METHODS: We developed and validated an internally controlled one-step single-tube real-time RT-PCR in terms of sensitivity, linearity, precision, and specificity for simultaneous detection of EVs and EV-A71. Subsequently, the assay was then applied on throat and rectal swabs sampled from 434 HFMD patients.
RESULTS: The assay was evaluated using both plasmid DNA and viral RNA and has shown to be reproducible with a maximum assay variation of 4.41 % and sensitive with a limit of detection less than 10 copies of target template per reaction, while cross-reactivity with other EV serotypes was not observed. When compared against a published VP1 nested RT-PCR using 112 diagnostic throat and rectal swabs from 112 children with a clinical diagnosis of HFMD during 2014, the multiplex assay had a higher sensitivity and 100 % concordance with sequencing results which showed EVs in 77/112 (68.8 %) and EV-A71 in 7/112 (6.3 %). When applied to clinical diagnostics for 322 children, the assay detected EVs in throat swabs of 257/322 (79.8 %) of which EV-A71 was detected in 36/322 (11.2 %) children. The detection rate increased to 93.5 % (301/322) and 13.4 % (43/322) for EVs and EV-A71, respectively, when rectal swabs from 65 throat-negative children were further analyzed.
CONCLUSION: We have successfully developed and validated a sensitive internally controlled multiplex assay for rapid detection of EVs and EV-A71, which is useful for clinical management and outbreak control of HFMD.
MATERIALS AND METHODS: The different primer sets were developed using bioinformatics software DNASTAR. The E. coli cells were used for recombinant protein expression.
RESULTS: The NiV 'G' region primers were designed and amplified for 1 kb fragment and cloned. The NiV 'G' fragments were sub-cloned in pET-28(+) B and pGEX-5x-1. Recombinant protein thus obtained in soluble form in both the cases was essayed using western blot. The result showed the protein expression yield was more in pET-28(+) B with low stability and vice versa for pGEX-5x-1.
CONCLUSION: The antibodies raised from the protein can be used as diagnostic reagent for detection of NiV. Thus, a new diagnostic technique can be industrialized.
DESIGN: Studies on the association between CT values and smear status were included in a descriptive systematic review. Authors of studies including smear, culture and Xpert results were asked for individual-level data, and receiver operating characteristic curves were calculated.
RESULTS: Of 918 citations, 10 were included in the descriptive systematic review. Fifteen data sets from studies potentially relevant for individual-level data meta-analysis provided individual-level data (7511 samples from 4447 patients); 1212 patients had positive Xpert results for at least one respiratory sample (1859 samples overall). ROC analysis revealed an area under the curve (AUC) of 0.85 (95%CI 0.82-0.87). Cut-off CT values of 27.7 and 31.8 yielded sensitivities of 85% (95%CI 83-87) and 95% (95%CI 94-96) and specificities of 67% (95%CI 66-77) and 35% (95%CI 30-41) for smear-positive samples.
CONCLUSION: Xpert CT values and smear status were strongly associated. However, diagnostic accuracy at set cut-off CT values of 27.7 or 31.8 would not replace smear microscopy. How CT values compare with smear microscopy in predicting infectiousness remains to be seen.