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Several commercial Zika virus (ZIKV) serology assays have been developed since the recognition of ZIKV outbreaks as a Public Health Emergency of International Concern in 2016. However, test interpretation for ZIKV serology can be challenging due to antibody cross-reactivity with other flaviviruses like dengue virus (DENV). Therefore, we sought to evaluate the performance of eight commercially available ZIKV IgM and IgG assays across three testing platforms, namely, immunochromatographic tests (ICT), ELISAs and immunofluorescence tests (IIFT). The test panel comprised of 278 samples, including acute and convalescent sera or plasma from ZIKV-confirmed, DENV-confirmed, non-ZIKV and non-DENV patients, and residual sera from healthy blood donors. The ZIKV IgM and IgG serology assays yielded higher test sensitivities of 23.5% - 97.1% among ZIKV convalescent samples as compared to 5.6% - 27.8% among ZIKV acute samples; the test specificities were 63.3% - 100% among acute and convalescent DENV, non-DENV samples. Among the ELISAs and IIFTs, the Diapro ZIKV IgM ELISA demonstrated high test sensitivity (96%) and specificity (80%) when tested on early convalescent samples, while the Euroimmun ZIKV IgG ELISA yielded the highest test specificity of 97% - 100% on samples from non-ZIKV patients and healthy blood donors. For rapid ICTs, the LumiQuick IgM rapid ICT yielded low test sensitivity, suggesting its limited utility. We showed that commercial ZIKV IgM and IgG serology assays have differing test performances, with some having moderate to high test sensitivities and specificities when used in a dengue endemic setting, although there were limitations in IgG serology.

Background Dengue is a global public health challenge which requires accurate diagnostic methods for surveillance and control. The gold standard for detecting dengue neutralizing antibodies (nAbs) is the plaque reduction neutralization test (PRNT), which is both labor-intensive and time-consuming. This study aims to evaluate three alternative approaches, namely, the MTT-based (or (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) microneutralization assay, the xCELLigence real-time cell analysis (RTCA), and the immuno-plaque assay-focus reduction neutralization test (iPA-FRNT). Methods Twenty-two residual serum samples were tested for DENV-2 nAbs using all four assays at three neutralization endpoints of 50%, 70% and 90% inhibition in virus growth. For each neutralization endpoint, results were compared using linear regression and correlation analyses. Test performance characteristics were further obtained for iPA-FRNT using 38 additional serum samples. Results Positive correlation of DENV-2 neutralization titers for the MTT-based microneutralization assay and the PRNT assay was only observed at the neutralization endpoint of 50% ( r  = 0.690). In contrast, at all three neutralization end points, a linear trend and positive correlation of DENV-2 neutralization titers for the xCELLigence RTCA and the PRNT assays were observed, yielding strong or very strong correlation ( r  = 0.829 to 0.967). This was similarly observed for the iPA-FRNT assay ( r  = 0.821 to 0.916), which also offered the added advantage of measuring neutralizing titers to non-plaque forming viruses. Conclusion The xCELLigence RTCA and iPA-FRNT assays could serve as suitable alternatives to PRNT for dengue serological testing. The decision to adopt these methods may depend on the laboratory setting, and the utility of additional applications offered by these technologies.

Chikungunya virus (CHIKV) is an arthropod-borne virus responsible for recent epidemics in the Asia Pacific regions. A customized gene expression microarray of 18,760 transcripts known to target Aedes mosquito genome was used to identify host genes that are differentially regulated during the infectious entry process of CHIKV infection on C6/36 mosquito cells. Several genes such as epsin I (EPN1), epidermal growth factor receptor pathway substrate 15 (EPS15) and Huntingtin interacting protein I (HIP1) were identified to be differentially expressed during CHIKV infection and known to be involved in clathrin-mediated endocytosis (CME). Transmission electron microscopy analyses further revealed the presence of CHIKV particles within invaginations of the plasma membrane, resembling clathrin-coated pits. Characterization of vesicles involved in the endocytic trafficking processes of CHIKV revealed the translocation of the virus particles to the early endosomes and subsequently to the late endosomes and lysosomes. Treatment with receptor-mediated endocytosis inhibitor, monodansylcadaverine and clathrin-associated drug inhibitors, chlorpromazine and dynasore inhibited CHIKV entry, whereas no inhibition was observed with caveolin-related drug inhibitors. Inhibition of CHIKV entry upon treatment with low-endosomal pH inhibitors indicated that low pH is essential for viral entry processes. CHIKV entry by clathrin-mediated endocytosis was validated via overexpression of a dominant-negative mutant of Eps15, in which infectious entry was reduced, while siRNA-based knockdown of genes associated with CME, low endosomal pH and RAB trafficking proteins exhibited significant levels of CHIKV inhibition. This study revealed, for the first time, that the infectious entry of CHIKV into mosquito cells is mediated by the clathrin-dependent endocytic pathway.

MicroRNAs (MiRNAs) are small, non-coding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. We analyzed the differential expression of miRNAs in 119 endometrial carcinomas, measuring their expression in histological subtypes, molecular subtypes, and tumors with CTNNB1 mutations. Tumors were subdivided into histological and molecular subtypes as defined by The Cancer Genome Atlas. The expression levels of 352 miRNAs were quantified using the PanoramiR panel. Mir-449a, mir-449b-5p, and mir-449c-5p were the top three miRNAs showing increased expression in both endometrioid and de-differentiated carcinomas but were not significantly increased in serous and clear cell carcinomas. The miRNAs with the most increased expression in serous and clear cell carcinomas were miR-9-3p and miR-375, respectively. We also identified 62 differentially expressed miRNAs among different molecular subtypes. Using sequential forward selection, we built subtype classification models for some molecular subtypes of endometrial carcinoma, comprising 5 miRNAs for MMR-deficient tumors, 10 miRNAs for p53-mutated tumors, and 3 miRNAs for CTNNB1-mutated tumors, with areas under curves of 0.75, 0.85, and 0.78, respectively. Our findings confirm the differential expression of miRNAs between various endometrial carcinoma subtypes and may have implications for the development of diagnostic and prognostic tools.

The aim of this study was to (1) translate the Gastrointestinal Tract Instrument (GIT) 2.0 from English to Chinese and (2) validate both versions in a multi-ethnic systemic sclerosis cohort in Singapore (SCORE). The English GIT2.0 was translated to Chinese using a standard forward-backward translation approach. Psychometric evaluation of the GIT2.0 included internal consistency reliability (using Cronbach’s alpha), test-retest reliability (using intra-class correlation coefficient (ICC)), scale level factor analysis, and construct validity (using Spearman correlation) against the modified Scleroderma Health Assessment Questionnaire (S-HAQ) and the SF-36 v2. Most of the patients were females (88.6%) and Chinese (78.2%), with mean (SD) age of 51.0 (13.0) years and median disease duration of 4.5 years. We administered English ( n  = 146) and Chinese ( n  = 74) GIT2.0. The mean (SD) total GIT score was 0.29 (0.37). There was good internal consistency (Cronbach’s alpha >0.70 for all subscales) and good test-retest reliability for the scale and all subscales (ICC 0.71–0.92) except for “diarrhoea” (ICC = 0.54). Our hypothesised a priori construct validity was supported by moderate correlations between the total GIT score and S-HAQ GI subscale ( r  = 0.446), and the social functioning subscale and SF36v2 role-social domain ( r  = 0.337), and weak-to-moderate correlation between the emotional subscale and SF-36v2 role-emotional ( r  = 0.295) and mental health ( r  = 0.298) domains and mental component summary ( r  = 0.356). Exploratory factor analysis of the seven subscales yielded a two-factor solution explaining 69.63% of the total variance. This study provides evidence for the reliability and validity of the English and Chinese GIT2.0 to be used in Singapore for research and routine practice.