Cervical atypical glandular cells and false negative HPV testing: a dramatic reality of the wrong test at the right place.

BACKGROUND: Due to cervical cancer screening the number of squamous cancer have declined. The number of adenocarcinomas (ADCs) does appear to be rising. ADCs are often missed and human papillomavirus (HPV) testing could be helpful in detecting these abnormalities earlier. CASE: A 36-year-old woman, who had a normal smear three years earlier, had a pap smear with atypical glandular cells. The L1 HPV test showed that there was no HPV infection. Other HPV tests which looked at E6 and E7 showed an infection with HPV 16. Due to unknown reasons, no action was taken regarding the atypical glandular cells. Two years later the patient was diagnosed with a FIGO Stage IVb ADC of the cervix. The L1 HPV test was still negative and the E6/E7 HPV test was still positive. Despite several multiple treatment modalities she succumbed of her disease two years later leaving behind a young family. CONCLUSION: HPV test looking only at L1 can give false negative results if the virus is integrated in the human genome.

Clinical validation of a type-specific real-time quantitative human papillomavirus PCR against the performance of hybrid capture 2 for the purpose of cervical cancer screening.

To be acceptable for use in cervical cancer screening, a new assay that detects DNA of high-risk human papillomavirus (hrHPV) types must demonstrate high reproducibility and performance not inferior to that of a clinically validated HPV test. In the present study, a real-time quantitative PCR (qPCR) assay targeting the E6 and E7 genes of hrHPV was compared with Hybrid Capture 2 (hc2) in a Belgian cervical cancer screening setting. In women >30 years old, the sensitivity and specificity for intraepithelial neoplasias of grade 2 or worse (93 cases of cervical intraepithelial neoplasias of grade 2 or worse (CIN2+) and 1,207 cases of no CIN or CIN1) were 93.6% and 95.6%, respectively, and those of hc2 were 83.9% and 94.5%, respectively {relative sensitivity of qPCR/hc2 = 1.12 [95% confidence interval (CI), 1.01 to 1.23]; relative specificity = 1.01 [95% CI, 0.99 to 1.03]}. A score test showed that the sensitivity (P < 0.0001) and specificity (P < 0.0001) of the qPCR assay were not inferior to those of hc2 at the required thresholds of 90% and 98%, respectively. The overall agreement of hrHPV positivity between the two runs of the qPCR tests was 98.7% (95% CI, 97.5 to 99.4%), with a kappa value of 0.96 (95% CI, 0.83 to 1.00). The qPCR assay used in this study can be considered a reliable HPV assay that fulfills the clinical validation criteria defined for use in cervical cancer screening.

Late-onset Pompe disease (LOPD) in Belgium: clinical characteristics and outcome measures.

BACKGROUND: Late-onset Pompe disease (LOPD) is a rare, hereditary, progressive disorder that is usually characterized by limb-girdle muscle weakness and/or respiratory insufficiency. LOPD is caused by mutations in the acid alpha-glucosidase (GAA) gene and treated with enzyme replacement therapy (ERT). METHODS: We studied the clinical, brain imaging, and genetic features of the Belgian cohort of late-onset Pompe disease patients (N = 52), and explored the sensitivity of different outcome measures, during a longitudinal period of 7 years (2010-2017), including the activity limitations ActivLim score, 6 min walking distance (6MWD), 10 m walk test (10MWT), MRC sum score, and forced vital capacity (FVC) sitting/supine. RESULTS: In Belgium, we calculated an LOPD prevalence of 3.9 per million. Mean age at onset of 52 LOPD patients was 28.9 years (SD: 15.8 y), ranging from 7 months to 68 years. Seventy-five percent (N = 39) of the patients initially presented with limb-girdle weakness, whereas in 13% (N = 7) respiratory symptoms were the only initial symptom. Non-invasive ventilation (NIV) was started in 37% (N = 19), at a mean age of 49.5 years (SD: 11.9 y), with a mean duration of 15 years (SD: 10.2 y) after symptom onset. Brain imaging revealed abnormalities in 25% (N = 8) of the patients, with the presence of small cerebral aneurysm(s) in two patients and a vertebrobasilar dolichoectasia in another two. Mean diagnostic delay was 12.9 years. All patients were compound heterozygotes with the most prevalent mutation being c.-32-13 T > G in 96%. We identified two novel mutations in GAA: c.1610_1611delA and c.186dup11. For the 6MWD, MRC sum score, FVC sitting and FVC supine, we measured a significant decrease over time (p = 0.0002, p = 0.0001, p = 0.0077, p = 0.0151), which was not revealed with the ActivLim score and 10MWT (p > 0.05). CONCLUSIONS: Awareness on LOPD should even be further increased because of the long diagnostic delay. The 6MWD, but not the ActivLim score, is a sensitive outcome measure to follow up LOPD patients.

Time has come to include Human Papillomavirus (HPV) testing in sperm donor banks.

HPV is well known as a potential cause of cervical cancer. Less well known is its link to temporal subfertility that is caused by binding of infectious virions to the spermatozoa's head which induces sperm-DNA damage and causes a reduction in clinical pregnancy rates in women receiving HPV positive semen. This impact on the global fertility burden remains greatly underestimated and underexplored. This risk of reduced fertility due to infectious HPV in sperm is especially important when donor sperm insemination is considered, since testing for the presence of HPV virions before use seems warranted. We tested 514 donor sperm samples from 3 different sperm banks for 18 different HPV types. Overall 3.9% (20/514) of tested donor sperm was positive for HPV, with different prevalence among the 3 different sperm banks (3.6% bank A, 3.1% bank B and 16.7% bank C). Also the HPV virion per spermatozoon ratio in donor samples was similar across the different sperm banks (95% CI 0,01 to 1,07 HPV virions/spermatozoon). When HPV positive donor sperm was used, no clinical pregnancies resulted, whereas when HPV negative donor sperm was used the clinical pregnancy rate was 14.6%. From both a cost/benefit and a safety point of view we recommend that donor sperm should always be tested for HPV before using it for insemination.

Correlation between laminin-5 immunohistochemistry and human papillomavirus status in squamous cervical carcinoma.

BACKGROUND: Human papillomavirus (HPV) plays a critical role in the carcinogenesis of squamous cervical carcinoma. Integration of viral DNA into the host genome is a major contributing factor to malignant transformation. Viral load may influence integration. AIMS: To compare HPV status (type, viral load, integration status) between normal samples, carcinoma in situ and invasive carcinoma in order to elucidate the role of HPV in progression to invasive lesions. METHODS: The study population comprised 10 biopsy samples from each diagnostic group. Laminin-5 immunohistochemistry was performed to distinguish invasive carcinoma from non-invasive high-grade lesions. Real-time PCR was used to detect specific HPV types, viral load and integrated HPV, with quantification of viral E2 and E6 genes. RESULTS: Invasive carcinomas contained a higher number of laminin-5 immunoreactive cells as compared to non-invasive lesions. Almost all samples contained HPV, with a higher viral load and copy number of HPV16 integrated in E2 in cases of laminin-5 immunoreactivity and cases of invasive carcinoma. High HPV16 viral load was associated with more integrated copies in E2. CONCLUSIONS: HPV is important in progression from carcinoma in situ to invasive carcinoma. Viral load and HPV integration influence the development of cervical cancer towards invasiveness. Overall HPV status may be more predictive of patient outcome and may influence patient management.

Human Papillomavirus (HPV) virion induced cancer and subfertility, two sides of the same coin.

In the natural history of HPV infections, the HPV virions can induce two different pathways, namely the infec- tious virion producing pathway and the clonal transforming pathway. An overview is given of the burden that is associated with HPV infections that can both lead to cervical cancer and/or temporal subfertility. That HPV infections cause serious global health burden due to HPV-associated cancers is common knowledge, but that it is also responsible for a substantial part of idiopathic subfertility is greatly underestimated. The bulk of the detected HPV DNA whether in men or women is however infectious from origin. Because the dissociation between HPV viruses and HPV virions or infection and disease remains difficult for clinicians as well as for HPV detection, we propose a review of the different effects caused by the two different HPV virion induced pathways, and highlight the mechanisms that are responsible for causing transient subfertility and cancer.

Ki-67 immunocytochemistry in liquid based cervical cytology: useful as an adjunctive tool?

AIMS: To test the ability of Ki-67 to detect cytological lesions in a screening setting and its use as a surrogate marker of human papillomavirus (HPV) infection. METHODS: A study of liquid based cytology, HPV DNA testing by MY09/MY11 consensus polymerase chain reaction (PCR), type specific PCRs, and Ki-67 immunocytochemistry on a randomly selected series of 147 patients. RESULTS: Comparison of the number of Ki-67 immunoreactive cells/1000 cells in the different cytological groups showed that the HSIL group yielded a significantly higher mean count than did the other groups. The number of Ki-67 immunoreactive cells/1000 cells was significantly higher in HPV-16 positive samples than in samples containing infections with other high risk types. Receiver operating characteristic curves indicated a test accuracy (area under curve) of 0.68, 0.72, and 0.86 for atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesions (LSIL), and high grade squamous intraepithelial lesions (HSIL), respectively. Thresholds for 95% sensitivity were 0.07, 0.08, and 0.15 Ki-67 immunopositive cells/1000 cells for ASCUS, LSIL and HSIL, respectively. The threshold for 95% specificity was 1.9 Ki-67 immunopositive cells/1000 cells. CONCLUSIONS: Ki-67 immunocytochemistry can be applied to liquid based cytology. The accuracy and diagnostic indices of the test are good when compared with those of other techniques. As part of a panel of screening procedures, it could be used as an adjunct to liquid based cytology to identify HSIL, and as a surrogate marker of HPV-16 infection.

The effects of combined conventional treatment, oral antioxidants and essential fatty acids on sperm biology in subfertile men.

We evaluated the effects of combined conventional treatment, oral antioxidants (N-acetyl-cysteine or vitamins A plus E) and essential fatty acids (FA) on sperm biology in an open prospective study including 27 infertile men. The evaluation included sperm characteristics, seminal reactive oxygen species (ROS), FA of sperm membrane phospholipids, sperm oxidized DNA (8-OH-dG), and induced acrosome reaction (AR). Treatment did not improve sperm motility and morphology, nor decrease the concentration of round cells and white blood cells in semen. Sperm concentration increased in oligozoospermic men (7.4+/-1.3 to 12.5+/-1.9 million/ml). Treatment significantly reduced ROS (mean+/-SEM) (775.3+/-372.2 to 150.3+/-105.2 x 10(3)counts/10 second) and 8-OH-dG (45.3+/-10.4 to 16. 8+/-3.3 fmol/microg DNA). Treatment increased the AR (55.1+/-2.2 to 71.6+/-2.2%), the proportion of polyunsaturated FA of the phospholipids, and sperm membrane fluidity. The overall pregnancy rate was 4.5% in 134 months. The per month pregnancy rate tended to be higher in partners of (ex)-smokers (7.15%, n=14,70 months) than in never-smokers (1.6%, n=13,64 months) (OR:4.57, 95% Cl:0.55-38.1).

The relation between reactive oxygen species and cytokines in andrological patients with or without male accessory gland infection.

The presence of various cytokines, namely hepatocyte growth factor (HGF), interleukin-1 receptor antagonist (IL-1 RA), and interleukins (IL-1 alpha, IL-6, and IL-8), as well as the production of reactive oxygen species (ROS) was investigated in seminal plasma of fertile and infertile patients in order to evaluate the possible value of measuring these substances for the diagnosis of male accessory gland infection, and to assess the possible relationship between oxidative stress and cytokines during leucocytospermia and male accessory gland infection (MAGI). Our findings indicate that all of the measured cytokines seem to be produced locally as well as by white blood cells (WBC) and that, due to the presence of higher numbers of WBC, accessory gland infection may exert a deleterious effect on sperm quality through the production of ROS and/or of particular cytokines such as IL-1 alpha, IL-1 RA, and IL-8. The most specific marker for a sensitivity of 95% in discriminating between cases with or without MAGI is the measurement of IL-6 in seminal plasma. In the absence of WBC several cytokines are constitutively produced and correlate with sperm concentration (HGF, IL-8), alpha-glucosidase (IL-6), and gamma-glutamyltransferase activity (HGF). The measurement of these cytokines in semen may provide clinically useful information for the diagnosis of male accessory gland infection, as well as in the absence of WBC where it can provide information about certain mechanisms of male reproductive function and dysfunction.

Hormonal regulation of inhibin B secretion by immature rat sertoli cells in vitro: possible use as a bioassay for estrogen detection.

The influences of follicle-stimulating hormone (FSH), gonadal steroids, and culture time were studied in relation to inhibin B production by Sertoli cells of immature rats cultured in vitro. Sertoli cell-enriched cultures were established from 18-day-old rats and were maintained in medium supplemented with insulin, transferrin, and epidermal growth factor at 34 degrees C. A recently developed ELISA for the measurement of inhibin B was used to assess the effects of recombinant human FSH (rh FSH), testosterone (T), and estradiol (E2) on inhibin B production and accumulation in the culture media of Sertoli cell-enriched cultures and to optimize the cell culture system to serve as a bioassay for the detection and quantification of estrogens and estrogenlike substances. Prolonging the incubation time (24, 48, or 72 hours) of Sertoli cells with control medium without rh FSH, T, or E2 resulted in a time-dependent increase of inhibin B production. Incubation with rh FSH (1, 2.5, 5, or 10 U/L) caused a dose- and time-dependent increase of inhibin B production by Sertoli cells (but not by cultured Leydig cells), reaching a plateau at 5 U/L rh FSH. Addition of T in concentrations of 2.88, 5, or 50 ng/ml to medium without rh FSH and E2 significantly lowered the daily production rate of inhibin B (P < 0.05). In contrast, addition of E2 (0.01 and 0.1 ng/ml) caused a dose-responsive increase in inhibin B production after 24 and 48 hours. The relative increment of inhibin B production induced by E2 was maximal after 24 hours in the presence of 2.5 U/L rh FSH (acting synergistically) and in the absence of T. When these conditions are implemented, the Sertoli cell culture system may serve as a bioassay for estrogenic substances, and it may reflect the possibly harmful effect they may have on spermatogenesis.

Levels of hepatocyte growth factor/scatter factor (HGF/SF) in seminal plasma of patients with andrological diseases.

Hepatocyte growth factor/scatter factor (HGF/SF) has all the characteristics of a molecule suitable for functioning in regulatory networks of motility, such as the spermatogenic epithelium, where spermatogenic cells must migrate between the cells of Sertoli, and it exerts its effect through binding of its high-affinity receptor (c-met). Considering the findings that c-met receptor is expressed in the human testis and on spermatozoa, and that HGF/SF in seminal plasma consists of pro-HGF/SF, mature alphabeta-HGF/SF, and less active forms of HGF/SF, we investigated the concentration and biological activity of HGF/SF in seminal plasma and their correlation with parameters of spermatogenesis to obtain better insight into mechanisms that may be involved in the pathogenesis of male infertility. We also evaluated the potential value of assessment of hepatocyte growth factor concentration and its bioactivity for the diagnosis of certain pathological conditions of male reproduction. We studied the concentration and biological activity of HGF/SF in seminal plasma of normal men and of patients with a range of andrological diseases or conditions by measuring HGF/SF in seminal plasma by enzyme-linked immunosorbent assay and by scatter assay using Madin-Darby canine kidney epithelial cells. We identified three sources of HGF/SF in seminal plasma. In samples from vasectomized men (n = 30; 2.01 ng/ml) and in split ejaculate samples (n = 6; 1e fraction 2.75 ng/ml, 2e fraction 1.62 ng/ml), a prostatic origin can be certified. This HGF/SF has low biological activity (133.3 U/ml). In inflammation of the accessory sex glands (n = 40), a high amount of HGF/SF (3.04 ng/ml) can be generated by white blood cells and has moderate scatter activity (426.7 U/ml). In normozoospermic samples, there is a lower amount of HGF/SF (1.12 ng/ml), with strong scatter activity (1280.0 U/ml). Finally, the clear difference between the low amount of HGF/SF (1.06 ng/ml) with poor scatter activity (106.6 U/ml) in oligozoospermic samples (n = 28) and the high amount of HGF/SF (3.35 ng/ml) with strong scatter activity (853.3 U/ml) in samples from men with azoospermia of primary testicular failure (n = 18) suggests a mainly testicular origin, with different activity in different pathological conditions.

The clinical and biologic significance of serum inhibins in subfertile men.

Inhibin B is a marker of spermatogenesis and Sertoli cell function. The objective of this study was to evaluate the biologic significance of inhibins in subfertile men and the usefulness of inhibin B for the detection of male reproductive dysfunction. Forty-seven subfertile men were evaluated by semen analysis and clinical examination. In addition to semen analysis and hormone determinations, inhibins A and B (Serotec) in all 47 and inhibin A in 25 of these samples using another kit (Biosource) were measured. Higher inhibin B (median, range: 160.3, 81.8-328.5 pg/mL vs. 94.9, 15.6-389.7 pg/mL, P = 0.024) and lower FSH (P = 0.001) were detected in men with sperm concentrations > or =20 million/mL (n = 9), compared to oligozoospermia (sperm concentration <20 million/mL, n = 38). Inhibin B correlated significantly negatively with FSH, LH, and E2, and patient's age and positively with sperm concentration, testicular volume, and TSH. Multiple regression analysis indicated FSH, LH, E2, TSH, and age as the independent variables for inhibin B with a coefficient of determination (R) of 0.53. Simultaneous measurement of both FSH and inhibin B identified more cases with oligozoospermia than either hormone alone. Taking into account the body mass index, the age of the patient, and the indirect mixed antiglobin reaction (MAR) test result in addition to FSH and inhibin B led to the correct semen classification in 45 out of 47 cases. The simultaneous measurement of FSH and inhibin B, taking into account age, body mass index, and the indirect MAR test result appears accurate in identifying subfertility. Inhibin A is detectable in some subfertile men but its significance is not clear.

MedCalc: a new computer program for medical statistics.

In recent years, the use and abuse of statistics in the medical literature has extensively been reviewed. Amongst others, the importance of the P-value has been challenged and the use of misleading graphics, including 3-dimensional displays, has been criticized. The ease of access to more complex statistical procedures, since the introduction of several statistical software packages for personal computers, has been identified as one of the factors involved in the misuse of statistics. Therefore, we have developed a new computer program that includes those statistical procedures commonly encountered in the medical literature and in statistical textbooks for medical researchers. More complex statistical analyses are not implemented in the software. If researchers with limited statistical training require more sophisticated statistical analyses, they should refer to a statistician, not to a more complete statistical software package.

Cervical cancer screening: which HPV test should be used--L1 or E6/E7?

Cervical cancer can and should be a historical disease. The reality, however, is that every year more than half a million women are diagnosed with cervical cancer and a quarter of a million die of this disease. The causal factor for cervical cancer is a persistent HPV infection and therefore a vaccine was developed: prophylactic HPV vaccination will reduce cervical cancer by 70%. Screening based on cytology will miss more than 40% of the abnormalities. The introduction of vaccination should lead to the reintroduction of cervical cancer screening based on HPV detection. Primary HPV screening followed by cytology will detect almost all abnormalities. Not all HPV tests, however, are the same! Clinicians are generally not aware that there is a huge difference among HPV tests. If a low grade lesion progresses to a high grade or invasive cancer, their HPV is likely to integrate. During integration L1 expression can be lost, but E6/E7 expression will always remain present. If the viral HPV is completely integrated then a L1 test looking for only L1 expression will miss this (pre)cancer, while the E6/E7 test will not miss it. HPV tests used in cervical cancer screening should be based on the early (E) and the late (L) genes in order not to miss the abnormality.

Human papillomavirus, lichen sclerosus and penile cancer: a study in Belgium.

PURPOSE: The prevalence of penile cancer varies between 1.5 (industrialized countries) and 4.5 per 100,000 men (non-industrialized countries). Predominant histological subtype is squamous cell carcinoma (SCC). Human papillomavirus (HPV) is found in 40-46% of cases: penile cancer is considered to behave as vulvar cancer. Non HPV related risk factors are lack of circumcision, phimosis, chronic inflammation, and smoking. The role of lichen sclerosus (LS) is unclear. Clinical diagnosis is difficult and treatment often mutilating. Preventive measures can be taken since the risk factors are known: the use of the prophylactic HPV vaccines may contribute. We measured the prevalence of HPV and LS in penile cancer in Belgium. MATERIALS AND METHODS: We found 76 samples of penile lesions in the archives of the departments of Histology of four university hospitals in Belgium. Real-time PCR of type-specific HPV DNA was performed targeting 18 HPV types. PRINCIPAL RESULTS: Patients with penile intraepithelial neoplasia (PeIN) were 56.1 years of age: patients with invasive penile cancer (IPC) 68.5 (p=0.009). Fifty-five samples (55/76) were adequate for HPV targeting. Overall HPV DNA was 70.9%: 89.5% in samples of PeIN (n=19) and 61.1% in samples of IPC (n=36). Invasive penile cancer samples were less likely to be HPV infected (p=0.028). HPV 16 was most prevalent: 48.3%: 20% PeIN, and 28.3% IPC. HPV DNA of the types, included in the prophylactic vaccines, was found in 33% of PeIN and 31.7% of IPC samples. Thrice, low risk HPV (lrHPV) types 6 (1 IPC) and 11 (1 PeIN, 1 IPC) were solely present. There was no difference in the presence of LS between HPV positive and HPV negative samples (p=0.944). CONCLUSIONS: Prevalence of HPV DNA in penile lesions in Belgium is high. However, the prophylactic vaccines may contribute to primary prevention of only a subset of cases. The role of LS remains unclear.

Cervical cytology biobanking: quality of DNA from archival cervical Pap-stained smears.

AIMS: Cervical cytology biobanking is a feasible concept in cervical pathology and could be an indispensable tool for fundamental and applied molecular biological research. PCR is a powerful molecular technique that can be performed on a variety of cervical sample types including Pap-stained cervical smears. However, since the quality of DNA from such specimens is inferior to that from fresh tissue, the correct processing methods are required. This study evaluates three commercial isolation methods and one digestion procedure for their ability to obtain DNA suitable for PCR from fixed and stained Pap smears. METHODS: The High Pure PCR Template Preparation kit, the NucliSENS easyMAG system, the QIAamp DNA Mini Kit and crude proteinase K digestion were used to obtain DNA for subsequent PCR applications. Amplification of beta-globin was performed to verify the presence and integrity of target DNA. The influence of PCR inhibitors and extent of DNA fragmentation were analysed. RESULTS: All commercial DNA isolation techniques provided DNA suitable for PCR amplification, and DNA isolated from 10-year-old archival smears yielded amplicons up to 400 base pairs. Conversely, crude proteinase K digestion limited the amplicon size to 300 bp and did not consistently yield amplifiable digests, as these were contaminated with PCR-inhibiting factors and debris. CONCLUSION: The study indicates that commercial DNA isolation techniques are suitable for PCR amplification of DNA isolated from archival smears, yielding amplicons up to 400 base pairs. Proteinase K digestion is not suitable to obtain amplifiable DNA from fixed and stained Pap-stained smears.

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

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

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

OBJECTIVE: Liquid-based cytology (LBC) for cervical screening is becoming increasingly used. Together with SurePath LBC, various collecting devices can be utilized, among which the Cervex-Brush is the most widely used. The new Rovers Cervex-Brush Combi combines the advantages of the Cervex-Brush with the EndoCervex-Brush increasing sampling of the endocervical canal. The objective of this study was to analyse and to compare the Cervex-Brush Combi with the Cervex-Brush for the collection of squamous and endocervical cells, human papillomavirus (HPV) typing/quantification and disease detection in SurePath LBC. METHODS: Using either the Cervex-Brush or the Cervex-Brush Combi 100 consecutive SurePath LBC samples were collected using each brush type. All 200 slides were read by the FocalPoint and screened by guided screening using slide wizards. The viral load of HPV type 16 E7, 18 E7, 31 E6, 33 L1, 33 E6, 35 E4, 39 E7, 45 E7, 51 E6, 52 L1, 52 E7, 53 E6, 56 E7, 58 L1, 58 E6, 59 E7, 66 E6 and 68 E7 was determined using a TaqMan-based real-time quantitative PCR analysis. RESULTS: The mean number of sampled squamous cells did not differ between the two brush types (54 963 versus 54 595 cells). The use of the Cervex-Brush Combi, however, resulted in a two- to threefold increase in the number of sampled endocervical cells (P < 0.00001). Using the Cervex-Brush Combi slightly more lesions were detected (three versus two low-grade squamous intraepithelial lesions), and resulted in the detection of more atypical squamous cells of undetermined significance (six versus three). In the Cervex-Brush group, 60% (3/5) of abnormal smears were positive for oncogenic HPV types, whereas 66.7% (6/9) of abnormal smears in the Cervex-Brush Combi group tested positive. The median HPV viral load for samples taken with the Cervex-Brush Combi was 0.1825 copies/cell and was significantly higher than in samples taken with the Cervex-Brush (0.0042 copies/cell) (P = 0.02). CONCLUSION: Sampling with the Cervex-Brush Combi resulted in the collection of the same amount of squamous cells, but in a two to threefold harvest of endocervical cells. This led to the detection of a higher viral load for oncogenic HPV and an increase in the number of detected abnormal smears.

Purification and characterization of hepatocyte growth factor (HGF) from human seminal plasma.

Naturally occurring forms of hepatocyte growth factor/scatter factor (HGF/SF) have been purified by heparin-Sepharose chromatography, followed by cation exchange chromatography from a pool of human seminal plasma. Using an enzyme-linked immunosorbant assay, MDCK scatter assay, and Western blot analysis, it was found that, after heparin-Sepharose chromatography, human HGF/SF present in seminal plasma eluted in two different fractions, between 0.72 and 0.85 M NaCl (fraction I) and between 0.95 and 1.10 M NaCl (fraction II). Further purification of fraction I by cation exchange chromatography resulted again in two fractions which eluted at 0.2-0.4 and at 0.6-0.8 M NaCl. The fraction which eluted at 0.2-0.4 M NaCl consisted of two biologically less active heavy chains of the heterodimeric form of HGF/SF (107.1 U/ng immunoreactive HGF), with approximate molecular weights of 65 and 62 kDa under reducing conditions. The second fraction, which eluted at 0.6-0.8 M NaCl, revealed three bands with molecular weights of 87, 65 and 62 kDa, respectively. The 87 kDa form is thought to be a single chain precursor of HGF/SF devoid of biological activity. After subjecting fraction II to cation exchange chromatography, only one major peak eluted between 0.9 and 1.0 M NaCl, and consisted of two biologically active heavy chains of the heterodimeric form of HGF/SF (708.3 U/ng immunoreactive HGF), with approximate molecular weights of 65 and 62 kDa under reducing conditions. Nonreducing conditions for both fraction I and fraction II revealed only one band with a molecular weight of 68 kDa. The ratio ofpro-HGF/SF and less biologically active HGF/ SF (fraction I) over mature heterodimeric HGF/SF (fraction II) was approximately 1:3, in seminal plasma from sperm donors. In seminal plasma, pro-HGF/SF represents an 87 kDa glycoprotein which, apparently, is converted by limited proteolysis into several bands with molecular weights of 65 and 62 kDa. This is the first report showing the presence of pro-HGF/SF and heterodimeric mature HGF/SF, as well as less biologically active forms of HGF/SF in human seminal plasma.

Cervical cytological screening and human papillomavirus DNA testing in Flanders.

The causal relationship between genital human papillomavirus (HPV) infection and cervical dysplasia/carcinoma has been recognised for some time. The aim of this study was to document the occurrence and distribution of HPV infection in the five provinces of the Flemish region in Belgium and to correlate the HPV DNA test results with the cytological results on simultaneously performed thin layer preparations of cervical cells. Out of a total screened group of 105107 samples, 1978 samples with cytological abnormalities were tested for HPV DNA using the MY09/MY11 consensus PCR. The mean age of the whole group was 36.9 years. The LSIL group, with a mean age of 33.6 years, was significantly younger than the other groups. There was no significant difference in HPV prevalence among the provinces. In four out of five provinces the HPV prevalence reached 100% in high-grade lesions. There is a significant increase in predominance of high-risk HPV types, with increasing abnormal cytology (17.9% WNL < 51.1% ASCUS < 83.8% LSIL < 97.2% HSIL). Three peaks of HPV DNA positivity were observed, a first at 22 yrs (82%), a second at 47 yrs (60%) and a third in women older than 65 yrs (52%). These results shed more light on HPV prevalence in Flanders and show that the MY09/MY11 consensus primer based detection system is very suitable for the detection of HPV infections in Flanders.