The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis.

BACKGROUND: Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. METHODS: To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. RESULTS: dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. CONCLUSIONS: TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.

Genital ulcers and concomitant complaints in men attending a sexually transmitted infections clinic: implications for sexually transmitted infections management.

BACKGROUND: Although genital herpes has emerged as the most common cause of genital ulcers in Southern Africa, treatment for herpes is not available routinely in the region. This study was performed to determine the etiology of genital ulcers in men in Durban and assess other sexually transmitted infections-related symptoms, presentation, and treatment patterns in this group. METHODS: Polymerase chain reaction (PCR) tests were performed on specimens from consecutive male patients with genital ulcers to detect sexually transmitted pathogens. PCR was also performed for the detection of Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis on urethral specimens from consecutive subjects with dysuria or urethral discharge. Antibody tests for syphilis and herpes simplex virus type-2 (HSV-2) and human immunodeficiency virus antibodies were performed. RESULTS: Of 162 patients enrolled with genital ulcers, 77.7% were human immunodeficiency virus-positive and 84.6% had antibodies to HSV-2. PCR results showed the following prevalences: HSV-2 53.7%, lymphogranuloma venereum 13.6%, Treponema pallidum 3.7%, Hemophilus ducreyi 1.2%, mixed infections 6.2%, and no pathogens identified 33.3%. One case of donovanosis was diagnosed clinically. In men with HSV-2 ulcers, delay before attendance recorded for 68 men was 1 to 3 days (24%), 4 to 7 days (47%), 8 to 14 days (12%), 15 to 30 days (12%), and >30 days (6%). History-taking using prompting increased the sensitivity but decreased the specificity and positive predictive value of reported genital ulceration when assessed against ulcers seen on examination. CONCLUSIONS: Men at risk of genital ulcers should be asked about relevant symptoms with and without prompting and examined clinically to maximize the likelihood of correct diagnosis and treatment. The finding of a high prevalence of HSV-2 and associated dysuria cautions against providing empirical treatment for gonorrhoea and chlamydia in ulcer patients with dysuria but without urethral discharge. Innovative strategies to limit the burden of HSV-2 infection in this population are required.

Association between HIV and subpreputial penile wetness in uncircumcised men in South Africa.

OBJECTIVES: To describe the prevalence and characteristics of subpreputial penile wetness and to investigate the association between current levels of penile wetness and HIV infection. METHODS: Male attenders at a sexually transmitted infections clinic in Durban, South Africa were enrolled and treated for their presenting sexually transmitted infection complaint. They were asked to return after 14 days when a structured questionnaire was administered, and the degree of wetness of the glans penis and coronal sulcus was assessed clinically. RESULTS: Six hundred and fifty men were enrolled, and 488 (75%) returned. Three hundred eighty-six uncircumcised men were included for statistical analysis of whom 215 (56%) were HIV positive. One hundred ninety-six (50.8%) had no penile wetness, and 190 (49.2%) had penile wetness. In the adjusted analysis, penile wetness was associated with younger age, low level of attained education, low income, higher lifetime numbers of sexual partners, and not washing after sex. The prevalence of HIV was greater in those with penile wetness 126 of 190 (66.3%) compared with 90 of 196 (45.9%) with no penile wetness, crude prevalence odds ratio 2.32 (95% confidence interval [CI], 1.54-3.50, P=or<0.001), crude prevalence relative risk 1.44 (95% CI, 1.23-1.63, P=or<0.001), and adjusted for predictors of HIV, confounders and herpes type 2 antibodies, odds ratio 2.38 (95% CI, 1.42-3.97, P=or<0.001), and relative risk 1.46 (95% CI, 1.19-1.68, P=or<0.001). CONCLUSIONS: This is the first study to show an association between subpreputial penile wetness and HIV. Consideration should be given to providing advice about improving penile hygiene in uncircumcised men in areas where HIV is a significant problem. Good penile hygiene should also be promoted at the community level to become a desirable social norm.

Molecular epidemiology of recently emergent ciprofloxacin-resistant Neisseria gonorrhoeae in South Africa.

OBJECTIVES: Syndromic management guidelines for male urethritis syndrome and female discharge syndrome (nonpregnant) in South Africa advocate the use of ciprofloxacin for potential infection with Neisseria gonorrhoeae. In 2003, reports of clinical failure of gonorrhea following ciprofloxacin treatment prompted a clinic-based surveillance to detect the presence of resistant isolates. STUDY: Urethral samples for the isolation of N gonorrhoeae were obtained from consecutive male patients with urethral discharge attending the largest sexually transmitted disease clinic in KwaZuluNatal. Molecular typing of isolates was performed by means of N gonorrhoeae multiantigen sequence typing (NG-MAST). RESULTS: Of 139 isolates, 31 (22%) were resistant to ciprofloxacin (minimum inhibitory concentration >or=1 mg/l). NG-MAST revealed novel, as well as previously described, sequence types (ST). The largest cluster of our isolates belonged to ST 217. This ST has been reported among ciprofloxacin-resistant isolates from Europe. CONCLUSION: : The results underscore the urgency of review of the current treatment guidelines for discharge disease in KwaZuluNatal.