HIV screening among patients seeking care at Xuanwu Hospital: A cross-sectional study in Beijing, China, 2011-2016.

OBJECTIVES: One-third of people living with HIV in China are still unaware of their status, so we sought to better understand HIV testing in the general hospital setting in China. METHODS: A cross-sectional study was conducted using the electronic medical records of all patients who attended Xuanwu Hospital in Beijing, January 1, 2011 to December 31, 2016. HIV screening and detection rates and characteristics of patients diagnosed with HIV were assessed. RESULTS: Overall, 235,961 patients were screened, for a screening rate of 1.4%. Although most were outpatients (98.4%), screening rate was higher among inpatients (70.0% versus 0.4%), and highest in internal medicine (36.1%) and surgery (33.3%) departments. A total of 140 patients were diagnosed with HIV, for a detection rate of 5.93 per 10,000. Detection rates were highest among outpatients (9.34 per 10,000), and patients attending the dermatology and sexually transmitted infection (STI) department (153.85 per 10,000). Most diagnoses were made among males (91.4%), aged 20-39 (67.1%), who reported becoming infected through homosexual contact (70.0%). CONCLUSIONS: HIV screening in China's general hospitals needs to be improved. More focus should be placed on screening outpatients, especially in the dermatology and STI department, and young men.

The characteristics of screening and confirmatory test results for HIV in Xi'an, China.

OBJECTIVES: Individuals with recent or acute HIV infection are more infectious than those with established infection. Our objective was to analyze the characteristics of detection among HIV infections in Xi'an. METHODS: A 4th-generation kit (Architect HIV Ag/Ab Combo) and three 3rd-generationEIA kits (WanTai, XinChuang and Livzon) were used for HIV screening. Overall, 665 individuals were identified as positive and were tested by western blotting (WB). The characteristics of the screening and confirmatory tests were analyzed, including the band patterns, the early detection performance and the false-positive rates. RESULTS: In total, 561 of the 665 patients were confirmed as having HIV-1 infection, and no HIV-2 specific band was observed. Among these 561 WB-positive cases, reactivity to greater than or equal to 9 antigens was the most commonly observed pattern (83.18%), and the absence of reactivity to p17, p31 and gp41 was detected in 6.44%, 5.9% and 2.86% of the cases, respectively. Two cases were positive by the 4th-generation assay but negative by the 3rd-generation assay for HIV screening and had seroconversion. The false-positive rate of the Architect HIV Ag/Ab Combo (22.01%) was significantly higher than those of WanTai (9.88%), XinChuang (10.87%) and Livzon (8.93%), p<0.05. CONCLUSION: HIV infection in Xi'an is mainly caused by HIV-1, and individuals are rarely identified at the early phase. Although the false-positive rate of the 4th-generation assay was higher than that of the 3rd-generation assay, it is still recommended for use as the initial HIV screening test for high-risk individuals. In Xi'an, a 3rd-generation assay for screening could be considered.

Generation of a recombinant Gag virus-like-particle panel for the evaluation of p24 antigen detection by diagnostic HIV tests.

BACKGROUND: Detection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens. METHODS: We generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection. RESULTS: Our VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection. CONCLUSIONS: The HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.

In vivo biotinylation and capture of HIV-1 matrix and integrase proteins.

This report describes the adaptation of the biotin ligase BirA-biotin acceptor sequence (BAS) labeling system to biotinylate specific human immunodeficiency virus 1 (HIV-1) proteins in vivo. Two HIV-1 clones were constructed, with the BAS introduced into the matrix region of gag or the integrase region of pol. Specific biotinylation of target proteins in virions was observed when molecular clones were co-expressed with BirA. Both BAS-containing viruses propagated in SupT1 T-cells although replication of the integrase clone was delayed. Further studies demonstrated that the integrase insertion yielded an approximate 40% reduction in single-round infectivity as assessed on MAGI-5 indicator cells, as well as in the in vitro integration activity of preintegration complexes extracted from acutely infected C8166-45 T-cells. Biotinylation of the integrase BAS tag furthermore rendered this virus non-infectious. The matrix viral clone by contrast displayed wild-type behavior under all conditions tested. These results therefore establish a system whereby biotinylated matrix protein in the context of replication-competent virus could be used to label and capture viral protein complexes in vivo.

Human immunodeficiency virus (HIV) antigens and RNA in HIV-seronegative women with cervical intraepithelial neoplasia.

While investigating whether proteins retrieved by cervicovaginal lavages (CVL) from women with cervical intraepithelial neoplasia (CIN) might correlate with risk of progression to invasive cervical cancer, we unexpectedly identified HIV gag and env glycoprotein in CVL from women with HIV-negative serology. HIV antigens were consistently identified by mass spectrometry (MS) in CVL from 4 women but were absent in CVL from the remaining 16 women. HIV serologies of all 20 patients were negative for both HIV-1 and HIV-2 antibodies. To validate the unexpected MS findings we performed Western blot (WB) and immunoaffinity chromatography (IC) analysis of CVL for HIV proteins, viral load assays of paired CVL and blood samples, and immunohistochemical HIV p24 expression in cervical biopsy specimens. WB analysis of CVL for prostate-specific antigen (PSA) was performed to exclude semen contamination as the source of HIV proteins. WB and IC results demonstrated the presence of HIV-1 gp41 and p24 antigens in four CVL that were identified by MS to have the HIV proteins. Despite negative serology, HIV RNA in CVL and HIV p24 in cervix biopsies were detected in patients with HIV antigen-positive CVL. HIV p24-positive CVL were PSA negative. All 20 subjects remained HIV seronegative throughout the study. Women with HIV proteins and RNA were comparatively older. Our findings suggest that CVL HIV proteins in women with CIN could be markers for unrecognized HIV exposure or subclinical infection. Proteomic screening of cervical secretions may be useful in identifying seronegative women exposed to HIV and/or at risk for AIDS.

Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial.

BACKGROUND: Observational data and non-human primate challenge studies suggest that cell-mediated immune responses might provide control of HIV replication. The Step Study directly assessed the efficacy of a cell-mediated immunity vaccine to protect against HIV-1 infection or change in early plasma HIV-1 levels. METHODS: We undertook a double-blind, phase II, test-of-concept study at 34 sites in North America, the Caribbean, South America, and Australia. We randomly assigned 3000 HIV-1-seronegative participants by computer-generated assignments to receive three injections of MRKAd5 HIV-1 gag/pol/nef vaccine (n=1494) or placebo (n=1506). Randomisation was prestratified by sex, adenovirus type 5 (Ad5) antibody titre at baseline, and study site. Primary objective was a reduction in HIV-1 acquisition rates (tested every 6 months) or a decrease in HIV-1 viral-load setpoint (early plasma HIV-1 RNA measured 3 months after HIV-1 diagnosis). Analyses were per protocol and modified intention to treat. The study was stopped early because it unexpectedly met the prespecified futility boundaries at the first interim analysis. This study is registered with ClinicalTrials.gov, number NCT00095576. FINDINGS: In a prespecified interim analysis in participants with baseline Ad5 antibody titre 200 or less, 24 (3%) of 741 vaccine recipients became HIV-1 infected versus 21 (3%) of 762 placebo recipients (hazard ratio [HR] 1.2 [95% CI 0.6-2.2]). All but one infection occurred in men. The corresponding geometric mean plasma HIV-1 RNA was comparable in infected male vaccine and placebo recipients (4.61 vs 4.41 log(10) copies per mL, one tailed p value for potential benefit 0.66). The vaccine elicited interferon-gamma ELISPOT responses in 75% (267) of the 25% random sample of all vaccine recipients (including both low and high Ad5 antibody titres) on whose specimens this testing was done (n=354). In exploratory analyses of all study volunteers, irrespective of baseline Ad5 antibody titre, the HR of HIV-1 infection between vaccine and placebo recipients was higher in Ad5 seropositive men (HR 2.3 [95% CI 1.2-4.3]) and uncircumcised men (3.8 [1.5-9.3]), but was not increased in Ad5 seronegative (1.0 [0.5-1.9]) or circumcised (1.0 [0.6-1.7]) men. INTERPRETATION: This cell-mediated immunity vaccine did not prevent HIV-1 infection or reduce early viral level. Mechanisms for insufficient efficacy of the vaccine and the increased HIV-1 infection rates in subgroups of vaccine recipients are being explored.

[Assay for simultaneous detection of HIV p24 antigen and anti-HIV antibody].

OBJECTIVE: To develop a rapid assay for simultaneous detection of HIV p24 antigen (Ag) and anti-HIV antibody (Ab). METHODS: HIV-1 gp41 antigen and HIV-2 gp36 antigen were expressed by recombinant baculovirus insect system and purified by immunochromatography. p24 monoclonal antibody (mAb) was obtained from p24 hybridoma cell line. Purified antigen and mAb were dot blotted to nitrocellular membrane; 20 nm colloidal gold-anti-human IgG ab and p24 ab complex were used for this test. Previously detected 39 sera specimens were tested in this study to compare with the result of HIV test with commercial HIV test kit. RESULTS: 20 mg/L purified gp41 Ag and gp36 Ag were obtained from recombinant baculovirus-insect cell system; 1.5 mg/L p24 mAb was obtained from p24 mAb hybridoma cell line. Compared the test result of 39 sera with commercial HIV test kits, consistency rate was 100%. CONCLUSIONS: The rapid assay for simultaneous detection of HIV p24 antigen and anti-HIV antibody provides a simple, sensitive and reliable test for HIV diagnosis.

Recombinant fusion proteins for haemagglutination-based rapid detection of antibodies to HIV in whole blood.

Recombinant fusion proteins, consisting of a monovalent anti-human RBC monoclonal antibody B6, and conserved immunodominant peptide of HIV-1 envelope glycoprotein gp41 or HIV-2 envelope glycoprotein gp36, have been designed and purified after over-expression in E. coli. These fusion proteins are Fab-based and were obtained by assembling the light chain with Fd (variable domain and the first constant domain of the heavy chain) or Fd fusions containing HIV-derived peptide, and following a protocol of in vitro denaturation of inclusion bodies and subsequent renaturation to assemble functional Fab. Using a multistep column chromatographic procedure, monomeric Fab and Fab fusion proteins containing HIV-derived peptide were purified to high degree, free of aggregates. The yield of various proteins on the laboratory scale (1-2 l of shake flask culture) was in the range of tens of milligram. Purified anti-human RBC Fab fusion proteins containing sequences derived from HIV-1 gp41 and HIV-2 gp36 were highly specific for detection of antibodies to HIV-1 and HIV-2, respectively. The described design, expression and purification protocols will make it possible to produce specific recombinant reagents in large quantities for agglutination-based rapid detection of antibodies to HIV in whole blood.

Gag-derived proteins of HIV-1 isolates from Indian patients: cloning, expression, and purification of p17 of B- and C-subtypes.

A simple and efficient method for expression in Escherichia coli and purification of matrix protein, p17, of human immunodeficiency virus type 1 (HIV-1) of both B- and C-subtypes is described. DNA sequences encoding p17 of B- and C-subtype were cloned from respective gag sequences. The gag sequences were obtained by PCR amplification using DNA extracted from peripheral blood lymphocytes of an HIV-1 infected patient from India. A T7-promoter-based expression system was optimized for expression of p17 in soluble form. p17 (B- and C-subtype) was purified to near homogeneity using conventional chromatographic techniques. Purification of p17 (C-subtype) is described for the first time with yield of 7.7 mg from a 1-liter culture. The yield of p17 (B-subtype) is 14.7 mg from a 1-liter culture, which is severalfold better than that reported earlier. N-terminal sequencing and CD spectra of the purified proteins, p17B and p17C, show that the proteins are properly processed and well-folded. The immunoreactivity of both types of p17 to sera from HIV-infected individuals is comparable.

Retrovirus in salivary glands from patients with Sjögren's syndrome.

AIMS: To investigate the possibility of an immune response to retroviral antigens or of detecting retrovirus in Sjögren's syndrome. METHODS: Retroviruses were sought in labial salivary glands and peripheral blood mononuclear cells from patients with Sjögren's syndrome by immunoblotting assay, immunohistochemical assay, polymerase chain reaction (PCR), reverse transcriptase (RT) activity assay, and transmission electron microscopy. RESULTS: Sera from five of 15 patients with Sjögren's syndrome (33%) reacted against p24 group specific antigen (gag) of human immunodeficiency virus (HIV). Labial salivary gland biopsy specimens from seven of the 15 patients with Sjögren's syndrome (47%) contained an epithelial cytoplasmic protein reactive with a monoclonal antibody to p24 of HIV. PCR was performed to detect HIV and human T lymphotropic virus type I (HTLV-I) genes from salivary gland tissues and peripheral blood mononuclear cells from patients with Sjögren's syndrome. Mn2+ dependent, Mg2+ independent RT activity was detected in the salivary gland tissues in three of 10 patients. A-type-like retroviral particles were observed in epithelial cells of salivary glands by transmission electron microscopy. Target genes for HIV and HTLV-I were not found in any of the salivary gland tissues or peripheral blood mononuclear cells from Sjögren's syndrome patients. CONCLUSIONS: The data suggest the presence of an unknown retrovirus similar to HIV in the salivary gland which might be involved in the pathogenesis of a subpopulation in Sjögren's syndrome.

Detección de antígeno del virus de la inmunodeficiencia humana libre y en complejos inmunes / Detection of free and complexed human immunodeficiency virus antigen

El objetivo de este trabajo es aumentar la sensibilidad de la detección de antígeno de VIH en pacientes infectados a través de un proceso de disociación de complejos inmunes que no reduzca la reactividad antigénica. Se realizó una etapa de precipitación con PEG 12 por ciento (concentración final 2 por ciento), tratamiento ácido disociante y neutralización, previo a la aplicación de un método convencional de captura de antígeno. Se mostró una relación significativa entre el tratamiento con PEG disociante y la detección de antígeno (X2: 13,97, p < 0,001). De 105 muestras, 27 sueros que resultaron negativos por el método estándar fueron positivos con el tratamiento. La cantidad de antígeno en 35 muestras previamente positivas se incrementó en promedio 2,3 veces luego del tratamiento

Detección de antígeno del virus de la inmunodeficiencia humana libre y en complejos inmunes / Detection of free and complexed human immunodeficiency virus antigen

El objetivo de este trabajo es aumentar la sensibilidad de la detección de antígeno de VIH en pacientes infectados a través de un proceso de disociación de complejos inmunes que no reduzca la reactividad antigénica. Se realizó una etapa de precipitación con PEG 12 por ciento (concentración final 2 por ciento), tratamiento ácido disociante y neutralización, previo a la aplicación de un método convencional de captura de antígeno. Se mostró una relación significativa entre el tratamiento con PEG disociante y la detección de antígeno (X2: 13,97, p < 0,001). De 105 muestras, 27 sueros que resultaron negativos por el método estándar fueron positivos con el tratamiento. La cantidad de antígeno en 35 muestras previamente positivas se incrementó en promedio 2,3 veces luego del tratamiento (AU)

[Laboratory diagnosis of HIV infection]. / Labordiagnostik der HIV-Infektion.

Recent use of modern methods in the field of molecular biology has provided new insights into the genetic and structural nature of HIV, which are now increasingly being applied to the diagnostic work-up. For the reliable laboratory diagnosis of HIV infection, three different possibilities are available: detection of specific antibodies against viral proteins (anti HIV AB), detection of the virus itself (HIV antigen) and detection of viral nucleic acid using in vitro amplification techniques. In this overview, we compare clinical and diagnostic parameters of an HIV infection, and discuss the current state of the art of an HIV infection and the future prospects offered by new molecular biological methods such as the use of recombinant antigens or the polymerase chain reaction (PCR) for the detection of viral nucleic acid.

Overproduction, purification, and diagnostic use of the recombinant HIV-1 Gag proteins, the precursor protein p55 and the processed products p17, p24, and p15.

HIV-1 Gag protein precursor p55, and its processed products, p17, p24, and p15 were overproduced in Escherichia coli and purified to near homogeneity. To study the antigenic properties and the potentiality as the diagnostic and prognostic reagents, varying amounts of the purified Gag proteins were dotted onto the polyvinylidene difluoride membrane and reacted with 40 sera of HIV-1-infected individuals (35 AC, 1 ARC, and 4 AIDS patients) and 10 sera of normal healthy donors. p55 reacted with 40 (100%) sera of HIV-1 carriers, while p17, p24, and p15 reacted with 37 (92.5%), 35 (87.5%) and 34 (85%) of the 40 sera of HIV-1 carriers, respectively. On the whole, the reaction of p55 was especially strong and that of p15 was the weakest. p55 showed the strongest reaction among the four Gag proteins with all specimens, and it showed a positive reaction with a carrier serum with which none of the processed Gag proteins showed a positive reaction. Therefore, p55 is the most useful antigen among the four Gag proteins for detection of the Gag antibodies and may even be one of the most useful antigens for the diagnosis of HIV-1 infection.

Infección primaria por el VIH: características clínicas y serológicas / Primary HIV infection: clinical and serological characteristics

An acute clinical picture of variable intensity may occur during the initial primary phase of HIV infection, it may however pass unnoticed. We report 12 seronegative subjects (11 male homosexuals, 1 female heterosexual, aged 18 to 44 years old), that sembling an acute clinical picture preceding seroconversion. All had a sudden beginning, reduration were variable, lasting a mean of 14 (range 5-44) days an remaining asymptomatic thereafter. Most patients presented a discrete leukopenia with lymphopenia at the expense of CD4 lymphocytes, followed by an absolute lymphocytosis in some, with an increase in CD8 lymphocytes. All became positive for HIV; circulating HIV antigen was identified in 3 and IgM anti-HIV antibodies were detected during the symptomatic period by third generation ELISA in other 3. It is concluded that the clinical picture of primary HIV infection has identificable clinical serological and immunological features and its recognition has diagnostic and preventive implications

Influence of MA internal sequences, but not of the myristylated N-terminus sequence, on the budding site of HIV-1 Gag protein.

HIV-1 Gag protein intracellular transport and budding was investigated by altering the sequence of the MA domain, which directly bears an essential N-terminal myristyl adduct and forms the viral matrix after Gag proteolysis in mature virions. We found that removal of a substantial MA internal segment did not abolish the assembly and budding of Gag particles, but rather diverted these events to intracellular cisternae. The internally deleted Gag was further modified by substituting either of two heterologous myristylated N-termini for the natural one: amino acids 1-12 from v-Src oncoprotein (for which a membrane-bound intracellular receptor has been postulated), or amino acids 1-12 from Poliovirus polyprotein (for which no membrane-targeting function has been demonstrated). Both Src-Gag and Polio-Gag chimerae exhibited transport and processing characteristics similar to those of the MA-deleted Gag. These results are discussed with respect to the possible transport pathway of HIV-1 Gag.