A novel method for the preparation of reproducible, stable, and non-infectious quality control materials for Chlamydia trachomatis nucleic acid detection.

Chlamydia trachomatis (CT) is a significant sexually transmitted pathogen known to evoke severe complications, including infertility. Nucleic acid amplification tests (NAATs) are recommended by the World Health Organization to detect CT infection. Furthermore, the establishment of methods, performance validation, internal quality control, and external quality assessment for CT NAATs necessitate the utilization of quality control materials (QCs). QCs are specimens or solutions that are analyzed for quality control purposes in a test system. In this study, we established a novel cell line that stably integrates CT amplification target sequences for producing QCs for CT NAATs. Utilizing clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology, we integrated the CT plasmid-mediated sequence (comprising the full length of the cryptic plasmid and the major outer membrane protein gene, 9,136 bp) into the MUC4 gene of HEK293T cells. Positive clones were screened through flow cytometric sorting, single-cell culture, and PCR-based identification, followed by the establishment of stable cell lines. These cells were then processed using optimized cell preservation procedures to prepare QCs. The sequence insertion copy number was confirmed by real-time quantitative PCR. This novel CT QCs demonstrate excellent clinical applicability, non-infectiousness, quantifiability, and stability. With an integrated sequence exceeding 9 kb in length, it offers exceptional flexibility for adapting to new kit developments. Furthermore, maintaining a well-defined copy number and stable shelf life, the QCs closely aligns with the quality control requirements of CT NAATs. This study presents an innovative method for preparing QCs for CT nucleic acid detection, making a valuable contribution to improving the performance of CT NAATs.IMPORTANCEUntreated CT infections impose significant burdens on individuals and communities, underscoring the importance of early and accurate testing via CT NAATs for disease control. QCs are instrumental in identifying testing process issues. Hence, we developed a cell line integrating CT-amplified target sequences as readily accessible non-infectious QCs. These QCs boast several advantages: the integration of over 9 kb of CT sequence allows for broad applicability, allowing flexible adaptation to the development of new kits. Confirming the CT sequence copy number provides a reliable basis for QC concentration preparation and kit detection limit evaluation. Optimized preservation protocol enhances QC stability during storage, facilitating convenient shipment to clinical laboratories at ambient temperatures. In summary, our novel CT QCs offer a powerful tool for improving CT NAAT performance and present a fresh perspective on QC preparation for detecting nucleic acids from intracellular parasitic pathogens.

Key performance evaluation of commercialized multiplex rRT-PCR kits for respiratory viruses: implications for application and optimization.

Respiratory tract infections (RTIs) caused by viruses are prevalent and significant conditions in clinical settings. Accurate and effective detection is of paramount importance in the diagnosis, treatment, and prevention of viral RTIs. With technological advancements, multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been developed and extensively adopted for the diagnosis of viral RTIs. Given the potential challenges in the detection performance of multiplex assays, this study evaluated the analytical sensitivity and competitive interference of the six most commonly used multiplex rRT-PCR kits for detection of respiratory viruses in China. The results revealed that the limits of detection were variable across the viruses and kits. Most of the evaluated multiplex kits demonstrated comparable or enhanced analytical sensitivity compared with singleplex kits for clinically significant viruses, including human adenovirus (HAdV)-3, HAdV-7, Omicron BA.5, H1N1pdm09, H3N2, B/Victoria, respiratory syncytial virus subtype A, and respiratory syncytial virus subtype B, whereas multiplex kits showed relatively less analytical sensitivity for human rhinovirus-B72, human metapneumovirus-A2, parainfluenza virus (PIV)-1, and PIV-3. In addition, most multiplex kits successfully identified co-infections when one analyte was present at a low concentration and another analyte was present at a high concentration. IMPORTANCE: The complexity and severity of viral respiratory tract infections (RTIs) emphasize the pivotal role of precise diagnosis for viral RTIs in guiding effective public health responses and ensuring appropriate medical interventions, given the substantial population at risk. This study highlights the necessity and importance of evaluating the analytical validity of multiplex real-time reverse transcription polymerase chain reaction assays, offering valuable insights into their optimization and application.

New practice of BCR::ABL1 standardization system based on p210 and p190 BCR::ABL1 reference materials.

Quantification of BCR::ABL1 monitors minimal residual disease, thus critical for patient stratification. While significant progress has been made in enhancing the accuracy of p210 BCR::ABL1 quantification, no equivalent standardization has been conducted for p190 BCR::ABL1. Therefore, we developed p190 BCR::ABL1 reference materials to calibrate the quantitative process through an innovative plasmid-based calibration strategy. Then, we further explored the use of p190 and p210 reference materials to standardize tests in 159 laboratories across China and assessed their detection capability utilizing quality assessment samples. Results suggested that after calibration, the coefficient of variation of detection results decreased from 50.8 %-57.4 % to 24.9 %-36.4 % for p190, and from 37.6 %-49.0 % to 19.1 %-28.5 % for p210. The percentage of laboratories within ± 2-fold of the target values increased from 77.1 %, 76.4 %, 73.2 %, and 74.5 % to 94.3 %, 95.5 %, 92.4 %, and 91.1 % for p190 samples 2023S21-2023S24, and from 72.3 %, 86.2 %, 79.2 %, and 81.1 % to 98.1 %, 99.4 %, 98.1 %, and 96.2 % for p210 samples 2023S11-2023S14. Overall, our study successfully developed and employed p190 and p210 reference materials to promote accuracy and comparability of BCR::ABL1 quantification among laboratories.

A real-world multi-center RNA-seq benchmarking study using the Quartet and MAQC reference materials.

Translating RNA-seq into clinical diagnostics requires ensuring the reliability and cross-laboratory consistency of detecting clinically relevant subtle differential expressions, such as those between different disease subtypes or stages. As part of the Quartet project, we present an RNA-seq benchmarking study across 45 laboratories using the Quartet and MAQC reference samples spiked with ERCC controls. Based on multiple types of 'ground truth', we systematically assess the real-world RNA-seq performance and investigate the influencing factors involved in 26 experimental processes and 140 bioinformatics pipelines. Here we show greater inter-laboratory variations in detecting subtle differential expressions among the Quartet samples. Experimental factors including mRNA enrichment and strandedness, and each bioinformatics step, emerge as primary sources of variations in gene expression. We underscore the profound influence of experimental execution, and provide best practice recommendations for experimental designs, strategies for filtering low-expression genes, and the optimal gene annotation and analysis pipelines. In summary, this study lays the foundation for developing and quality control of RNA-seq for clinical diagnostic purposes.

Diagnostic performance of mpox virus (MPXV) real-time PCR assays: multicenter assessment and extended sensitivity analysis.

PURPOSE: To comprehensively investigate the diagnostic performance of routinely used assays in MPXV testing, the National Center of Clinical Laboratories in China conducted a nationwide external quality assessment (EQA) scheme and an evaluated nine assays used by ≥ 5 laboratories in the EQA. METHODS: MPXV virus-like particles with 2700, 900 and 300 copies/mL were distributed to 195 EQA laboratories. For extended analysis, triple-diluted samples from 9000 to 4.12 copies/mL were repeated 20 times using the assays employed by ≥ 5 laboratories. The diagnostic performance was assessed by analyzing EQA data and calculating the limits of detection (LODs). RESULTS: The performance was competent in 87.69% (171/195) of the participants and 87.94% (175/199) of the datasets. The positive percentage agreements (PPAs) were greater than 99% for samples at 2700 and 900 copies/mL, and 95.60% (761/796) for samples at 300 copies/mL. The calculated LODs for the two clades ranged from 228.44 to 924.31 copies/mL and were greater than the LODs specified by the respective kits. EasyDiagnosis had the lowest calculated LODs and showed superior performance in EQA, whereas BioGerm and Sansure, with higher calculated LODs, did not perform well in EQA. CONCLUSION: This study provides valuable information from the EQA data and evaluation of the diagnostic performance of MPXV detection assays. It also provided insights into reagent optimization and enabled prompt public health interventions for the outbreak.

External quality assessment for molecular detection of Ureaplasma urealyticum in China.

BACKGROUND AND AIMS: A pilot external quality assessment (EQA) scheme for molecular detection of Ureaplasma urealyticum (UU) was conducted by the National Center for Clinical Laboratories (NCCL) to evaluate the testing capabilities of clinical laboratories and the actual performance of DNA-based nucleic acid amplification tests (NAAT) and RNA-based NAATs when applied in clinical settings. MATERIALS AND METHODS: The EQA panel contained twelve lyophilized samples, including positive samples containing inactivated cell culture supernatants of UU at different concentrations and sterile saline for negative samples. The positive samples were further divided into three groups of high, moderate and low concentrations. The panels were distributed to the participants and the datasets were analyzed according to the qualitative results. RESULTS: A total of 365 laboratories participated in the EQA scheme, and 360 results submitted by 338 laboratories were collected, of which 96.11 % (346/360) of the returned results and 95.86 % (324/338) of the laboratories were deemed competent. The positive percentage agreement (PPA) was ≥ 97.5 % for high and moderate concentration samples, but varied significantly for low concentration samples, decreasing from 86.94 % to 51.94 % as the sample concentration decreased. Additionally, for low concentration samples, RNA-based NAAT showed higher PPAs than DNA-based NAATs, but these results were specific to UU supernatants used in this study. CONCLUSION: Most of UU detection assays employed by the participants were generally consistent with their estimated limit of detection (LOD), and the majority of participants can reliably detect UU samples with high and moderate concentrations, while the poor analytical performance for low concentration samples requires further improvement and optimization.

Enhancing the quality of panel-based tumor mutation burden assessment: a comprehensive study of real-world and in-silico outcomes.

Targeted panel-based tumor mutation burden (TMB) assays are widely employed to guide immunotherapy for patients with solid tumors. However, the accuracy and consistency of this method can be compromised due to the variability in technical details across different laboratories, particularly in terms of panel size, somatic mutation detection and TMB calculation rules. Currently, systematic evaluations of the impact of these technical factors on existing assays and best practice recommendations remain lacking. We assessed the performance of 50 participating panel-based TMB assays involving 38 unique methods using cell line samples. In silico experiments utilizing TCGA MC3 datasets were performed to further dissect the impact of technical factors. Here we show that the panel sizes beyond 1.04 Mb and 389 genes are necessary for the basic discrete accuracy, as determined by over 40,000 synthetic panels. The somatic mutation detection should maintain a reciprocal gap of recall and precision less than 0.179 for reliable psTMB calculation results. The inclusion of synonymous, nonsense and hotspot mutations could enhance the accuracy of panel-based TMB assay. A 5% variant allele frequency cut-off is suitable for TMB assays using tumor samples with at least 20% tumor purity. In conclusion, this multicenter study elucidates the major technical factors as sources of variability in panel-based TMB assays and proposed comprehensive recommendations for the enhancement of accuracy and consistency. These findings will assist clinical laboratories in optimizing the methodological details through bioinformatic experiments to enhance the reliability of panel-based methods.

RLP system: A single-tube two-step approach with dual amplification cascades for rapid identification of EGFR T790M.

BACKGROUND: The detection of cancer gene mutations in biofluids plays a pivotal role in revolutionizing disease diagnosis. The presence of a large background of wild-type sequences poses a challenge to liquid biopsy of tumor mutation genes. Suppressing the detection of wild-type sequences can reduce their interference, however, due to the minimal difference between mutant and wild-type sequences (such as single nucleotide variants differing by only one nucleotide), how to suppress the detection of wild-type sequences to the greatest extent without compromising the sensitivity of mutant sequence detection remains to be explored. SIGNIFICANCE: The RLP system addresses the incompatibility between RPA and RT-PCR reactions through a physical separation strategy. Besides, due to the remarkable flexibility of locked nucleic acid probes, the RLP system emerges as a potent tool for detecting mutations across diverse genes. It excels in sensitivity and speed, tolerates plasma matrix, and is cost-effective. This bodes well for advancing the field of precision medicine. RESULTS: The recombinase-assisted locked nucleic acid (LNA) probe-mediated dual amplification biosensing platform (namely RLP), which combines recombinase polymerase amplification (RPA) and LNA clamp PCR method in one tube, enabling highly sensitive and selective detection of EGFR T790M mutation under the help of well-designed LNA probes. This technique can quantify DNA targets with a limit of detection (LoD) at the single copy level and identify point mutation with mutant allelic fractions as low as 0.007 % in 45 min. Moreover, RLP has the potential for the direct detection of plasma samples without the need for nucleic acid extraction and the cost of a single test is less than 1USD. Furthermore, the RLP system is a cascading dual amplification reaction conducted in a single tube, which eliminates the risk of cross-contamination associated with opening multiple tubes and ensures the reliability of the results.

External quality assessment for detection of colorectal cancer by Septin9 DNA methylation in clinical laboratories.

BACKGROUND AND AIMS: The incidence and mortality rate of colorectal cancer (CRC) are increasing worldwide. Septin9 methylated (mSEPT9) DNA in circulation can be used as a non-invasive detection method to assist in the early diagnosis of CRC; however, the detection methods and procedures are complicated. This study aimed to evaluate the ability of clinical laboratories to detect Septin9 methylation in plasma cell-free DNA (cfDNA). MATERIALS AND METHODS: We prepared a sample panel consisting of positive and negative Septin9 methylation cells and CRC cells. Three positive samples with different methylation levels, one negative sample and one duplicate sample, two samples containing interference, three different CRC cell samples, and a fictitious case report were included. The panel was distributed to 59 laboratories for mSEPT9 analysis, result comparison, and scoring. RESULTS: The sample panel, validated by National Medical Products Administration (NMPA)-approved tests and targeted bisulfite sequencing, met expectations and could be used for external quality assessment (EQA). Among the 59 laboratories, 55 (93.22%) correctly reported the mSEPT9 results for all samples, while four (6.79%) reported 15 false negatives and were considered improvable. All false negatives originated from four laboratories using laboratory-developed tests (LDTs), with three failing to detect weakly positive samples, samples containing interference, and samples from different CRC cells, and one reported erroneous results on all positive samples. CONCLUSION: Our results illustrated that the detection of mSEPT9 in cfDNA is satisfactory in China. EQA is indispensable because it can help improve the diagnostic capability and quality management of the laboratories, and provide suggestions for the problems existing in mSEPT9 detection.

Comparison of analytical sensitivity of DNA-based and RNA-based nucleic acid amplification tests for reproductive tract infection pathogens: implications for clinical applications.

Currently, DNA-based nucleic acid amplification tests (NAATs) and RNA-based NAATs are employed to detect reproductive tract infection (RTI) pathogens including Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU). Although evaluations of DNA-based NAATs have already existed, the comparison of the two methods is scarce. Thus, we compared the limits of detection (LODs) of DNA-based and RNA-based NAATs on the same experimental conditions. Inactivated culture supernatants of CT, NG, and UU with determined pathogen DNA and RNA load were used to evaluate LODs of seven DNA kits and one RNA kit. The LODs of the seven DNA kits for CT, NG, and UU ranged between 38-1,480, 94-20,011, and 132-2,011 copies/mL, respectively. As for RNA kits, they could detect samples at RNA concentrations of 3,116, 2,509, and 2,896 copies/mL, respectively. The RNA concentrations of CT, NG, and UU were 40, 885, and 42 times that of corresponding pathogen DNA concentrations in the employed supernatants, so RNA kits could detect pathogen DNA concentrations as low as 78 copies/mL, 3 copies/mL, and 69 copies/mL, respectively, but the level of pathogen load that the RNA tests could detect was primarily dependent on the infectious phase and transcriptional level of RNA. Thus, a schematic of bacterial dynamics during the period of reproductive tract infections was provided, which suggests that in terms of the analytical sensitivity of pathogen detection, RNA tests are more suitable for detecting active infection and recovery phase, while DNA tests are more suitable for detection in the early stage of infection. IMPORTANCE Reproductive tract infections have considerable effects on the health of humans. CT, NG , and UU are common pathogens. Although evaluation of DNA-based tests has already existed, the comparison between DNA-based and RNA-based tests is rare. Therefore, this study compared the limits of detection of the two tests on the same experimental conditions. Results suggested that most DNA-based NAATs could detect CT, NG, and UU at DNA concentrations lower than 1,000 copies/mL, while RNA-based NAATs could detect bacteria at RNA concentrations around 3,000 copies/mL. Considering the copy number of RNA per bacterium is dynamic through the growth cycle, further comparison is combined with a schematic of bacterial dynamics. Results suggested that in terms of the analytical sensitivity of pathogen detection, RNA tests are more suitable for detecting active infection and recovery phase, while DNA tests are more suitable for detection in the early stage of infection.

Evaluation of noninvasive prenatal screening for copy number variations among screening laboratories.

OBJECTIVE: To evaluate the current situation of expanded noninvasive prenatal screening (NIPS) for copy number variations (CNVs) in laboratories in China, the National Center of Clinical Laboratories conducted an externalqualityassessment (EQA) program. METHODS: The EQA panel consisted of 12 artificial samples associated with different syndromes, which were mixed with maternal plasma collected from pregnant women and enzyme-digested cell-free DNA (cfDNA) from cell lines with different fetal fractions (FFs) ranging from 5% to 15%. The panel was validated by next-generation sequencing and distributed to laboratories, along with questionnaires and case scenarios. RESULTS: Sixty-nine laboratories participated in the EQA program, and 91.30% (63/69) of laboratories correctly identified all samples. A total of 7.25% (5/69) of the laboratories reported false-negative results, and 2.90% (2/69) of the laboratories reported unexpected CNVs. The correct rates of the 22q11.2 deletion syndrome, Cri-du-chat syndrome, 1p36 deletion syndrome and Angelman/Prader-Willi syndrome samples were 97.46%, 98.55%, 100%, and 100%, respectively. With the increase in the FF, deletion size, and read depth, the detection rate increased. For results reports, only five laboratories reported FF values, one laboratory reported the CNV classification type, and none reported sensitivity, specificity, positive predictive values, and negative predictive values. CONCLUSION: The detection capabilities of NIPS for CNVs still need to be improved and standardized, and FF, deletion size, and read depth are factors that affect the detection rate.

Validation and benchmarking of targeted panel sequencing for cancer genomic profiling.

OBJECTIVES: To validate a large next-generation sequencing (NGS) panel for comprehensive genomic profiling and improve patient access to more effective precision oncology treatment strategies. METHODS: OncoPanScan was designed by targeting 825 cancer-related genes to detect a broad range of genomic alterations. A practical validation strategy was used to evaluate the assay's analytical performance, involving 97 tumor specimens with 25 paired blood specimens, 10 engineered cell lines, and 121 artificial reference DNA samples. RESULTS: Overall, 1107 libraries were prepared and the sequencing failure rate was 0.18%. Across alteration classes, sensitivity ranged from 0.938 to more than 0.999, specificity ranged from 0.889 to more than 0.999, positive predictive value ranged from 0.867 to more than 0.999, repeatability ranged from 0.908 to more than 0.999, and reproducibility ranged from 0.832 to more than 0.999. The limit of detection for variants was established based on variant frequency, while for tumor mutation burden and microsatellite instability, it was based on tumor content, resulting in a minimum requirement of 20% tumor content. Benchmarking variant calls against validated NGS assays revealed that variations in the dry-bench processes were the primary cause of discordances. CONCLUSIONS: This study presents a detailed validation framework and empirical recommendations for large panel validation and elucidates the sources of discordant alteration calls by comparing with "gold standard measures."

Assessing the Quality of Metagenomic Next-Generation Sequencing for Pathogen Detection in Lower Respiratory Infections.

BACKGROUND: Laboratory-developed metagenomic next-generation sequencing (mNGS) assays are increasingly being used for the diagnosis of infectious disease. To ensure comparable results and advance the quality control for the mNGS assay, we initiated a large-scale multicenter quality assessment to scrutinize the ability of mNGS to detect pathogens in lower respiratory infections. METHODS: A reference panel containing artificial microbial communities and real clinical samples was used to assess the performance of 122 laboratories. We comprehensively evaluated the reliability, the source of false-positive and false-negative microbes, as well as the ability to interpret the results. RESULTS: A wide variety of weighted F1-scores was observed across 122 participants, with a range from 0.20 to 0.97. The majority of false positive microbes (68.56%, 399/582) were introduced from "wet lab." The loss of microbial sequence during wet labs was the chief cause (76.18%, 275/361) of false-negative errors. When the human context is 2 × 105 copies/mL, most DNA and RNA viruses at titers above 104 copies/mL could be detected by >80% of the participants, while >90% of the laboratories could detect bacteria and fungi at titers lower than 103 copies/mL. A total of 10.66% (13/122) to 38.52% (47/122) of the participants could detect the target pathogens but failed to reach a correct etiological diagnosis. CONCLUSIONS: This study clarified the sources of false-positive and false-negative results and evaluated the performance of interpreting the results. This study was valuable for clinical mNGS laboratories to improve method development, avoid erroneous results being reported, and implement regulatory quality controls in the clinic.

Performance evaluation of SARS-CoV-2 antigen detection in the post-pandemic era: multi-laboratory assessment.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen detection is an indispensable tool for epidemic surveillance in the post-pandemic era. Faced with irregular performance, a comprehensive external quality assessment (EQA) scheme was conducted by the National Center for Clinical Laboratories (NCCL) to evaluate the analytical performance and status of SARS-CoV-2 antigen tests. METHODS: The EQA panel included ten lyophilized samples containing serial 5-fold dilutions of inactivated SARS-CoV-2-positive supernatants of the Omicron BA.1 and BA.5 strains and negative samples, which were classified into "validating" samples and "educational" samples. Data were analyzed according to qualitative results for each sample. RESULTS: A total of 339 laboratories in China participated in this EQA scheme, and 378 effective results were collected. All validating samples were correctly reported by 90.56 % (307/339) of the participants and 90.21 % (341/378) of the datasets. The positive percent agreement (PPA) was >99 % for samples with concentrations of 2 × 107 copies/mL but was 92.20 % (697/756) for 4 × 106 copies/mL and 25.26 % (382/1,512) for 8 × 105 copies/mL samples. Colloidal gold was the most frequently used (84.66 %, 320/378) but showed the lowest PPAs (57.11 %, 1,462/2,560) for positive samples compared with fluorescence immunochromatography (90 %, 36/40) and latex chromatography (79.01 %, 335/424). Among 11 assays used in more than 10 clinical laboratories, ACON showed a higher sensitivity than other assays. CONCLUSIONS: The EQA study can help to validate whether it's necessary to update antigen detection assays for manufacturers and provide participants with information about the performance of assays to take the first step toward routine post-market surveillance.

Analysis of Clinical Laboratory Detecting Challenging Variants from Exome Sequencing Using Simulated Patient-Parent Trio Sample: Pilot Study for Neurodevelopmental Disorder de Novo Variants.

To date, there has been no systematic analysis for the clinical laboratory in detecting technically challenging variants using the trio-based exome sequencing (ES) approach. Here, we present an interlaboratory pilot proficiency testing study that used synthetic patient-parent specimens to assess the detection of challenging variants with de novo dominant inheritance modes for neurodevelopmental disorders using various trio-based ES. In total, 27 clinical laboratories that performed diagnostic exome analyses participated in the survey. One of the 26 challenging variants was identified by all laboratories, whereas all 26 variants were identified by only nine laboratories. The lack of identification of mosaic variants was often due to the bioinformatics analysis that excluded the variant. For missing intended heterozygous variants, probable root causes were related to the technical bioinformatics pipeline and variant interpretation and reporting. For each missing variant, there may be more than one probable reason from the different laboratories. There was considerable variation in interlaboratory performance for detecting challenging variants using trio-based ES. This finding may have important implications for the design and validation of tests for different variant types in clinical laboratories, especially for technically challenging variants, and necessary workflow modification can potentially improve trio-based ES performance.

The mechanism of Girdin in degenerative brain disease caused by high glucose stimulation.

Girdin, as an actin-binding protein, plays a major role in maintaining the stability of the actin skeleton structure and affects the growth, development, and migration of neurons. This study discusses the mechanism of Girdin in brain degeneration caused by high glucose stimulation. We examined the expression of Girdin in diabetic patients. The positive expression rate of Girdin in the diabetic group was 17.2% (5/29), which was obviously lower than the positive expression rate of 83.3% (20/24) in the non-diabetic group. We examined the expression of Girdin and its signaling pathway-related proteins Akt and STAT3 in hippocampal neurons induced by high glucose. The results showed that, in contrast to the control group (glucose concentration = 25 mmol/L), the expression of Girdin in the high-glucose group (glucose concentration = 225 mmol/L) was reduced (P < 0.05); the phosphorylation levels of Akt and STAT3 related to Girdin signaling pathway were also reduced (P < 0.05). Under high-glucose stimulation, the structure of neurons is abnormal, such as the reduction or disappearance of dendritic spines, and the number of neurons is reduced. In addition, Girdin and Akt were less expressed in neurons and synapses, especially the most obvious reduction in synaptic terminals. The activity of Girdin and its signaling pathway-related proteins Akt and STAT3 decreased in neurons under high glucose stimulation, indicating that the mechanism of Girdin in brain degeneration caused by high glucose stimulation was closely related to the Akt and STAT3 pathways. Graphic Abstract: The mechanism of Girdin in degenerative brain disease caused by high glucose stimulation. This article discusses the mechanism of Girdin in brain degeneration induced by high glucose stimulation. The expression of Girdin in the diabetic group was significantly lower than that in the non-diabetic group. The expression of Girdin and its signaling pathway-related proteins Akt and STAT3 in hippocampal neurons was significantly reduced under high glucose stimulation. Under high glucose stimulation, the structure of neurons is abnormal and the number decreases; synapses become shorter. It indicates that the mechanism of brain degeneration caused by high glucose stimulation by Girdin is closely related to the Akt and STAT3 pathways.

Evaluation of Four Strategies for SARS-CoV-2 Detection: Characteristics and Prospects.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed an enormous burden on the global public health system and has had disastrous socioeconomic consequences. Currently, single sampling tests, 20-in-1 pooling tests, nucleic acid point-of-care tests (POCTs), and rapid antigen tests are implemented in different scenarios to detect SARS-CoV-2, but a comprehensive evaluation of them is scarce and remains to be explored. In this study, 3 SARS-CoV-2 inactivated cell culture supernatants were used to evaluate the analytical performance of these strategies. Additionally, 5 recombinant SARS-CoV-2 nucleocapsid (N) proteins were also used for rapid antigen tests. For the wild-type (WT), Delta, and Omicron strains, the lowest inactivated virus concentrations to achieve 100% detection rates of single sampling tests ranged between 1.28 × 102 to 1.02 × 103, 1.28 × 102 to 4.10 × 103, and 1.28 × 102 to 2.05 × 103 copies/mL. The 20-in-1 pooling tests ranged between 1.30 × 102 to 1.04 × 103, 5.19 × 102 to 2.07 × 103, and 2.59 × 102 to 1.04 × 103 copies/mL. The nucleic acid POCTs were all 1.42 × 103 copies/mL. The rapid antigen tests ranged between 2.84 × 105 to 7.14 × 106, 8.68 × 104 to 7.14 × 106, and 1.12 × 105 to 3.57 × 106 copies/mL. For the WT, Delta AY.2, Delta AY.1/AY.3, Omicron BA.1, and Omicron BA.2 recombinant N proteins, the lowest concentrations to achieve 100% detection rates of rapid antigen tests ranged between 3.47 to 142.86, 1.74 to 142.86, 3.47 to 142.86, 3.47 to 142.86, and 5.68-142.86 ng/mL, respectively. This study provided helpful insights into the scientific deployment of tests and recommended the full-scale consideration of the testing purpose, resource availability, cost performance, result rapidity, and accuracy to facilitate a profound pathway toward the long-term surveillance of coronavirus disease 2019 (COVID-19). IMPORTANCE In the study, we reported an evaluation of 4 detection strategies implemented in different scenarios for SARS-CoV-2 detection: single sampling tests, 20-in-1 pooling tests, nucleic acid point-of-care tests, and rapid antigen tests. 3 SARS-CoV-2-inactivated SARS-CoV-2 cell culture supernatants and 5 recombinant SARS-CoV-2 nucleocapsid proteins were used for evaluation. In this analysis, we found that for the WT, Delta, and Omicron supernatants, the lowest concentrations to achieve 100% detection rates of single sampling tests ranged between 1.28 × 102 to 1.02 × 103, 1.28 × 102 to 4.10 × 103, and 1.28 × 102 to 2.05 × 103 copies/mL. The 20-in-1 pooling tests ranged between 1.30 × 102 to 1.04 × 103, 5.19 × 102 to 2.07 × 103, and 2.59 × 102 to 1.04 × 103 copies/mL. The nucleic acid POCTs were all 1.42 × 103 copies/mL. The rapid antigen tests ranged between 2.84 × 105 to 7.14 × 106, 8.68 × 104 to 7.14 × 106, and 1.12 × 105 to 3.57 × 106 copies/mL. For the WT, Delta AY.2, Delta AY.1/AY.3, Omicron BA.1, and Omicron BA.2 recombinant N proteins, the lowest concentrations to achieve 100% detection rates of rapid antigen tests ranged between 3.47 to 142.86, 1.74 to 142.86, 3.47 to 142.86, 3.47 to 142.86, and 5.68 to 142.86 ng/mL, respectively.

Reprogramming of Human B Cells from Secreting IgG to IgM by Genome Editing.

B lymphocytes are activated and regulated by their interactions with T cells, a process that results in one-way class switching of immunoglobulins (ig) from IgM to IgG, IgE, or IgA. In this study, we show the application of clustered regularly interspaced short palindromic repeat-Cas9-induced nonhomologous end joining in B cells to achieve reverse-directional Ig class switching. By electroporating Cas9 and guide RNA and a Cµ encoding donor into cells, we engineered IgG-secreting human B cell lines to switch to express IgM antibody. This approach offers a new potential path for the production of IgM antibodies.

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.

Adaptation of ACMG-ClinGen Technical Standards for Copy Number Variant Interpretation Concordance.

This study aimed to evaluate inter-laboratory classification concordance for copy number variants (CNVs) with a semiquantitative point-based scoring metric recommended by the American College of Medical Genetics and Genomics (ACMG) and Clinical Genome Resources (ClinGen). A total of 234 CNVs distributed by the National Center of Clinical Laboratories (NCCLs), and 72 CNVs submitted by different laboratories, were distributed to nine clinical laboratories performing routine clinical CNV testing in China and independently classified across laboratories. The overall inter-laboratory complete classification concordance rate of the 234 distributed CNVs increased from 18% (41/234) to 76% (177/234) using the scoring metric compared to the laboratory's previous method. The overall inter-laboratory complete classification concordance rate of the 72 submitted CNVs was 65% (47/72) using the scoring metrics. The 82 variants that initially did not reach complete concordance classification and 1 additional CNV deletion were reviewed; 34 reached complete agreement, and the overall post-review complete concordance rate was 85% (260/306). Additionally, the overall percentage of classification discordance possibly impacting medical management [i.e., pathogenic (P) or likely pathogenic (LP) vs. variant of uncertain significance (VUS)] was 11% (35/306). The causes of initial and final discordance in the classification were identified. The ACMG-ClinGen framework has promoted consistency in interpreting the clinical significance of CNVs. Continuous training among laboratories, further criteria and additional clarification of the standards, sharing classifications and supporting evidence through public database, and ongoing work for dosage sensitive genes/regions curation will be beneficial for harmonization of CNVs classification.