P24 Ag Research Articles

A More Comprehensive Clinical and Laboratory Characterization of 61 Acute HIV Infection Patients in Southwest China

Acute HIV infection (AHI), i.e., the early stage of HIV infection, plays an important role in immune system failure and HIV transmission, but most AHI patients are missed due to their non-specific symptoms. To facilitate the identification of patients with high AHI risk and reduction of missed diagnosis, we characterized 61 AHI patients in a Southwest China hospital with 4300 beds; specifically, we characterized their general clinical characteristics, evolution in results of a novel HIV screening assay called Elecsys® HIV Duo, and by programming, we analyzed the ability of all routine laboratory tests (e.g., routine blood analysis) to identify AHI patients. Among 61 AHI patients, 85.2% were male and the median age was 42 (interquartile range, 25-62) years. A total of 61.9% of patients visit the emergency department first during AHI. Clinical presentation of AHI patients included fever, fatigue, chills, rash, and various respiratory, digestive, and nervous system symptoms. Two of three results from Elecsys® HIV Duo show clear evolution trends: HIV P24 antigen decreased while HIV antibody increased in consecutive samples of nearly all patients. High fluorescence lymphocytes have a very high positive likelihood ratio (LR+) of 10.33 and a relatively high "rate of out-of-range tests" of 56.8% (21 in 37 patients who received this test had a result outside the reference range). In addition, we identified more than ten tests with LR+ greater than two. In summary, the emergency department is important for AHI screening. The evolution of HIV P24 Ag and HIV Ab and those laboratory tests with a high "rate of out-of-range tests" or high LR+ may aid the AHI identification and missed diagnosis reduction.

Toddler With Fever and Limp.

Toddler With Fever and Limp.

Clinical Application Evaluation of Elecsys® HIV Duo Assay in Southwest China.

BackgroundHIV/AIDS continues to be a serious health concern of morbidity and mortality globally, and novel HIV testing is still an important component of diagnosing HIV earlier and reducing the spread of HIV. The Elecsys® HIV Duo assay is a 4th generation assay that can detect both HIV-1 p24 antigen (Ag) and HIV-1/2 antibody (Ab) in parallel and show the subresults for the Ab and Ab units.ObjectivesTo evaluate the clinical performance of the Elecsys® HIV Duo assay on the new cobas E 801 analyzer using a large number of clinical samples from a population in southwest China.MethodsWe collected testing results and information from all patients in a large general hospital. All eligible clinical specimens were first analyzed using the Elecsys® HIV Duo assay. The test results are given either as reactive or nonreactive as well as in the form of a cutoff index (COI). All initially reactive specimens were retested in duplicate with a 3rd-generation kit. Supplementary tests were divided into Ab confirmation tests and HIV-1 nucleic acid tests. GraphPad Prism and Python were used for plotting, and SPSS 21.0 software was used for statistical analysis.ResultsA total of 186391 specimens were received, and 436 patients were confirmed to be positive for HIV. Among the 86 cases with contact history available, there were more males than females, and heterosexual transmission was the most common route of HIV infection. The Elecsys® HIV Duo assay displayed 99.94%, 99.93% and 99.98% specificity for inpatient, outpatient and physical examination patients, respectively. The median COI ratios of the false-positive group were significantly lower than those of the true-positive group.ConclusionsThe Elecsys® HIV Duo test (Cobase801 analyzer) differentiates the detection of HIV-1 p24 Ag and HIV-1/2 Ab with high specificity and facilitates the diagnosis of patients with early HIV infection. Therefore, the Elecsys®HIV Duo test is used for differentiation of antigen and antibody reactivity, making it suitable for routine clinical diagnosis.

Use of fourth-generation rapid combined antigen and antibody diagnostic tests for the detection of acute HIV infection in a community centre for men who have sex with men, between 2016 and 2019.

To assess the use of fourth-generation rapid diagnostic tests in identifying acute infection of Human Immunodeficiency Virus (HIV). BCN Checkpoint promotes sexual health among men who have sex with men (MSM), with a focus on diagnosing HIV early, initiating combined antiretroviral treatment (cART) promptly, and recommending regular repeat testing for those who have tested negative. This cross-sectional study included all test results obtained at the centre between 25 March 2016 and 24 March 2019. The Alere™ HIV Combo (now rebranded to Determine™ HIV Ultra, from Abbott) was used to detect p24 antigen (p24 Ag) and/or immunoglobulin M (IgM) and G (IgG) antibodies to HIV-1/HIV-2 (HIV Ab). Rapid polymerase chain reaction (PCR) confirmatory testing and Western blot (WB) were performed for clients with a positive rapid test result. Confirmed HIV cases were promptly referred to the HIV unit for care and cART prescription. A total of 12,961 clients attended BCN Checkpoint during the study and 27,298 rapid tests were performed. 450 tests were found to be reactive, of which 430 confirmed as HIV-positive, representing a prevalence of 3.32%. Four confirmed cases (0.93%) were detected as "p24 Ag only", nine (2.09%) as "both p24 and HIV Ab" and 417 (96.98%) as "HIV Ab only". The "p24 Ag only" group had a 1-log higher viral load than the other groups and initiated treatment on the following working day. Overall, there were 20 false-positive results (0.07% and 4.44% of total and reactive tests, respectively), of which 10 positive for "p24 Ag only" and 10 for "HIV Ab only". Four Acute HIV Infections (AHI), with very high viral loads, have been detected with the "p24 Ag only" while the HIV Ab were still absent. Referral to the HIV unit and initiation of cART on the following working day contributed to improving persons' health and to reduce HIV transmission chain.

MO170“SWEET HYPOXIA” WITH ACUTE KIDNEY INJURY: THE UNPREDICTABILITY OF ACUTE HYPOXIC RESPIRATORY FAILURE IN COVID-19 INFECTION - A COMMUNITY HOSPITAL EXPERIENCE

Background and AimsSevere COVID-19 infection may result in hypoxemic respiratory failure necessitating invasive mechanical ventilation. We revisit the phenomenon of asymptomatic patients despite very low pulse oximetry readings, the so-called “sweet hypoxia” or “happy hypoxia” or “silent hypoxemia”. We describe for the first time, the sequential chest radiographic images of the progressive radiological trajectory of COVID-19 pneumonia.Method Case Report. ResultsA 62-year old hypertensive obese Caucasian male, an ex-smoker, was diagnosed with mild community-acquired pneumonia in mid-March 2020, following evaluation for low grade fever. He had traveled to Florida and Texas in the previous month. He tested positive for COVID-19 by RT-PCR. A week later, he was admitted to a Community Hospital with one day history of new shortness of breath and loose stools. Vital signs were stable. Pulse oximeter was 96% on room air. He was fatigued with few bibasilar lung crackles. CBC was normal. Creatinine was 1.0 mg/dL. Abnormal laboratory: sodium 131 mmol/L, AST 50 iu/L, ALT 96 iu/L. Chest radiograph revealed new patchy left lower lobe airspace infiltrate (Figure 1B). EKG showed regular sinus rhythm of 96/min, QT interval 445 msec and PVCs. Treatment included nasal cannula oxygen, IV fluids, IV Azithromycin and IV Ceftriaxone. He improved the next day, requested discharge home, vital signs were stable, pulse oximetry was 91% on room air, sodium had normalized at 137 mmol/L and he was discharged home on Azithromycin 500 mg daily x 3 days and Cefdinir 300 mg BID x 5 days.MO170 Figure 1:Sequence of chest radiographs, normal in November 2019 (A), before initial hospital admission - March 20, 2020 @ 02:10:10 (B), and just before intubation in the Emergency Department following the second .admission - March 22, 2020 @ 03:32:01 (C).He cheerfully went home. Later that night he quickly developed worsening dyspnea. He was readmitted about 18 hours post-discharge. Temperature 99.40F, blood pressure 161/101, pulse 100/min. He was tachypneic and pulse oximetry was 82% on room air. This improved to 93% on 4.5 LPM nasal cannula oxygen. Initial EKG was normal. New pertinent laboratory data: Bicarbonate 17 mmol/L, phosphorus 5.5 mg/dL, calcium 7.2 mg/dL, creatinine 1.1 mg/dL, BNP 31 pg/mL and lactic acid 1.2 mmol/L. PTT was 28.3 sec. HIV-1 p24 AG, HIV-1 AB, HIV-2 AB, HbSAG and Hepatitis C AB were negative. Chest radiograph showed worsening bilateral infiltrates (Figure 1C). He very quickly desaturated in the ED down to 81% despite high flow oxygen therapy. He was promptly intubated (Figure 2A). Oxygenation immediately improved. He was transferred to the ICU on IV Vancomycin and IV Cefepime. He developed septic shock and required IV Norepinephrine. With worsening chest radiographs, (Figures 2B & 2C), he was transferred to a tertiary medical center. On transfer, pertinent new data: creatinine 1.38 mg/dL, albumin 2.8 g/dL, Ferritin 2,573 ng/mL, LDH 534 u/L, CRP 6.0 mg/L, INR 1.2, D-Dimer 1.04, procalcitonin 0.38 ng/mL, WBC 13.3 x 109/L. EKG showed sinus bradycardia. Urine Legionnaire AG and Strep. Pneumonia AG were negative. IV Azithromycin 500 mg daily and IV Ceftriaxone 2 gm daily were administered for 8 days. Chloroquine phosphate 500 mg 2x daily was added. IV Norepinephrine was continued. IV fluids were withheld. The head of the bed was elevated to >300. DVT prophylaxis with SQ Enoxaparin and Vitamin C were administered. New blood cultures remained negative. COVID-19 RT-PCR after 3 days remained positive. He was extubated after 4 days and discharged home after 9 days with normalized creatinine of 1.03 mg/dL.ConclusionWe have for the first time demonstrated the sequential chest radiographic images of the progressive radiological trajectory of COVID-19 pneumonia. The place of non-invasive ventilation demands further study. The so-called “sweet hypoxia” or “happy hypoxia” or “silent hypoxemia” in COVID-19 is revisited – indeed, it is not exactly limited to COVID-19 patients. The need to mitigate lung barotrauma is mandatory. Finally, prognostication of pneumonia in COVID-19 is unpredictable. Too early premature discharge from the hospital is strongly discouraged.

Effect of HIV pre-exposure prophylaxis (PrEP) on detection of early infection and its impact on the appropriate post-PrEP deferral period.

HIV antivirals for pre-exposure prophylaxis (PrEP) are known to affect detection of early HIV infection through suppression of viral load and delayed seroconversion. To cover potential delay in HIV detection associated with PrEP use by blood donors in the context of international reductions in sexual activity-based deferral periods, we analysed the available data to determine the appropriate minimum post-PrEP deferral period for blood donation. Published cases of incident HIV infection when PrEP use was objectively demonstrable were identified, consisting principally of seroconverters from the Partners PrEP study (a clinical trial of PrEP efficacy). Data were reviewed to determine the impact of PrEP on the detection of HIV RNA, p24 Ag and seroconversion delay. Nucleic acid testing (NAT) detected early HIV infection in the presence of PrEP prior to or in concordance with serological testing in approximately 90% of cases. Undetectable HIV RNA would rebound to detectable levels within two months of PrEP cessation. PrEP delayed p24 antigen detection and antibody seroconversion by about 7days. Even when daily PrEP is continued, it is likely that the majority of early HIV infections are detectable by individual donation (ID)-NAT, with p24 Ag or antibody seroconversion occurring conservatively within four weeks of exposure. HIV RNA levels also rebound rapidly in the absence of PrEP. In Australia, a three-month deferral period for blood donation after the last dose of PrEP provides an appropriate safety margin to mitigate the residual risk of transfusion-transmitted HIV.

The Relationship Between HIV Antibody Titer, HIV Viral Load, HIV p24 Antigen, and CD4 T-cell Count Among Iranian HIV-positive Patients.

This study aimed to simultaneously measure and assess the correlation between the available HIV infection parameters including HIV antibody, p24 Antigen, CD4 cell count, and viral load at the different stages of HIV disease among HIV-positive individuals in Iran. Fifty HIV-positive individuals were classified into three stages (1, 2, and 3) according to the HIV disease stages classification, available in Control of Disease and Prevention (CDC) guideline. 10 ml of the venous blood sample was collected to run the tests for HIV antibody and p24 Ag levels, CD4 cell counts, and viral load. Pearson's correlation test was employed to calculate the coefficients for the in-between correlation of different HIV parameters in each stage. Of 50 participants, 17 (34%), 25 (50%), and 8 (16%) patients belonged to stages 1, 2, and 3, respectively. Sexual relationship was the main route of HIV transmission among the patients (36%); however, injecting drug use (20%) was also reported frequently. There was no significant correlation between the parameters of HIV disease in different stages in the present study. The findings showed no correlation between HIV parameters in the present study. Considering the fact that the association of HIV antibodies with HIV disease progression in infected individuals is independent of HIV-1 RNA levels, combined measurement of HIV-1 RNA and CD4 cell counts should be routinely carried out in HIV infected patients follow up.

Validation of Spiked Postmortem Blood Samples from Cornea Donors on the Abbott ARCHITECT and m2000 Systems for Viral Infections

Background: Transplantation of human corneal tissue is associated with the potential risk of transmittance of viral infections. In accordance with European directives and federal laws, in Germany each tissue donor has to be tested for infectious diseases such as hepatitis B and C virus (HBV and HCV) and human immunodeficiency virus (HIV) infection. However, most of the currently available CE-marked serologic and nucleic acid screening systems are only validated for antemortem blood. Methods: Twenty related and paired ante- and postmortem blood samples from cornea donors were obtained and subsequently analyzed for hepatitis B surface antigen (HBsAg), hepatitis B antibody (anti-HBc), anti-HCV, HCV RNA, anti-HIV-1/2, and HIV p24 Ag using Abbott test systems. The sera were also spiked with reference materials in concentrations giving low and high positivity for HBV, HCV, and HIV markers. Results: The spiked ante- and postmortem sera from related donors showed similar results for HBsAg, anti-HBc, anti-HCV, HCV RNA, anti-HIV, and HIV p24 Ag, indicating a high stability of viral markers in cadaveric specimens. Three cornea donors had a medical history of HBV infection and revealed anti-HBc at similar levels in the ante- and postmortem sera. In addition, there was a single postmortem sample demonstrating a weak signal of anti-HIV-1 and HIV-1 p24 Ag. False-positive or false-negative results were not detected. The results obtained with the Abbott ARCHITECT analyzer and Abbott RealTime HCV PCR showed no significant differences. Conclusion: The analyzed screening assays are suitable for the detection of infectious markers of HBV, HCV, and HIV at similar levels in spiked ante- and postmortem sera from cornea donors.

Performance of the Alere Determine™ HIV-1/2 Ag/Ab Combo Rapid Test with algorithm-defined acute HIV-1 infection specimens

BackgroundThe capacity of HIV Antigen/Antibody (Ag/Ab) immunoassays (IA) to detect HIV-1 p24 antigen has resulted in improved detection of HIV-1 infections in comparison to Ab-only screening assays. Since its introduction in the US, studies have shown that the Determine HIV-1/2 Ag/Ab Combo assay (Determine Ag/Ab) detects HIV infection earlier than laboratory-based IgM/IgG-sensitive IAs, but its sensitivity for HIV-1 p24 Ag detection is reduced compared to laboratory-based Ag/Ab assays. However, further evaluation is needed to assess its capacity to detect acute HIV-1 infection. ObjectiveTo assess the performance of Determine Ag/Ab in serum from acute HIV-1 infections. Study designSelect serum specimens that screened reactive on a laboratory-based Ag/Ab IA or IgM/IgG Ab-only IA, with a negative or indeterminate supplemental antibody test and detectable HIV-1 RNA were retrospectively tested with Determine Ag/Ab. Results were compared with those of the primary screening immunoassay to evaluate concordance within this set of algorithm-defined acute infections. ResultsOf 159 algorithm-defined acute HIV-1 specimens, Determine Ag/Ab was reactive for 105 resulting in 66.0% concordance. Of 125 that were initially detected by a laboratory-based Ag/Ab IA, 81 (64.8%) were reactive by Determine Ag/Ab. A total of 34 acute specimens were initially detected by a laboratory-based IgM/IgG Ab-only IA and 24 (70.6%) of those were reactive by Determine Ag/Ab. ConclusionsDue to their enhanced sensitivity, laboratory-based Ag/Ab IAs continue to be preferred over the Determine Ag/Ab as the screening method used by laboratories conducting HIV diagnostic testing on serum and plasma specimens.

Evaluation of the performance of a new automated HIV combination assay

BackgroundMore and more countries test for HIV infection using combination assays that simultaneously detect p24 antigen and HIV antibodies. ObjectiveTo assess the performance of a new HIV combo assay: LIAISON® XL murex HIV Ab/Ag HT. Study designThe assays were examined with a total of 3090 samples that included 769 selected HIV antibody-negative samples, 1849 unselected HIV samples, 15 HIV-1 p24 Ag reference samples, 90 primary HIV-1 infection (PHI) samples, 167 HIV-1 antibody-positive samples (well characterized of groups M and O), 95 HIV-1 antibody-positive samples and 105 HIV-2 antibody-positive samples. ResultsThe specificity of the LIAISON® XL murex HIV Ab/Ag HT was 99.7%. The analytical sensitivity of Ag p24 of the LIAISON® XL murex HIV Ab/Ag HT was 0.58IU/mL and 9.93pg/mL when using WHO and French national standards, respectively. All screened HIV subtypes was identified by this assay. Also, 90 PHI specimens were detected by this screening assay. ConclusionThe sensitivity and specificity of the LIAISON® XL murex HIV Ab/Ag HT assay are high. Hence the assay offers automated high-throughput screening with ability to detect primary infection.

Seroprevalence of hepatitis B, hepatitis C, human immunodeficiency virus, Treponema pallidum, and co-infections among blood donors in Kyrgyzstan: a retrospective analysis (2013\u20132015)

BackgroundPost-Soviet Kyrgyzstan has experienced a major surge in blood-borne infections, but data from adequately powered, up-to-date studies are lacking. We thus examined a) the seroprevalences of hepatitis B virus surface antigen (HBsAg), HIV-1 p24 antigen and antibodies against hepatitis C virus (anti-HCV), human immunodeficiency viruses (anti-HIV-1/2, HIV-1 group O), and Treponema pallidum among blood donors in Kyrgyzstan and assess their distribution according to sex, age, and provinces of residence; b) trends in the respective seroprevalences; and c) co-infection rates among the pathogens studied.MethodsSerological screening was performed on 37 165 blood donors at the Republican Blood Centre in Bishkek, Kyrgyzstan, between January 2013 and December 2015. We applied poststratification weights to control for sampling bias and used logistic regression analyses to examine the association of seropositivity and co-infections with sex, age, provinces of residence, and year of blood donation.ResultsTwenty nine thousand and one hundred forty-five (78%) donors were males and 8 020 (22%) were females. The median age was 27 years (range: 18 – 64). The prevalences of HBsAg, anti-HCV, HIV (p24 Ag and anti-HIV), and anti-T. pallidum were 3.6% (95%CI: 3.4 – 3.8%), 3.1% (3.0 – 3.3%), 0.78% (0.69 – 0.87%), and 3.3% (3.1 – 3.5%), respectively. Males were more likely to be seropositive for HBsAg than females (OR: 1.63; 95%CI: 1.40 – 1.90), but less likely to be seropositive for anti-HCV (0.85; 0.74 – 0.98) and HIV (0.65; 0.49 – 0.85). Prevalences were lower in the capital than in the other provinces. There was a decreasing trend in the seroprevalences of HBsAg, anti-HCV, and anti-T. pallidum from 2012 to 2015 (P-value for trend, P = 0.01, P < 0.0001, P < 0.0001, respectively), while the seroprevalence of HIV increased (P = 0.049). One hundred eighty donors (0.48%) were seropositive for multiple infections. The highest co-infection rate was observed between anti-T. pallidum and HBsAg (6.0%), followed by anti-HCV and anti-T. pallidum (5.2%), and HIV and anti-HCV (4.9%).ConclusionsThe data suggest that Kyrgyzstan can be reclassified from high to lower-intermediate HBsAg endemicity, whereas the high HIV prevalence with a rising trend is an alarming finding that needs to be urgently addressed by public health authorities. The observed co-infections suggest common risk factors but also common preventive interventions.

Point-Of-Care p24 Infant Testing for HIV May Increase Patient Identification despite Low Sensitivity.

The long delay in returning test results during early infant diagnosis of HIV (EID) often causes loss-to-follow-up prior to antiretroviral treatment (ART) initiation in resource-limited settings. A point-of-care (POC) test may help overcome these challenges. We evaluated the performance of the LYNX p24 Antigen POC test in Mozambique. 879 HIV-exposed infants under 18 months of age were enrolled consecutively at three primary healthcare clinics (PHC). Lancet heel-drawn blood was tested on-site by nurses using a prototype POC test for HIV Gag p24 antigen detection. Results of POC testing were compared to laboratory-based nucleic acid testing on dried blood spots. A comparison of the effect of sensitivity and timely test results return on successful diagnosis by POC and laboratory-based platforms was also calculated. The sensitivity and specificity of the LYNX p24 Ag test were 71.9%; (95% confidence interval [CI]: 58.5–83.0%) and 99.6% (95% CI: 98.9–99.9%), respectively. The predictive value of positive and negative tests were 93.2% (95% CI: 81.3–98.6%) and 97.9% (95% CI: 96.8–98.8%), respectively. Overall agreement was high (Cohen Kappa = 0.80; 95% CI: 0.71–0.89). Despite its lower sensitivity, the POC test had the potential to provide test results to up to 81% more patients compared to the laboratory-based test. This prototype POC p24 assay was feasible for use in PHCs but demonstrated low sensitivity for HIV detection. POC EID technologies that perform below standard recommendations may still be valuable diagnostic tools in settings with inefficient EID networks.

Effects of heme degradation products on reactivation of latent HIV-1.

Human immunodeficiency virus (HIV-1) infection can be currently controlled by combined antiretroviral therapy, but a sterilizing cure is not achievable as this therapy does not target persistent HIV-1 in latent reservoirs. Therefore, different latency reversal agents are intensively explored in various models. We have previously observed that heme arginate, a drug approved for human use, reveals a strong synergism with PKC inducers in reactivation of the latent provirus. Heme is physiologically decomposed by heme oxygenases into 3 degradation products: iron (Fe2+), carbon monoxide (CO) and biliverdin which is further converted to bilirubin by biliverdin reductase. In this paper, we have studied the effects of individual heme-degradation products on latent HIV-1 reactivation in ACH-2 cells harboring integrated HIV-1 provirus and in H12 clone of Jurkat cells harboring HIV-minivirus expressing EGFP. We employed addition of ascorbate to generate Fe2+, resulting in increased expression of both HIV-1 p24 Ag and EGFP in PMA-stimulated ACH-2 and H12 cells, respectively, as characterized on RNA and protein levels. On the other hand, addition of a CO-donor or bilirubin decreased the p24 expression. The reactivation of latent HIV-1 by iron or heme arginate was inhibited by antioxidant N-acetyl cysteine, or by an iron chelator desferrioxamine, suggesting that the effects were mediated by iron- or heme-induced redox stress. Finally, we demonstrated the stimulatory effects of heme arginate and PMA on HIV-1 expression in peripheral blood mononuclear cells of HIV-infected patients cultured ex vivo. These results may constitute a new direction in the latent HIV-1 reactivation and therapy.

Virological and immunological characteristics of HIV-infected individuals at the earliest stage of infection

BackgroundThe challenges of identifying acute HIV infection (AHI) have resulted in a lack of critical information on early AHI that constrains the development of therapeutics that are designed to eradicate HIV from the infected host. MethodsAHI participants were recruited from the Thai Red Cross Anonymous Clinic in Bangkok, Thailand into the RV254/SEARCH010 protocol and categorised according to Fiebig stages as follows: Fiebig I (HIV-RNA+, p24 Ag−, HIV IgM−) and Fiebig II–IV (HIV-RNA+, p24 Ag + or −, HIV IgM− or +, Western blot- or indeterminate). Proviral and viral burden and immune activation levels were compared between Fiebig stage groups at the time of AHI. CD4 and CD4/CD8 ratio were also compared between groups before and up to 96 weeks of ART. ResultsMedian age was 27 years and 96% were male. Fiebig I individuals had lower median HIV-DNA in mononuclear cells from blood (3 vs. 190 copies/106 cells) and gut (0 vs. 898 copies/106 cells), and lower HIV-RNA in blood (4.2 vs. 6.2 log10 copies/mL), gut (1.7 vs. 3.1 log10 copies/mg) and cerebrospinal fluid (2.0 vs. 3.8 log10 copies/mL), when compared to Fiebig II–IV individuals (all P<0.01). Median plasma sCD14 level was lower (1.1 vs. 1.6 μg/mL) in Fiebig I individuals as was the frequency of CD8+HLADR+CD38+ T cells in blood (7.6 vs. 14.9%, both P<0.05). The median plasma interleukin 6 levels were similar between stages (0.6 in Fiebig I vs. 0.5 pg/mL in Fiebig II–IV, P>0.05). The frequencies of CD4+HLA-DR+CD38+ T cells were also similar between these stages (2.1 vs. 2.6%, P>0.05). Median CD4 count and CD4/CD8 ratio were higher in Fiebig I: 508 vs. 340 cells/mm3 and 1.1 vs. 0.7, respectively (both P<0.001). After ART, CD4 cell count normalised by week 24 in Fiebig I and week 48 in Fiebig II–IV. However, CD4/CD8 ratio was lower in both groups after 96 weeks of ART compared to healthy Thais (P=0.02). ConclusionsCompared to later AHI stages, Fiebig I was associated with lower HIV burden in blood and tissue compartments, lower immune activation and higher CD4 and CD4/CD8 ratio. ART in Fiebig I–IV resulted in normalisation of CD4 cell count within the first year, supporting the benefit of early ART. However, the CD4/CD8 ratio was not normalised after 2 years of ART in all AHI stages, suggesting some degree of persistent immunological dysfunction even when ART was instituted as early as Fiebig I.

ID: 139: Reactivation of latent HIV-1 by iron-mediated redox stress

HIV pathogenesis includes dysregulated immune responses and cytokines production, resulting in an increased redox stress. Cytokines, namely TNF-alpha, and redox-stress promote replication of HIV-1. We have previously observed that heme arginate, a drug approved for human use in treatment of hepatic porphyria, can reactivate the latent provirus in synergism with PKC inducers like TNF-alpha, phorbol myristate acetate (PMA), prostratin or bryostatin-1, while inhibiting HIV-1 replication during the acute infection. Heme is physiologically decomposed by action of heme oxygenases into 3 degradation products: iron (Fe2+), carbon monoxide (CO) and biliverdin that is further converted to bilirubin by biliverdin reductase. Therefore, we have studied the effects of heme degradation products on latent HIV-1 reactivation when added individually to ACH-2 cells harboring integrated HIV-1 provirus or to a clone of Jurkat cells harboring HIV-minivirus expressing EGFP. Addition of iron increased expression of both HIV-1 p24 Ag and EGFP, while addition of a CO-donor or bilirubin decreased the PMA-stimulated p24 and EGFP expression. The reactivation was inhibited by antioxidants N-acetyl cysteine and vitamin E or by an iron chelator desferrioxamine, decreasing iron-induced redox stress. Desferrioxamine also inhibited the effect of heme arginate on latent HIV-1 reactivation. Redox stress was shown to affect epigenetic mechanisms regulating gene expression as well as to activate redox-sensitive transcription factors. Therefore, we propose a model in which redox-modulating agents induce chromatin remodeling, affect binding of specific transcription factors to HIV-LTR and allow HIV-1 expression. These results may point towards a new direction in the latent HIV-1 reactivation and therapy.

Herpes simplex virus type 2-infected dendritic cells produce TNF-α, which enhances CCR5 expression and stimulates HIV production from adjacent infected cells.

Prior HSV-2 infection enhances the acquisition of HIV-1 >3-fold. In genital herpes lesions, the superficial layers of stratified squamous epithelium are disrupted, allowing easier access of HIV-1 to Langerhans cells (LC) in the epidermis and perhaps even dendritic cells (DCs) in the outer dermis, as well as to lesion infiltrating activated T lymphocytes and macrophages. Therefore, we examined the effects of coinfection with HIV-1 and HSV-2 on monocyte-derived DCs (MDDC). With simultaneous coinfection, HSV-2 significantly stimulated HIV-1 DNA production 5-fold compared with HIV-1 infection alone. Because <1% of cells were dually infected, this was a field effect. Virus-stripped supernatants from HSV-2-infected MDDCs were shown to enhance HIV-1 infection, as measured by HIV-1-DNA and p24 Ag in MDDCs. Furthermore these supernatants markedly stimulated CCR5 expression on both MDDCs and LCs. TNF-α was by far the most prominent cytokine in the supernatant and also within HSV-2-infected MDDCs. HSV-2 infection of isolated immature epidermal LCs, but not keratinocytes, also produced TNF-α (and low levels of IFN-β). Neutralizing Ab to TNF-α and its receptor, TNF-R1, on MDDCs markedly inhibited the CCR5-stimulating effect of the supernatant. Therefore, these results suggest that HSV-2 infection of DCs in the skin during primary or recurrent genital herpes may enhance HIV-1 infection of adjacent DCs, thus contributing to acquisition of HIV-1 through herpetic lesions.

Assessment of immune response against HIV-1 P24 antigen using Bacillus thuringiensis serovar dakota (H15) activated delta-endotoxin as an adjuvant (P4491)

Abstract Bacillus thuringiensis var. dakota activated δ-endotoxin (BtDEAP) had many biological activity. Our previous experience recorded that BtDEAP had anti-malignancy activity in-vitro and in-vivo. There were many thoughts about the mode of action and one of them was their capability of immune modulation against tumor cell Ag. This work assesses the immune response in BALB/c mice immunized with HIV-1 P24 Ag individually and/or in combination with BtDEAP, complete Freund's adjuvant (CFA). The evaluation of the immune response against immunogenic preparations was carried out through estimating the immunoregulatory cytokines (IL-1α, Il-1β, IL-4, IL-10 and TNF-α) in the supernatant of splenocytes from mice that were immunized with HIV-1 P24 Ag alone and/or in combination with BtDEAP, CFA in relation to splenocytes from control mice. Also, the total IG level in the serum of tested and control mice were appraised. The results obtained conducted that the total IG level was raised remarkably by using CFA in comparing with BtDEAP while HIV-1 P24 Ag alone record the minimal raise in IG levels. These results was accompanied with raised levels of IL-4 and IL-10 by using CFA combination with HIV-1 P24 while IL-1 and TNF-α raised by immunization using BtDEAP in combination with HIV-1 P24 Ag over that recorded by HIV-1 P24 Ag alone. Based on the described above, BtDEAP, seems to be playing a significant role in preferential induction of Th1-dependent immune response against HIV-1 P24 Ag.

Improved quantification of HIV-1-infected CD4 + T cells using an optimised method of intracellular HIV-1 gag p24 antigen detection

The capacity of CD8+ T cells to inhibit HIV-1 replication in vitro strongly correlates with virus control in vivo. Post-hoc evaluations of HIV-1 vaccine candidates suggest that this immunological parameter is a promising benchmark of vaccine efficacy. Large-scale analysis of CD8+ T cell antiviral activity requires a rapid, robust and economical assay for accurate quantification of HIV-1 infection in primary CD4+ T cells. Detection of intracellular HIV-1 p24 antigen (p24 Ag) by flow cytometry is one such method but it is thought to be less sensitive and quantitative than p24 Ag ELISA. We report that fixation and permeabilisation of HIV-infected cells using paraformaldehyde/50% methanol/Nonidet P-40 instead of a conventional paraformaldehyde/saponin-based protocol improved their detection across multiplicities of infection (MOI) ranging from 10−2 to 8×10−5, and by nearly two-fold (p<0.001) at the optimal MOI tested (10−2). The frequency of infected cells was strongly correlated with p24 Ag release during culture, thus validating its use as a measure of productive infection. We were also able to quantify infection with a panel of HIV-1 isolates representing the major clades. The protocol described here is rapid and cost-effective compared with ELISA and thus could be a useful component of immune monitoring of HIV-1 vaccines and interventions to reduce viral reservoirs.

ARCHITECT® HIV Ag/Ab Combo assay: Correlation of HIV-1 p24 antigen sensitivity and RNA viral load using genetically diverse virus isolates

BackgroundHIV antigen/antibody (Ag/Ab) combination assays represent a significant advancement in assays used for diagnosing HIV infection based on their ability to detect acute and chronic infections. During acute HIV infection (AHI), detection depends on assay sensitivity for p24 Ag. ObjectiveTo directly compare the Ag sensitivity of the ARCHITECT® HIV Ag/Ab Combo assay to RNA viral load using cell culture supernatants of virus isolates. HIV-1 isolates allow correlation in the total absence of an antibody response to infection and across genetically diverse HIV-1 group M strains. MethodsThirty-five HIV-1 isolates comprising subtypes A–D, F and G, CRF01_AE, CRF02_AG, and unique recombinant forms were evaluated. Cell-free culture supernatant for each isolate was diluted to four levels and tested in the HIV Combo assay to determine a signal to cutoff ratio and the RealTime® HIV-1 assay to quantify RNA. The RNA copies/mL at the HIV Combo assay cutoff was determined. ResultsThe median RNA copies/mL at the HIV Combo assay cutoff was 57,900 for individual virus isolates (range 26,440–102,400). A single plot of all the data gave a value of 58,500RNA copies/mL. An analysis of data published for acute HIV infection in human subjects gave a similar result; HIV Combo detected 97% of AHIs with RNA copies/mL>30,700. ConclusionsBased on analysis of virus isolates, the ARCHITECT HIV Combo assay can detect p24 Ag when RNA is above approximately 58,000copies/mL. The correlation of viral load and Ag sensitivity was consistent across genetically diverse HIV-1 group M strains.

Evaluation of two indigenous rapid and two ELISA assays for the diagnosis of HIV infection India

Purpose: Human immunodeficiency virus (HIV) diagnostic tests are being used extensively in India. However, the evaluation data on these assays are very limited. The present study evaluates indigenous HIV test kits manufactured in India. Materials and Methods: A total of 200 characterised specimens were assayed with Comb AIDS - RS Advantage HIV 1+2 Immunodot Test, Enzaids HIV 1+2 ELISA test, Enzaids Duet HIV Antigen+antibody ELISA test and Signal HIV Flow Through HIV 1+2 test kits. Performance characteristics of these assays were calculated. Results: Sensitivity, specificity, positive predictive value, negative predictive value and efficiency of all the assays were 100% except for Signal HIV Flow Through HIV 1+2 test kit. The specificity, positive predictive value and efficiency of the Signal HIV Flow Through HIV 1+2 test kit were 98.9%, 98.9% and 99.4%, respectively. The Enzaids Duet HIV kit was found to be extremely sensitive in detecting p24 Ag with the sensitivity of 1.5 pg/mL. Conclusions: To conclude, selection of better diagnostic assay is very much important to resolve discrepancies in HIV diagnosis. All these assays under evaluation in this report have got excellent performance characteristics and much suitable to use in serial testing algorithms in use for resources limited settings.