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1.  An evaluation of three processing methods and the effect of reduced culture times for faster direct identification of pathogens from BacT/ALERT blood cultures by MALDI-TOF MS 
Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three different processing methods for the rapid direct identification of bacteria from positive blood culture bottles were compared. In total, 101 positive aerobe BacT/ALERT bottles were included in this study. Aliquots from all bottles were used for three bacterial processing methods, i.e. the commercially available Bruker’s MALDI Sepsityper kit, the commercially available Molzym’s MolYsis Basic5 kit and a centrifugation/washing method. In addition, the best method was used to evaluate the possibility of MALDI application after a reduced incubation time of 7 h of Staphylococcus aureus- and Escherichia coli-spiked (1,000, 100 and 10 colony-forming units [CFU]) aerobe BacT/ALERT blood cultures. Sixty-six (65%), 51 (50.5%) and 79 (78%) bottles were identified correctly at the species level when the centrifugation/washing method, MolYsis Basic 5 and Sepsityper were used, respectively. Incorrect identification was obtained in 35 (35%), 50 (49.5%) and 22 (22%) bottles, respectively. Gram-positive cocci were correctly identified in 33/52 (64%) of the cases. However, Gram-negative rods showed a correct identification in 45/47 (96%) of all bottles when the Sepsityper kit was used. Seven hours of pre-incubation of S. aureus- and E. coli-spiked aerobe BacT/ALERT blood cultures never resulted in reliable identification with MALDI-TOF MS. Sepsityper is superior for the direct identification of microorganisms from aerobe BacT/ALERT bottles. Gram-negative pathogens show better results compared to Gram-positive bacteria. Reduced incubation followed by MALDI-TOF MS did not result in faster reliable identification.
doi:10.1007/s10096-011-1480-y
PMCID: PMC3364411  PMID: 22080416
2.  Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods 
To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay.
doi:10.1007/s10096-010-1090-0
PMCID: PMC3034886  PMID: 20972809
3.  Evaluation of MeningoFinder, a Novel Multiplex Ligation-Dependent Probe Amplification Assay for Simultaneous Detection of Six Virus Species Causing Central Nervous System Infections▿  
Journal of Clinical Microbiology  2009;47(8):2620-2622.
A multiplex ligation-dependent probe amplification assay for simultaneous detection of six virus species was developed and tested on clinical cerebrospinal fluid (CSF) samples. The assay, termed MeningoFinder, showed an accordance of 97%, concordance of 96%, interlaboratory sensitivity of 90%, and interlaboratory specificity of 94% compared to PCRs.
doi:10.1128/JCM.02436-08
PMCID: PMC2725638  PMID: 19553572
4.  Preoperative radiochemotherapy is successful also in patients with locally advanced rectal cancer who have intrinsically high apoptotic tumours 
Annals of Oncology  2008;19(12):2026-2032.
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/mm2 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.
doi:10.1093/annonc/mdn428
PMCID: PMC2733117  PMID: 18664561
apotosis; locally advanced rectal carcinoma; radiochemotherapy
5.  Multicenter Comparison of Molecular Methods for Detection of Legionella spp. in Sputum Samples▿  
Journal of Clinical Microbiology  2007;45(10):3390-3392.
Legionellosis can be diagnosed by PCR using sputum samples. In this report, the methods of nine laboratories for 12 sputum samples with Legionella pneumophila and Legionella longbeachae are compared. We conclude that (i) liquefaction prevents PCR inhibition, (ii) the employed mip gene PCRs detected L. pneumophila only, and (iii) the 16S rRNA gene PCR detected both Legionella species and is preferred for the diagnosis of legionellosis.
doi:10.1128/JCM.00505-07
PMCID: PMC2045317  PMID: 17670926
6.  Quality control in diagnostic molecular pathology in the Netherlands; proficiency testing for patient identification in tissue samples 
Journal of Clinical Pathology  2004;57(7):717-720.
Aims: To describe the evolution of proficiency testing for molecular diagnostic pathology with respect to determining unambiguously the patient identity of tissue samples by microsatellite analysis.
Method: Four rounds of quality control exchanges of samples from different patients were sent with the purpose of identifying the correct origin of these samples. The samples were either paraffin wax embedded sections on glass, sections in tubes, or isolated DNA. Blinded samples were distributed to all participating laboratories. No restrictions to the method and short tandem repeat markers used for identification were imposed.
Results: In four subsequent rounds the number of participating laboratories increased from three to 10. The numbers of samples tested increased in time from five to 12. The microsatellite markers used by the different laboratories showed little overlap. In the first three rounds, in which isolated DNA was provided, all samples were accurately classified irrespective of the microsatellite markers used. In the last round, which also included paraffin wax embedded sections, a small number of laboratories experienced problems, either with amplification or incorrect classification of a few samples.
Conclusion: Proficiency testing was useful, and showed country wide high quality and correct identification of (patient) samples with molecular techniques for diagnostic purposes.
doi:10.1136/jcp.2003.011973
PMCID: PMC1770359  PMID: 15220364
molecular pathology; quality control; proficiency testing; sample identification; microsatellite analysis
7.  Epstein-Barr virus in gastric carcinomas and gastric stump carcinomas: a late event in gastric carcinogenesis 
Journal of Clinical Pathology  2004;57(5):487-491.
Background: To determine at what stage during gastric carcinogenesis Epstein-Barr virus (EBV) enters the gastric epithelial cells, the presence of EBV was investigated in two pathogenetically related but distinct forms of adenocarcinoma of the stomach—gastric carcinoma of the intact stomach (GCIS) and gastric stump carcinoma (GSC)—and their presumed precursor lesions.
Patients and methods: Eleven patients with EBV positive GCIS and eight patients with EBV positive GSC, demonstrated by the highly sensitive EBV encoded RNA 1/2 (EBER1/2) RNA in situ hybridisation (RISH) technique, were studied. Paraffin wax embedded tissue available from preoperative gastric biopsies and tumour adjacent tissue from the resection specimens containing normal gastric mucosa, inflamed gastric mucosa, and preneoplastic lesions (intestinal metaplasia and dysplasia) was investigated by EBER1/2 RISH, in addition to EBV nuclear antigen 1 (EBNA-1) and latent membrane protein 1 (LMP-1) immunohistochemistry (IHC).
Results: In both GCIS and GSC and their precursor lesions EBER1/2 transcripts were restricted to the carcinoma cells. In addition, positivity of EBNA-1 IHC was also restricted to the tumour cells. IHC for LMP-1 was negative in all cases tested.
Conclusions: The absence of EBER1/2 transcripts in preneoplastic gastric lesions (intestinal metaplasia and dysplasia) and their presence in two distinct types of gastric carcinoma strongly suggest that EBV can only infect neoplastic gastric cells and thus is a late event in gastric carcinogenesis.
doi:10.1136/jcp.2003.014068
PMCID: PMC1770299  PMID: 15113855
Epstein-Barr virus; gastric carcinogenesis; adenocarcinoma; stump carcinoma; late event
8.  Prevalence and determinants of Chlamydia trachomatis infections in women from Bogota, Colombia 
Sexually Transmitted Infections  2003;79(6):474-478.
Objectives: Chlamydia trachomatis infection in the cervix and uterus has been hypothesised to be a co-factor for cervical cancer. We performed a cross sectional study in Bogota, Colombia, where cervical cancer rates are high, to determine the prevalence and determinants of C trachomatis infection, and in particular its association with human papillomavirus (HPV).
Methods: 1829 low income sexually active women were interviewed and tested for C trachomatis, using an endogenous plasmid PCR-EIA, and for 37 HPV types, using a general primer GP5+/6+ mediated PCR-EIA.
Results: The overall prevalence of C trachomatis was 5.0%, and it did not differ substantially between women with normal (5.0%) and those with abnormal (5.2%) cervical cytology. Women infected with any HPV type (15.1%) had a slightly increased risk of being simultaneously infected with C trachomatis (adjusted OR 1.3, 95% CI: 0.8 to 2.4). This association was stronger when multiple HPV infections (adjusted OR 2.5, 95% CI: 1.1 to 5.9) were present. No other lifestyle or reproductive characteristics were clearly associated with risk of C trachomatis infection.
Conclusions: HPV infected women, particularly women with multiple HPV infections, are at increased risk of being infected with C trachomatis.
doi:10.1136/sti.79.6.474
PMCID: PMC1744784  PMID: 14663124
9.  HPV type concordance in sexual couples determines the effect of condoms on regression of flat penile lesions 
British Journal of Cancer  2005;92(8):1388-1392.
We earlier demonstrated, in a randomised clinical trial, that the regression time of flat penile lsions in male sexual partners of women with cervical intraepithelial neoplasia (CIN) was shorter in men who used condoms compared to those who did not. To further evaluate this finding, we examined whether the effect of condom use on the regression of flat penile lesions depends on the presence of human papillomavirus (HPV) type concordance in sexual couples, as determined in cervical and penile scrapes by GP5+/6+ PCR testing. A Cox model with time-dependent covariates showed a beneficial effect of condoms on regression of flat penile lesions in concordant couples (hazard ratio 2.63, 95% CI 1.07–6.48) but not in those who were nonconcordant. When both partners harboured different HPV types, no effect of condoms was found (hazard ratio 0.90, 95% CI 0.27–2.96). Delayed regression of flat penile lesions was associated with either stable lesions or with new penile lesions developing at sites surrounding pre-existing lesions suggesting reinfection of the penile epithelium. We conclude that condom use blocks sexual HPV transmission by preventing reinfection and development of new penile lesions in men who are susceptible to the same type as present in the female partner.
doi:10.1038/sj.bjc.6602524
PMCID: PMC2361997  PMID: 15812547
condom use; flat penile lesions; HPV type concordance
10.  Specific and Sensitive Detection of Neisseria gonorrhoeae in Clinical Specimens by Real-Time PCR 
Journal of Clinical Microbiology  2005;43(11):5653-5659.
Early diagnosis of Neisseria gonorrhoeae infections is important with regard to patients' health and infectivity. We report the development of a specific and sensitive TaqMan assay for the detection of N. gonorrhoeae in clinical samples. The target sequence is a 76-bp fragment of the 5′ untranslated region of the opa genes that encode opacity proteins. A panel of 448 well-defined N. gonorrhoeae isolates was used to evaluate and optimize the assay. The method employs two minor-groove binding probes, one of them recognizing a newly identified sequence in the opa genes. Testing a large panel of related and unrelated microorganisms revealed that other Neisseria strains and other microorganisms tested negative in the opa test. With a lower detection limit of one genome per reaction, the opa test appeared more sensitive than both the COBAS AMPLICOR (Roche Diagnostics Nederland BV, Almere, The Netherlands) and a LightCycler 16S rRNA test. Analysis of a panel of 122 COBAS AMPLICOR-positive samples revealed that 68% were negative in both the 16S rRNA test and the opa assay (confirming that the COBAS AMPLICOR test produces false positives), while 30% were positive in both assays. Three samples were opa positive and 16S rRNA negative, which may be due to the higher sensitivity of the opa assay. We conclude that the opa gene-based real-time amplification assay offers a sensitive, specific, semiquantitative, and reliable assay suitable for the detection of N. gonorrhoeae in clinical specimens and/or for confirmation of less specific tests.
doi:10.1128/JCM.43.11.5653-5659.2005
PMCID: PMC1287782  PMID: 16272500
11.  Comparison of Three Different PCR Methods for Quantifying Human Papillomavirus Type 16 DNA in Cervical Scrape Specimens 
Journal of Clinical Microbiology  2005;43(9):4868-4871.
We compared real-time LightCycler and TaqMan assays and the GP5+/6+ PCR/enzyme immunoassay (EIA) to assess the human papillomavirus type 16 (HPV16) load in cervical scrape specimens. Both real-time PCR assays determined the HPV16 load in scrape specimens similarly. The level of agreement between these assays and the GP5+/6+ PCR/EIA was low (P = 0.004), suggesting that the latter method is not suited for quantifying HPV16 DNA.
doi:10.1128/JCM.43.9.4868-4871.2005
PMCID: PMC1234115  PMID: 16145162
12.  Primary screening for high risk HPV by home obtained cervicovaginal lavage is an alternative screening tool for unscreened women 
Journal of Clinical Pathology  2002;55(6):435-439.
Background/Aims: Self sampling is considered an adjuvant tool to facilitate the participation of women in cervical cancer screening programmes. This study aimed to evaluate whether cervicovaginal lavage could be an alternative for the cervical smear in cytology and human papillomavirus (HPV) testing and to assess the acceptance of the self sampling device by women.
Methods: Fifty six women with abnormal cervical cytology (very mild dyskaryosis or worse) and 15 women with normal cervical cytology obtained a self collected cervicovaginal lavage at home and filled in a questionnaire on the use of the device. At the colposcopy clinic the gynaecologist performed the same procedure followed by a cervical smear for cytology and HPV DNA testing.
Results: The self sampling device was acceptable to 88% of the women. The concordance between the cytology results in the smear and the lavage by the doctor and the patient was 54% and 41%, respectively (κ = 0.28 and 0.14). The concordance between high risk HPV detection in the smear and the lavage by the doctor and the patient was 93% and 78%, respectively (κ = 0.82 and 0.53). Ninety one per cent of the women with high grade cervical intraepithelial neoplasia (CIN) had a high risk HPV positive test in the smear, compared with 91% and 81% in the lavages taken by the doctor and the patient, respectively.
Conclusions: HPV DNA testing by home obtained samples is useful as a screening tool for cervical cancer, whereas cervical cytology by self sampling is not. Although the sensitivity for high grade CIN by high risk HPV testing in the lavage by the patient is not significantly lower than that in the cervical smear, self sampling for HPV DNA is a feasible alternative method in women who decline to participate in population based cervical cancer screening programmes. However, participation in the screening programme remains the best option.
PMCID: PMC1769675  PMID: 12037026
human papillomavirus; cervicovaginal lavage; self sampling; cervical cytology; cervical dysplasia
13.  Risk factors for genital HPV DNA in men resemble those found in women: a study of male attendees at a Danish STD clinic 
Sexually Transmitted Infections  2002;78(3):215-218.
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.
doi:10.1136/sti.78.3.215
PMCID: PMC1744457  PMID: 12238658
14.  Evaluation of Conventional and Real-Time PCR Assays Using Two Targets for Confirmation of Results of the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae Test for Detection of Neisseria gonorrhoeae in Clinical Samples 
Journal of Clinical Microbiology  2005;43(5):2231-2235.
Two conventional PCR-enzyme immunoassays (PCR-EIAs) and two real-time PCR assays (LightCycler system; Roche Diagnostics) were evaluated as confirmation assays with cppB and 16S rRNA genes as targets. Of 765 male and female genitourinary and nasopharyngeal specimens positive for Neisseria gonorrhoeae in the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR test (Roche Diagnostics), 229 (30%) were confirmed positive; 13 of these (5.7%) were lacking the cppB gene. Of the 534 samples (70%) that could not be confirmed, 81 (15%) showed a positive crossing point. However, melting curve analysis revealed an aberrant melting temperature in the LightCycler 16S rRNA assay; therefore, these samples were considered non-N. gonorrhoeae Neisseria species. Both of the 16S rRNA assays performed well, with positive predictive values of 99.1% and 100% for the PCR-EIAs and the real-time assays, respectively, and a negative predictive value of 99.8% for both. The cppB assays were compromised by the absence of the cppB gene in 5.7% of the N. gonorrhoeae-positive samples, resulting in negative predictive values of 96.8% and 97.6% for the PCR-EIAs and the real-time assays, respectively. Therefore, the 16S rRNA gene is preferable to the cppB gene as a target for confirmation assays. The melting curve analysis of the real-time assays provides useful additional information.
doi:10.1128/JCM.43.5.2231-2235.2005
PMCID: PMC1153731  PMID: 15872247
15.  Multicenter Validation of the cppB Gene as a PCR Target for Detection of Neisseria gonorrhoeae 
Journal of Clinical Microbiology  2004;42(9):4332-4334.
The cppB gene is often used as a target for detection of Neisseria gonorrhoeae by PCR. Using a coded panel of 500 DNA samples, we determined that the cppB gene is missing in 5.8% of N. gonorrhoeae strains, and therefore we consider the cppB gene to be an unsuitable target.
doi:10.1128/JCM.42.9.4332-4334.2004
PMCID: PMC516305  PMID: 15365038
16.  Notice of Redundancy 
doi:10.1136/sti.77.4.301
PMCID: PMC1744327  PMID: 11463940
17.  Cost effectiveness analysis of a population based screening programme for asymptomatic Chlamydia trachomatis infections in women by means of home obtained urine specimens 
Sexually Transmitted Infections  2001;77(4):276-282.
Objectives: To evaluate the cost effectiveness of a systematic screening programme for asymptomatic Chlamydia trachomatis infections in a female inner city population. To determine the sensitivity of the cost effectiveness analysis to variation in the probability of developing sequelae.
Methods: A decision tree was constructed to evaluate health effects of the programme, such as averted sequelae of chlamydial infection. Cost effectiveness from a societal perspective was estimated for screening by means of a ligase chain reaction on mailed, home obtained urine specimens, in a population with a C trachomatis test prevalence of 2.9%. An extensive sensitivity analysis was performed for the probability of sequelae, the percentage of preventable pelvic inflammatory disease (PID), and the discount rate.
Results: The estimated net cost of curing one woman, aged 15–40 years, of a C trachomatis infection is US$1210. To prevent one major outcome (PID, tubal factor infertility, ectopic pregnancy, chronic pelvic pain, or neonatal pneumonia), 479 women would have to be screened. The net cost of preventing one major outcome is $15 800. Changing the probability of PID after chlamydial infection from 5% to 25% decreases the net cost per major outcome averted from $28 300 to $6380, a reduction of 78%. Results were less sensitive to variations in estimates for other sequelae. The breakeven prevalence of the programme ranges from 6.4% for the scenario with all probabilities for complications set at the maximum value to a prevalence of 100% for probabilities set at the minimum value.
Conclusions: Systematic screening of all women aged 15–40 years for asymptomatic C trachomatis infections is not cost effective. Although the results of the analyses are sensitive to variation in the assumptions, the costs exceed the benefits, even in the most optimistic scenario.
Key Words: cost effectiveness analysis; Chlamydia trachomatis; screening
doi:10.1136/sti.77.4.276
PMCID: PMC1744325  PMID: 11463928
18.  Low grade squamous intra-epithelial lesions and human papillomavirus infection in Colombian women 
British Journal of Cancer  2002;87(12):1417-1421.
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
doi:10.1038/sj.bjc.6600650
PMCID: PMC2376287  PMID: 12454771
LSIL; HPV; C. trachomatis; Colombia; risk factors
19.  Low diagnostic accuracy of selective screening criteria for asymptomatic Chlamydia trachomatis infections in the general population 
Sexually Transmitted Infections  2000;76(5):375-380.
Objectives: To develop and validate selective screening criteria for asymptomatic Chlamydia trachomatis infections in the general population.
Methods: 11 505 people, aged 15–40 years, registered in 16 general practices in Amsterdam were invited to return by mail a home obtained first void urine sample and a questionnaire. Participants were randomly allocated into a development group (75%) or a validation group (25%). C trachomatis infection was determined by the ligase chain reaction. In the development group a set of criteria was identified by means of stepwise logistic regression analysis. The diagnostic accuracy (area under the ROC curve; AUC) and sensitivity, and the corresponding percentage of people selected for screening were calculated. The criteria developed in this study were applied to the validation group.
Results: The prevalence of asymptomatic C trachomatis infections among men was found to be 2.4% (1.7–3.0), and among women 2.8% (2.2–3.4). Screening men, based on Surinam/Antillean origin and painful micturition, yielded an AUC of 0.58 (0.55–0.60). Screening women, based on Surinam/Antillean origin, new sex partner in the previous 2 months, and unmarried/not cohabiting, yielded an AUC of 0.67 (0.65–0.69). Application of the criteria for men to the validation group yielded an AUC of 0.53 (0.48–0.57); by screening 10% of the men, 15% of the cases were detected. The AUC of the criteria for women in the validation group was 0.58 (0.54–0.61); by screening 51% of the women, 63% of the cases were detected.
Conclusion: The prevalence of asymptomatic C trachomatis infections in Amsterdam is less than 3%. No suitable selective screening criteria for the general population could be identified.
Key Words: screening; prevalence; Chlamydia trachomatis
doi:10.1136/sti.76.5.375
PMCID: PMC1744221  PMID: 11141855
20.  Microorganisms in the aetiology of atherosclerosis 
Journal of Clinical Pathology  2000;53(9):647-654.
Recent publications have suggested that infective pathogens might play an important role in the pathogenesis of atherosclerosis. This review focuses on these microorganisms in the process of atherosclerosis. The results of in vitro studies, animal studies, tissue studies, and serological studies will be summarised, followed by an overall conclusion concerning the strength of the association of the microorganism with the pathogenesis of atherosclerosis. The role of the bacteria Chlamydia pneumoniae and Helicobacter pylori, and the viruses human immunodeficiency virus, coxsackie B virus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus, and measles virus will be discussed.
Key Words: atherosclerosis • Chlamydia pneumoniae • Helicobacter pylori
doi:10.1136/jcp.53.9.647
PMCID: PMC1731245  PMID: 11041053
21.  Prevalence and determinants of HPV infection among Colombian women with normal cytology 
British Journal of Cancer  2002;87(3):324-333.
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
doi:10.1038/sj.bjc.6600442
PMCID: PMC2364213  PMID: 12177803
HPV; Colombia; epidemiology; cervix uteri
22.  High-risk human papillomavirus clearance in pregnant women: trends for lower clearance during pregnancy with a catch-up postpartum 
British Journal of Cancer  2002;87(1):75-80.
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<0.005). Such a phenomenon was not found in non-pregnant women. Pregnant women showed a trend for increased high-risk human papilloma virus clearance during the third trimester and postpartum compared to non-pregnant women (hazard ratios 3.3 (0.8–13.7) and 4.6 (1.6–12.8), respectively). These results suggest a lowered immune-response against human papilloma virus during the first two trimesters of pregnancy with a catch-up postpartum.
British Journal of Cancer (2002) 87, 75–80. doi:10.1038/sj.bjc.6600367 www.bjcancer.com
© 2002 Cancer Research UK
doi:10.1038/sj.bjc.6600367
PMCID: PMC2364279  PMID: 12085260
human papillomavirus; pregnancy; follow-up study; natural history; prevalence
23.  Prevalence of Chlamydia trachomatis in urine of male patients with ankylosing spondylitis is not increased 
Annals of the Rheumatic Diseases  2000;59(4):300-302.
OBJECTIVE—To compare the prevalence of Chlamydia trachomatis infections in ankylosing spondylitis (AS) patients with controls, using DNA amplification assays in urine specimens.
METHODS—The prevalence of C trachomatis infections was assessed in 32 male AS patients and 120 age and sex matched controls. Urine specimens were tested by ligase chain reaction and polymerase chain reaction. In addition, blood samples of AS patients were tested on serum antibodies to C trachomatis (IgA and IgG) by a specific peptide based solid phase enzyme immunoassay. A questionnaire was used to assess the differences in sexual behaviour and ethnic origin between the two groups. AS patients were also asked about disease characteristics.
RESULTS—No significant differences were found between cases and controls in the prevalence of C trachomatis infections. No associations were found between C trachomatis antibodies and disease characteristics, except for acute anterior uveitis (AAU). Four of eight (50%) AS men positive for IgG had a history of AAU in comparison with three of 24 (12.5%) IgG negative men (OR = 7.0; 95% confidence intervals: 1.1, 44.1).
CONCLUSION—The prevalence of Chlamydia trachomatis infections, as detected by commercially available DNA amplification assays in urine specimens, in AS patients is not higher compared with male controls of the same age. However, there seems to be an association between specific antibodies to C trachomatis and AAU.


doi:10.1136/ard.59.4.300
PMCID: PMC1753118  PMID: 10733479
24.  Activity of the EBNA1 promoter associated with lytic replication (Fp) in Epstein-Barr virus associated disorders 
Molecular Pathology  2001;54(2):98-102.
Background/Aims—In Epstein-Barr virus (EBV) positive cell lines that are stably infected, three different promoters are known to direct the transcription of EBV nuclear antigen 1 (EBNA1). These are located in the BamHI-C, BamHI-Q, and BamHI-F regions of the viral genome (Cp, Qp, and Fp, respectively). Fp is activated upon induction of the viral lytic cycle. The aim of this study was to investigate the activity of Fp in EBV associated diseases.
Methods—Using reverse transcriptase polymerase chain reaction, a qualitative analysis of EBNA1 promoter usage in various EBV associated diseases was performed.
Results—Fp driven transcription was detected in the context of primary infection and/or lytic replication; at least a portion of the Fp driven transcripts encoded EBNA1. Qp driven EBNA1 transcripts were detected in most samples across the range of disorders tested. Cp driven EBNA1 transcripts were detected in the context of immune suppression and in samples containing EBV positive (non-neoplastic) lymphoid cells.
Conclusions—These results confirm the previously proposed “housekeeping” function of the Qp promoter.
PMCID: PMC1187010  PMID: 11322171
Epstein-Barr virus; Epstein-Barr virus nuclear antigen 1; BamHI-F region
25.  Microarray techniques in pathology: tool or toy? 
Molecular Pathology  2000;53(6):289-294.
Microarray technology allows the simultaneous analysis of up to thousands of different genes in histological or cytological specimens. Although microarray technology has so far mainly been applied in the research setting, its clinical application in pathology is expected in the foreseeable future. This paper presents an overview of the technical “ins and outs” of microarray technology, and discusses several putative applications in diagnostic pathology, which include tumour classification, the prediction of responses to certain chemotherapeutical or hormonal agents, the biological staging of tumours, the risk assessment of premalignant lesions, and the detection of microorganisms.
PMCID: PMC1186982  PMID: 11193046
microarray technology; diagnostic pathology; comparative genomic hybridisation; gene sequencing; gene expression

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