in both groups, i.e., losses of chromosome 17, 12q, and loss of 1p. Interestingly, gains of 13q (10/34) and 3q (5/34) and loss of 1q (5/34) were solely observed in EBV-negative gastric carcinomas. Conclusions: These data indicate that EBV-carrying and EBV-negative gastric carcinomas have different pathogenetic pathways in which EBV might play a crucial role.

Posttransplant lymphoproliferative disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)–associated complication in transplantation recipients that is caused by iatrogenic suppression of T-cell function. The diagnostic value of weekly EBV DNA load monitoring was investigated in prospectively collected unfractionated whole blood and serum samples of lung transplantation (LTx) recipients with and without PTLD. In PTLD patients, 78% of tested whole blood samples were above the cut-off value of quantitative competitive polymerase chain reaction (Q-PCR) (greater than 2000 EBV DNA copies per mL blood), with the majority of patients having high viral loads before and at PTLD diagnosis. Especially in a primary EBV-infected patient and in patients with conversion of immunosuppressive treatment, rapid increases in peripheral blood EBV DNA load diagnosed and predicted PTLD. In non-PTLD transplantation recipients, only 3.4% of the whole blood samples was above the cut-off value (P

We assessed a possible role for high-risk human papillomavirus (HPV) testing in the policy after treatment for cervical intraepithelial neoplasia (CIN) 2 or 3 (moderate to severe dysplasia). According to the Dutch guidelines follow-up after treatment consists of cervical cytology at 6, 12 and 24 months. Colposcopy is only performed in case of abnormal cervical cytology. In this observational study 184 women treated for CIN 2 or 3 were prospectively monitored by cervical cytology and high-risk HPV testing 3, 6, 9, 12 and 24 months after treatment. Post-treatment CIN 2/3 was present in 29 women (15.8%). A positive high-risk HPV test 6 months after treatment was more predictive for post-treatment CIN 2/3 than abnormal cervical cytology (sensitivity 90% and 62% respectively, with similar specificity). At 6 months the negative predictive value of a high-risk HPV negative, normal smear, was 99%. Largely overlapping, partly different groups of women with post-treatment CIN 2/3 were identified by HPV testing and cervical cytology. Based on these results we advocate to include high-risk HPV testing in monitoring women initially treated for CIN 2/3. In case of a high-risk HPV positive test or abnormal cervical cytology, colposcopy is indicated. All women should be tested at 6 and 24 months after treatment and only referred to the population-based cervical cancer screening programme when the tests are negative on both visits. © 2001 Cancer Research Campaign http://www.bjcancer.com

To determine whether certain Chlamydia trachomatis serovars are preferentially associated with a symptomatic or an asymptomatic course of infection, C. trachomatis serovar distributions were analyzed in symptomatically and asymptomatically infected persons. Furthermore, a possible association between C. trachomatis serovars and specific clinical symptoms was investigated. C. trachomatis -positive urine specimens from 219 asymptomatically infected men and women were obtained from population-based screening programs in Amsterdam. Two hundred twenty-one C. trachomatis -positive cervical and urethral swabs from symptomatically and asymptomatically infected men and women were obtained from several hospital-based departments. Serovars were determined using PCR-based genotyping, i.e., restriction fragment length polymorphism analysis of the nested-PCR-amplified omp1 gene. The most prevalent C. trachomatis serovars, D, E, and F, showed no association with either a symptomatic or asymptomatic course of infection. The most prominent differences found were (i) the association of serovar Ga with symptoms in men ( P = 0.0027), specifically, dysuria ( P < 0.0001), and (ii) detection of serovar Ia more often in asymptomatically infected people (men and women) ( P = 0.035). Furthermore, in women, serovar K was associated with vaginal discharge ( P = 0.002) and serovar variants were found only in women ( P = 0.045).

In this retrospective study Chlamydia pneumoniae and Mycoplasma pneumoniae infections were detected by polymerase chain reaction (PCR) in samples (n = 457) from children presenting with acute respiratory infection to general practitioners during 1992-97. Samples were collected in autumn and winter, and from 1994 onwards in spring and summer also. Overall, C. pneumoniae and M. pneumoniae were detected in throat or nasal samples by PCR in 3.1% and 2.4% of the cases, respectively. The proportion of both C. pneumoniae and M. pneumoniae infections varied between 0% and 6.9% over the years studied, whereas seasonal proportions varied from 1.8 to 9.1% and 1.2 to 4.5%, respectively. For both microorganisms the lowest proportion was detected during winter and the highest in summer. C. pneumoniae could already be detected by PCR in patients under 4 y of age, an observation not made in sero-epidemiological studies. In conclusion, both C. pneumoniae and M. pneumoniae infections play a minor role in children presenting with acute respiratory infection.

The use of mailed, home-obtained urine specimens could facilitate screening programs for the detection of asymptomatic Chlamydia trachomatis infections. Since transport time could have an adverse effect on the sensitivity of C. trachomatis detection by PCR, the influence of DNA degradation on amplification was monitored over the course of 1 week. Therefore, urine specimens were aliquoted on the day of collection or arrival. Two groups of urine specimens were investigated. Group I contains first-void C. trachomatis -positive and -negative urine samples. DNA degradation was monitored in group I samples for 7 days at room temperature (RT) and at 4°C by amplifying different lengths of the human β-globin gene and the C. trachomatis plasmid target. DNA degradation was observed only for the larger human β-globin fragments at days 5 to 7 at RT. In contrast, at 4°C all targets could be amplified. Urine specimens were also frozen and thawed before aliquoting to mimic freezing during transport. This resulted in a lower sensitivity for the detection of C. trachomatis after thawing and 3 to 4 days at RT. In addition, mailed, home-obtained C. trachomatis -positive urine specimens (group II) were analyzed for 7 days after arrival by two commercially available C. trachomatis detection systems (PCR and ligase chain reaction [LCR]). The C. trachomatis plasmid target in mailed, home-obtained urine specimens could be amplified by both PCR and LCR after 1 week of storage and/or transport at RT. In conclusion, our findings indicate that mailed, home-obtained urine specimens are suitable for the sensitive detection of asymptomatic C. trachomatis infections by amplification methods, even if the transport time is up to 1 week at RT. These findings support the feasibility and validity of screening programs based on mailed, home-obtained urine specimens. Larger studies should be initiated to confirm our results.

A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per genome equivalent in cervical scrapings. The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP5+/6+ PCR, and β-globin DNA. The two competitive PCRs involve co-amplification of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region. The accuracy of quantification by combining the two competitive PCR assays was validated on mixtures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lines. Comparison of this fully quantitative PCR assay with two semi-quantitative HPV PCR assays on a series of crude cell suspensions from HPV 16 containing cervical scrapings revealed remarkable differences in the calculated relative HPV load between samples. We found evidence that correction for both intertube variations in PCR efficiency and number of input cells/integrity of DNA significantly influence the outcome of studies on viral DNA load in crude cell suspensions of cervical scrapings. Therefore, accurate measurements on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach. © 1999 Cancer Research Campaign

AIM: To investigate the value of RNA detection by nucleic acid sequence based amplification (NASBA) for the monitoring of Chlamydia trachomatis infections after antibiotic treatment. METHODS: Cervical smears (n = 97) and urine specimens (n = 61) from 25 C trachomatis positive female patients were analysed for the presence of C trachomatis 16S ribosomal RNA (rRNA) by NASBA and C trachomatis plasmid DNA by the polymerase chain reaction (PCR) before and up to five weeks after antibiotic treatment. RESULTS: Chlamydia trachomatis RNA was found in all cervical smears taken before antibiotic treatment (n = 24) and in two smears taken one week after antibiotic treatment; no C trachomatis RNA was detected after two weeks or more. In contrast, C trachomatis DNA was found in all such specimens before treatment, and 21 of 25, six of 21, and five of 20 smears were found to be positive at one, two, and three weeks after treatment, respectively. After four weeks, only one of six smears was positive, and this smear had been negative in the two preceding weeks. Of the 61 urine samples investigated, C trachomatis DNA and C trachomatis RNA were found in all before treatment (n = 15), whereas one week after treatment four of 15 were C trachomatis DNA positive and C trachomatis RNA was detected in one sample only. CONCLUSIONS: These data show that RNA detection by NASBA can be used successfully to monitor C trachomatis infections after antibiotic treatment. Furthermore, it might be possible to use urine specimens as a test of cure because neither C. trachomatis DNA or RNA could be detected two weeks or more after treatment.

Two cocktails of digoxigenin-labeled human papillomavirus (HPV) type-specific oligonucleotide probes and an enzyme immunoassay (EIA) were used as a basis to developed a group-specific detection method for 14 high-risk (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and 6 low-risk (types 6, 11, 40, 42, 43, and 44) HPVs, following a general primer GP5+/bioGP6(+)-mediated PCR. The sensitivity of this high-risk/low-risk (HR/LR) HPV PCR-EIA ranged from 10 to 200 HPV copies, depending on the HPV type. Comparison of HR/LR HPV PCR-EIA with radioactive Southern blot hybridization using a general probe on the same PCR products derived from 417 cytomorphologically abnormal cervical scrapings resulted in an overall agreement of 96% between the two methods. Complete concordance between group-specific HR/LR detection and individual typing results for both single and multiple infections indicate the strong specificity of this HR/LR HPV PCR-EIA. Multiple infections could be predicted by comparing PCR-EIA optical density values of the cocktail probes with one of the individual oligonucleotide probes. This novel HR/LR PCR-EIA allows accurate and rapid identification of high-risk and low-risk HPV types in cervical scrapings and will facilitate HPV detection in HPV mass-screening programs.

Background Breast cancer is a heterogeneous disease with various histological features and molecular markers. These are utilized for the prediction of clinical outcome and therapeutic decision making. In addition to well established markers such as HER2 overexpression and estrogen and progesterone receptor (ER and PR) status, chromosomal instability is evolving as an important hallmark of cancers. The HER2/TOP2A locus is of great importance in breast cancer. The copy number variability at this locus has been proposed to be a marker for the degree of chromosomal instability. We therefore developed a Single Nucleotide Polymorphism (SNP) assay to evaluate allelic imbalance at the HER2/TOP2A locus in three different entities of primary breast tumors. Methods Eleven SNPs were carefully selected and detected by real time PCR using DNA extracted from paired (histologically normal and tumor) paraffin-embedded tissues. Primary breast tumors of 44 patients were included, 15 tumors with HER2 overexpression, 16 triple negative tumors, defined by the absence of HER2 overexpression and a negative ER and PR status and 13 ER and PR positive tumors without HER2 overexpression. As controls, histologically normal breast tissues from 10 patients with no breast tumor were included. Results Allelic imbalance was observed in 13/15 (87 %) HER2 positive tumors, the remaining 2 being inconclusive. Of the 16 triple negative tumors, 12 (75 %) displayed instability, 3 (19 %) displayed no instability, and 1 was inconclusive. Of the 13 hormone receptor positive tumors, 5 (38 %) displayed allelic imbalance, while 8 did not. Conclusions We conclude that the SNP assay is suitable for rapid testing of allelic (im)balance at the HER2/TOP2A locus using paraffin-embedded tissues. Based on allelic imbalance at this locus, both triple negative and ER and PR positive breast tumors can be subcategorized. The clinical relevance of the allelic (im)balance status at the HER2/TOP2A locus in breast cancer is subject of future study. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2086062232155220 Electronic supplementary material The online version of this article (doi:10.1186/s13000-015-0289-x) contains supplementary material, which is available to authorized users.

The prevalence rates and serovar distributions of Chlamydia trachomatis cervical infections were investigated in two different groups of women. Group I consisted of 393 asymptomatic young women (aged 17 to 30 years) who were invited to participate in a C. trachomatis screening program. Group II consisted of 734 randomly selected patients (aged 17 to 68 years) attending an inner-city gynecological outpatient clinic. C. trachomatis was detected in cervical scrapes by PCR specific for endogenous plasmid. These plasmid PCR-positive samples were subsequently subjected to genotyping by C. trachomatis-specific omp1 PCR-based restriction fragment length polymorphism analysis (J. Lan, J. M. M. Walboomers, R. Roosendaal, G. J. van Doornum, D. M. MacLaren, C. J. L. M. Meijer, and A. J. C. van den Brule, J. Clin. Microbiol. 31:1060-1065, 1993). The overall prevalence rates of C. trachomatis found in patients younger than 30 years were 9.2 and 11.8% in groups I and II, respectively. A clear age dependency was seen in group II, with the highest prevalence rate (20%) found in patients younger than 20 years, while the rate declined significantly after 30 years of age (5.9%). In women younger than 30 years, the genotyping results showed that serovars E, I, and D (in decreasing order) were frequent in group I, while serovars F, E, and G (in decreasing order) were predominantly found in group II. The study shows that C. trachomatis infections are highly prevalent in asymptomatic young women. The different serovar distributions found most likely reflect the different compositions of the study groups, but additional analysis of the case histories of individual patients suggests that certain serovars might be associated with symptomatic (i.e., serovar G) or asymptomatic (i.e., serovars D and I) infections.

OBJECTIVE--To investigate C trachomatis serovars in contact-traced heterosexual partners. METHODS--Urogenital Chlamydia trachomatis isolates (n = 112) derived from 35 heterosexual patients (index patients) and their 37 chlamydia positive partners (contact patients) were differentiated into serovars by genotyping with restriction fragment length polymorphism (RFLP) analysis of the PCR amplified omp1 gene. In order to investigate whether different strains within the frequently prevalent serovar F were transmitted, two pairs of serovar F (n = 4) were further analysed by genomic DNA fingerprinting with arbitrary primer PCRs (AP-PCRs). RESULTS--Identical C trachomatis serovars were found in 31 of the 35 pairs, serovars E, F, D, and G being most prevalent. In the remaining four pairs different serovars (either D, E, F or G) were found between the index and the contact patients. By AP-PCR analysis the strains of serovar F were found to be identical between the index and the contact patients, but were different between the two pairs in all AP-PCRs used. CONCLUSION--A majority of heterosexual partners, once traced positive for C trachomatis infections, are infected with identical serovars. Identical strains of serovar F found in partners as found by DNA fingerprinting confirms the sexual transmission of C trachomatis.

AIMS--To examine the role of Chlamydia trachomatis in ectopic pregnancy by detection of DNA in archival salpingectomy specimens, and in their preceding cervical specimens and endometrial biopsies, by using the polymerase chain reaction (PCR). METHODS--Archival paraffin embedded salpingectomy tissues (n = 48) from 37 women with ectopic pregnancy were examined for the presence of C trachomatis plasmid and omp1 DNA by PCR. In addition, preceding cervical specimens (n = 58) stored either as cervical cell suspensions or as archival cervical smears, and preceding endometrial biopsies (n = 18), taken 0-5.8 years before the ectopic pregnancy, were examined by PCR for the presence of C trachomatis. RESULTS--C trachomatis DNA was detected in only one of the 48 salpingectomy specimens from 37 women. However, in six of the 37 women, C trachomatis DNA was detected in the genital specimens (cervix and/or endometrial) taken before salpingectomy. C trachomatis infections were mostly found in endometrial or cervical specimens taken more than three years before ectopic pregnancy. No chlamydial DNA was found in endometrial or cervical specimens taken at the same time of the ectopic pregnancy. CONCLUSIONS--Although no C trachomatis DNA was found in salpingectomy specimens, several women with ectopic pregnancy had C trachomatis infections in endometrial and cervical specimens in the past. This suggests that at least in these cases the ectopic pregnancy is a late post-inflammatory complication of an ascending C trachomatis infection resulting in a scarred fallopian tube.

The efficiency of a freeze-thaw method, a proteinase K/Tween 20 lysis method and a guanidinium isothiocyanate/silica beads method for DNA extraction from fixed and Papanicolaou-stained cells from the cervical cancer cell line Siha was measured by beta-globin polymerase chain reaction (PCR). The GTC/silica beads method, which appeared superior, revealed a human papillomavirus (HPV) general primer-mediated PCR sensitivity of 50-500 copies of HPV 16 per sample using dilutions of fixed and stained Siha cells. Application to archival cervical smears (n = 116) revealed that the yield and size of amplifiable DNA decreases with storage time. The longer the storage time, the more repetitions of the whole procedure, including the lysis step, were required to extract sufficient amplifiable DNA. In this way, an overall beta-globin PCR positivity for 98% of the smears was reached. Further analysis revealed that a maximum size of 200 bp could be amplified from smears stored for up to 9 years. The method was validated by demonstrating by PCR the same HPV types in archival smears and corresponding cervical biopsies of cervical cancer patients. In conclusion, the GTC/silica beads method appears suitable to process archival cervical smears for HPV detection by PCR. provided that stepwise adjustments are made until beta-globin PCR positivity is obtained and primers are chosen which amplify a maximum of about 200 bp. Images Figure 1 Figure 2 Figure 3

The aim of this study was to investigate the distribution of 27 mucosotropic human papillomavirus (HPV) genotypes (HPV 6, 11, 13, 16, 18, 30, 31, 32, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61 and 66) in cytomorphologically abnormal cervical scrapes (Pap IIIa-Pap IV; n = 1,373) using the polymerase chain reaction (PCR) method on crude cell suspensions. The scrapes were analyzed for the presence of HPV DNA by HPV general-primer-mediated PCR (GP-PCR), which allows the detection of a broad spectrum of HPV types at the subpicogram level. Subsequently, 2 HPV typing procedures based on either type-specific PCR (for HPV 6, 11, 16, 18, 31 and 33) or characterization of GP-PCR products by hybridization (for HPV 13, 30, 32, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61 and 66) were applied. Increasing total HPV prevalence was found with increasing severity of dysplasia from 71% in Pap IIIa to 100% in Pap IV scrapes (carcinoma in situ). The scrapes which were positive by type-specific PCR included 47% cases of Pap IIIa, 71% cases of Pap IIIb and 90% cases of Pap IV. Moreover, 12% of Pap IIIa scrapes, 6% of Pap IIIb scrapes and 8% of Pap IV scrapes revealed positivity for one or more of the remaining HPV types, as determined by successive hybridizations of the GP-PCR products. Taking the typing data together, we noted that the level of HPV heterogeneity decreased from 22 different HPV types (HPV 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 54, 55, 56, 58, 59, 61 and 66) detected in the group of Pap IIIa scrapes to 13 (HPV 6, 11, 16, 18, 31, 33, 35, 45, 51, 52, 58, 59 and 61) and 10 HPV genotypes (HPV 6, 16, 18, 31, 33, 45, 51, 52, 54 and 58) in the Pap IIIb and Pap IV classes, respectively. An increasing prevalence rate from Pap IIIa to Pap IV was found for HPV 16, 18, 31, 33, 45 and 54. The prevalence rate of identified HPV genotypes increased from 59% in Pap IIIa to 98% in Pap IV, indicating that almost all high-risk HPV genotypes related to cervical cancer in The Netherlands have been characterized.

We examined the effect of a questionnaire addressing sexual behaviour on participation in a systematic screening programme for asymptomatic Chlamydia trachomatis infections. Furthermore, we compared participation among persons requested to mail a home-obtained urine sample directly to the laboratory and persons requested to bring a sample to the physician's office. Seven hundred and fifty men and women were randomly assigned to receive a questionnaire with or without intimate questions and to deliver or mail the samples. The inclusion of questions about sexual behaviour did not affect participation among both men and women. Among women there was no difference in participation between delivering or mailing the sample. Among men delivering the sample, participation was 18% (95% confidence interval [CI]: 5-32) lower. This study shows that questions on sexual behaviour can be included in a screening questionnaire without adversely affecting participation. Furthermore, mailing the specimens is the most efficient strategy for men, when screening for C. trachomatis by means of home-obtained urine specimens.