Christopher Sonnex

Chlamydia trachomatis Load at Matched Anatomic Sites: Implications for Screening Strategies

ABSTRACT Urethral and endocervical swabs and self-collected vaginal swabs (SCVSs) and urine specimens are all used as samples for diagnosis of urogenital infection with Chlamydia trachomatis. We have now determined chlamydial organism load in matched specimens from different anatomic sites and examined its relation to clinical signs and symptoms in men and women. Organism load was measured with assays based on the ligase chain reaction or real-time PCR analysis. The mean organism loads in 58 infected men were 1,200 and 821 elementary bodies (EBs) per 100 μl of sample for first-void urine (FVU) and urethral swabs, respectively (P > 0.05). Organism load in FVU samples or urethral swabs was positively associated with symptoms (P < 0.01) and clinical signs (P < 0.01) in men. The mean organism loads in 73 infected women were 2,231, 773, 162, and 47 EBs/100 μl for endocervical swabs, SCVSs, urethral swabs, and FVU samples, respectively (P < 0.001 for each comparison). Only the presence of multiple symptoms or clinical signs was associated with organism load in women. These results show that FVU is a suitable noninvasive sample type for men, given the fact that its chlamydial load did not differ significantly from that of urethral swabs. Given their higher organism load compared with FVU, SCVSs are the preferred noninvasive sample type for women.

Development of Real-Time PCR Assays for Genotyping of Chlamydia trachomatis

ABSTRACT We have developed and validated a nested real-time PCR (NRT-PCR) for the genotyping of Chlamydia trachomatis and used it specifically for the typing of either eight genovars from D to K or three genovars of lymphogranuloma venereum (LGV). The 11 probes used in the NRT-PCR correctly identified the DNA from D to K and LGV reference strains and did not cross-react with the DNA from 26 strains representing the bacterial pathogens and commensals of the oropharynx, genital tract, and rectum. The NRT-PCR had a 95% probability of detection at four genome copies (confidence interval, three to six copies) of C. trachomatis per reaction. One hundred cervical and urethral swab specimens containing C. trachomatis DNA from 63 women and 37 men were used to validate the method. The results from the NRT-PCR and the DNA sequencing of amplicons generated from the omp1 gene showed 100% correlation for these samples. The assay also identified the LGV-II genotype in 24 of 48 rectal swab specimens containing C. trachomatis DNA that were obtained from men having sex with men. The Sexually Transmitted Bacteria Reference Laboratory, London, independently confirmed these results using group-specific LGV real-time PCR and restriction fragment length polymorphism analysis. Compared with the NRT-PCR, non-NRT-PCR was found to be less sensitive: it typed C. trachomatis DNA in only 80% of the genital samples and 90% of the rectal swab samples. This is the first successful demonstration of the use of real-time PCR for the genotype-specific typing of C. trachomatis strains that cause sexually transmitted diseases.

Detection of specific HPV subtypes responsible for the pathogenesis of condylomata acuminata

BackgroundThe low-risk human papillomavirus types 6 and 11 are responsible for approximately 90% of anogenital wart cases, with approximately 190,000 new and recurrent cases reported in the UK in 2010. The UK has recently selected the quadrivalent HPV vaccine, which conveys protection against both HPV6 and HPV 11, as part of its immunisation programme for 2012 and it is expected that this will reduce disease burden in the UK. The aims of the study were to evaluate current strategies used for the monitoring of HPV infection in genital warts and to assess the suitability of laser-capture microdissection (LCM) as a technique to improve the understanding of the natural history of HPV types associated with genital wart lesions.MethodsDNA and RNA were extracted from whole wart, surface swabs and LCM sections from 23 patients. HPV types present were determined using the Linear Array HPV Genotyping Test (Roche), with HPV DNA viral load and mRNA expression investigated using qPCR and qRT-PCR, respectively.ResultsResults indicated that swabbing the surface of warts does not accurately reflect potential causative HPV types present within a wart lesion, multiple HPV types being present on the surface of the wart that are absent in the lower layers of tissue isolated by LCM. Although it was shown that HPV DNA viral load does not directly correlate with HPV mRNA load, the presence of both DNA and mRNA from a single HPV type suggested a causative role in lesion development in 8/12 (66.6%) of patients analysed, with dual infections seen in 4/12 (33.3%) cases. HPV 6 and HPV 11 were present in more than 90% of the lesions examined.ConclusionsSurface swabbing of warts does not necessarily reflect the causative HPV types. HPV type specific DNA and mRNA loads do not correlate. HPV 6 and 11 were likely to be causally involved in over 90% of the lesions. Dual infections were also found, and further studies are required to determine the biological and clinical nature of dual/multiple infections and to establish the relationship of multiple HPV types within a single lesion.

Genital chlamydial infection: association between clinical features, organism genotype and load

The association between the clinical features of genital chlamydial infection and organism genotype and load was evaluated. Chlamydial DNA was detected and quantified in genital swabs from 233 (7 %) of 3384 consecutive patients attending a genitourinary medicine clinic. The chlamydia-positive subcohort comprised 132 (57 %) females and 101 (43 %) males. Clinical features were present in 33 % women and 72 % men. The chlamydial load was found to be higher in women (median load: 5.6 log) than men (median load: 3.5 log). Single variable analysis failed to show a significant association between chlamydial load and clinical features (P value = 0.3). Owing to the limited amount of clinical material, information on chlamydial genotypes was available for 70 % (n = 162) of chlamydia-positive patients. However, multivariable analysis of these samples did show a significant association between chlamydial load and clinical features (P value = 0.02). This discrepancy is most probably due to the difference in the amount of data analysed by single variable (data from 233 patients) and multivariable (data from 162 patients) analysis. The distribution of chlamydia genotypes was as follows: type E (46 %), F (22 %), D (8 %), K (8 %), G (7 %), J (4 %), I (1 %) and H (0.6 %). No statistically significant association was observed between chlamydial genotype and clinical features in either single variable (P value = 0.6) or multivariable (P value = 0.4) analysis. These findings suggest that chlamydial load and diversity in the ompA gene plays little, if any, role in the pathogenesis of genital chlamydial infection.

Prevalence, clinical features and quantification of genital non-viral infections.

Summary We conducted a study of the prevalence, clinical features and microscopy findings, by retrospective case-notes survey, of six non-viral organisms, among 1718 attendees at a genitourinary (GU) medicine clinic in England. An in-house assay for six non-viral infections was used and quantitation of ureaplasmas performed. The prevalences of the six organisms were: Chlamydia trachomatis (CT), 7.1%; Neisseria gonorrhoeae (NG), 0.6%; Mycoplasma genitalium (MG), 1.0%; Trichomonas vaginalis, 0.2%; Ureaplasma urealyticum, 16.1%; Ureaplasma parvum, 35.6%. Among men (but not women) there were significantly raised odds ratios compared with that for U. parvum, for the symptom of discharge with CT, 7.30; MG, 6.43; NG 19.29; dysuria with CT, 5.89 and MG, 5.95; and the microscopy finding of >4 pus cells per high power field with: CT, 7.22; MG, 4.58 and NG 22.31. Evaluation of a possible link between quantitation of U. urealyticum and urethritis did not confirm research findings elsewhere.

Molecular epidemiology of genital human papillomavirus and Chlamydia trachomatis among patients attending a genitourinary medicine clinic - : will vaccines protect?

High-risk subtypes of human papillomavirus (HPV) are the main causative agents of cervical cancer, for which Chlamydia trachomatis (CT) may sometimes be a co-factor. Vaccines have been developed against some subtypes of human papillomavirus and a vaccine against CT is in development. The objective of this study was to determine the prevalence of the subtypes of HPV and CT in genitourinary (GU) medicine clinic attenders. In total, 1000 consecutive patients attending the GU clinic participated in this anonymized point-prevalence study. Urethral swabs from 437 men and urethral plus cervical swabs as a single specimen from 563 women were tested for the subtypes of both organisms. Nested major outer membrane protein (MOMP) polymerase chain reaction detected CT chromosomal DNA in 44/437 (10%) of the men and 73/563 (13%) of the women. Genotypes E, F, and D were the most common. In all, 55/437 (13%) of men and 244/563 (43%) of women were infected with at least one high-risk HPV type. In conclusion, the new HPV vaccines, Gardasil and Cervarix, would have protected against 58% and 45%, respectively, of the high-risk subtypes found in women in this population. The rate of high-risk HPV infection (43%) found in women in this study raises concern.

Testing for ‘threads’ and leucocyte esterase in first-void urine to exclude the diagnosis of non-specific urethritis in asymptomatic men

Recent evidence suggests that asymptomatic nonspecific urethritis (NSU), which is not routinely tested for, is a clinically significant pathology.The aim of this pilot study was to determine if testing for urinary threads, leucocyte esterase (LE) or both in asymptomatic men is a good screening tool for NSU. Of the126 asymptomatic men, 8% met microscopic criteria for the diagnosis of NSU. The positive predictive value for NSU was 71% (95% confidence interval (CI): 29.3-95.5%) and the negative predictive value was 96% (95% CI: 92.8-99.5%). The absence of threads and negative LE makes urethritis highly unlikely, making urinary chlamydia (Chlamydia trachomatis) and gonorrhoea (Neisseria gonorrhoeae) testing sufficient. Incidental findings of further pathology occurred in 7%.

Reply to Lascar et al., December 2011.

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