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Objective Based on the current difficulties in early diagnosis of HBV-related hepatocellular carcinoma (HBV-HCC), we assessed the values of preoperative serum fibrinogen-like protein 1 (FGL1) by itself and in combination with alpha-fetoprotein (AFP) for the diagnosis of HBV-HCC. Methods We used ELISA and chemiluminescence assays to detect the serum levels of FGL1 and AFP, respectively. Results Serum FGL1 level in the HBV-HCC group was significantly higher than in the chronic HBV (CHBV) group, the liver cirrhosis (LC) group, and the healthy control (HC) group. Serum FGL1 had an outstanding performance in distinguishing AFP-negative HBV-HCC from different control conditions. In the patients with AFP-negative HBV-HCC, the sensitivity of serum FGL1 was high. Moreover, serum FGL1 had a stronger performance than AFP in distinguishing early-stage HBV-HCC. Conclusions Serum FGL1 is significantly elevated among patients with HBV-HCC, including those with negative AFP and with disease at an early stage. Hence, serum FGL1 may serve as a potential diagnostic marker in the early diagnosis of HBV-HCC. [ABSTRACT FROM AUTHOR]

Background: The two‐spotted spider mite (TSSM), Tetranychus urticae (Acari: Tetranychidae), is a cosmopolitan phytophagous pest in agriculture and horticulture. It has developed resistance to many acaricides by target‐site mutations. Understanding the status and evolution of resistant mutations in the field is essential for resistance management. Here, we applied a high‐throughput Kompetitive allele‐specific polymerase chain reaction (KASP) method for detecting six mutations conferring resistance to four acaricides of the TSSM. We genotyped 3274 female adults of TSSM from 43 populations collected across China in 2017, 2020, and 2021. Results: The KASP genotyping of 24 testing individuals showed 99% agreement with Sanger sequencing results. KASP assays showed that most populations had a high frequency of mutations conferring avermectin (G314D and G326E) and pyridaben (H92R) resistance. The frequency of mutation conferring bifenazate (A269V and G126S) and etoxazole (I1017F) resistance was relatively low. Multiple mutations were common in the TSSM, with 70.2% and 24.6% of individuals having 2–6 and 7–10 of 10 possible resistant alleles, respectively. No loci were linked in most populations among the six mutations, indicating the development of multiple resistance is mainly by independent selection. However, G314D and I1017F on the nuclear genome deviated from Hardy–Weinberg equilibrium in most populations, indicating significant selective pressure on TSSM populations by acaricides or fitness cost of the mutations in the absence of acaricide selection. Conclusion: Our study revealed that the high frequency of TSSMs evolved multiple resistant mutations in population and individual levels by independent selection across China, alarming for managing multiple‐acaricides resistance. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: The diamondback moth (DBM) Plutella xylostella has developed resistance to almost all insecticides used to control it. Populations of DBM in temperate regions mainly migrate from annual breeding areas. However, the distribution pattern of insecticide resistance of DBM within the context of long-distance migration remains unclear. RESULTS: In this study, we examined the frequency of 14 resistance mutations for 52 populations of DBM collected in 2010, 2011, 2017 and 2018 across China using a high-throughput KASP genotyping method. Mutations L1041F and T929I conferring pyrethroid resistance, and mutations G4946E and E1338D conferring chlorantraniliprole resistance were near fixation in most populations, whereas resistant alleles of F1020S, M918I, A309V and F1845Y were uncommon or absent in most populations. Resistance allele frequencies were relatively stable among different years, although the frequency of two mutations decreased. Principal component analysis based on resistant allele frequencies separated a southern population as an outlier, whereas the immigrants clustered with other populations, congruent with the migration pattern of northern immigrants coming from the Sichuan area of southwestern China. Most resistant mutations deviated from Hardy–Weinberg equilibrium due to a lower than expected frequency of heterozygotes. The deviation index of heterozygosity for resistant alleles was significantly higher than the index obtained from single nucleotide polymorphisms across the genome. These findings suggest heterogeneous selection pressures on resistant mutations. CONCLUSION: Our results provide a picture of resistant mutation patterns in DBM shaped by insecticide usage and migration of this pest, and highlight the widespread distribution of resistance alleles in DBM. [ABSTRACT FROM AUTHOR]

Sporotrichosis is the most important subcutaneous mycosis that affects humans and animals worldwide. The mycosis is caused after a traumatic inoculation of fungal propagules into the host and may follow an animal or environmental transmission route. The main culprits of sporotrichosis are thermodimorphic Sporothrix species embedded in a clinical clade, including S. brasiliensis, S. schenckii, S. globosa, and S. luriei. Although sporotrichosis occurs worldwide, the etiological agents are not evenly distributed, as exemplified by ongoing outbreaks in Brazil and China, caused by S. brasiliensis and S. globosa, respectively. The gold standard for diagnosing sporotrichosis has been the isolation of the fungus in vitro. However, with the advance in molecular techniques, molecular assays have complemented and gradually replaced the classical mycological tests to quickly and accurately detect and/or differentiate molecular siblings in Sporothrix. Nearly all techniques available for molecular diagnosis of sporotrichosis involve PCR amplification, which is currently moving towards detecting Sporothrix DNA directly from clinical samples in multiplex qPCR assays. From an epidemiological perspective, genotyping is key to tracing back sources of Sporothrix infections, detecting diversity in outbreak areas, and thus uncovering finer-scale epidemiological patterns. Over the past decades, molecular epidemiological studies have provided essential information to policymakers regarding outbreak management. From high-to-low throughput genotyping methods, MLSA, AFLP, SSR, RAPD, PCR-RFLP, and WGS are available to assess the transmission dynamics and sporotrichosis expansion. This review discusses the trends in the molecular diagnosis of sporotrichosis, genotyping techniques applied in molecular epidemiological studies, and perspectives for the near future. [ABSTRACT FROM AUTHOR]

Simple Summary: The western flower thrips (WFT) Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) is an important invasive pest in agriculture and forestry. It has developed resistance to a frequently used pesticide spinetoram world widely, including the invading area of China. However, the mechanism of resistance to spinetoram is unclear in China. In this study, we found the presence of the G275E mutation in the nicotinic acetylcholine receptor Foα6 in the early invading populations, which has now increased to a high frequency in China. There was a correlation between the frequency of the G275E mutation and resistance to spinetoram as characterized by median lethal concentration. Our results showed that G275E mutation is one of the mechanisms conferring spinetoram resistance in invading populations in China, as in many other countries. Our study highlights the rapid spread of the G275E mutation in China in the 2009–2021 period. The western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) is an important invasive pest worldwide. Field-evolved resistance to the pesticide spinetoram is an increasing problem in the chemical control of this pest. Here, we examined changes in the frequency of a genetic mutation associated with spinetoram resistance, the G275E mutation in the acetylcholine receptor Foα6, in 62 field populations collected from 2009 to 2021 across areas of China invaded by this pest. We found a low frequency of the G275E mutation in populations collected at the early invasion stage, in contrast to a high frequency in native USA populations. However, the frequency of the G275E mutation has increased to a high level in recently collected populations, with the mutation becoming fixed in some populations. There was a correlation between the frequency of the G275E mutation and resistance to spinetoram as characterized by median lethal concentration, although two populations were outliers. These results showed that G275E mutation is one of the mechanisms conferring spinetoram resistance in many invading populations in China. Ongoing dispersal of the WFT may have facilitated a rapid increase in the G275E mutation across China. Our study highlights the rapid evolution of pesticide resistance in an invasive species and points to a useful marker for molecular diagnostics of spinetoram resistance. [ABSTRACT FROM AUTHOR]

Bovine theileriosis caused by several Theileria species including Theileria annulata, Theileria parva, Theileria orientalis, Theileria mutans, and Theileria sinensis is a significant hemoprotozoan tick-borne disease. Among these, Theileria species, T. annulata, which causes tropical theileriosis (TT), is regarded as one of the most pathogenic and is responsible for high mortality. At present, most conventional diagnostic methods for tropical theileriosis are time-consuming and laborious and cannot distinguish newfound T. sinensis in China. Therefore, a high sensitivity and specificity real-time quantitative PCR method based on the TA19140 target molecule was developed, and the method was found to be specific for T. annulata. No cross-reaction was observed with T. sinensis, T. orientalis, Babesia bovis, Babesia bigemina, or Hyalomma anatolicum which is negative for T. annulata. A total of 809 field samples from different regions of China were analyzed by using the developed qPCR and conventional PCR. The positive samples for T. annulata detected by real-time qPCR and conventional PCR were 66/809 (8.16%) and 20/809 (2.47%), respectively, and all positive amplicons by qPCR were confirmed by Sanger sequencing. The results showed that the developed qPCR for the T. annulata 19,140 gene was more sensitive than conventional PCR. In addition, we first discovered that TA19140 was mainly expressed at the schizont and merozoite stages of T. annulata by relative quantification. The protein encoded by the TA19140 gene may be used as a potential diagnostic antigen for tropical theileriosis. In conclusion, a real-time quantitative PCR diagnostic method targeting the TA19140 gene was successfully established and could be used for both the quantitative and qualitative analysis of T. annulata infection from cattle and vector ticks, which will greatly help to control and diagnosis of tropical theileriosis. [ABSTRACT FROM AUTHOR]

Objectives: Comprehensive data using Next-Generation Sequence (NGS) and fluorescence in situ hybridization (FISH) for detecting MET amplification is limited in Chinese patients, we evaluating NGS performance both in tissue and plasma samples using FISH as reference. We also sought to find optimal thresholds value for NGS in detecting MET amplification via bioinformatics methods., Method: Patients progressed after 1st-, 2nd-, or 3rd-generation (G) EGFR-TKIs were enrolled. Tissue biopsy samples were performed for MET amplification detection via both NGS and FISH. Paired plasma samples were collected for MET amplification detection by NGS. The sensitivity, specificity and agreement were analyzed between NGS and FISH., Results: 116 eligible patients were analyzed. 44 patients were male. 82 patients were after 3rd generation EGFR-TKI. MET amplification was detected in 43 (37.1 %) patients by FISH, including 19 (16.4 %) polysomy and 24 (20.7 %) focal amplification. The positive rate of MET amplification in post 3rd generation EGFR-TKI and post 1st/2ndgeneration EGFR-TKI resistant patients was 42.7 % (35/82), and 23.5 % (8/34). The sensitivity, specificity and agreement of detecting MET amplification by NGS in tissue were 39.5 % (17/43), 98.6 % (72/73) and 76.7 % (89/116), respectively, 66.7 % (16/24), 98.6 % (72/73) and 90.7 % (88/97) for focal MET amplification in tissue and 29.2 % (7/24), 94.5 % (69/73), 78.4 % (76/97) for focal amplification in plasma. Results were shown in the table below., Conclusion: NGS is an alternative method for MET focal amplification detection in tissue. While the sensitivity of NGS testing in plasma needs further improvement to maximize identification of patients with potential benefit from dual-targeted therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To follow the revision of the fourth edition of WHO classification and the recent progress on the management of diffuse gliomas, the joint guideline committee of Chinese Glioma Cooperative Group (CGCG), Society for Neuro-Oncology of China (SNO-China) and Chinese Brain Cancer Association (CBCA) updated the clinical practice guideline. It provides recommendations for diagnostic and management decisions, and for limiting unnecessary treatments and cost. The recommendations focus on molecular and pathological diagnostics, and the main treatment modalities of surgery, radiotherapy, and chemotherapy. In this guideline, we also integrated the results of some clinical trials of immune therapies and target therapies, which we think are ongoing future directions. The guideline should serve as an application for all professionals involved in the management of patients with adult diffuse glioma and also a source of knowledge for insurance companies and other institutions involved in the cost regulation of cancer care in China and other countries. [ABSTRACT FROM AUTHOR]

Putative mechanisms underlying spinosyn resistance have been identified in controlled studies on many species; however, mechanisms underlying field-evolved resistance and the development of a molecular diagnostic method for monitoring field resistance have lagged behind. Here, we examined levels of resistance of melon thrips, Thrips palmi Karny (Thysanoptera:Thripidae), to spinetoram as well as target site mutations in field populations across China to identify potential mechanisms and useful molecular markers for diagnostic and quantifying purposes. In resistant populations, we identified the G275E mutation, which has previously been linked to spinosyns resistance, and F314V mutation, both located in the α 6 subunit of the nicotinic acetylcholine receptor. There was a strong correlation between levels of spinetoram resistance and allele frequency of G275E mutation in field-collected populations (r 2 = 0.84) and those reared under laboratory conditions for two to five generations (r 2 = 0.91). LC50 ranged from 0.12 to 0.66 mg/liter in populations without G275E mutation, whereas it ranged from 33.12 to 39.91 mg/liter in most populations with a G275E mutation frequency more than 90%. Our results indicate that the field-evolved resistance of T. palmi to spinetoram in China is mainly conferred by the G275E mutation. The frequency of the G275E mutation provides a useful diagnostic for quantifying resistance levels in field populations of T. palmi. [ABSTRACT FROM AUTHOR]

The roll-out of molecular diagnostic tools continues to be the most important shift in the tuberculosis diagnostic landscape. The aim of this study was to develop a novel external quality assessment (EQA) panels for molecular TB diagnostics. In addition, we also assessed the performance of the laboratories with the EQA panels in China. Dried Mycobacterium tuberculosis (MTB) DNA in the chelex resin was designed as part of an EQA program. The storage of genomic DNA in the chelex resin layer had no effect on the stability of genomic DNA, even after 12 weeks of storage. Seventy-one laboratories have participated in EQA of molecular diagnostics for TB diagnosis in 2018. GeneXpert (74.6%, 53/71) was the most predominant molecular method, followed by GeneChip (32.3%, 23/71), MeltPro (22.5%, 16/71), and TB-LAMP (7.0%, 5/71). Out of 105 EQA panels, 103 EQA results (98.1%) achieved perfect scores, whereas the other two (1.9%) had satisfactory scores. There were a total of two false-negative results reported from two laboratories with local LAMP, respectively. In conclusion, we firstly develop feasible EQA panels for molecular diagnostics for tuberculosis in China. Our data demonstrate that a majority of participating laboratories are able to produce perfect results with molecular diagnostics in China, giving us important hints for the implementation of molecular diagnostics. [ABSTRACT FROM AUTHOR]

Background: Cell-free circulating DNA has been known for many years, but this knowledge has not been beneficial for diagnosis. In this meta-analysis, we examine the diagnostic role of circulating cell-free DNA in HCC patients to find a reliable biomarker for the early detection of HCC., Materials and Methods: We performed a systematic literature search using Science Direct, Web of Science, PubMed/Medline, Scopus, Google Scholar, and Embase, up to April 1st, 2022. Meta-Disc V.1.4 and Comprehensive Meta-Analysis V.3.3 software calculated the pooled specificity, sensitivity, area under the curve (AUC), diagnostic odds ratio (DOR), positive likelihood ratio (PLR), negative likelihood ratio (NLR) Q*index, and summary receiver-- operating characteristic (SROC) for the role of cfDNA as a biomarker for HCC patients. Moreover, the subgroup analyses have been performed based on sample types (serum/plasma) and detection methods (MS-PCR/methylation)., Results: A total of 7 articles (9 studies) included 697 participants (485 cases and 212 controls). The overall pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and area under the curve (AUC) were 0.706 (95% CI: 0.671 - 0.739), 0.905 (95% CI: 0.865 - 0.937), 6.66 (95% CI: 4.36 - 10.18), 0.287 (95% CI: 0.185 - 0.445), 28.40 (95% CI: 13.01 - 62.0), and 0.93, respectively. We conducted a subgroup analysis of diagnostic value, which showed that the plasma sample had a better diagnostic value compared to the serum., Conclusion: This meta-analysis showed that cfDNA could be a fair biomarker for diagnosing HCC patients., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: The diamondback moth (DBM) Plutella xylostella has developed resistance to almost all insecticides used to control it. Populations of DBM in temperate regions mainly migrate from annual breeding areas. However, the distribution pattern of insecticide resistance of DBM within the context of long-distance migration remains unclear., Results: In this study, we examined the frequency of 14 resistance mutations for 52 populations of DBM collected in 2010, 2011, 2017 and 2018 across China using a high-throughput KASP genotyping method. Mutations L1041F and T929I conferring pyrethroid resistance, and mutations G4946E and E1338D conferring chlorantraniliprole resistance were near fixation in most populations, whereas resistant alleles of F1020S, M918I, A309V and F1845Y were uncommon or absent in most populations. Resistance allele frequencies were relatively stable among different years, although the frequency of two mutations decreased. Principal component analysis based on resistant allele frequencies separated a southern population as an outlier, whereas the immigrants clustered with other populations, congruent with the migration pattern of northern immigrants coming from the Sichuan area of southwestern China. Most resistant mutations deviated from Hardy-Weinberg equilibrium due to a lower than expected frequency of heterozygotes. The deviation index of heterozygosity for resistant alleles was significantly higher than the index obtained from single nucleotide polymorphisms across the genome. These findings suggest heterogeneous selection pressures on resistant mutations., Conclusion: Our results provide a picture of resistant mutation patterns in DBM shaped by insecticide usage and migration of this pest, and highlight the widespread distribution of resistance alleles in DBM. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Background: At present, the hereditary hearing loss homepage, ( https://hereditaryhearingloss.org/ ), includes 258 deafness genes and more than 500 genes that have been reported to cause deafness. With few exceptions, the region-specific distributions are unclear for many of the identified variants and genes., Methods: Here, we used a custom capture panel to perform targeted sequencing of 518 genes in a cohort of 879 deaf Chinese probands who lived in Yunnan. Mutation sites of the parents were performed by high-throughput sequencing and validated by Sanger sequencing., Results: The ratio of male to female patients was close to 1:1 (441:438) and the age of onset was mainly under six. Most patients (93.5%) were diagnosed with moderate to severe deafness. Four hundred and twenty-eight patients had variants in a deafness gene, with a detection rate of 48.7%. Pathogenic variants were detected in 98 genes and a number of these were recurrent within the cohort. However, many of the variants were rarely observed in the cohort. In accordance with the American College of Medical Genetics and Genomics, pathogenic, likely pathogenic and variants of uncertain significance accounted for 34.3%, 19.3% and 46.4% of all detected variants, respectively. The most common genes included GJB2, SLC26A4, MYO15A, MYO7A, TMC1, CDH23, USH2A and WFS1, which contained variants in more than ten cases. The two genes with the highest mutation frequency were GJB2 and SLC26A4, which accounted for 28.5% (122/428) of positive patients. We showed that more than 60.3% of coding variants were rare and novel. Of the variants that we detected, 80.0% were in coding regions, 17.9% were in introns and 2.1% were copy number variants., Conclusion: The common mutation genes and loci detected in this study were different from those detected in other regions or ethnic groups, which suggested that genetic screening or testing programs for deafness should be formulated in accordance with the genetic characteristics of the region., (© 2023. The Author(s).)

The western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), is an invasive species and currently occurs in only a few areas in China. An easy, accurate and developmental-stage independent method to identify F. occidentalis would be a valuable tool to facilitate pest management decision making and, more importantly, to provide an early warning so actions can be taken to prevent its introduction into non-infested areas. Morphological identification of thrips adults and, to a lesser extent, of second-stage larvae is the main method currently available to identify F. occidentalis. Molecular identification, however, can be easily carried out by a non-thrips-specialist with a little training. In this study, DNA sequence data [within the mitochondrial cytochrome oxidase I gene ( COI)] and polymerase chain reaction (PCR) were utilized to develop a molecular diagnostic marker for F. occidentalis. A primer set and PCR cycling parameters were designed for the amplification of a single marker fragment (340 bp) of F. occidentalis COI mtDNA. Specificity tests performed on 28 thrips species, efficacy tests performed on five immature developmental stages as well as on male and female adults and tests on primer sensitivity all demonstrated the diagnostic utility of this marker. Furthermore, the primer set was tested on seventeen F. occidentalis populations from different countries and invaded areas in China and proved to be applicable for all geographic populations. It was used successfully to clarify the distribution of F. occidentalis in the Beijing metro area. These results suggested that this diagnostic PCR assay provides a quick, simple and reliable molecular technique for the identification of F. occidentalis. [ABSTRACT FROM AUTHOR]

In December 2019, an outbreak of atypical pneumonia (Coronavirus disease 2019 -COVID-19) associated with a novel coronavirus (SARS-CoV-2) was reported in Wuhan city, Hubei province, China. The outbreak was traced to a seafood wholesale market and human to human transmission was confirmed. The rapid spread and the death toll of the new epidemic warrants immediate intervention. The intra-host genomic variability of SARS-CoV-2 plays a pivotal role in the development of effective antiviral agents and vaccines, as well as in the design of accurate diagnostics. We analyzed NGS data derived from clinical samples of three Chinese patients infected with SARS-CoV-2, in order to identify small- and large-scale intra-host variations in the viral genome. We identified tens of low- or higher- frequency single nucleotide variations (SNVs) with variable density across the viral genome, affecting 7 out of 10 protein-coding viral genes. The majority of these SNVs (72/104) corresponded to missense changes. The annotation of the identified SNVs but also of all currently circulating strain variations revealed colocalization of intra-host as well as strain specific SNVs with primers and probes currently used in molecular diagnostics assays. Moreover, we de-novo assembled the viral genome, in order to isolate and validate intra-host structural variations and recombination breakpoints. The bioinformatics analysis disclosed genomic rearrangements over poly-A / poly-U regions located in ORF1ab and spike (S) gene, including a potential recombination hot-spot within S gene. Our results highlight the intra-host genomic diversity and plasticity of SARS-CoV-2, pointing out genomic regions that are prone to alterations. The isolated SNVs and genomic rearrangements reflect the intra-patient capacity of the polymorphic quasispecies, which may arise rapidly during the outbreak, allowing immunological escape of the virus, offering resistance to anti-viral drugs and affecting the sensitivity of the molecular diagnostics assays., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel human coronavirus that causes coronavirus disease 2019 (COVID-19), was first discovered in December 2019 as the cause of an outbreak of pneumonia in the city of Wuhan, Hubei province, China. The clinical presentation of COVID-19 is fairly nonspecific, and symptoms overlap those of other seasonal respiratory infections concurrently circulating in the population. Furthermore, it is estimated that up to 80% of infected individuals experience mild symptoms or are asymptomatic, confounding efforts to reliably diagnose COVID-19 empirically. To support infection control measures, there is an urgent need for rapid and accurate molecular diagnostics to identify COVID-19-positive patients. In the present study, we evaluated the analytical sensitivity and clinical performance of the following four SARS-CoV-2 molecular diagnostic assays granted emergency use authorization by the FDA using nasopharyngeal swabs from symptomatic patients: the New York SARS-CoV-2 Real-time Reverse Transcriptase (RT)-PCR Diagnostic Panel (modified CDC) assay, the Simplexa COVID-19 Direct (Diasorin Molecular) assay, GenMark ePlex SARS-CoV-2 (GenMark) assay, and the Hologic Panther Fusion SARS-CoV-2 (Hologic) assay. This information is crucial for both laboratories and clinical teams as decisions on which testing platform to implement are made., (Copyright © 2020 Zhen et al.)

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is a globally distributed zoonotic disease of livestock. While F. hepatica and F. gigantica have temperate and tropical distributions, respectively, parasite sympatry occurs in parts of Asia and Africa. A growing protein demand has the potential to facilitate the translocation of parasites from endemic to non-endemic areas, via associated international livestock movements. Such is the case in Southeast Asia, where livestock trade from F. hepatica-endemic countries into China and Vietnam may account for detection of F. hepatica hybrid/introgressed forms. Of particular importance is Lao People's Democratic Republic, which acts as a major livestock thoroughfare for the region. Our ability to understand the impacts of livestock-associated Fasciola spp. movements on local animal and human health is hindered by a lack of ante-mortem diagnostic tools allowing species differentiation. Molecular tools have been developed for Fasciola spp. differentiation, however those rely on access to pure DNA from adult specimens, limiting their application to post-mortem use. Our aim was to detect and differentiate F. hepatica from the endemic F. gigantica in local smallholder cattle in a region of Southeast Asia with frequent livestock trafficking. To do this we designed and validated ante-mortem molecular assays for Fasciola spp. differentiation targeting single-nucleotide polymorphisms (SNPs) within ITS1 and lsrRNA. We then deployed these SNP genotyping assays to diagnose Fasciola spp. infection in 153 local cattle from 27 villages in Northern Laos. We demonstrate the presence of F. hepatica DNA, confirmed by qualitative Sanger and quantitative Illumina amplicon sequencing of ITS1 and lsrRNA, and highlight the shortfalls of Sanger sequencing for Fasciola spp. identification due to the preferential amplification of F. gigantica nucleotides in mixed DNA samples. The outlined protocol enables rapid surveillance of faecal samples for the presence of Fasciola species eggs, their co-infection and/or infection with F. hepatica/F. gigantica hybrids., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background Evaluating the tumor RAS/BRAF status is important for treatment selection and prognosis assessment in metastatic colorectal cancer (mCRC) patients. Correction of artifacts from library preparation and sequencing is essential for accurately analyzing circulating tumor DNA (ctDNA) mutations. Here, we assessed the analytical and clinical performance of a novel amplicon-based next-generation sequencing (NGS) assay, Firefly™, which employs a concatemer-based error correction strategy. Methods Firefly assay targeting KRAS/NRAS/BRAF/PIK3CA was evaluated using cell-free DNA (cfDNA) reference standards and cfDNA samples from 184 mCRC patients. Plasma results were compared to the mutation status determined by ARMS-based PCR from matched tissue. Samples with a mutation abundance below the limit of detection (LOD) were retested again by droplet digital polymerase chain reaction (ddPCR) or NGS. Results The Firefly assay demonstrated superior sensitivity and specificity with a 98.89% detection rate at an allele frequency (AF) of 0.2% for 20 ng cfDNA. Generally, 40.76% and 48.37% of the patients were reported to be positive by NGS of plasma cfDNA and ARMS of FFPE tissue, respectively. The concordance rate between the two platforms was 80.11%. In the pre-treatment cohort, the concordance rate between plasma and tissue was 93.33%, based on the 17 common exons that Firefly™ and ARMS genotyped, and the positive percent agreement (PPA) and negative percent agreement (NPA) for KRAS/NRAS/BRAF/PIK3CA were 100% and 99.60%, respectively. Conclusions Total plasma cfDNA detected by Firefly offers a viable complement for mutation profiling in CRC patients, given the high agreement with matched tumor samples. Together, these data demonstrate that Firefly could be routinely applied for clinical applications in mCRC patients.

Background Quality indicators (QIs) are crucial tools in measuring the quality of laboratory services. Based on the general QIs of the Working Group "Laboratory Errors and Patient Safety (WG-LEPS)" of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), specific QIs have been established in order to monitor and improve the quality of molecular diagnostics, and to assess the detection level of associated disease. Methods A survey was conducted on 46 independent commercial laboratories in China, investigated using questionnaires and on-site inspections. Specific QIs established were mainly based on the specific laboratory work-flow for molecular diagnoses. The specific QI results from three volunteer laboratories were collected and used to validate their effectiveness. Results Of the 46 laboratories participating in the study, 44 (95.7%), conducted molecular diagnostics. Of 13 specific established QIs, six were priority level 1, and seven, priority level 3. At pre-evaluation of data from the three volunteering laboratories, it was found that the newly classified specific QIs had outstanding advantages in error identification and risk reduction. Conclusions Novel specific QIs, a promising tool for monitoring and improving upon the total testing process in molecular diagnostics, can effectively contribute to ensuring patient safety.