The Zika virus (ZIKV) epidemic is an ongoing public health concern. ZIKV is a flavivirus reported to be associated with microcephaly, and recent work in animal models demonstrates the ability of the virus to cross the placenta and affect fetal brain development. Recent findings suggest that the virus preferentially infects neural stem cells and thereby deregulates gene expression, cell cycle progression, and increases cell death. However, neuronal stem cells are not the only brain cells that are susceptible to ZIKV and infection of other brain cells may contribute to disease progression. Herein, we characterized ZIKV replication in astrocytes, and profiled temporal changes in host microRNAs (miRNAs) and transcriptomes during infection. We observed the deregulation of numerous processes known to be involved in flavivirus infection, including genes involved in the unfolded protein response pathway. Moreover, a number of miRNAs were upregulated, including miR-30e-3p, miR-30e-5p, and, miR-17-5p, which have been associated with other flavivirus infections. This study highlights potential miRNAs that may be of importance in ZIKV pathogenesis.

We have developed a novel microsphere-based genotyping method for 46 mucosal human papillomavirus (HPV) types. HPV DNA was amplified by PCR using general primers and typed by hybridization to HPV type-specific probes coupled to sortable microspheres based on the Luminex xMAP technology. Hybridization to each probe was specific for each HPV type without cross-hybridization and sensitive enough to allow typing of HPV contained in clinical specimens. The method was validated with direct sequencing and the Roche Linear Array genotyping method.A bout 50 human papillomavirus (HPV) types are associated with infections of the genital, anal, and oropharyngeal mucosae (2,8,10). A few of these are known to be high-risk oncogenic types and the cause of cervical cancer (16), other anogenital cancers, and head and neck malignancies (3,8). Detection of high-risk HPV types collectively is being considered as a screening method for cervical cancer, with the promise of improving the sensitivity and cost-effectiveness of cervical cancer screening programs (6). In recent years, there is also mounting evidence for the utility of type-specific identification of HPV type 16 (HVP16) and HPV18, as these two types are significantly associated with persistent infection and lesion progression, thus conferring a higher risk for cancer than other oncogenic types (15). As HPV16 and -18 account for 70% of the cervical cancers worldwide (2), two HPV vaccines targeting these types have been developed and shown to be highly efficacious in preventing both persistent infection with the types and the associated dysplastic changes in the cervical epithelium that lead to malignant transformation (12,22). Since these vaccines are type specific, it is important to know the distribution of the various HPV types in a population, as well as to have a surveillance system in place to monitor vaccine efficacy over time and any unexpected shifts in the frequency of HPV types not covered by the vaccines. The above highlights the need for and the importance of using type-specific tests for HPV.Numerous HPV typing methods based on a variety of detection platforms have been described in the literature, and some are commercially available (reviewed in reference 19). At the Canadian National Microbiology Laboratory (NML), we have developed a Luminex-based method (NML Luminex assay) to meet our need for sensitive and extensive typing of HPV in the context of epidemiological and molecular surveillance studies at the national level. The assay is based on the xMAP platform (Luminex Corporation, Austin, TX), and it detects 46 mucosal types. In this report, we describe its design and performance.HPV DNA either cloned in plasmids (for validation of the probes) or from clinical cervical cancer specimens (for assay validation) was amplified by a nested PCR method using the PGMY primers for the first step (5, 11) and the GP5ϩ/GP6ϩ primers (7) for the second step. The GP6ϩ primer (Invitrogen, Burlington, ON, Canada) carried a 5= biotin label and phosphorothioate bonds in the f...

The World Health Organization (WHO) has targeted measles for global eradication through mass immunization. For effective monitoring of eradication targets, high quality surveillance is needed. The detection of IgM antibodies, specific to the measles virus, with the use of commercial enzyme-linked immunosorbent assays (ELISA or EIA) is broadly used within the WHO global measles and rubella laboratory network for laboratory confirmation, and in particular ELISA kits manufactured by Siemens (Enzygnost® kits) have been primarily used. Spurred by the discontinuation of these kits, this study aims to report on the clinical sensitivity and specificity of comparable commercial ELISA kits and one automated chemiluminescent immunoassay (CLIA) method. A panel of 239 serum samples was assembled that included sera from confirmed measles cases (n=50) and probable post-MMR vaccine response (n=2). Measles negative sera (n=187) were collected from individuals presenting with other fever and rash illnesses. A total of 7 ELISA kits were evaluated (Euroimmun native antigens and recombinant nucleoprotein, IBL, Clin-Tech Microimmune, NovaTec NovaLisa®, Serion and Siemens Enzygnost®) and one CLIA method (DiaSorin LIAISON® XL). The ELISA kits included two IgM capture methods and five indirect methods. Calculated sensitivities and specificities ranged from 75.0% to 98.1% and 86.6% to 99.5% respectively. The parvovirus B19 IgM positive sera were noted to cause false positive results particularly for the ELISA kits from Serion and NovaLisa®; specificities for this subset of samples ranged from 51.4% to 100.0%. The capture IgM ELISA methods provided the best combination of sensitivity and specificity.

Rubella and congenital rubella syndrome are caused by the rubella virus and are preventable through vaccination, making disease eradication possible. Monitoring of progress towards global eradication and local elimination requires high quality, sensitive disease surveillance that includes laboratory confirmation of cases. Previous evaluations of anti-rubella IgM detection methods resulted in the broad adoption of Enzygnost (most recently manufactured by Siemens) enzyme-linked immunosorbent assay (ELISA or EIA) kits within WHO’s global measles and rubella laboratory network but they have been discontinued. This study evaluates seven comparable ELISA methods from six manufacturers (Trinity Biotech, Euroimmun, Clin-Tech, NovaTec and Virion\Serion) as well as one automated chemiluminescent assay (CLIA) from Diasorin. These methods consisted of three IgM capture methods and five indirect ELISA methods. A panel of 238 sera was used for the evaluation that included 38 archival rubella IgM positive sera and 200 sera collected from symptomatically similar cases, such as measles, dengue, parvovirus B19 and roseola. With this panel of sera, the sensitivity of the methods ranged from 63.2% to 100% and the specificity from 80.0% to 99.5%. No single method had both sensitivity and specificity >90%, unless sera with equivocal results were considered to be presumptive positive. Some methods, particularly the Serion ELISA, had a large number of false positives with parvovirus B19 IgM positive sera as well as sera from confirmed measles cases. The performance characteristics identified in this evaluation serve as a reminder to not rely solely on rubella IgM results for case confirmation in elimination settings.

Background. Population-specific epidemiologic data on human Papillomavirus infection, which are limited in most of the SubSaharan African countries, are necessary for effective cervical cancer prevention. This study aimed to generate population-specific data on human Papillomavirus infections, and determine which of these, self-collected and provider-collected specimens, gives a higher estimate of the prevalence of human Papillomaviruses, including vaccine and non-vaccine-type human Papillomavirus. Methods. In this cross-sectional study, following a questionnaire-based collection of epidemiological data, self-, and provider-collected specimens, obtained from women 15−65 years of age, were analysed for human Papillomavirus types by a nested-multiplex polymerase chain reaction, and for cervical lesions by Pap testing. HPV data were categorised according to risk type and vaccine types for further analysis. Results. The difference between the overall human Papillomavirus infection prevalences obtained with the self-collected specimens, 43.1% (95% CI of 38.0–51.0%) and that with the provider-collected samples, 23.3% (95% CI of 19.0–31.0%) were significant (p≤0.001). The prevalence of quadrivalent vaccine-type human Papillomaviruses was 12.3% with self-collected specimens, but 6.0% with provider-collected specimens. For the nonavalent vaccine-types, the prevalences were 26.6% and 16.7% respectively. There were multiple infections involving both vaccine-preventable and nonvaccine preventable high-risk human Papillomavirus genotypes. Conclusion. The Akuse subdistrict can, therefore, be said to have a high burden of human Papillomavirus infections, which included nonvaccine types, as detected with both self-collected and provider-collected specimens. These imply that self-collection is to be given a higher consideration as a means for a population-based high-risk human Papillomavirus infections burdens assessment/screening. Additionally, even with a successful implementation of the HPV vaccination, if introduced in Ghana, there is still the need to continue with the screening of women.