Anal infections with concomitant Chlamydia trachomatis genotypes among men who have sex with men in Amsterdam, the Netherlands.

<p>Abstract</p> <p>Background</p> <p>Lymphogranuloma venereum (LGV) proctitis is caused by <it>Chlamydia trachomatis </it>(Ct) genotype L and is endemic among men who have sex with men (MSM) in western society. Genotype L infections need to be distinguished from non-LGV (genotypes A-K) Ct infections since they require prolonged antibiotic treatment. For this purpose, an in-house developed <it>pmpH </it>based LGV polymerase chain reaction (PCR) test is used at the Amsterdam STI outpatient clinic. We investigated retrospectively the anal Ct genotype distribution, and the frequency of concomitant genotype infections in MSM infected with LGV and non-LGV Ct infections. To detect concomitant Ct genotype infections, the <it>pmpH </it>LGV PCR and genoTyping Reverse Hybridization Assay (Ct-DT RHA) were used.</p> <p>Methods</p> <p>A total of 201 Ct positive rectal swabs from MSM were selected, which were previously diagnosed as either LGV (n = 99) or non-LGV Ct infection (n = 102) according to the algorithm of Ct detection by the commercially available Aptima Combo 2 assay followed by an in-house <it>pmpH </it>LGV PCR. The samples were retested with the commercially available Ct-DT RHA, which differentiates between 14 major genotypes and is able to detect concomitant Ct genotypes.</p> <p>Results</p> <p>Excellent genotyping agreement was observed between the Ct-DT RHA and the <it>pmpH </it>LGV PCR (Kappa = 0.900, 95%CI = 0.845-0.955, McNemar's p = 1.000). A concomitant non-LGV genotype was detected in 6/99 (6.1%) LGV samples. No additional LGV infections were observed with the Ct-DT RHA among the non-LGV Ct group. In the non-LGV group genotype G/Ga (34.3%) was seen most frequent, followed by genotype D/Da (22.5%) and genotype J (13.7%). All LGV infections were caused by genotype L2.</p> <p>Conclusions</p> <p>Concomitant non-LGV genotypes do not lead to missed LGV proctitis diagnosis. The <it>pmpH </it>LGV PCR displayed excellent agreement with the commercially available Ct-DT genotyping RHA test. The genotypes G/Ga, D/Da and J were the most frequent non-LGV Ct strains in MSM.</p

Evaluation of a Novel Multiplex Human Papillomavirus (HPV) Genotyping Assay for HPV Types in Skin Warts

Public Health and primary careMinor Ailment

Sequence analysis of the 5' untranslated region in isolates of at least four genotypes of hepatitis C virus in The Netherlands

The RNAs of hepatitis C virus (HCV) isolates from 62 patients with chronic HCV infection were analyzed by direct sequencing of the 5' untranslated region. Two important sequence motifs were recognized: one between positions -170 and -155 and the other between positions -132 and -117. These motifs are partly complementary. All three previously published genotypes were observed; 34 (55%) isolates were classified as type 1 (including prototype [from the United States] and HCV-BK [from Japan] sequences), 11 (18%) were classified as type 2 (including HC-J6 and HC-J8), and 12 (19%) were classified as type 3 (including EB1); one patient was infected with genotypes 1 and 2. Four (6%) isolates showed aberrant sequences and were therefore provisionally classified as genotype 4. These results indicate the significance of sequence variation among the 5' untranslated regions of different HCV genotypes and indicate that this region could possibly be used for consistent genotyping of HCV isolates

Influence of volume of sample processed on detection of Chlamydia trachomatis in urogenital samples by PCR

In the present study, it was demonstrated that the sensitivity of the PCR for the detection of Chlamydia trachomatis is influenced by the volume of the clinical sample which is processed in the PCR. An adequate sensitivity for PCR was established by processing at least 4%, i.e., 80 microliters, of the clinical sample volume per PCR. By using this preparation procedure, 1,110 clinical samples were evaluated by PCR and by cell culture, and results were compared. After discordant analysis, cell culture resulted in a sensitivity of 79.1% and PCR resulted in a sensitivity of 92.7%. Furthermore, it was shown that treatme

Evaluation of Clearview and Magic Lite tests, polymerase chain reaction, and cell culture for detection of Chlamydia trachomatis in urogenital specimens

The Clearview Chlamydia test (CV; Unipath Ltd., Bedford, United Kingdom), the Magic Lite Chlamydia test (ML; CIBA Corning, Medfield, Mass.), a polymerase chain reaction (PCR), and cell culture (CC) were evaluated for detection of Chlamydia trachomatis in urogenital specimens. Specimens were collected from 283 men and 724 women visiting the outpatient clinic for Sexually Transmitted Diseases at the University Hospital Rotterdam, Rotterdam, The Netherlands. ML, PCR, and CC were all performed on the same sample to prevent swab-to-swab variability. CV was performed on a separate sample. Analysis of discordant results was performed by application of the following confirmatory assays: first, PCR on th

Rapid genotyping of hepatitis C virus RNA-isolates obtained from patients residing in Western Europe

Two rapid genotyping methods for hepatitis C virus (HCV), the line probe assay (Inno‐LiPA) and the subtype‐specific core amplification system [Okamoto et al., (1992b) Journal of General Virology 73:673‐679], were applied to 58 HCV isolates which were typed as type 1 (n=37) and type 2 (n=21) by sequence analysis of the 5′ untranslated region (5′UTR). The line probe assay targets the 5′UTR and recognized 12 subtype 1a, 25 subtype 1b, 18 subtype 2a, 2 subtype 2b and 1 subtype 2d in accordance with sequence analysis of this region. Subtype‐specific core amplification revealed 7 discrepancies among the 37 type 1 isolates when compared to LiPA. A different subtype was observed in 3 isolates (la versus 1b), 2 isolates remained untyped and 2 isolates showed a coinfection of subtype la and 1b. The first 5 discrepancies were confirmed by sequence analysis of the core region whereas the coinfection could not be confirmed. Of the 21 type 2 isolates only one could be typed by subtype‐specific core amplification. HCV RNA was detected in all 21 cases after the general first round of polymerase chain reaction (PCR). Direct sequencing of the core region indicated sequence variation as a source of failure. It is concluded that LiPA results are conclusive for typing of HCV. However, LiPA is hampered occasionally for subtyping by lack

Development of a species-specific polymerase chain reaction assay for Gardnerella vaginalis

The nucleotide sequence of the region between the 16S and 23S rRNA genes of the facultative anaerobic bacteriumGardnerella vaginalishas been determined, together with the 5′ proximal 500 nucleotides of the 23S rRNA gene. Regions suited for the development of specific, probe-confirmable polymerase chain reaction (PCR) assays were selected. PCR assays were evaluated with respect to sensitivity and specificity, the latter in comparison with a number ofG. vaginalisreference strains and closely related species likeBifidobacteriumspp. In an initial diagnostic study it appeared that the PCR test detectedG. vaginalisin 40% of women irrespective of their clinical status. Ten out of 11 patients suffering from bacterial vaginosis as defined on the basis of clinical parameters were carryingG. vaginalis

Expression of p16 and HPV E4 on biopsy samples and methylation of FAM19A4 and miR124-2 on cervical cytology samples in the classification of cervical squamous intraepithelial lesions

The decision to treat a cervical squamous intraepithelial lesion (SIL) by loop electrosurgical excision procedure (LEEP) relies heavily on a colposcopy-directed biopsy showing high-grade (H)SIL. Diagnosis is often supported by p16, an immunohistochemical (IHC) biomarker of high-risk (hr)HPV E7 gene activity. Additional potential markers include methylation of tumor suppressor genes FAM19A4/miR124-2 in cervical cytology for advanced transforming HSIL and the IHC marker HPV E4 for productive, potentially regressing lesions. In 318 women referred for colposcopy, we investigated the relationship between staining patterns of p16 and E4 IHC in the worst biopsy, and the relation of these to FAM19A4/miR124-2 methylation status in cytology. E4-positive staining decreased with increasing SIL/CIN grade from 41% in LSIL to 3% in HSIL/CIN3. E4 positivity increased with grade of p16 when p16 expression was limited to the lower two third of the epithelium (r = 0.378), but fell with expression over. Loss of E4 expression in the worst lesion was associated with the methylation of FAM19A4/miR124-2. We also examined whether these biomarkers can predict the histological outcome of the LE