Fifteen years of the proficiency testing program for HIV-1 viral load testing laboratories in China, 2005-2019.

BACKGROUND: HIV-1 viral load (VL) testing is essential for monitoring the effects of antiretroviral therapy in HIV-infected patients. In order to identify factors that impact testing performance of HIV-1 VL testing laboratories, this study performed a retrospective analysis on data from the domestic HIV-1 VL proficiency testing (PT) program in China during 2005 to 2019. METHODS: Analysis was conducted on testing results of 155 PT panel specimens that were distributed to HIV-1 VL testing laboratories nationwide during 2005 to 2019. Follow-up on-site inspection records on unqualified laboratories were also analyzed. RESULTS: By 2019, 267 HIV-1 VL testing laboratories in China enrolled in the national PT assessment. Overall, HIV-1 VL testing performance has been consistently good after 2012, with less than 5% of participants reporting an unqualified score. Unsatisfactory equipment conditions and poor laboratory administration were the two main reasons causing unqualified scores in the PT assessment. Interestingly, we found that HIV-1 VL testing laboratories had better performance in the CDC system than in hospitals. In analysis on the variance of specimen testing results by different assays, we found that variation in results existed across different assay platforms, and HIV-1 VL testing assays based on real-time PCR technology showed comparatively smaller inter-laboratory variability. CONCLUSIONS: To maintain high performance in HIV-1 VL testing laboratories, it is important to strengthen laboratory management and preserve equipment conditions. Due to the variation of testing results among different assay platforms, we suggest using the same assay platform for longitudinal monitoring of patient viral load.

Lack of HIV-1 integrase inhibitor resistance among 392 antiretroviral-naïve individuals in a tertiary care hospital in Beijing, China.

: Reports of resistance to integrase strand transfer inhibitors (INSTIs) are now not uncommon. We analyzed the HIV int gene from plasma of antiretroviral-naïve individuals during acute and chronic HIV-1 infection. No individual with major INSTI mutations was identified. Two individuals harbored INSTI accessory mutations E157Q/T97A were detected for the first time. Our results emphasize the need to consider testing for INSTI resistance at baseline as this class of drugs is increasingly used in clinical routine.

The Transmission and Evolution of HIV-1 Quasispecies within One Couple: a Follow-up Study based on Next-Generation Sequencing.

Next-generation sequencing (NGS) has been successfully used to trace HIV-1 infection. In this study, we investigated the transmission and evolution of HIV-1 quasispecies in a couple infected through heterosexual behavior. A heterosexual couple in which both partners were infected with HIV-1 was followed up for 54 months. Blood samples including whole-blood and plasma samples, were collected at various time points. After HIV-1 subtyping, NGS (Miseq platform) was used to sequence the env region of the HIV-1 quasispecies. Genetic distances were calculated, and phylogenetic trees were generated. We found both partners were infected with HIV-1 subtype circulating recombinant form (CRF), CRF65_cpx. The quasispecies distribution was relatively tightly clustered in the phylogenetic tree during early infection. Over time, the distribution of HIV-1 quasispecies gradually became more dispersed at 12th months, with a progressive increase in gene diversity. By 37th months, the sequences obtained for both partners formed different clusters in the phylogenetic tree. These results suggest that the HIV-1 contact tracing results generated by the Miseq platform may be more reliable than other conventional sequencing methods, which can provide important information about the transmission and evolution of HIV-1. Our findings may help to better target preventative interventions for promoting public health.

Short Communication: Investigating a Chain of HIV Transmission Events Due to Homosexual Exposure and Blood Transfusion Based on a Next Generation Sequencing Method.

This study investigates a chain of HIV transmission events due to homosexual exposure and blood transfusion in China. The MiSeq platform, a next generation sequencing (NGS) system, was used to obtain genetic details of the HIV-1 env region (336 base pairs). Evolutionary analysis combined with epidemiologic evidence suggests a transmission chain from patient T3 to T2 through homosexual exposure and subsequently to T1 through blood transfusion. More importantly, a phylogenetic study suggested a likely genetic bottleneck for HIV in homosexual transmission from T3 to T2, while T1 inherited the majority of variants from T2. The result from the MiSeq platform is consistent with findings from the epidemiologic survey. The MiSeq platform is a powerful tool for tracing HIV transmissions and intrapersonal evolution.

[Contact tracing of a possible case of HIV sexual transmission by using Miseq platform].

OBJECTIVE: An approach for analysis of HIV quasispecies using Miseq high-throughput sequencing platform (hereinafter referred to as Miseq platform) was established and applied to contact tracing for a possible case of HIV sexual transmission. METHODS: Four plasma specimens were collected from 2 HIV infections (P1 and P2) suspected to be involved in the sexual transmission and 2 local HIV infections as controls (P3 and P4). The RNAs were extracted from the specimens and then reverse-transcribed into cDNA. After HIV subtyping, Miseq platform was performed to detect and sequence the HIV quasispecies (352 bp) in each specimen. The frequency of quasispecies was counted and ranked. Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated by using the top 5, 20, 100, 500, and all quasispecies, respectively. RESULTS: The subtypes of HIV from all 4 specimens were CRF01_AE. 23 788 to 37 397 cleaned sequences representing 1 229 to 1 412 unique HIV quasispecies were obtained from these specimens by using Miseq platform. The average genetic distance (3.5%-4.5%) between quasispecies from specimens P2 and P1 was significantly lower than that (10.3%-19.6%) between quasispecies from P2 and the controls (P3 or P4). Phylogenetic tree analysis indicated that sequences from specimens P1 and P2 clustered together while sequences from P3 and P4 exhibited completely independent clusters. When the top 20 or more quasispecies from each specimen were analyzed, sequences from P1 showed a paraphyletic relationship with those from P2, which may indicated that the direction of HIV transmission was from P1 to P2. CONCLUSION: With the feature of convenient and economic operation, Miseq platform has high practical value in contact tracing of possible HIV transmission.

[Molecular investigation of a possible case of HIV transmission after a blood transfusion].

OBJECTIVE: A molecular technique based on quasispecies analysis for tracing postexposure HIV transmission was applied in an investigation of a possible case of HIV transmission after blood transfusion. METHODS: Sixteen plasma specimens were collected from 3 HIV infections (T1-T3) involved in a possible HIV transmission chain and 13 HIV/AIDS (C1-C13) controls. The RNAs were extracted and then amplified by RT-PCR, the PCR products were cloned and sequenced.BioEdit 6.0.7 and MEGA 4.0 software were used to analyze gene sequences, calculate gene dispersion ratio and construct phylogenetic tree. RESULTS: The sequences of 13 specimens were successfully obtained.The HIV strains from T1, T2 and T3 were CRF07_BC recombinants, those from 5 out of the 6 controls lived in the same city with T2 and T3 were CRF07_BC recombinants as well, while those from 4 controls living in the same city with T1 were CRF01_AE recombinants. Compared with the clone sequences from T1, the mean gene dispersion ratio of T2 was the least (2.0%), followed by C12 (2.8%) , T3 (2.9%) and others. The phylogenetic tree showed that all clones from T1, T2, T3 and C12 might cluster together,and implied that the direction of HIV transmission was from T3 to T2, and then to T1. CONCLUSION: The results support the possible epidemiological clue that HIV was transmitted from T3 to T2, and then to T1, indicating that molecular epidemiological investigation could provide more direct evidence for tracing postexposure HIV transmission.

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen.

BACKGROUND: HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. METHOD: A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. RESULTS: The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. CONCLUSIONS: When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3-4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.

[Comparison of three HIV antibody confirmatory assay kits in confirming early HIV infection].

OBJECTIVE: This study was to compare the performance of three HIV antibody confirmatory assay kits in confirming early HIV infection. METHODS: Five HIV antibody-positive plasma specimens were ten-fold serially diluted and then detected by ELISA. The above diluted specimens were detected with the following three HIV antibody confirmatory assay kits to analyze their sensitivity, including Wantai-RIBA (Recombinant immunoblot assay, Beijing Wantai Biological Pharmacy, China), MP-WB (HIV Blot 2.2 WB, MP Biomedicals Asia Pacific Pte. Ltd., Singapore) and INNO-LIA (INNO-LIA(TM) HIV I/II Score, Innogenetics N.V., Belgium), respectively. These kits were further used to detect 48 ELISA-reactive specimens from 11 sets of HIV seroconversion specimens (a total of 48 samples) which were previously detected as HIV antibody-positive by ELISA. RESULTS: When 5 samples were diluted to 100 fold, Wantai-RIBA still can detect them positive. Among the 48 HIV antibody-positive specimens detected with ELISA, the confirmation positive rate for Wantai-RIBA, MP-WB and INNO-LIA were 97.92% (47/48), 81.25% (39/48) and 91.67% (44/48), respectively. There was statistically significant difference between the confirmatory results of Wantai-RIBA and MP-WB (χ(2) = 6.13, P < 0.05), as well as between those of INNO-LIA and MP-WB (χ(2) = 5.48, P < 0.05); however, there was no statistically significant difference between those of Wantai-RIBA and INNO-LIA (χ(2) = 1.33, P > 0.05). For other six HIV seroconversion panels containing indeterminate specimens, the average seroconversion period of time for Wantai-RIBA, MP-WB and INNO-LIA were 0.7, 13.3 and 3.7 days, respectively. CONCLUSION: Compared with MP-WB, Wantai-RIBA and INNO-LIA could reduce the window period to confirm early HIV infection.

Quality assurance in the HIV/AIDS laboratory network of China.

BACKGROUND: In 2009, there were 8273 local screening laboratories, 254 confirmatory laboratories, 35 provincial confirmatory central laboratories and 1 National AIDS Reference Laboratory (NARL) in China. These laboratories were located in Center for Disease Control and Prevention (CDC) facilities, hospitals, blood donation clinics, maternal and child health (MCH) hospitals and border health quarantine health-care facilities. METHODS: The NARL and provincial laboratories provide quality assurance through technical, bio-safety and managerial training; periodic proficiency testing; on-site supervisory inspections; and commercial serologic kit evaluations. RESULTS: From 2002 to 2009, more than 220 million HIV antibody tests were performed at screening laboratories, and all reactive and indeterminate samples were confirmed at confirmatory laboratories. The use of highly technically complex tests, including CD4 cell enumeration, viral load, dried blood spot (DBS)-based early infant diagnosis (EID), drug resistance (DR) genotyping, HIV-1 subtyping and incidence assays, have increased in recent years and their performance quality is closely monitored. CONCLUSION: China has made significant progress in establishing a well-coordinated HIV laboratory network and QA systems. However, the coverage and intensity of HIV testing and quality assurance programmes need to be strengthened so as to ensure that more infected persons are diagnosed and that they receive timely prevention and treatment services.

Human immunodeficiency virus-1 genotypic drug resistance among volunteer blood donors in Yunnan, China.

BACKGROUND: Drug resistance profiles of human immunodeficiency virus-1 (HIV-1) in treatment-naïve infections have been reported in developed countries. However, little is known in developing countries, including China, especially in treatment-naïve volunteer blood donors. STUDY DESIGN AND METHODS: Fifty-two HIV-1-positive samples of blood donors were collected from 2005 to 2006 in Yunnan, China. Recent and long-term infections were distinguished by the HIV-1 subtypes B, E, and D immunoglobulin G-capture enzyme immunoassay assay. The nucleotide sequences of pol genes were amplified and sequenced. Phylogenetic tree and drug resistance analyses were performed. RESULTS: Of 49 samples successfully analyzed, circulating strains were circulating recombinant form (CRF)08_BC (51.0%), CRF07_BC (24.5%), CRF01_AE (20.4%), and B (4.1%). No protease inhibitors (PI) major drug resistance mutation (DRM) was detected. Six samples (12.2%) displayed seven minor PI DRMs. Nine samples (18.4%) displayed 10 nucleoside reverse transcriptase inhibitor DRMs, and DRMs to nonnucleoside reverse transcriptase inhibitors were present in one sample (2.0%). There was only one sample of the 49 (2.0%) in which the DRMs were of sufficient magnitude to result in a clinical change to drug susceptibility, but even in this sample, the clinical effect of these DRMs was predicted to be low. Significant differences were not observed between the long-term and recent infected population. Differences in DRMs were not observed between peripheral blood mononuclear cells and plasma within an individual. CONCLUSIONS: CRF_BC was the dominant subtype circulating in HIV-1-infected donors in Yunnan. Prevalence of genotypic drug resistances among donors in Yunnan was low in this study. Surveillance on HIV-1 infections among blood donors should be continued in China.

Early infant human immunodeficiency virus type 1 detection suitable for resource-limited settings with multiple circulating subtypes by use of nested three-monoplex DNA PCR and dried blood spots.

The early detection of human immunodeficiency virus type 1 (HIV-1) infection in infants is complicated by the persistence of maternal antibodies and by diverse HIV-1 subtypes. We developed a nested, three-monoplex HIV-1 DNA PCR (N3M-PCR) assay to detect diverse HIV-1 subtypes in infants born to infected mothers. We optimized the test for use with dried blood spot (DBS) samples for ease of storage and transport from rural China to central laboratories. Six pairs of primers were designed that targeted env, gag, and pol genes, and the test was run in three reactions with an analytical sensitivity of 10 copies DNA per reaction to cover nine HIV-1 subtypes, A, B, C, D, F, G, CRF01-AE, CRF08-BC, and CRF07-BC. The assay performance was evaluated on 347 DBS specimens from 151 exposed infants in four diverse provinces of China in which multiple subtypes were circulating. The results of this test were compared to those of HIV antibody enzyme immunoassay and Western blotting confirmation for the infants at > or =18 months of age or to convincing clinical and epidemiologic data for deceased infants. The sensitivity of the N3M-PCR assay was 30.0% (3/10) for infants at 48 h after birth, 91.7% (11/12) at 1 to 2 months of age, and 93.7% (15/16) at 3 to 6 months of age. The specificity was 100% (94/94) at all three time points. The PCR reproducibility in the three DNA regions was 100% for samples at 48 h after birth, 96.7% at 1 to 2 months, and 100% at 3 to 6 months of age. The HIV-1 DNA N3M-PCR assay on DBSs offers a simple and affordable approach for early infant HIV-1 diagnosis in regions with diverse HIV-1 circulating subtypes.

[Study on the influence of biological characteristics to HIV/HCV co-infection among HIV infected illegal blood donors in China].

OBJECTIVE: To study the influence of biological characteristics on HCV/HIV co-infection, including HIV-1 sequence distance, HIV-1 viral load and CD4+ count. METHODS: HIV-1 sequence distance was calculated by Clustal W and Phylip software while HIV-1 viral load being tested by NASBA and CD4+ count was tested using Epics XL of Coulter. Significance was determined by t-test using SPSS 12.0. RESULTS: The mean HIV-1 genetic distances were 7.95% and 15.73% (P < 0.001) between those with HCV co-infection and those without. Their mean HIV-1 viral loads were 4.61 and 4.45 (P = 0.522) and their mean CD4I T counts were 308 and 251 (P = 0.161), respectively. CONCLUSION: Data showed that in the study group, the HIV/HCV co-infection had an influence on the HIV sequence distance, but did not have major impact on HIV-1 viral load and their mean CD4+ T count.

[Comparison of NucliSens HIV-1 QT and Amplicor HIV-1 monitor 1.5 in detecting HIV-1 viral load].

OBJECTIVE: To compare the results of detecting HIV-1 load by using NucliSens HIV-1 QT and Amplicor HIV-1 monitor 1.5 assays. METHODS: Eighty-two clinical samples were collected and HIV viral load was determined with the above-mentioned two methods. RESULTS: The number of samples in which values obtained by NucliSens HIV-1 QT and Amplicor HIV-1 monitor 1.5 differed by <0.5 log10 RNA copies/ml and in which the viral load was undetectable accounted for 88.9 percent of the measures. The correlation coefficient between the two methods was 0.956 in 56 samples of Deltalog10 VL<0.5. CONCLUSION: The results of HIV-1 viral load determination with the two methods are highly comparable.

Clinical comparison of branched DNA and reverse transcriptase-PCR and nucleic acid sequence-based amplification assay for the quantitation of circulating recombinant form_BC HIV-1 RNA in plasma.

OBJECTIVE: To investigate the correlation between three viral load assays for circulating recombinant form (CRF)_BC. DESIGN: Recent studies in HIV-1 molecular epidemiology, reveals that CRF_BC is the dominant subtype of HIV-1 virus in mainland China, representing over 45% of the HIV-1 infected population. The performances of nucleic acid sequence-based amplification (NASBA), branched DNA (bDNA) and reverse transcriptase polymerase chain reaction (RT-PCR) were compared for the HIV-1 viral load detection and quantitation of CRF_BC in China. METHODS: Sixteen HIV-1 positive and three HIV-1 negative samples were collected. Sequencing of the positive samples in the gp41 region was conducted. The HIV-1 viral load values were determined using bDNA, RT-PCR and NASBA assays. Deming regression analysis with SPSS 12.0 (SPS Inc., Chicago, Illinois, USA) was performed for data analysis. RESULTS: Sequencing and phylogenetic analysis of env gene (gp41) region of the 16 HIV-1 positive clinical specimens from Guizhou Province in southwest China revealed the dominance of the subtype CRF_BC in that region. A good correlation of their viral load values was observed among three assays. Pearson's correlation between RT-PCR and bDNA is 0.969, Lg(VL)RT-PCR = 0.969 * Lg(VL)bDNA + 0.55; Pearson's correlation between RT-PCR and NASBA is 0.968, Lg(VL)RT-PCR = 0.968 * Lg(VL)NASBA + 0.937; Pearson's correlation between NASBA and bDNA is 0.980, Lg(VL)NASBA = 0.980 * Lg(VL)bDNA - 0.318. CONCLUSION: When testing with 3 different assays, RT-PCR, bDNA and NASBA, the group of 16 HIV-1 positive samples showed the viral load value was highest for RT-PCR, followed by bDNA then NASBA, which is consistent with the former results in subtype B. The three viral load assays are highly correlative for CRF_BC in China.

[Study on the use of dried blood spot in sequencing and subtyping HIV-1 DNA genome].

OBJECTIVE: To use dried blood spot (DBS) in studying the sequence and subtype analysis of HIV-1 genome. METHODS: 2 ml whole blood containing EDTA anticoagulant from 20 HIV-1 infected patients were collected, then 80 microl blood was used to propare DBS. QIAamp Blood Mini kit and 10% Chelex100 resin extracted DNA genome from whole blood and DBS as well as nested PCR amplified specifically HIV-1 Gp41 region from the two kinds of DNA extraction. Software MAGE 3.0 was used to study the sequence and subtype of the PCR products. RESULTS: Eligible 18 paired samples were analysed to show that 16 of them belonged to C54A, 97CNGX-7F, 98CN006 subtypes. The other two samples might belong to B. CN._. RL42 and B. US. 90WEAU160. CONCLUSION: Data showed that there were parallel results between the whole blood and DBS samples including subtype analysis, position of mutation and types of amino acid sequencing. Since DBS itself facilitated the collection, transportation and storage, it could be used as a measure to collect blood sample in resource limited area and to develop molecular epidemiologic research as well as early diagnosis on infant exposed to HIV.

[Heterogeneity of HIV strains isolated from different tissues of 3 AIDS patients].

BACKGROUND: To reveal the characteristics of genotype and phenotype of HIV strains in blood and some tissues of AIDS patients. METHODS: The virus was isolated from peripheral blood mononuclear cell (PBMC),cerebrospinal fluid (CSF)and lymph nodes of 3 AIDS patients by coculture with PBMC stimulated by PHA for 72 hours from uninfected donor. The cytopathic effect of the HIV isolates was determined in cultured MT2 cell line. The env gene sequences form proviral DNA were analyzed by GCG software. RESULTS: In one patient,there were differences between the strains from blood and different tissues both in genotype and phenotype. The biological phenotypes of two strains from CSF were non syncytium (NSI) type, their env sequences were similar to standard CNS tropic strain (SF162). CONCLUSIONS: The viral heterogeneity exists in different body compartments within an infected individual. The neurotropic isolate which is similar to international standard strain exists in some AIDS patients in China.