Molecular typing of enteroviruses: comparing 5'UTR, VP1 and whole genome sequencing methods.

Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5' untranslated region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5'UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS). We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5'UTR in a subset of samples. Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5'UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared. Of the 74/118 (63%) samples that were successfully typed using both the 5'UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used. Genotyping with WGS and VP1 is more accurate than 5'UTR. Genotyping by the 5'UTR method is very sensitive, but less specific.

Circulating human papillomavirus DNA detection in Barrett's dysplasia and esophageal adenocarcinoma.

There is evidence to suggest that human papillomaviruses (HPV) are associated with Barrett's dysplasia and esophageal adenocarcinoma. In other HPV-linked cancers such as cervical and oropharyngeal cancer, circulating HPV DNA is a potential biomarker to assist in tumor diagnosis and management. This study aimed to determine whether circulating HPV DNA was detectable in patients with Barrett's dysplasia and esophageal adenocarcinoma, and if so, whether there is any correlation with esophageal tissue HPV status. Plasma from 138 patients representing esophageal adenocarcinoma (N = 41), Barrett's dysplasia (N = 48) and hospital controls (N = 49) were analyzed for the presence of circulating HPV DNA using droplet-digital PCR targeting the E7 gene of HPV types 16 and 18. Circulating HPV DNA was detected in 11/138 (8.0%) study subjects including 1/49 (2.0%) hospital controls, 4/48 (8.3%) Barrett's dysplasia patients, and 6/41 (14.6%) esophageal adenocarcinoma patients. Detection of circulating HPV DNA was higher in patients with HPV-positive esophageal tissue (6/35, 17.1%) compared to those with HPV-negative specimens (5/103; 4.9%) (OR = 4.06; 95% CI 1.15-14.25; P = 0.020). The highest rates of detection occurred in esophageal adenocarcinoma patients, particularly those with invasive tumors that had breached the esophageal submucosa, had regional lymph node involvement or metastatic disease. Circulating HPV DNA was detectable in a subset of Barrett's dysplasia and esophageal adenocarcinoma patients. Detection was associated with tissue HPV positivity and possibly disease severity.

Transmission of hepatitis C virus to recipients of parenteral vitamin therapy in a primary care facility.

BACKGROUND AND OBJECTIVES: The Australian prevalence of hepatitis C virus (HCV) is approximately 1%, with the majority of cases acquired through injecting drug use. However, occasionally HCV infection occurs in healthcare settings. Three new HCV infections were identified amongst patients attending a general practice in Sydney, Australia, specialising in parenteral vitamin therapy. STUDY DESIGN: An investigation was conducted to identify the source of infection and mechanism of transmission. Molecular analysis was conducted by sequencing the HCV NS5A, Core and NS5B regions. RESULTS: Two sources were identified using molecular epidemiology - a genotype 3a case was the source for a case acquired in late 2004 and a genotype 1b case the source for one case acquired in late 2006 and another in early 2007. The common risk factor was parenteral vitamin C therapy. CONCLUSIONS: Inadequate infection control was apparent and likely to have resulted in blood contamination of the healthcare workers, their equipment, the clinic environment and parenteral medications. Molecular and clinical epidemiology clearly identified parenteral transmission of HCV, highlighting the risks of blood contamination of parenteral equipment and use of multi-dose flasks on more than one patient.