5%, n=138) when compared to 5% (74.6%, n=138). Our analysis confirms that the implementation of new methods for detecting circulating cell-free tumor DNA in cell-free plasma is associated with poor performance. It is important to apply optimal detection methods as well as to extensively validate new methods for cell-free tumor DNA testing before treatment decisions are made. Circulating tumor DNA (ctDNA) measurements can be used to estimate tumor burden, but avoiding false-positives is a challenge. We evaluated digital next-generation sequencing (NGS) as a ctDNA detection method. Plasma KRAS and GNAS hotspot mutation levels were measured in 140 subjects including 67 with pancreatic ductal adenocarcinoma, and 73 healthy and disease controls. To limit chemical modifications of DNA that yield false-positive mutation calls, plasma DNA was enzymatically pre-treated, after which DNA was aliquoted for digital detection of mutations (up to 384 aliquots/sample) by PCR and NGS. A digital NGS score of two standard deviations above the mean in controls was considered positive. 37% of patients with pancreatic cancer, including 31% of patients with Stage I/II disease had positive KRAS codon 12 ctDNA scores; only one patient had a positive GNAS mutation score. Two disease control patients had positive ctDNA scores. Low normal-range digital NGS scores at mutation hot-spots were found at similar levels in healthy and disease controls, usually at sites of cytosine deamination, and were likely the result of chemical modification of plasma DNA and NGS error, rather than true mutations. Digital NGS detects mutated ctDNA in patients with pancreatic cancer with similar yield to other methods. The detection of low-level, true-positive ctDNA is limited by frequent low-level detection of false-positive mutation cells in plasma DNA from controls. Polyglutamine spinocerebellar ataxias (SCAs) constitute a group of autosomal dominantly inherited neurodegenerative disorders with considerable phenotypic overlap. A definitive diagnosis relies on the detection of a mutation in each associated locus, comprising the abnormal expansion of the trinucleotide CAG in coding exons. The assessment of single nucleotide polymorphisms (SNPs) associated with the CAG expansion in the context of SCAs is also relevant for the improvement of molecular diagnosis and for the generation of novel therapeutic strategies. Here we focused on Machado-Joseph disease (MJD)/SCA3, aiming to develop a protocol for the accurate determination of the CAG length in exon 10 of the human ATXN3 gene and to characterize flanking polymorphisms. A single pair of primers was designed and validated, and two complementary PCR-based methods were established. In method I, PCR amplicons were cloned and sequenced, allowing the assessment of three SNPs in the vicinity of the CAG repeat (C987GG/G987GG, TAA1118/TAC1118 and C1178/A1178), which can constitute potential targets for personalized gene-based therapies. Method II combines PCR, capillary electrophoresis and a size correction formula, enabling a time and cost-effective determination of the number of CAGs. The established protocol paves the way to overcome technical difficulties related to the molecular characterization of the CAG motif and intragenic polymorphisms in the context of MJD/SCA3 and may prove its utility when applied to other polyglutamine SCAs.  https://www.selleckchem.com/JAK.html  OBJECTIVES This study aimed to evaluate whether tenofovir prophylaxis for mothers with high viral loads in late pregnancy is a cost-effective way to prevent mother-to-child HBV transmission in China. METHODS A decision tree-Markov model was constructed for a cohort of infants born to HBV surface antigen-positive mothers in China, 2016. The expected cost and effectiveness were compared between the current active-passive immunoprophylaxis strategy and the tenofovir prophylaxis strategy and the incremental cost-effectiveness ratio was calculated. One-way and multi-way probabilistic sensitivity analyses were performed. RESULTS For 100,000 babies born to mothers positive for hepatitis B surface antigen, tenofovir prophylaxis strategy would prevent 2,213 perinatal HBV infections and gain 931 quality-adjusted life years, compared with the current active-passive immunoprophylaxis strategy. The incremental cost-effectiveness ratio was ￥59,973 ($9,087) per QALY gained. This result was robust over a wide range of assumptions. CONCLUSIONS Tenofovir prophylaxis for mothers with high viral loads in late pregnancy was more cost-effective than the current active-passive immunoprophylaxis alone. Embedding tenofovir prophylaxis for mothers with high virus loads into the present hepatitis B prevention strategies should be considered to further prevent mother-to-child hepatitis B transmission in China. OBJECTIVE Japanese encephalitis virus infection (JEV) remains a leading cause of neurological infection in Asia, largely involving individuals living in remote areas with limited access to treatment centres and diagnostic facilities. Laboratory confirmation is fundamental for the justification and implementation of vaccination programmes. We sought to review the literature on historical developments and current diagnostic capability worldwide, to identify knowledge gaps and instil urgency to address them. METHODS Searches were performed in Web of Science and PubMed using the text word term 'Japanese encephalitis' up to 13th October 2019. Studies reporting laboratory-confirmed symptomatic JE cases in humans were included, and data on details of diagnostic tests were extracted. A JE case was classified according to confirmatory levels (1-4), where level 1 represented the highest level of confidence. FINDINGS 20,212 published JE cases were identified from 205 studies. 15,167 (75%) of these positive cases were confirmed with the lowest confidence diagnostic test (level 3 or 4, or level 4). Only 109 (53%) of the studies reported contemporaneous testing for dengue-specific antibodies. CONCLUSION A fundamental pre-requisite for the control of JE is lacking --- that of a simple and specific diagnostic procedure that can be adapted for point-of-care tests and readily used throughout JE endemic regions of the world.