Screening of selected male blood donors for p24 antigen of human immunodeficiency virus type 1. The Transfusion Safety Study Group

BACKGROUND. The p24 antigen of human immunodeficiency virus type 1 (HIV-1) is sometimes detected before antibody (anti-HIV-1) is detectable in the serum of recently infected persons. This has led to the consideration of p24-antigen testing for routine screening of blood donors. METHODS. To estimate how many HIV-infected seronegative donors would be identified if p24-antigen screening was introduced, we tested selected donations from a repository of 200,000 serum samples from voluntary donors that was established in late 1984 and early 1985. The 8597 serum samples selected for p24-antigen screening were chosen because their donors had demographic characteristics known to be associated with a high prevalence of seropositivity. RESULTS. The prevalence of anti-HIV-1 antibodies in the 1984-1985 serum samples selected for p24-antigen screening was 1.54 percent--more than 100 times the 0.012 percent prevalence in present-day donations in the United States. The antigen was detected in 15 of 132 serum samples (11.4 percent) from donors who had already been confirmed as seropositive. No instance of confirmed positivity for p24 antigen was found among the 8465 seronegative serum samples. CONCLUSIONS. These data indicate that the yield of screening for p24 antigen in volunteer donors to identify HIV-1 carriers would be negligible. We therefore recommend against routine screening with currently available p24-antigen assays.     

Ultrasensitive p24 antigen assay for diagnosis of perinatal human immunodeficiency virus type 1 infection

We evaluated an ultrasensitive p24 antigen enzyme immunosorbent assay on 802 plasma specimens from 582 infants and children of 0 to 180 days of age. Overall sensitivity and specificity were 91.7% and 98.5%, respectively. After exclusion of infants of less than 7 days of age, the sensitivity and specificity were 93.7% and 98.3%, respectively.     

Clinical utility of an enhanced human immunodeficiency virus type 1 p24 antigen capture assay

The presence of p24 core antigen in the serum of individuals with human acquired immunodeficiency syndrome has been used as one of the important prognostic markers of HIV-1 infection and also as an end point in evaluating antiviral drugs and vaccines. Unfortunately the majority of p24 antigen present in serum exists as an antigenantibody complex and is not detected with the commercial kits currently available to measure p24 antigen. In this study, we report a simple procedure utilizing treatment of serum samples with glycine buffer (pH 1.85) to dissociate antigen-antibody complexes prior to assaying for p24 antigen. A 300% increase in the number of p24-reactive samples and a 3- to 12-fold increase in the quantity of antigen detected were observed when samples were pretreated with 1.5M glycine buffer (pH 1.85) for 1 hr. Glycine treatment of samples did not result in nonspecific positive tests and samples previously shown to be reactive remained positive. In reconstruction experiments the release of antigen was found to be inversely proportional to the amount of p24 antibody present in the serum. The percentage of HIV-1-infected patients positive for p24 antigen was clearly a function of CD4 count. Forty-nine percent of patients with more than 500 CD4 cells and 100% of patients with less than 200 CD4 were p24 positive. The improved sensitivity for detection of p24 provided by this procedure enhances our understanding of the pathogenesis of AIDS by showing that the majority of patients with HIV-1 infection is p24 positive and facilitates the analysis of data obtained in clinical trials involving anti-HIV compounds.     

Evaluation of an indirect immunofluorescence assay for confirmation of human immunodeficiency virus type 1 antibody in U.S. blood donor sera.

An indirect immunofluorescence assay (IFA) was evaluated as a confirmatory test for antibody to human immunodeficiency virus type 1 in U.S. blood donor sera previously found to be repeatedly reactive by enzyme immunoassay. IFA results were 100% concordant with a licensed Western blot (immunoblot) for 53 negative and 49 positive samples. Four samples which exhibited antibody to viral proteins from more than one gene, yet were indeterminate by Western blot by the manufacturer's criteria, were also reactive by IFA, whereas 49 additional indeterminate samples were IFA negative.     

Deterioration of detectable human immunodeficiency virus serum p24 antigen in samples stored for batch testing.

Virologic measurements are becoming important surrogate markers for therapeutic efficacy in clinical trials with human immunodeficiency virus (HIV)-infected subjects. One such marker which is inexpensive and easily evaluated is the HIV p-24 antigen. To determine the storage stability of p24 antigen assayed by enzyme-linked immunosorbent assay of serum collected during clinical trials, a retrospective analysis was performed. The p24 antigen results were available from four Adult or Pediatric AIDS Clinical Trials Group protocols: studies 047, 050, 128, and 213. Paired samples (n = 930) which were assayed by ELISA for p24 antigen both in real time and in batch were analyzed for agreement. Batch and real-time values were correlated; however, there was a lack of agreement which increased with prolonged storage time of batched samples and greater p24 antigen levels. The p24 antigen values were significantly lower in the batched samples, which had a maximum storage time of 1,548 days. The degradation rate of p24 antigen per year was 0.052 log10 for samples with less than 30 pg/ml, 0.197 log10 for those with 30 to 100 pg/ml, and 0.245 log10 for those with > 100 pg/ml. Due to degradation over time, use of p24 antigen values from batch assays with long-term storage could bias study results toward a lack of treatment effect. On the basis of these results we make the following recommendations. (i) Samples should be assayed either in real time by laboratories undergoing quality assurance or in batch with short-term storage (less than 1 year). (ii) When real-time assays are to be performed, the serum samples should not be stored at 4 degrees C, but should be frozen immediately after processing and stored frozen until tested.     

The use of viral culture and p24 antigen testing to diagnose human immunodeficiency virus infection in neonates. The HIV Infection in Newborns French Collaborative Study Group.

BACKGROUND. Early diagnosis of human immunodeficiency virus (HIV) infection in infants born to infected mothers is important for the infants' medical care, but the presence of maternal antibodies makes serologic tests uninformative. METHODS. In a cohort study of 181 infants born to HIV-infected mothers, we assessed the diagnostic value of HIV viral culture and testing for the presence of p24 antigen. The infants were tested at birth, again during the first 3 months, then followed and tested at the age of at least 18 months. RESULTS. Of the 181 infants, 3 died of HIV infection and 37 were seropositive after the age of 18 months. Viral cultures at birth were positive in 19 of the 40 infected infants and in none of the uninfected infants, yielding a sensitivity of 48 percent (95 percent confidence interval, 32 to 63 percent) and a specificity of 100 percent (95 percent confidence interval, 97 to 100 percent). By the age of three months, 30 of the 40 infants (75 percent) had positive cultures; again, there were no false positive results among the infants who were tested a second time, of the 141 who remained uninfected. The sensitivity of testing for p24 antigen at birth was only 18 percent, with a specificity of 100 percent. The presence of p24 antigen at birth was associated with the development of early and severe HIV-related disease (P less than 0.04). CONCLUSIONS. Viral culture at birth can correctly identify about half of newborns with HIV infection. The fact that this usually sensitive technique fails to identify about half the ultimately infected neonates suggests that vertical transmission of HIV may occur late in pregnancy or during delivery.     

Sensitivity of immune complex-dissociated p24 antigen testing for early detection of human immunodeficiency virus in infants.

Several investigators have suggested that early diagnosis of human immunodeficiency virus (HIV) infection in infants could be accomplished with a modified, more-sensitive, acid-dissociated p24 antigen enzyme-linked immunosorbent assay (ELISA) technique (p24 antigen immune complex dissociation [ICD]). We compared detection of HIV infection by HIV culture, PCR, and p24 antigen ICD assays in 46 infants by using samples collected independently. The detection sensitivity of the p24 antigen ICD assay was 0% with cord blood samples (2 HIV-positive infants), 38% with plasma samples from infants under 3 months of age (8 HIV-positive infants), and 58% overall (12 HIV-positive infants). By contrast, the sensitivities of HIV culture and PCR were 50% for cord blood samples, 75% for plasma samples from infants under 3 months of age, and 83% overall. These results indicate that the p24 antigen ICD does not offer the sensitivity necessary for this assay to be used as an indicator of HIV infection in infants.     

Immunohistochemical localization of human immunodeficiency virus p24 antigen in placental tissue.

As human immunodeficiency virus (HIV) infection spreads into the heterosexual population, perinatally acquired HIV infection will increase in incidence, and knowledge of the mechanism of this transfer is important. We have used immunoperoxidase techniques to detect HIV p24 antigen in formalin-fixed, paraffin-embedded placental tissue from nine known HIV serologically positive mothers. In four of these cases we have detected evidence or viral antigen in placental Hofbauer cells, vascular endothelium, or intermediate trophoblast. The implications for understanding the mode of transfer of infection to the fetus are discussed.     

Performance of a quantitative human immunodeficiency virus type 1 p24 antigen assay on various HIV-1 subtypes for the follow-up of human immunodeficiency type 1 seropositive individuals

The heat-denatured signal-amplified p24 antigen assay is a low-cost test allowing the determination of plasma levels of HIV-1 p24 antigen in infected patients. This assay may be appropriate for monitoring disease progression in HIV seropositive patients in developing countries. Only a few data on the clinical validation of the test are available for HIV-1 non-subtypes B viruses that represent the vast majority of virus circulating in Africa. The present study was undertaken to evaluate and compare the performance of a heat-denatured signal-amplified p24 assay for the determination of p24 viral load in the plasma of individuals infected with different subtypes of HIV-1 and using the RT-PCR-based RNA viral load test as the gold standard. A total of 120 plasma samples from individuals infected with HIV-1 strains belonging to group M (subtypes A-->H) and group O, as well as recombinant strains, were tested in parallel with the heat-denatured signal-amplified p24 assay and the RNA viral load. Plasma p24 levels appeared to be correlated significantly with the plasma RNA viral loads (R=0.751, P<0.0001). The heat-denatured p24 antigen assay was capable of measuring the plasma level of p24 derived from all the HIV-1 subtypes and recombinants selected for this study, in contrast to the RNA viral load test which lacked sensitivity towards HIV-1 group O. The heat-denatured signal-amplified p24 assay is a reliable, sensitive and a more affordable tool that can be used for the follow-up of patients infected with B and non-B subtypes as well as recombinant forms of HIV-1 in developing countries.     

Using a simplified human immunodeficiency virus type 1 p24 antigen assay to diagnose pediatric HIV-infection in Malawi

BackgroundThere is a worldwide need for a pediatric HIV-1 diagnostic test that has a high diagnostic accuracy, is technically simple and cost efficient. The Up24 HIV-1 assay, which requires both the HIV-1 p24 ELISA and the ELAST signal amplification kit, has previously been shown to be a robust tool to diagnose pediatric HIV-1 from dried whole blood spots (DBS) (Cachafeiro et al., JCM 2009;47:459–62¹³). In order to make the assay more accessible to a resource-limited clinical setting, we eliminated the ELAST system, which simplified the Up24 assay, reduced its cost, and tested the accuracy of the modified assay in a rural Malawian hospital.ObjectivesIn this proof of concept study, we tested the ability of a simplified Up24 antigen assay, without ELAST, to detect HIV-1 on DBS obtained via heel prick from 6-week-old Malawian infants.Study designA case–control study of DBS collected from 113 HIV-infected and 109 HIV-negative infants, using the HIV-1 DNA PCR assay as the reference standard.ResultsThe simplified HIV-1 Up24 assay had a sensitivity and specificity of 84% and 98%, respectively. When HIV-1 prevalence is 15%, the positive- and negative-predictive values are 89% and 97%, respectively.ConclusionThe simplified Up24 assay has a good positive- and a robust negative-predictive values, is easier to perform and has a reduced cost compared to both HIV DNA PCR and Up24 assays. With additional testing, the simplified Up24 assay has the potential to increase global access to pediatric HIV-1 diagnostics.     

HIV核心抗原p24原核系统的高效表达、纯化及活性鉴定

目的　在原核表达系统中对HIVp2 4抗原基因进行克隆和高效表达、纯化并鉴定其活性。方法　利用PCR技术从HIV-1全基因质粒 (B2N)中扩增p2 4抗原基因 ,并克隆入T载体中。通过酶切消化后连接到表达载体pRSET上 ,用此连接产物转化大肠埃希菌BL2 1,经IPTG诱导 ,表达p2 4抗原。利用固定化金属离子 (Ni2 )配体亲和层析技术从表达蛋白中纯化目的蛋白。并运用双酶切技术、SDS-PAGE电泳、WesternBlot(WB)及ELISA法分别对插入基因片段的正确性、表达产物的活性及特异性进行检测。结果　PCR产物约为 6 90bp ,与预期p2 4抗原全基因片段大小一致。重组质粒T-p2 4和pRSET-p2 4经BamHⅠ和HindⅢ双酶切 ,其插入的外源基因片段均为 6 90bp。将纯化前与纯化后的蛋白作SDS PAGE电泳 ,均可见一条约 2 4× 10 3的外源基因表达带 ,与计算的相对分子质量相符。经WB和ELISA试验 ,证明基因工程表达的p2 4抗原具有较高的特异性及活性。结论　成功构建了HIVp2 4表达载体pRSET-p2 4 ,并在原核细胞中高效表达 ,其表达产物具有良好的特异性及活性     

Variability and immunogenicity of human immunodeficiency virus type 1 p24 gene quasispecies

Despite the conserved nature of the human immunodeficiency virus type 1 (HIV-1) gag gene, multiple quasispecies of the p24 gene coexist in HIV-1-infected patients. We cloned and sequenced 31 p24 genes from four HIV-1-infected patients. The intrapatient homology between the p24 genes ranged from 97.1 to 99.1%, whereas the interpatient homology ranged from 91.5 to 93.8%, suggesting a host-specific evolution. Synonymous and nonsynonymous nucleotide changes were evenly distributed in the p24 gene, with 27 and 28%, respectively, located within host human leukocyte antigen class I recognition sites. This would suggest only a minor influence from the host cytotoxic T-cell response on the evolution of the p24 gene. The importance of minor variations within p24 was analyzed by designing DNA-based immunogens from two distinct p24 quasispecies genes simultaneously derived from one patient. In plasmid-immunized H-2(b), H-2(d), and H-2(k) haplotype mice, a clear influence from the host major histocompatibility complex was noted on the immune responses, fully consistent with those noted when a recombinant p24 protein is used as the immunogen. The two p24 DNA immunogens did not differ in their immunogenicity, indicating that the limited genetic variability (<1%) had little influence on the immune responses.     

Detection of p24 antigen with and without immune complex dissociation for longitudinal monitoring of human immunodeficiency virus type 1 infection.

Sequential specimens obtained from 87 multicenter AIDS cohort study participants were tested by three p24 antigen tests. They included a polyclonal enzyme immunoassay (EIA), a monoclonal EIA, and a monoclonal EIA after immune complex dissociation (ICD) of specimens. Subjects were grouped into two categories defined by real-time testing with the polyclonal EIA: 39 had become positive for p24 antigen (antigen converters) during follow-up, and 48 had progressed to AIDS without detectable antigenemia. Twenty-four (61%) antigen converters were positive by ICD-monoclonal EIA about 1 year earlier than by monoclonal EIA. In contrast, only 12 (25%) patients who progressed to AIDS without detectable antigenemia became positive by ICD-p24 EIA before developing AIDS. Thus, the main benefit of ICD treatment may be to detect p24 antigenemia approximately 1 year before the regular assay rather than to identify additional antigenemic people. Quantitative plasma RNA levels were also determined in longitudinal samples from 20 antigen converters and 7 men who developed AIDS without antigenemia. Although mean human immunodeficiency virus type 1 RNA levels were higher in antigen-positive than in antigen-negative samples (P = 0.002), more than half (11 of 20) of the antigen converters had no measurable change in human immunodeficiency virus type 1 RNA associated with change to antigen positivity.     

Evaluation of the ultrasensitive human immunodeficiency virus type 1 (HIV-1) p24 antigen assay performed on dried blood spots for diagnosis of HIV-1 infection in infants

The diagnostic accuracy of the modified p24 antigen assay performed on pediatric dried blood spots was evaluated. Samples analyzed within 6 weeks of collection yielded no false-positive results (specificity, 100%) and few false-negative results (sensitivity, 96.5% to 98.3%). Laboratory services with limited resources should assess this option for routine infant diagnosis.     

A sensitive radioimmunoassay of human immunodeficiency virus type-1 p24 antigen in serum

We describe a sensitive and specific radioimmunoassay (RIA) for HIV-1 p24. The test called cgeacren-p24-VIH RIA is a transposition of signal-amplification-boosted of head-denatured plasma ELISA developed by Schupbach to a RIA performed with in-house reagents. This modified assay is based on the use radiolabeled tracer to decrease the lower limit of p24 antigen detection from 0.5 pg/ml [JCM, 2003, 1016–1022] to 0.05 pg/ml by RIA method. The RIA technique is based on the competitive binding of the serum HIV-1 p24 and radiolabeled p24 preparation. They compete for binding to an antibody specific present in a substoichiometric amount. Complex of radiolabeled and unlabeled p24 antigen were separated from unbound species by reaction with an anti-rabbit sheep immunoglobulin as second antibody [10]. This paper describes, for the first time, the successful production of polyclonal anti sera specific for HIV-1 p24 and the development of a sensitive RIA for measuring HIV-1 p24 in serum. The second part of this study will concern the clinical evaluation of this “cgeacren-HIV-1 p24RIA” kit.     

Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group

BACKGROUND AND METHODS: We examined the influence of viral load in relation to other risk factors for the heterosexual transmission of human immunodeficiency virus type 1 (HIV-1). In a community-based study of 15,127 persons in a rural district of Uganda, we identified 415 couples in which one partner was HIV-1-positive and one was initially HIV-1-negative and followed them prospectively for up to 30 months. The incidence of HIV-1 infection per 100 person-years among the initially seronegative partners was examined in relation to behavioral and biologic variables. RESULTS: The male partner was HIV-1-positive in 228 couples, and the female partner was HIV-1-positive in 187 couples. Ninety of the 415 initially HIV-1-negative partners seroconverted (incidence, 11.8 per 100 person-years). The rate of male-to-female transmission was not significantly different from the rate of female-to-male transmission (12.0 per 100 person-years vs. 11.6 per 100 person-years). The incidence of seroconversion was highest among the partners who were 15 to 19 years of age (15.3 per 100 person-years). The incidence was 16.7 per 100 person-years among 137 uncircumcised male partners, whereas there were no seroconversions among the 50 circumcised male partners (P<0.001). The mean serum HIV-1 RNA level was significantly higher among HIV-1-positive subjects whose partners seroconverted than among those whose partners did not seroconvert (90,254 copies per milliliter vs. 38,029 copies per milliliter, P=0.01). There were no instances of transmission among the 51 subjects with serum HIV-1 RNA levels of less than 1500 copies per milliliter; there was a significant dose-response relation of increased transmission with increasing viral load. In multivariate analyses of log-transformed HIV-1 RNA levels, each log increment in the viral load was associated with a rate ratio of 2.45 for seroconversion (95 percent confidence interval, 1.85 to 3.26). CONCLUSIONS: The viral load is the chief predictor of the risk of heterosexual transmission of HIV-1, and transmission is rare among persons with levels of less than 1500 copies of HIV-1 RNA per milliliter.     

HIV-1 p24 antigen testing in blood banks: results from Saudi Arabia

HIV-1 p24 antigen testing was introduced to increase sensitivity in the early detection of HIV infection in blood donors. Since the introduction of HIV-1 p24 antigen testing in Saudi Arabia, we have failed to detect a single positive case. Over a three-year period, only four indeterminates were detected out of 24,654 blood donors. All four proved negative by confirmatory testing. Based on this experience, we believe that resources would be better directed to pooled nucleic acid testing and that p24 serological testing should be abandoned.