External quality assessment for laboratory testing of HLA-B*15:02 allele in relation to carbamazepine therapy.

BACKGROUND: Due to the significant risk of developing Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), the use of carbamazepine is not recommended in patients carrying the human leukocyte antigen B (HLA-B) *15:02 allele. In an effort to guarantee reliable community-based HLA-B*15:02 testing throughout China, a HLA-B*15:02 genotyping external quality assessment (EQA) program was set up. METHODS: In 2016, 10 genomic DNA samples with known HLA-B*15:02 allele status were sent to 37 laboratories from 16 provinces with a request for routine HLA-B*15:02 screening. The samples were validated using Sanger sequencing by a reference laboratory. Both genotyping results and clinical written reports were evaluated. RESULTS: Thirty-six of the participating laboratories correctly identified the HLA-B*15:02 allele status for all EQA samples. However, one lab failed to identify any positive challenges. The overall analytical sensitivity was 97.3% (180/185 challenges; 95% confidence interval: 93.8%-99.1%) and the analytic specificity was 100% (185/185; 95% confidence interval: 98.0%-100%). A review of the written reports showed that the clinical reporting for HLA-B*15:02 detection should be improved. Some essential information was missing, most notably laboratory information/contact, therapeutic recommendations, and methodology. CONCLUSION: External quality assessment is valuable in assessing and improving the quality of laboratory testing of HLA-B*15:02 allele.

A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V mutation testing.

BACKGROUND: KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations. METHODS: We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS. RESULTS: We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively. CONCLUSION: The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material.

Quality Assessment of Reporting Performance for EGFR Molecular Diagnosis in Non-Small Cell Lung Cancer.

BACKGROUND: Reports serve as a bridge between laboratories and clinicians, help synthesize an overwhelming amount of raw data into evidence-based medicine, and play a significant role in designing clinical treatments. In an effort to guarantee high-quality epidermal growth factor receptor (EGFR) gene mutation testing and reporting performance, the National Center for Clinical Laboratories launched a proficiency testing (PT) scheme reflecting clinical practices in China since 2014. This study focuses on the quality assessment of gene mutation reports. MATERIALS AND METHODS: Fifty-three laboratories that submitted reports in both 2014 and 2016 EGFR gene mutation PT schemes were selected for report analysis and comparison according to predefined evaluation criteria. RESULTS: The average score for reports from 2014 was 14 out of 30 points. The overall scores for reports from 2016 improved substantially, yielding an average score of 20 out of 30 points. Among the evaluation criteria, general items were well documented in the reports. However, items specific to molecular diagnosis were far from satisfactory, and some items were even missing. CONCLUSION: The quality assessment of clinical written reports from 2014 and 2016 demonstrates that substantial improvements have been made in overall reporting performance. However, not all statements pertaining to important elements met expectations. To continue education, repeated PT schemes need to be executed in a timely fashion to expose and address existing shortcomings in clinical reports. There remains ample room for improvement towards generating concise, comprehensive, and readable reports. IMPLICATIONS FOR PRACTICE: This article compares the quality of clinical gene mutation reports submitted in 2014 to those submitted in 2016 epidermal growth factor receptor proficiency testing schemes, exposes the existing shortcomings, and discusses ways to communicate results more effectively in the future. The findings demonstrate that notable progress was observed in the overall reporting performance. However, key points specific to molecular diagnosis were far from expectation, and some items were even missing. Standardization needs to be emphasized to improve the report format and content. This article provides a reference that laboratories can use to write concise, comprehensive, and readily accessible clinical reports.

Synthetic Circulating Cell-free DNA as Quality Control Materials for Somatic Mutation Detection in Liquid Biopsy for Cancer.

BACKGROUND: Detection of somatic genomic alterations in tumor-derived cell-free DNA (cfDNA) in the plasma is challenging owing to the low concentrations of cfDNA, variable detection methods, and complex workflows. Moreover, no proper quality control materials are available currently. METHODS: We developed a set of synthetic cfDNA quality control materials (SCQCMs) containing spike-in cfDNA on the basis of micrococcal nuclease digestion carrying somatic mutations as simulated cfDNA and matched genomic DNA as genetic background to emulate paired tumor-normal samples in real clinical tests. Site-directed mutagenesis DNA that contained 1500-2000 bases with single-nucleotide variants or indels and genomic DNA from CRISPR/Cas9 edited cells with EML4-ALK rearrangements was fragmented, quantified, and added into micrococcal nuclease-digested DNA derived from HEK293T cells. To prove their suitability, the SCQCMs were compared with patient-derived plasma samples and validated in a collaborative study that encompassed 11 laboratories. RESULTS: The results of SCQCM analysis by next-generation sequencing showed strong agreement with those of patient-derived plasma samples, including the size profile of cfDNA and the quality control metrics of the sequencing data. More than 95% of laboratories correctly detected the SCQCMs with EGFR T790M, L858R, KRAS G12D, and a deletion in exon 19, as well as with EML4-ALK variant 2. CONCLUSIONS: The SCQCMs were successfully applied in a broad range of settings, methodologies, and informatics techniques. We conclude that SCQCMs can be used as optimal quality controls in test performance assessments for circulating tumor DNA somatic mutation detection.

Evaluation of an individual-donation nucleic acid amplification testing algorithm for detecting hepatitis B virus infection in Chinese blood donors.

BACKGROUND: This multicenter study was performed to evaluate the efficiency of a multiplex individual-donation nucleic acid amplification technology (ID-NAT) and discriminatory testing algorithm for detecting hepatitis B virus (HBV) infection in Chinese blood donors. STUDY DESIGN AND METHODS: A total of 1,205,796 hepatitis B surface antigen (HBsAg)-nonreactive donations from 10 blood centers were tested by ID-NAT using the Ultrio assay. Multiplex Ultrio-reactive donations were tested in the discriminatory tests as well as in quantitative polymerase chain reaction (qPCR) and in supplemental electrochemiluminescence immunoassays for HBsAg, hepatitis B surface antibody (anti-HBs), hepatitis B e antigen, and antibody to hepatitis B core antigen (anti-HBc). Meanwhile, a control group of 4317 Ultrio-nonreactive donations was tested for anti-HBc and anti-HBs. RESULTS: Of all donations, 2033 (0.17%) were reactive in the multiplex Ultrio assay. Among 1776 further tested samples, 548 (30.9%) were HBV discriminatory assay (dHBV)-reactive, while 1214 (68.4%) were nonreactive. Of 472 Ultrio+ and dHBV+ samples 86.2% were qPCR positive compared to 15.0% in 1046 Ultrio+ and dHBV- samples. The proportion of anti-HBc+ and anti-HBs- (potentially infectious) donations was higher in 409 Ultrio+ and dHBV+ than in 1028 Ultrio+ and dHBV- samples (51.3% vs. 31.1%, p < 0.001). The yield rate of Ultrio+, dHBV+, and qPCR+ donations was estimated at 1 in 2500, but at 1 in 1100 when all supplemental tests were taken into account assuming that 44% of detected donations by Ultrio were false reactive. CONCLUSIONS: A quarter of HBsAg-negative Ultrio+ and dHBV- donations in China are likely given by potentially infectious low-viral-load occult carriers. Although this has no implication for blood safety, the testing algorithm needs to be redesigned to more efficiently discriminate between true and false NAT reactivity.

What is the meaning of a nonresolved viral nucleic acid test-reactive minipool?

BACKGROUND: This study aimed at analyzing the prevalence of hepatitis B virus (HBV) DNA among hepatitis B surface antigen (HBsAg)-negative donations by cobas TaqScreen MPX test (Roche Molecular Systems) and discussing the meaning of a reactive minipool (MP) that does not resolve to an individual donation (ID)-reactive result. STUDY DESIGN AND METHODS: Nucleic acid amplification testing (NAT) was performed in 12 Chinese blood centers on 826,044 serologic negative donations in MPs of six. MP-reactive pools that were resolved to ID-reactive donations were confirmed by Roche TaqMan viral load assays. Antibody to hepatitis B surface antigen and antibody to hepatitis B core antigen (anti-HBc) results were also analyzed. Cycle threshold (Ct) values of reactive MPs were analyzed in relation to the probability of pool resolution. RESULTS: A total of 1267 of 137,674 pools were reactive, of which 839 donations were reactive by ID-NAT. The MP6 HBV NAT-yield rate lay between 1 in 1600 and 1 in 1000. At MP Ct values equal or below 37, the probability of pool resolution was approximately 80%. The prevalence of anti-HBc in ID-reactive donations was 81%. The proportion of reactive pools that could not be resolved was 36%. The prevalence of anti-HBc in donations implicated in nonresolved MPs was significantly higher than those in nonreactive MPs (48% vs. 37%, p = 0.016). CONCLUSION: The anti-HBc data suggest that approximately 10% of nonresolved MPs contain HBV DNA from a low-viral-load occult carrier. We consider ID-NAT resolution testing in duplicate to minimize HBV transmission risk associated with transfusing nonreactive donations implicated in reactive MPs.

Improvements in CYP2C9 Genotyping Accuracy Are Needed: A Report of the First Proficiency Testing for Warfarin-related CYP2C9 and VKORC1 Genotyping in China.

Warfarin is the most commonly used oral anticoagulant in clinical practice. The cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex 1 (VKORC1) genotypes have been confirmed to be associated with warfarin dose requirements. Accurate genotyping results are of particular importance for obtaining reliable genotype-guided warfarin dosing information. This study aims to determine analytic performance of laboratories offering CYP2C9 and VKORC1 testing in China. A proficiency panel of 15 validated cell samples covering common CYP2C9 and VKORC1 genetic polymorphisms was provided to 31 participating laboratories, and their genotyping results were evaluated. Fourteen data sets (45.2%) performed well with the entire panel of samples, and 17 data sets (54.8%) reported at least one genotyping error. For VKORC1 (-1639G>A), participating laboratories were 100% successful in detecting genotypes of GG, GA, and AA. For CYP2C9, participants were greater than 90% successful in detecting genotypes of *1/*1, *1/*2, *1/*3, *2/*3, and *3/*3. However, 15 laboratories failed to detect rarely encountered variant genotype *2/*2. The poor performance of CYP2C9 genotyping may be because of the limitation of methodologies used for detecting CYP2C9*2 allele. The proficiency testing survey highlighted the need for improving genotyping accuracy for some laboratories in this field.

Using a novel microRNA delivery system to inhibit osteoclastogenesis.

Previously, we developed a novel microRNA (miRNA) delivery system based on bacteriophage MS2 virus-like particles (MS2 VLPs). In this current study, we used this system to transport miR-146a into human peripheral blood mononuclear cells (PBMCs), and demonstrated the inhibition of osteoclastogenesis in precursors. Two cytokines, receptor activator of NF-κB ligand (RANKL), and macrophage-colony stimulating factor (M-CSF) were used to induce osteoclastogenesis. MS2 VLPs were transfected into PBMCs. qRT-PCR was applied to measure expression levels of miR-146a and osteoclast (OC)-specific genes. Western blot (WB) was conducted to evaluate miR-146a downstream target proteins: epidermal growth factor receptor (EGFR) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). The formation and activity of OCs were assessed by cytochemical staining and bone resorption assay, respectively. In PBMCs treated with MS2-miR146a VLPs, qRT-PCR assays showed increased expression of miR-146a (p < 0.01) and decreased expression of all four OC-specific genes (p < 0.05). WB results indicated decreased expression of EGFR (p < 0.01) and TRAF6 (p < 0.05). The number of OCs decreased markedly and bone resorption assay demonstrated inhibited activity. This miR-146a delivery system could be applied to induce overexpression of miR-146a and to inhibit the differentiation and function of OCs.

External quality assessment for Avian Influenza A (H7N9) Virus detection using armored RNA.

An external quality assessment (EQA) program for the molecular detection of avian influenza A (H7N9) virus was implemented by the National Center for Clinical Laboratories (NCCL) of China in June 2013. Virus-like particles (VLPs) that contained full-length RNA sequences of the hemagglutinin (HA), neuraminidase (NA), matrix protein (MP), and nucleoprotein (NP) genes from the H7N9 virus (armored RNAs) were constructed. The EQA panel, comprising 6 samples with different concentrations of armored RNAs positive for H7N9 viruses and four H7N9-negative samples (including one sample positive for only the MP gene of the H7N9 virus), was distributed to 79 laboratories in China that carry out the molecular detection of H7N9 viruses. The overall performances of the data sets were classified according to the results for the H7 and N9 genes. Consequently, we received 80 data sets (one participating group provided two sets of results) which were generated using commercial (n = 60) or in-house (n = 17) reverse transcription-quantitative PCR (qRT-PCR) kits and a commercial assay that employed isothermal amplification method (n = 3). The results revealed that the majority (82.5%) of the data sets correctly identified the H7N9 virus, while 17.5% of the data sets needed improvements in their diagnostic capabilities. These "improvable" data sets were derived mostly from false-negative results for the N9 gene at relatively low concentrations. The false-negative rate was 5.6%, and the false-positive rate was 0.6%. In addition, we observed varied diagnostic capabilities between the different commercially available kits and the in-house-developed assays, with the assay manufactured by BioPerfectus Technologies (Jiangsu, China) performing better than the others. Overall, the majority of laboratories have reliable diagnostic capacities for the detection of H7N9 virus.

Inhibition of the TLR4/NF-κB pathway promotes the polarization of LPS-induced BV2 microglia toward the M2 phenotype.

This study aimed to investigate whether the inhibition of the TLR4/NF-κB pathway can promote lipopolysaccharide (LPS)-induced microglial polarization from the M1 to M2 phenotype, and thus exert neuroprotection. LPS-induced microglia were used as a model for inflammation in vitro. TLR4-specific inhibitor resatorvid (TAK-242) and NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) were used to verify the effect of the TLR4/NF-κB pathway on microglia activation and polarization. Cell proliferation was measured by cell counting, and nitric oxide (NO) and reactive oxygen species (ROS) release was measured using the Griess reagent and ROS kit, respectively. Immunofluorescence and RT-qPCR analyses were used to detect the expression of microglial activation markers, phenotypic markers, related pathway molecules, and inflammatory factors. TLR4 specific inhibitor TAK-242 and NF-κB inhibitor PDTC alleviated LPS-induced microglia over-activation by inhibiting the TLR4/NF-κB pathway, and reduced LPS-stimulated cell proliferation and the release of NO, ROS, TNF-a, and IL-6 and IL-1ß. Meanwhile, TAK-242 and PDTC promoted LPS-induced polarization of microglia from M1 to M2 phenotype, decreased the expression of microglial activation marker Iba1 and M1 phenotypic markers (TNF-a and CD86), and increased the expression of M2 phenotypic markers (Arg-1 and CD206). The mechanism may be related to inhibiting the TLR4/NF-κB pathway. The inhibition of the TLR4/NF-κB pathway can promote LPS-induced polarization of BV2 microglia from M1 phenotype to M2 phenotype.

Multicolor flow cytometry analysis of the proliferations of T-lymphocyte subsets in vitro by EdU incorporation.

EdU (5-ethynyl-2'-deoxyuridine) incorporation has proved advantageous in the studies of cell kinetics, DNA synthesis, and cellular proliferation in vitro and in vivo compared to [(3) H]thymidine incorporation and BrdU (5-bromo-2'-deoxyuridine) incorporation. Here, we describe a method that combines EdU incorporation and immunostaining with flow cytometric analysis to detect the proliferations of T lymphocyte subsets in vitro and optimized the assay's conditions. We found that the number of EdU(+) cells were associated with EdU concentration, incubation time, and the volume of Click reaction solution, the best EdU concentration 10-50 µM, the optimal incubation time 8-12 h and the proper volume of Click volume 100 µl for labeling 1 × 10(6) lymphocytes. Fixation was better to be performed before permeabilization, not together with. Furthermore, the permeabilization detergent reagent, PBS with 0.05% saponin was better than Tris buffer saline (TBS) with 0.1% Triton X-100. In addition, sufficient wash with PBS with 0.05% saponin has no influence on the staining of EdU(+) cells. Also, the lymphocytes incorporating EdU could be stored at 4°C, -80°C, and in liquid nitrogen up to 21 days. The present study will aid in optimization of flow cytometry assay to detect the proliferations of T cell subsets by EdU incorporation and the labeling of cell surface antigens.

[Construction and expression of virus-like particles containing rotavirus NSP3 gene].

OBJECTIVE: To construct and express ribonuclease-resistant virus-like particles containing rotavirus NSP3 gene by changing the affinity of MS2 bacteriophage coat protein pac site and to discuss the stability. METHODS: In the study, 1 049 bp rotavirus NSP3 gene fragments were amplified by PCR using the primers containing PvuI and KpnI restriction enzyme sites and the uridine at position -5 in the pac site was replaced with cytosine to increase the affinity. The gel-purified PCR-amplified DNA fragments and pACYC-MS2 vector were digested with PvuI and KpnI and then ligated to generate recombinant plasmid pACYC-MS2-NSP3. The expression vector was transformed into competent Escherichia coli strain TOP 10, and was verified by PCR and sequencing. The positive bacteria were transformed into competent E.coli strain BL21(DE3). Then the cells were lysed by ultrasonic disruption, virus like particles (VLPs) were harvested after purification and their stability was discussed. RESULTS: The expression plasmid containing mutant pac site (uridine at position -5 in the pac site replaced with cytosine) was constructed successfully and the ribonuclease-resistant virus-like particles containing rotavirus NSP3 gene were expressed successfully. The VLPs were resistant to ribonuclease and deoxyribonuclease, and were stable at -20 °C, 4 °C and room temperature(25 °C), respectively. CONCLUSION: The methods used to increase the affinity of pac site could successfully construct and express the VLPs. The VLPs containing rotavirus NSP3 gene are stable and could be used as surrogates for positive controls and standards in rotavirus real time fluorescent quantitative reverse transcription-PCR kits.

Multilaboratory assessment of metagenomic next-generation sequencing for unbiased microbe detection.

Introduction: Metagenomic next-generation sequencing (mNGS) assay for detecting infectious agents is now in the stage of being translated into clinical practice. With no approved approaches or guidelines available, laboratories adopt customized mNGS assays to detect clinical samples. However, the accuracy, reliability, and problems of these routinely implemented assays are not clear. Objectives: To evaluate the performance of 90 mNGS laboratories under routine testing conditions through analyzing identical samples. Methods: Eleven microbial communities were generated using 15 quantitative microbial suspensions. They were used as reference materials to evaluate the false negatives and false positives of participating mNGS protocols, as well as the ability to distinguish genetically similar organisms and to identify true pathogens from other microbes based on fictitious case reports. Results: High interlaboratory variability was found in the identification and the quantitative reads per million reads (RPM) values of each microbe in the samples, especially when testing microbes present at low concentrations (1 × 103 cell/ml or less). 42.2% (38/90) of the laboratories reported unexpected microbes (i.e. false positive problem). Only 56.7% (51/90) to 83.3% (75/90) of the laboratories showed a sufficient ability to obtain clear etiological diagnoses for three simulated cases combined with patient information. The analysis of the performance of mNGS in distinguishing genetically similar organisms in three samples revealed that only 56.6% to 63.0% of the laboratories recovered RPM ratios (RPM S. aureus /RPM S. epidermidis ) within the range of a 2-fold change of the initial input ratios (indicating a relatively low level of bias). Conclusion: The high interlaboratory variability found in both identifying microbes and distinguishing true pathogens emphasizes the urgent need for improving the accuracy and comparability of the results generated across different mNGS laboratories, especially in the detection of low-microbial-biomass samples.

Research on a new cationic polyacrylamide (CPAM) with a cationic microblock structure and its enhanced effect on sludge condition and dewatering.

Flocculation is one of the commonly used sludge conditioning methods in water supply plants, which can improve the sludge dewatering performance by reducing the specific resistance of sludge (SRF), decreasing the amount of sludge, and finally lowering the transportation cost and subsequent disposal cost of sludge. Therefore, it is particularly important to develop new and efficient flocculants. In this paper, the template copolymer of acryloxy trimethylammonium chloride (DAC) and acrylamide (AM) was successfully synthesized by microwave-template copolymerization (MV-TP) using sodium polyacrylate (NaPAA) as template. The template copolymer was analyzed by infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance hydrogen spectroscopy (1H NMR), and scanning electron microscopy (SEM). It was found that this template copolymer had obvious cationic microblock structure. In addition, the test results of association constant (KM) and polymerization kinetics showed that the MW-TP was assigned to free radical initiated polymerization and the polymerization mechanism was I Zip-up (ZIP). It confirmed the formation of cation fragment structure again. Due to its dense positive charges in this new cationic microblock structure, it greatly improved the functions of electric neutralization, electrical patching, and adsorption bridging. The cationic fragment structure in the template copolymer could help to generate large and dense floc structure and form stable drainage channels. Under external pressure, these large and compact floc structures had greater compressive resistance, which avoided deformation and blockage of drainage channels and voids. It was beneficial to reduce SRF and evidently enhanced sludge dewatering performance.

Reliable assessment of BRCA1 and BRCA2 germline variants by next-generation sequencing: a multicenter study.

BACKGROUND: BRCA1/2 gene mutation testing, based on next-generation sequencing (NGS), has been gradually applied in the clinic to serve as preventive early screening for predisposed individuals or to provide treatment options for patients with hereditary breast or ovarian cancers. Here, we evaluated the accuracy of NGS-based mutation detection in BRCA1/2 and the consistency in variant interpretation among clinical laboratories to find the possible reasons underlying inaccurate results and discrepant variant interpretation. METHODS: Laboratories were asked to use their routine procedures to detect six mimetic DNA samples with different BRCA1/2 germline variants. The results of variant detection were required to be submitted via a web-based evaluation system and were automatically scored, according to predefined criteria. The variant interpretation report, including the detailed clinical evidence, was summarized and analyzed for reasons underlying inconsistent results. RESULTS: Overall, only 55.2% (16/29) of laboratories, whose detection score was higher than 90 points, was found to be an acceptable detection capability level. 82.9% (29/35) of the errors were genotype errors. The variant classification results were generally consistent, and 77.8% (7/9) of the variants were given the consistent classification answer. Only two single nucleotide variants (SNVs) had a discrepant classification opinion across laboratories. CONCLUSIONS: The BRCA1/2 variant detection performance should be further improved, especially in reporting the correct genome coordinates. Inconsistent variant classification may be a result of the different clinical pieces of evidence collected by the laboratories. However, discordant clinical evidence also appeared within the same classification results. Therefore, our study provided clear clinical evidence assessment strategies for BRCA1/2 variants, which was aimed at obtaining a consistent variant classification strategy for providing accurate clinical reports to the clinicians.

Quality evaluation of molecular diagnostic tests for astrovirus, sapovirus and poliovirus: A multicenter study.

BACKGROUND: Astrovirus (AstV), Sapovirus (SaV) and Poliovirus (PV) are important pathogens that cause infections in children under five years of age. It is a very important task to systematically monitor and evaluate the diagnostic performance of these viruses in clinical laboratories. METHODS: In our study, we performed a multicenter evaluation study among 21 laboratories across China using simulated stool samples spiked with self-designed AstV, SaV and PV pseudoviral particles. RESULTS: The testing capability of 80.0% (16/20, AstV), 52.6% (10/19, SaV), and 25.0% (2/8, PV) of the participating laboratories were found to be "competent" in reporting correct results for all samples. The main type of errors were false negatives. None of the laboratories identified the subtypes of AstV and SaV, and six laboratories specifically identified the subtypes of PV. Lacking of well-trained personnel and adequate funding were the main challenges. From the questionnaire results, 55.6% laboratories (10/18) believe that training personnel could improve the laboratory testing performance. CONCLUSIONS: The laboratories showed a competent diagnostic performance for AstV, but inferior diagnostic performances for SaV and PV. Sensitivity of detection and the ability for virus typing should be improved clinically. Professional and standardized personnel training is urgently needed to further improve laboratory performance.

Multicenter assessment of microbial community profiling using 16S rRNA gene sequencing and shotgun metagenomic sequencing.

INTRODUCTION: Microbiome research based on high-throughput sequencing has grown exponentially in recent years, but methodological variations can easily undermine the reproducibility across studies. OBJECTIVES: To systematically evaluate the comparability of sequencing results of 16S rRNA gene sequencing (16Ss)- and shotgun metagenomic sequencing (SMs)-based microbial community profiling in laboratories under routine conditions. METHODS: We designed a multicenter study across 35 participating laboratories in China using designed mock communities and homogenized fecal samples. RESULTS: A wide range of practices and approaches was reported by the participating laboratories. The observed microbial compositions of the mock communities in 46.2% (12/26) of the 16Ss and 82.6% (19/23) of the SMs laboratories had significant correlations with the expected result (Spearman r>0.59, P <0.05). The results from laboratories with near-identical protocols showed slight interlaboratory deviations. However, a high degree of interlaboratory deviation was found in the observed abundances of specific taxa, such as Bacteroides spp. (range: 0.3%-53.5%), Enterococci spp. (range: 0.8%-43.9%) and Fusobacterium spp. (range: 0.1%-39.8%). SMs performed better than 16Ss in detecting low-abundance bacteria (B. bifidum). The differences in DNA extraction methods, amplified regions and bioinformatics analysis tools (taxonomic classifiers and database) were important factors causing interlaboratory deviations. Addressing laboratory contamination is an urgent task because various sources of unexpected microbes were found in negative control samples. CONCLUSIONS: Well-defined control samples, such as the mock communities in this study, should be routinely used in microbiome research for monitoring potential biases. The findings in this study will provide guidance in the choice of more reasonable operating procedures to minimize potential methodological biases in revealing human microbiota composition.

Continual Improvement of the Reliability of EML4-ALK Rearrangement Detection in Non-Small-Cell Lung Cancer: A Long-Term Comparison of ALK Detection in China.

The results of EML4-ALK testing are critical to manage ALK tyrosine kinase receptor inhibitor treatment. Thus, the accurate detection of ALK rearrangement is increasingly becoming a matter of serious concern. To address this issue, a long-term EML4-ALK proficiency testing (PT) scheme was launched in China in 2015, serving as an educational tool for assessing and improving the testing quality of EML4-ALK fusion detection. Responses across 20 different PT samples interrogating three different variants and wild-type samples were collected between 2015 and 2019. Performance was analyzed by evaluating the detection methods, kits, and pre-analytic practices used to further display the landscape of changing conditions of the reliability of EML4-ALK testing. During the 5 years, 3224 results reported from 988 laboratories were evaluated, with an overall error rate of 5.36%. Along with an increasing number of participating laboratories, the error rate within each of the different methods showed a significantly downward trend over the years. No obvious differences in the error rates were found regarding the testing methods or kit manufacturers. Moreover, the individual performance of the laboratories improved when they participated in more PT scheme rounds. The data demonstrated that the performance of individual Chinese laboratories for EML4-ALK testing continuously improved over time by participating PT schemes, regardless of their method. However, care must be taken in standardized operations and validations.

Challenges of Providing Concordant Interpretation of Somatic Variants in Non-Small Cell Lung Cancer: A Multicenter Study.

Background: Success of multiple-gene mutation tests by next-generation sequencing (NGS), associated with molecular targeting therapies for cancers, depending on the accuracy and consistency of interpreting variants. Here, we summarized reports from clinical laboratories for cases with non-small cell lung cancer (NSCLC) and discussed conflicting interpretations of somatic variants. Methods: Three mimetic DNA samples, containing six somatic mutations, were prepared based on three clinical case reports of NSCLC. Clinical reports and genetic testing questionnaires were collected from 67 laboratories enrolled in this investigation. Results: Thirty-four laboratories with correct variant results identified two variants, based on FDA approval of targeted drugs for the same tumor, consistently, with strong clinical significance, whereas the other variants were classified with conflicting interpretations. Discordant interpretations were reported for ERBB2 with three different classifications, including strong clinical significance (53.0%, 18/34), potential clinical significance (38.2%, 13/34), and unknown significance (8.8%, 3/34). In the variant therapeutic drug recommendation section, 32.4% of the laboratories (11/34) did not recommend all the available therapeutic drugs designated by the National Comprehensive Cancer Network (NCCN). In the remaining group of 33 laboratories with incorrect variant results, less correct classifications were acquired for the variants with strong clinical significance. Conclusions: Owing to numerous reasons, the interpretation of variants differed greatly, which might in turn lead to the inappropriate clinical care of patients with NSCLC. By analyzing the limitations of different databases used by laboratories, we integrated various types of databases with different levels of evidence to form a comprehensive and detailed variant interpretation pipeline, aiming to standardize the variant classification and provide accurate and sufficient therapeutic drug recommendation to clinicians for minimal-inappropriate therapeutic options.