Results from the second WHO external quality assessment for the molecular detection of respiratory syncytial virus, 2019-2020.

Background: External quality assessments (EQAs) for the molecular detection of human respiratory syncytial virus (RSV) are necessary to ensure the standardisation of reliable results. The Phase II, 2019-2020 World Health Organization (WHO) RSV EQA included 28 laboratories in 26 countries. The EQA panel evaluated performance in the molecular detection and subtyping of RSV-A and RSV-B. This manuscript describes the preparation, distribution, and analysis of the 2019-2020 WHO RSV EQA. Methods: Panel isolates underwent whole genome sequencing and in silico primer matching. The final panel included nine contemporary, one historical virus and two negative controls. The EQA panel was manufactured and distributed by the UK National External Quality Assessment Service (UK NEQAS). National laboratories used WHO reference assays developed by the United States Centers for Disease Control and Prevention, an RSV subtyping assay developed by the Victorian Infectious Diseases Reference Laboratory (Australia), or other in-house or commercial assays already in use at their laboratories. Results: An in silico analysis of isolates showed a good match to assay primer/probes. The panel was distributed to 28 laboratories. Isolates were correctly identified in 98% of samples for detection and 99.6% for subtyping. Conclusions: The WHO RSV EQA 2019-2020 showed that laboratories performed at high standards. Updating the composition of RSV molecular EQAs with contemporary strains to ensure representation of circulating strains, and ensuring primer matching with EQA panel viruses, is advantageous in assessing diagnostic competencies of laboratories. Ongoing EQAs are recommended because of continued evolution of mismatches between current circulating strains and existing primer sets.

Purification and analysis of DNases of Tritrichomonas foetus: evidence that these enzymes are glycoproteins.

Tritrichomonas foetus is the causative agent of trichomoniasis. In cattle, infection results in economic losses to the beef and dairy industries due to abortion and infertility. Soluble DNases of T. foetus that play a role in pathogenesis and are potential therapeutic targets, were extracted and purified utilising lectin affinity chromatography. The DNases were bound to and eluted from Concanavalin A (Con A)-sepharose indicating that they are glycoproteins with alpha-linked mannose or glucose residues. The nature of the glycans carried on the eluted proteins in the fraction containing DNase activity was assessed using an enzyme-linked lectin assay. The lectin binding studies predict the presence of both N- and O-type glycans. Manganese was a potent (33%) activator of the DNase(s) whereas zinc inhibited enzyme activity by approximately 66%. The DNase(s) had a pH optimum of 4 and a molecular weight of 160 kDa. The DNase(s) were able to completely degrade DNA from animal, plant, fungal, yeast and bacterial sources, but did not significantly degrade RNA.

Detection of human papillomavirus from archival tissues in cervical cancer patients in Mauritius.

BACKGROUND: Around half a million new cases of cervical cancer are diagnosed worldwide each year, accounting for almost 300,000 deaths. Development of cervical cancer can be multi-factorial, but high-risk human papillomaviruses (HPV) have been associated with the aetiology of cervical cancer. It is believed that HPV DNA integrates into the host DNA causing abnormal cell growth with cells becoming carcinogenic and spreading metastatically. In Mauritius, cervical cancer account for 65% of gynaecological cancers and 3.4% of the cervical cancers are diagnosed at the stage of carcinoma in situ. OBJECTIVES: To determine the prevalence of HPV in histological samples from patients with cervical cancer in Mauritius. STUDY DESIGN: DNA from archival cervical samples from a cohort of 65 patients suffering from cervical cancer and controls from Mauritius were tested for the presence of HPV using MY09/11 and GP5+/6+ primer sets. RESULTS: In a cohort of 65 patients from Mauritius, diagnosed with cervical cancer in the year 2000, 19% of cervical histology sections were found to be positive for the presence of high-grade HPV, exclusively HPV18 using MY09 and MY11 primers. Only 15% of the Mauritian population is over 50 years of age, whereas 66% (35) of the diagnosed cases of cervical cancer were seen in patients above 50 years with 50% (5) affected with HPV. These findings suggest that for an infection with HPV to develop into cancer may take years if not decades. Differences were noted using two different primer sets, MY09/11 and GP5+/6+. The latter produce a much smaller amplicon (150bp) compared to the former ( approximately 450bp). Seven additional positive cases were detected with the GP5+/6+ primer set, resulting in an apparent prevalence of 32% as compared to the 19% seen with the MY09/11 primer set. This may indicate that some degradation of the target DNA has occurred during processing and storage of histological samples. CONCLUSION: Using primer sets MY09/11 and GP5+/6+, only HPV type 18 was found in the Mauritian cohort with a prevalence of 32%.