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.

The chemical class of quinazoline compounds provides a core structure for the design of anticytomegaloviral kinase inhibitors.

HCMV is a member of the family Herpesviridae and represents a worldwide distributed pathogen with seropositivity rates in the adult population ranging between 40% and 90%. Notably, HCMV infection is a serious, sometimes life-threatening medical problem for newborns and immunosuppressed individuals, including transplant recipients and patients under antitumoral chemotherapy. Current standard therapy with valganciclovir has the disadvantage of inducing drug-resistant virus mutants and toxicity-related side effects. Our analysis stresses the earlier finding that kinase inhibitors of the quinazoline class exert an antiviral response by targeting the viral protein kinase pUL97 without inducing resistance. Therefore, quinazolines have been used as a core structure to gain insight in the mode of inhibitor-kinase interaction. Here, we demonstrate that (i) the novel quinazolines Vi7392 and Vi7453 are highly active against HCMV laboratory and clinically relevant strains including maribavir- and ganciclovir-resistant variants, (ii) antiviral activity is not cell-type specific and was also detected in a placental explant tissue model using a genetically intact HCMV strain (iii) the viral kinase pUL97 represents a target of the anticytomegaloviral activity of these compounds, (iv) induction of pUL97-conferring drug resistance was not detectable under single-step selection, thus differed from the induction of ganciclovir resistance, and (v) pUL97 drug docking simulations enabled detailed insights into specific drug-target binding properties providing a promising basis for the design of optimized kinase inhibitors. These novel findings may open new prospects for the future medical use of quinazoline drug candidates and the use of drug-target dynamic simulations for rational design of antivirals.

Viral factors influencing the outcome of human cytomegalovirus infection in liver transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) remains the leading viral cause of disease following orthotopic liver transplantation (OLT) despite the availability of antiviral agents for prophylaxis and therapy. OBJECTIVE: Examine the viral factors that influence the outcome of CMV infection following valganciclovir prophylaxis or laboratory-guided preemptive therapy in OLT recipients. STUDY DESIGN: The value of valganciclovir prophylaxis and laboratory-guided preemptive therapy for the prevention of CMV infection and disease was observed in 64 OLT recipients. Prophylaxis was given to all CMV seronegative recipients receiving a liver from a seropositive donor (D+R-; n=15), and all other recipients were randomised to receive either prophylaxis (n=24) or laboratory-guided preemptive therapy (n=25). Recipients were monitored for CMV DNAemia, viral load, emergence of antiviral resistant strains and co-infections. RESULTS: CMV end-organ disease and antiviral resistant strains only occurred in D+R- recipients despite the use of prophylaxis in these patients. The D+R- recipients commencing prophylaxis immediately following transplantation had better outcomes compared to those for whom prophylaxis was delayed due to renal impairment. Prophylaxis reduced the incidence of CMV DNAemia, persistent infection, and high viral loads for CMV seropositive (D-R+and D+R+) recipients, but laboratory-guided preemptive therapy effectively controlled CMV infection and prevented disease in these OLT recipients. CONCLUSION: Delaying the commencement of valganciclovir prophylaxis may be associated with worse outcomes for high-risk OLT recipients. Laboratory-guided pre-emptive therapy remains an alternative approach for seropositive recipients at lower risk of CMV disease.

Mycoplasma genitalium is associated with cervicitis and HIV infection in an urban Australian STI clinic population.

OBJECTIVES: To investigate the prevalence of the genital mollicutes, Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU) and Ureaplasma parvum (UP), and their associations with cervicitis in a sexually transmitted infection (STI) clinic population. Clinical correlates of MG infection were also assessed. METHODS: 527 women were enrolled in a cross-sectional study at two STI clinics in Sydney between June 2006 and January 2010. Genital mollicutes were detected by multiplex PCR testing of cervical swabs, and associations with cervicitis were analysed. Cervicitis was defined as >30 polymorphonuclear cells per high-power field in at least three non-adjacent fields of cervical mucus on Gram stain. RESULTS: MG was found in 4.0% of women, MH in 17.1%, UU in 14.1%, and UP in 51.8%. MG was the only mollicute associated with cervicitis (unadjusted prevalence ratio (PR) 1.85, 95% CI 1.52 to 2.26, p<0.0001), and this association remained after adjustment for Chlamydia trachomatis (CT) infection (adjusted PR 1.24 (95% CI 1.04 to 1.48), p=0.02). MG was significantly associated with women being HIV positive (p=0.03), but not with age, vaginal discharge, commercial sex work, being of culturally and linguistically diverse background, or concurrent CT infection. Two of the 21 women with MG had ectopic pregnancies. CONCLUSIONS: The authors recommend wider application of PCR testing for MG in STI services, particularly in high-risk women and those with cervicitis or HIV infection.

Diversity of antiviral-resistant human cytomegalovirus in heart and lung transplant recipients.

Immunocompromised transplant recipients are at high risk for human cytomegalovirus (CMV)-related infection and disease. Antiviral prophylaxis and treatment have reduced CMV morbidity and mortality, but at times promote development of antiviral-resistant CMV strains that can significantly contribute to adverse clinical outcomes in transplant recipients. We have investigated CMV genotypes in transplant recipients (bone marrow, stem cell, kidney, heart, lung, and liver) receiving antiviral prophylaxis or preemptive therapy or treatment, to determine the viral characteristics and clinical impact of antiviral-resistant CMV in these different groups. Antiviral-resistant CMV strains were detected by polymerase chain reaction sequencing of the CMV protein kinase (UL97) and viral DNA polymerase (UL54) genes from clinical specimens. A trend toward more frequent detection of multidrug resistance and co-circulation of multiple resistant strains was seen in heart and lung transplant recipients compared with other transplantation types. A greater diversity and number of UL97 and UL54 mutations were observed in heart and lung transplant recipients; whereas antiviral-resistant CMV infections in other transplant recipients were predominantly the result of a single mutant genotype. Furthermore, 43% (6/14) of CMV-positive heart and lung transplant recipients were infected with CMV strains containing UL54 mutations conferring multidrug resistance compared with only 6% (1/18) of CMV-positive recipients of other transplanted organs or stem cells. Emergence of CMV strains containing previously unrecognized UL54 mutations (F412S and D485N) also occurred in 1 lung and 1 heart transplant recipient. The development of these mutations under antiviral selective pressure, and clinical outcome of infection suggests these mutations are likely to confer antiviral resistance. Emergence of CMV antiviral resistance remains a significant issue in immunocompromised patients treated with antiviral agents, and emphasizes the relevance of regular antiviral resistance testing when designing optimal patient-management strategies.