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Dawei Shi,1,* Qiulong Zhou,2,* Sihong Xu,1 Yumei Zhu,3 Hui Li,4 Ye Xu2 1National Institutes for Food and Drug Control, Institute of Pathogen Biology at the Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China; 2Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, People’s Republic of China; 3Shenzhen Center for Chronic Disease Control, Shenzhen, People’s Republic of China; 4Tuberculosis Reference Laboratory, Henan Provincial Centers for Disease Control and Prevention, Zhengzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Hui Li, Tuberculosis Reference Laboratory, Henan Provincial Centers for Disease Control and Prevention, Zhengzhou, People’s Republic of China, Tel/Fax +86 371-68089049, Email lihui2005yan@126.com Ye Xu, Engineering Research Centre of Molecular Diagnostics of the Ministry of Education, State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University, Xiamen, People’s Republic of China, Tel +86 592 2187992, Fax +86 592 2187363, Email xuye@xmu.edu.cnPurpose: Pyrazinamide (PZA) is a critical component of standardized chemotherapy for tuberculosis (TB) and is recommended for the treatment of multidrug-resistant (MDR) TB. We aimed to characterize mutations in pncA of M. tuberculosis and evaluate their diagnostic accuracy for PZA susceptibility in China. We also combined genotypic methods with phenotypic susceptibility testing and pyrazinamidase (PZAse) activity to confirm PZA-resistant M. tuberculosis isolates.Results: An evaluation of 82 MDR M. tuberculosis strains revealed that 28.0% (23/82) were phenotypically resistant to 100 mg/L PZA and 15.9% (13/82) showed resistance to 300 mg/L PZA. Mutations in pncA were detected at 33 unique sites, and the majority were point mutations. No evident mutation hotspots or mutations affecting multiple amino acids were found, but the association between pncA mutations and PZA resistance was significant under 100 and 300 mg/L. The sensitivity of pncA mutation detection for predicting PZA susceptibility was 82.6% (19/23), and the specificity was 61.0% (36/59), based on 100 mg/L PZA, whereas the sensitivity was 84.6% (11/13) and the specificity was 55.1% (38/69), based on 300 mg/L PZA. All mutations identified in the highly PZA-resistant (300 mg/L) strains had an 80% loss relative to PZAse activity. No evident PZAse activity loss was observed in one synonymous mutation strain and the loss exceed 60% in all other strains.Conclusion: The association between pncA mutation and PZA resistance was significant. Relatively, the molecular method have shown better reliability than the phenotypic method for the detection of PZA resistance. This provides a theoretical basis for the clinical diagnosis of drug-resistant TB.Keywords: MDR, PZA, pncA, DST, Beijing genotype, enzymatic activity

Many factors have been identified as having the ability to affect the sensitivity of rapid antigen detection (RAD) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to identify the impact of sample processing on the sensitivity of the RAD tests. We explored the effect of different inactivation methods, viral transport media (VTM) solutions, and sample preservation on the sensitivity of four RAD kits based on two SARS-CoV-2 strains. Compared with non-inactivation, heat inactivation significantly impacted the sensitivity of most RAD kits; however, β-propiolactone inactivation only had a minor effect. Some of the VTM solutions (VTM2, MANTACC) had a significant influence on the sensitivity of the RAD kits, especially for low viral-loads samples. The detection value of RAD kits was slightly decreased, while most of them were still in the detection range with the extension of preservation time and the increase of freeze-thaw cycles. Our results showed that selecting the appropriate inactivation methods and VTM solutions is necessary during reagent development, performance evaluation, and clinical application.

Coxsackie virus B5 (CVB5), a main serotype in human Enterovirus B (EVB), can cause severe viral encephalitis and aseptic meningitis among infants and children. Currently, there is no approved vaccine or antiviral therapy available against CVB5 infection. Here, we determined the atomic structures of CVB5 in three forms: mature full (F) particle (2.73 Å), intermediate altered (A) particle (2.81 Å), and procapsid empty (E) particle (2.95 Å). Structural analysis of F particle of CVB5 unveiled similar structures of "canyon," "puff," and "knob" as those other EV-Bs. We observed structural rearrangements that are alike during the transition from F to A particle, indicative of similar antigenicity, cell entry, and uncoating mechanisms shared by all EV-Bs. Further comparison of structures and sequences among all structure-known EV-Bs revealed that while the residues targeted by neutralizing MAbs are diversified and drive the evolution of EV-Bs, the relative conserved residues recognized by uncoating receptors could serve as the basis for the development of antiviral vaccines and therapeutics.

Background Shotgun metagenomics has been used clinically for diagnosing infectious diseases. However, most technical assessments have been limited to individual sets of reference standards, experimental workflows, and laboratories. Methods A reference panel and performance metrics were designed and used to examine the performance of shotgun metagenomics at 17 laboratories in a coordinated collaborative study. We comprehensively assessed the reliability, key performance determinants, reproducibility, and quantitative potential. Findings Assay performance varied significantly across sites and microbial classes, with a read depth of 20 millions as a generally cost-efficient assay setting. Results of mapped reads by shotgun metagenomics could indicate relative and intra-site (but not absolute or inter-site) microbial abundance. Interpretation Assay performance was significantly impacted by the microbial type, the host context, and read depth, which emphasizes the importance of these factors when designing reference reagents and benchmarking studies. Across sites, workflows and platforms, false positive reporting and considerable site/library effects were common challenges to the assay's accuracy and quantifiability. Our study also suggested that laboratory-developed shotgun metagenomics tests for pathogen detection should aim to detect microbes at 500 CFU/mL (or copies/mL) in a clinically relevant host context (10^5 human cells/mL) within a 24h turn-around time, and with an efficient read depth of 20M. Funding This work was supported by National Science and Technology Major Project of China (2018ZX10102001).

Isothermal amplification methods are a promising trend in virus detection because of their superiority in rapidity and sensitivity. However, the generation of false positives and limited multiplexity are major bottlenecks that must be addressed. In this study, we developed a multiplex Argonaute (Ago)-based nucleic acid detection system (MULAN) that integrates rapid isothermal amplification with the multiplex inclusiveness of a single Ago for simultaneous detection of multiple targets such as SARS-CoV-2 and influenza viruses. Owing to its high specificity, MULAN can distinguish targets at a single-base resolution for mutant genotyping. Moreover, MULAN also supports portable and visible devices with a limit of detection of five copies per reaction. Validated by SARS-CoV-2 pseudoviruses and clinical samples of influenza viruses, MULAN showed 100% agreement with quantitative reverse-transcription PCR. These results demonstrated that MULAN has great potential to facilitate reliable, easy, and quick point-of-care diagnosis for promoting the control of infectious diseases.

Currently, quality control of glycoprotein in the human rabies vaccine is based on enzyme-linked immunosorbent assay (ELISA). However, ELISA does not match the needs of a modernised quality control system. For a long time, human rabies virus vaccine manufacturers have been devoted to seeking a detection platform that is sensitive, accurate, automatic, and feasible for practical applications. Therefore, our team invested major efforts into establishing a fully automated micromagnetic particle (MMP)-based chemiluminescence immunoassay (CLIA) platform. For vaccine quality control, MMP-coupled rabies virus glycoprotein monoclonal antibodies (S037) were used to capture the rabies virus. Another rabies virus glycoprotein antibody (S053) labelled with acridinium ester was added as a signal tracer. After pretreating the vaccine sample, the entire analysis was performed using a fully automated machine, which had a limited detection time (only 30 min) and eliminated manual error. Multiple experiments have identified the optimal conditions allowing valid and reliable assessment of vaccine potency. The CLIA platform has exhibited merits in terms of speed, robustness, high sensitivity (with a minimum detection value of 0.45 mIU/mL), considerable accuracy, and a wide linear range of detection (9.4-1200 mIU/mL). Furthermore, the results showed that the CLIA platform is consistent with the National Institutes of Health test and time-resolved fluorescent immunoassay (TRFIA) in quantitative analysis, and had a better analytic performance than TRFIA. Therefore, the CLIA platform presented here may be important for application in modern vaccine quality control.

Background Clinical shotgun metagenomics for pathogen detection has been used in diagnosing infectious diseases. However, its technical assessments have been limited to reference standards by individual labs, single experimental workflow and laboratory.Results Here we reported the design and development of a set of reference reagents and reporting metrics dedicated for clinical metagenomics by the National Institutes for Food and Drug Control (NIFDC) of China, and a joint evaluation study including 17 independent laboratories in cross-workflow and cross-platform settings. Our results showed that the performance of metagenomic assays was significantly impacted by the factors of microbial types, host context, and read depth, thus highlighting the importance to take these factors into consideration when designing reference reagents and benchmarking assays. Through this study, we found false positives to be a common challenge across centers, and considerable site and library effects to limited the assay’s quantitative value. Our multicenter study also provided practical guidance of performance that laboratory-developed shotgun pathogen metagenomics tests should aim to detect microbes at 500 CFU/mL (or copies/mL) in clinically relevant host context (10^5 human cells/mL) within a 24h turn-around time, and with a read depth of 20M reads or lower. This collaboration work provided a unique resource comprising nearly 600 billion reads (>5Tb) for technical evaluation in clinical and regulatory settings.Conclusion We demonstrated that the performance of metagenomic assays was significantly impacted by the microbial type, the host context and read depth, which emphasizes the importance to consider these factors when designing reference reagents and benchmarking studies. Across sites, workflows and platforms, false positive reporting and considerable site/library effects were common challenges to the assay’s accuracy and quantifiability. Our study also suggested practical guidance of performance that laboratory-developed shotgun pathogen metagenomics tests should aim to detect microbes at 500 CFU/mL (or copies/mL) in clinically relevant host context (10^5 human cells/mL) within a 24h turn-around time, and with a read depth of 20M reads or lower. This collaboration work provided a unique resource comprising nearly 600 billion reads (>5Tb) for technical evaluation in clinical and regulatory settings.

Metagenomic next-generation sequencing (mNGS) could be used for pathogen detection from nearly all types of clinical samples. Especially, the unique diagnostic capability of pathogen mNGS detecting unknown causative agent of infectious diseases makes this method become an importation complement and irreplaceable component for conventional routine laboratory test. However, the complexity of the testing process, the rapid product update, and the insufficiency in quality control and evaluation methods that all make clinical transformation, industry development, and regulation of this technology full of challenge and uncertainty. This review briefly introduces the technical advantages and challenges, and describes the general workflow and quality control steps in details. Finally, it focuses on current considerations regarding quality evaluation methods and standards for pathogen mNGS.病原宏基因组高通量测序技术理论上能够检测几乎所有病原体基因组核酸，且适用于几乎所有类型的临床样本，尤其适用于病原不明的疑难感染性疾病的诊断。因此该技术正逐渐成为实验室常规检测方法的重要补充和不可替代的项目。然而，基于该技术的诊断试剂不仅检测流程繁琐复杂、技术更新迭代速度较快，同时相关质量控制与评价的方法和标准也有待明确，这些因素均给该技术的临床转化应用、行业发展以及监管带来挑战和不确定性。文中简述了该技术的原理和优势，以及检测流程和关键质量控制环节，最后着重介绍了关于该技术的质量评价方法和标准的相关思考。.

The outbreak of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. To meet the urgent and massive demand for the screening and diagnosis of infected individuals, many in vitro diagnostic assays using nucleic acid tests (NATs) have been urgently authorized by regulators worldwide. A reference standard with a well-characterized concentration or titer is of the utmost importance for the study of limit of detection (LoD), which is a crucial feature for a diagnostic assay. Although several reference standards of plasmids or synthetic RNA have already been announced, a reference standard for inactivated virus particles with an accurate concentration is still needed to evaluate the complete procedure. Here, we performed a collaborative study to estimate the NAT-detectable units as a viral genomic equivalent quantity (GEQ) of an inactivated whole-virus SARS-CoV-2 reference standard candidate using digital PCR (dPCR) on multiple commercialized platforms. The median of the quantification results (4.6 × 105 ± 6.5 × 104 GEQ/mL) was treated as the consensus true value of GEQ of virus particles in the reference standard. This reference standard was then used to challenge the LoDs of six officially approved diagnostic assays. Our study demonstrates that an inactivated whole virus quantified by dPCR can serve as a reference standard and provides a unified solution for assay development, quality control, and regulatory surveillance.

SARS-CoV-2 is the seventh coronavirus known to infect humans and has caused an emerging and rapidly evolving global pandemic (COVID-19) with significant morbidity and mortality. To meet the urgent and massive demand for the screening and diagnosis of infected individuals, many in vitro diagnostic assays using nucleic acid tests (NATs) have been urgently authorized by regulators worldwide. The limit of detection (LoD) is a crucial feature for a diagnostic assay to detect SARS-CoV-2 in clinical samples, and a reference standard with a well-characterized concentration or titer is of the utmost importance for LoD studies. Although several reference standards of plasmids or synthetic RNA carrying specific genomic regions of SARS-CoV-2 have already been announced, a reference standard for inactivated virus particles with accurate concentration is still needed to evaluate the complete procedure including nucleic acid extraction and to accommodate customized primer-probe sets targeting different genome sequences. Here, we performed a collaborative study to estimate the NAT-detectable units as viral genomic equivalent quantity (GEQ) of an inactivated whole-virus SARS-CoV-2 reference standard candidate using digital PCR (dPCR) on multiple commercialized platforms. The median of the quantification results (4.6×105 ± 6.5×104 GEQ/mL) was treated as the consensus true value of GEQ of virus particles in the reference standard. This reference standard was then used to challenge the LoDs of six officially approved diagnostic assays. Our study demonstrates that an inactivated whole virus quantified by dPCR can serve as a reference standard and provides a unified solution for assay development, quality control, and regulatory surveillance.

A sophisticated regulatory framework has been constructed for Human immunodeficiency virus (HIV) diagnostics in China, which have developed over the past 30 years. China National Institutes for Food and Drug Control acts as the legal institution in this regulatory framework, launching important activities to ensure the quality of HIV diagnostics. These include the analysis of the main problems faced in developing domestic HIV diagnostics, by investigating the quality of HIV diagnostics and their development; exploring the key factors affecting the quality of HIV diagnostics, to determine the criteria for screening national reference samples; the development of new technologies and methods for preparing reference samples; and the establishment of nine types of national reference panels and nine national standards to evaluate the quality of HIV diagnostics. Based on these researches, a quality evaluation system was established, including nine types of national reference panels, nine national standards for HIV diagnostics, and five sample banks (HIV-positive sample bank, HIV-negative sample bank, common international genotype sample bank, seroconversion series sample bank, HIV virus bank) to evaluate the quality of HIV diagnostics in China. The regulatory framework and the quality evaluation system are pivotal in ensuring the quality of the HIV diagnostics licensed in China.

A compound control method is proposed in this paper to alleviate the effects of delay and nonlinear in coagulant dosing process of water treatment system and it merges the fuzzy control algorithm with the Cerebellar Model Arithmetic Control (CMAC). It is taken fuzzy CMAC revision control combined with expert control. According to the features of coagulant dosing process, by using the compound controller, an optimum dosing of coagulant is presented. The compound controller uses the turbidity and PH value of raw water as input variables and the proportional coefficient of coagulant as output variable. It is tested with the actual data from water works, and the simulation results show that the approach is efficient and feasible.

Transmission of antiretroviral drug-resistant strains of HIV-1 has a major impact on the success of HIV treatment regimens in most countries where antiretroviral therapy (ART) is available. It is now recommended that HIV-1 drug resistance be monitored in order to recommend appropriate therapy for infected ART-naïve individuals, or when choosing a new regimen for those patients not responding to ART. Commercial assays for the analysis of HIV-1 genotypic resistance have been approved by the US FDA. In China, several laboratories and research enterprises have been developing related assays, but at present, no systematic standardization or quality control for genotypic resistance assays has been established in China. A national reference panel is needed to evaluate and control the quality of HIV-1 genotype resistance assays in China.A panel of five HIV-1 stocks (G1, G2, G3, G4, and G5) with well-characterized drug resistance was used to evaluate the resistance-mutation inclusivity of HIV-1 clades prevalent in China. Six samples (GS1-GS6) were used to evaluate the limit of detection (LOD) for the viral load levels, and five samples (GSS1-GSS3, GSS5, and GSS6) were used to evaluate the LOD for the percentages of mutant species within the range of detection. The samples were evaluated by five separate laboratories using one or two methods each, generating seven datasets in all.In samples G1-G5, which were used to evaluate inclusivity of HIV-1 clades, 92.86-100%, 16.67-83.33% and 4.17-8.33% of the most, intermediately, and least common resistance mutations, respectively, were reported by the seven datasets. For the LOD samples, four of the seven datasets reported correct resistance mutations in samples with minimal viral loads of2 x 10(3) copies/mL, as well as in samples with40% of mutants and viral loads of about 1 x 10(4) copies/mL; the other three tests did not amplify the target region or identify the mutants.The results were quite variable between different tests. The panel of HIV-1 genotypic resistance could be used as a control for resistance testing in China.

In the present study, 277 clinical samples from untreated and treated HIV-1-infected patients with different clades were used to assess the agreement between the COBAS AmpliPrep/COBAS TaqMan HIV-1 test (CAP/CTM) and VERSANT HIV-1 RNA 3.0 Assay (bDNA). A qualitative comparison of the results of the two assays showed concordance for 255 positive and 15 negative samples (94.95%, kappa = 0.798). However, seven samples with viral loads close to the lower limit of detection for CAP/CTM were negative by bDNA. A significant correlation (r = 0.881, p < 0.001) was observed for 253 samples with viral loads within the dynamic ranges of the two assays, and Bland-Altman analysis showed good agreement (96.05%) between the two assays for these 253 samples [mean (+/-2 SD), 0.389(-0.385, 1.163)]. Furthermore, ART drugs had no impact on the performances of the two assays. For samples with different clades predominant in China, the fitted regression line differed significantly from the line of equality, although significant correlations (r = 0.850-0.891, p < 0.001) and good agreements (92.86-97.25%) were found for the two assays. The mean differences for clade B' and BC samples were significant (p < 0.01). Good precision for clade B' samples was achieved for the CAP/CTM (CV: 20.73%) and bDNA (CV: 12.19%) assays. Furthermore, for clades B', BC, and AE, both assays exhibited good linearities (r = 0.9773-0.9998). Thus, the CAP/CTM and bDNA assays could be useful for quantifying HIV-1 RNA in routine clinical samples and monitoring viral loads in treated and untreated HIV-infected patients in China.

Objective To evaluate the performance of the Abbott RealTime™ HIV-1 assay for measuring viral loads of the prevalent HIV-1 clades in China. Study design Serially diluted samples, as well as 521 clinical samples from 213 untreated HIV-1-infected individuals, 56 HIV-1-infected patients receiving ART, 60 HCV- and 57 HBV-infected patients and 135 healthy blood donors, were tested with RealTime™ and EasyQ™. Results Both assays exhibited linearity coefficients of >0.98. RealTime™ and EasyQ™ detected HIV-1 RNA in 87.36% and 86.99% of 269 HIV-1-seropositive samples, respectively. The correlation coefficients between the two assays for quantifying HIV-1 clades B′, BC and AE were 0.884, 0.813 and 0.881, respectively, and the mean differences between the two assays for clades B′, BC and AE were −0.087, 0.314 and 0.559 log 10 IU/mL, respectively. The correlation coefficient between the two assays for measuring samples from patients receiving ART was 0.945 (mean difference, 0.085 log 10 IU/mL). No false-positive samples were found among the 60 HCV-infected patients, 57 HBV-infected patients and 135 healthy blood donors. Conclusions The Abbott RealTime™ HIV-1 assay shows good linearity and specificity. ART drugs and HIV-1 clades B′ and BC do not affect the performance of the assay. However, clade AE may be less readily detected.

The immunological assays for detection of antibodies against SARS-CoV were developed in-house and some of them are available commercially. However, the antigens used in these assays differed. In order to validate the reliability of these assays, the standard panel should be established. In this study, we have expressed and purified severe acute respiratory syndrome (SARS) structural proteins and their fragments and developed indirect enzyme-linked immunosorbent assays (ELISAs) that detect antibodies against the SARS N, N1, N2, S1, SC, S2, and M proteins as well as the human coronavirus OC43 and 229E N proteins. These assays were used to screen 58 samples from SARS convalescent patients, 40 serial serum specimens from patients at different phases of SARS infection, and 88 plasma specimens from normal blood donors. The samples from normal blood donors were also tested for antibodies against other respiratory virus. The representative samples were chosen to comprise a reference panel of SARS antibodies that may be used for the detection of SARS. The panel is composed of 25 positive samples, 25 negative samples, 7 diluted samples for anti-N antibody, 6 diluted samples for anti-S antibody, and one sample for validating precision. Comparison of detection results with different SARS antibody assays indicated that our panel should differentiate the specificity and sensitivity of different assays.

Oxide mixtures MO–SDC, M = Cu, Ni, Co, SDC = Ce 0.9 Sm 0.1 O 1.95 were synthesized by employing a citrate/nitrate combustion technique. Two kinds of Carbon materials, activated carbon (AC) and vapor grown carbon fiber (VGCF) were homogeneously dispersed into the MO–SDC. The materials can be used as anodes to fabricate single cells using a uniaxial die-press method. The sintering temperature was studied to optimize cell performance. Experimental results showed that cells sintered at 700 °C had better performance. When the temperature was above 750 °C, the cells were severely distorted, and cannot be tested. Compared with the basic MO–SDC anode, AC and VGCF improve the solid oxide fuel cell (SOFC) anode properties, due to a change of the microstructures of the anode materials which enhance their electron conductivity. Single cell performances were evaluated by I–V measurements, and when 1.25 wt.%VGCF was introduced into the MO–SDC by ball-milling, termed: 1.25 wt.%VGCF–MO–SDC, the 1.25 wt.%VGCF–MO–SDC anode material could achieve the highest power density of up to 0.326 W cm −2 with H 2 as fuel. The calcination temperature of the MO–SDC dry gel also strongly influenced the electrochemical performance of the 1.25 wt.%VGCF–MO–SDC material. XRD spectra for each calcined temperature and the I–V measurement both suggest that calcinations at 550 °C for 1 h are suitable. 1.0 wt.%AC–MO–SDC and 1.25 wt.%VGCF–MO–SDC have similar performance when the cell was fed in methanol/3%H 2 O, and the corresponding power density was up to 0.253 W cm −2 . Traces of carbon were found in the off-gases.

Background and Objectives: Drug resistance in HIV-1 is one of the main causes of failure of antiretroviral therapy. Phenotypic detection of drug-resistant HIV-1 can provide guidance in selecting the optimal treatment regimen. Traditional phenotype assays are labor intensive and time consuming. Thus, a rapid and convenient phenotype assay with a single cycle of replication was developed and used in this study. Methods: Two restriction endonuclease sites, ApaI and AgeI, were inserted into the plasmid pSG3,DΔenv using site-directed mutagenesis. The reverse transcriptase and protease genes of HIV-1 were amplified from patients and cloned into the modified pSG3Δenv. Sixteen original recombinant pseudoviruses were generated. The phenotypic susceptibility of these 16 recombinant pseudoviruses to 12 antiretro viral drugs was determined using a luciferase reporter system, and the phenotype and genotype results were compared. Results: A modified phenotype assay with a single-cycle system was established, and its reproducibility and feasibility were validated. Approximately 89% of the phenotype results were in agreement with the genotype results; this slight disagreement may have been due to complex and multiple resistance mutations. The phenotype results showed that individual pseudoviruses with four thymidine analog mutations (TAMs) [M41L, T67N, L210W, and T215Y] in combination with various other mutations had different levels of resistance to nucleoside reverse transcriptase inhibitors (NRTIs). Mutations E44A, T69D, and V118I influenced the pattern of resistance of TAMs. The level of resistance to non-NRTIs (NNRTIs) was also variable when different NNRTI-resistance mutations were combined. Conclusion: The single-cycle pseudovirus phenotypic susceptibility detection system reflects HIV-1 drug resistance, especially for complex resistance mutants, and could be used to screen new antiretroviral candidates.

Background Several commercially available HIV-1 viral load assays based on real-time detection technology and automated platforms are available. It is not clear how the diversity of HIV-1 genotypes impacts the ability to consistently detect HIV-1 viral loads. Objectives To examine whether the diversity of HIV-1 genotypes impacts the ability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0), its version 1.0 (CAP/CTM v1.0) and the NucliSens EasyQ HIV-1 version 2.0 (EasyQ v2.0) assays to consistently determine the viral loads. Study design The three assays were used to measure the viral load in 178 plasma samples with diverse genotypes from treatment-naive patients. Results CAP/CTM v2.0 showed significant correlation and high agreement with CAP/CTM v1.0 and EasyQ v2.0. CAP/CTM v2.0 showed excellent detection of clade B samples compared with CAP/CTM v1.0 and EasyQ v2.0. However, significant differences were observed when using CAP/CTM v2.0 to test clade BC and AE samples. The HIV-1 load measured by CAP/CTM v2.0 differed by >0.5logIU/ml in 59.52% and 72.62% of clade BC samples, and in 57.14% and 85.71% of clade AE samples, compared with CAP/CTM v1.0 and EasyQ v2.0, respectively. CAP/CTM v2.0 was more precise (13.18%) than EasyQ v2.0 (29.21%), and both assays showed good linearity ( R ≥0.9926). Conclusions The three assays may not deliver consistent results for samples belonging to clades BC and AE. It is strongly suggested that the version of the HIV-1 viral load assay used initially is also used at follow-up.

Background and Objective: With the introduction of the ViroSeq™ HIV-1 Genotyping System (ViroSeq™ assay) into China, it is important to evaluate the impact of the diversity of HIV-1 genotypes found in China on the performance of the ViroSeq™ assay compared with an in-house method. Materials and Methods: A total of 318 plasma samples, collected from 206 HIV-1-infected patients receiving antiretroviral therapy and 112 treatment-naive HIV-1-infected patients, were used for evaluating the concordance of genotypes, genotypic resistance mutations, and phenotypic resistance between the ViroSeq™ assay and an in-house method for analyzing HIV-1 drug resistance in China. Results: A concordance of genotypes between the ViroSeq™ assay and the in-house method was observed for the 313 samples (98.4%), using the Stanford University HIV Drug Resistance Database (Version 6.0.5). The overall concordances of drug-resistance-related mutations (DRRMs) in the HIV-1 protease (PR) and reverse transcriptase (RT) coding sequences within the HIV-1 pol gene, scored by the ViroSeq™ assay and the in-house method, were 99.5% and 98.1%, respectively. Discrepancies between the two methods were found in 38 samples assayed for protease inhibitor (PI) DRRMs, 36 samples assayed for nucleoside reverse transcriptase inhibitor (NRTI) DRRMs, and 72 samples assayed for non-nucleoside reverse transcriptase inhibitor (NNRTI) DRRMs, and 100%, 88.9%, and 87.5% of the samples with discrepancies for PI, NRTI, and NNRTI DRRMs, respectively, were genotyped as subtype B. One NNRTI mutation (the RT mutation Y318F) was reported only by the ViroSeq™ assay, and this discrepancy resulted from the difference in the pol gene lengths generated by the two systems. Furthermore, the overall concordance of phenotypic resistance was 94.7% (301/318) between the two methods. Conclusion: The ViroSeq™ assay will be a useful tool for monitoring clinical drug resistance and for better management of HIV-1 patients receiving antiretro viral therapy in China.

Background and Objectives: Six HIV-1 viral load assays have been widely used in China. These include the Cobas Amplicor HIV-1 Monitor Version 1.5 (‘Amplicor’), Cobas AmpliPrep/Cobas TaqMan HIV-1 test Version 1.0 (‘CAP/CTM’), Versant HIV-1 RNA Version 3.0 (branched DNA [bDNA]-based assay; ‘Versant bDNA’), Abbott RealTime HIV-1 assay (‘Abbott RealTime’), NucliSens HIV-1 QT (nucleic acid sequencebased amplification assay; ‘NucliSens NASBA’), and NucliSens EasyQ HIV-1 Version 1.1 (‘EasyQ V1. 1’). Recently, an updated version of EasyQ V1.1, NucliSens EasyQ HIV-1 Version 2.0 (‘EasyQ V2.0’) was introduced into China. It is important to evaluate the impact of HIV-1 genotypes on the updated assay compared with the other commercial available assays in China. Methods: A total of 175 plasma samples with different HIV-1 clades prevalent in China were collected from treatment-naive patients. The viral loads of those samples were determined with the seven HIV-1 viral load assays, and the quantitative differences between them were evaluated. Results: Overall, EasyQ V2.0 exhibited a significant correlation (R = 0.769−0.850, p≤0.001) and high agreement (94.77−97.13%, using the Bland-Altman model) with the other six assays. Although no significant differences between EasyQ V2.0 and the other six assays were observed when quantifying clade B′ samples, there were statistically significant differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays when quantifying clade BC samples, and between EasyQ V2.0 and the Versant bDNA and Abbott RealTime assays when quantifying clade AE samples. For clade BC samples, the quantitative differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log10 IU/mL in approximately 50% of samples and exceeded 1 log10 IU/mL in approximately 15% of samples. For clade AE samples, the quantitative differences between EasyQ V2.0 and the CAP/CTM, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log10 IU/mL in approximately 50% of samples, and the differences between EasyQ V2.0 and CAP/CTM exceeded 1 log10 IU/mL in approximately 15% of samples. Conclusion: Genotypes may affect the quantification of HIV-1 RNA, especially in clade BC samples with respect to EasyQ V2.0 and the Amplicor, Versant bDNA, or Abbott RealTime assays, and in clade AE samples with respect to EasyQ V2.0 and the Versant bDNA or Abbott RealTime assays. It is therefore strongly suggested that, where possible, the HIV-1 viral load in infected patients be quantified at follow-up by the same version of the same assay that was used initially.

To determine whether genetic variability influences the ability to detect antibody, nine gp41 ectodomain recombinant proteins from human immunodeficiency virus type 1 (HIV-1) CRF07_BC, CRF01_AE and subtype B' were expressed in a bacterial expression system and purified. An indirect sandwich ELISA was developed with individual purified recombinant proteins. Plasma samples from 26 individuals infected with HIV-1 of different subtypes and four samples from the 1st international antibody reference panel were tested against each recombinant protein by ELISA. Heat-map and two-dimensional hierarchical clustering methods revealed that ELISA reactivity against antigens derived from the same subtypes clustered together. This suggests a similar reactivity pattern among infections of the same subtype, and thus the antigenicity of gp41 recombinant proteins may vary depending on the subtype, and subtype-related serotypes may exist among these antigens. Using association analysis methods, eight signature sites related to the subtype-specific reactivity patterns were identified. This study provides valuable information for the development of diagnostic assays with the ability to detect broadly cross-reactive antibodies induced by infection with different HIV-1 subtypes.

Objectives: To investigate the impact of genetically diverse HIV samples from China on the performance of the automated COBAS® AmpliPrep/COBAS TaqMan® HIV-1 Test (CAP/CTM). Methods: 185 samples from untreated HIV-1-infected patients were used to assess the performance of the CAP/CTM Test and COBAS Amplicor HIV-1 Monitor Test, v1.5 (Cobas). Results: A comparison of the qualitative results of both assays showed concordance for 1 negative and 184 positive samples (100%, ĸ = 1.000). A significant correlation (R = 0.862, p < 0.001) and high agreement (95.53%) was observed for 179 samples with viral loads within the dynamic ranges of both assays. For samples of clades predominant in China, the fitted regression line differed significantly from the line of equality, although significant correlations (R = 0.694–0.899, p < 0.05) and high agreements (91.30–95.35%) were found for the two assays. The mean differences for samples from clades B’ and BC were significant (p < 0.001) whilst no great difference (7.19–10.88%) between the quantitative values for both assays was found by plotting the regression line. Conclusion: The viral loads of different HIV genotypes in China measured by the CAP/CTM and Cobas assays were comparable.

Background Although Enfuvirtide (T20) has not been used in China, investigating the natural occurrence of primary resistance mutations in the HR1 region of the gp41 sequence from T20 naive patients would help in determining treatment strategies for AIDS patients. Objectives To investigate natural mutations in the HR1 region of the gp41 sequence in China and evaluate their biological function. Study design The amino acid sequences in the HR1 region of 27 isolates including subtypes AE, BC and B′ from Chinese T20 naive patients were analyzed. All 27 isolates were constructed into pseudoviruses and their susceptibility to T20 was measured by using recombinant virus assays. Results Mutations in a 10 amino acid motif, such as N42S and N42R, and those outside this motif, such as L33M, R46S, A50V, Q52H and L54M, were found in the 27 isolates. N in position 42 was mutated to S in all three AE subtype strains and 17 of 18 subtype BC strains, whilst it was mutated to R in all 6 subtypes B strains. Compared to the SF162 strain, the susceptibility of the mutants, except for L33M, to T20 was not significantly different. After L was mutated to M in position 33 of six strains by site directed mutagenesis, their IC50 values were increased by 3.7–7.9-fold. Conclusions Mutations outside a 10 amino acid motif in HR1, such as L33M, may cause T20 resistance.

In this study, 190 HIV-positive samples were collected from different regions of China. The HIV clades of 153 samples were determined successfully based on env sequencing. Specifically, 48, 5, 87, and 13 isolates belonged to clades B', B, BC, and AE, respectively. The viral loads in all samples were measured using three commercial assays, Amplicor HIV-1 monitor v1.5, Nuclisens HIV-1 QT and NucliSens EasyQ HIV-1 assays. The differences and linear correlations for individual assays were compared, with expected 1:1 relationships. Significant differences were found for the following viral loads: clade BC measured by any two assays (P0.001); clade AE between Amplicor 1.5 and Easy Q (P = 0.005); clade B' between Amplicor 1.5 and Nuclisens QT (P = 0.002); clade AE between Amplicor 1.5 and Nuclisens QT (P = 0.025); and clade B' between Amplicor 1.5 and EasyQ (P = 0.04). The largest mean difference in the log(10) values was 0.9518, which was found between Amplicor 1.5 and Nuclisens QT. However, the viral loads for clades AE and B' measured by EasyQ and Nuclisens QT, and those for clade B measured by any two assays did not differ significantly. The degrees of correlation for clades B and B' between any two assays (R0.8) were higher that those for clades AE and BC between any two assays (R0.7), except for clade AE between Amplicor 1.5 and Easy Q. Thus, the clade types, especially clades BC and AE, are most likely to impact on the quantitation of viral load using differentassays.