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Normal assay variation associated with bDNA tests for human immunodeficiency virus type 1 (HIV-1) RNA performed at two laboratories with different levels of test experience was investigated. Two 5-ml aliquots of blood in EDTA tubes were collected from each patient for whom the HIV-1 bDNA test was ordered. Blood was stored for no more than 4 h at room temperature prior to plasma separation. Plasma was stored at -70 degrees C until transported to the Central Pennsylvania Alliance Laboratory (CPAL; York, Pa.) and to the Hershey Medical Center (Hershey, Pa.) on dry ice. Samples were stored at < or =-70 degrees C at both laboratories prior to testing. Pools of negative (donor), low-HIV-1-RNA-positive, and high-HIV-1-RNA-positive plasma samples were also repeatedly tested at CPAL to determine both intra- and interrun variation. From 11 August 1999 until 14 September 2000, 448 patient specimens were analyzed in parallel at CPAL and Hershey. From 206 samples with results of > or =1,000 copies/ml at CPAL, 148 (72%) of the results varied by < or =0.20 log(10) when tested at Hershey and none varied by >0.50 log(10). However, of 242 specimens with results of <1,000 copies/ml at CPAL, 11 (5%) of the results varied by >0.50 log(10) when tested at Hershey. Of 38 aliquots of HIV-1 RNA pool negative samples included in 13 CPAL bDNA runs, 37 (97%) gave results of <50 copies/ml and 1 (3%) gave a result of 114 copies/ml. Low-positive HIV-1 RNA pool intrarun variation ranged from 0.06 to 0.26 log(10) while the maximum interrun variation was 0.52 log(10). High-positive HIV-1 RNA pool intrarun variation ranged from 0.04 to 0.32 log(10), while the maximum interrun variation was 0.55 log(10). In our patient population, a change in bDNA HIV-1 RNA results of < or =0.50 log(10) over time most likely represents normal laboratory test variation. However, a change of >0.50 log(10), especially if the results are >1,000 copies/ml, is likely to be significant.

Mechanisms that protect most high-risk HIV-1 seronegative (HRSN) persons are not well understood. Among hemophiliacs from the Multicenter Hemophilia Cohort Study who remained HIV-1 seronegative despite a high (94%) risk for acquisition of HIV-1 infection, only 7/43 were homozygous for the protective CCR5 Delta32 polymorphism. Among the remainder, neither CCR5 density nor beta-chemokine production, nor in vitro susceptibility to infection with the HIV-1 isolate JR-FL could distinguish HRSN hemophiliacs from healthy controls. When compared to lymphocytes of healthy controls not at risk for HIV-1 infection, diminished spontaneous lymphocyte proliferation was seen in lymphocytes of HRSN hemophiliacs as well as in lymphocytes of hemophiliacs not at risk for HIV-1 infection. Surprisingly sera/plasmas obtained from high-risk HIV-1 seropositve hemophiliacs prior to seroconversion more often contained alloreactive antibodies than date-matched sera/plasmas obtained from HRSN hemophiliacs. Thus alloreactivity may predispose to acquisition of HIV-1 infection after parenteral exposure., (Copyright 2000 Academic Press.)

A large collection of natural HIV-1 integrase (IN) sequences has not previously been described. We reasoned that analysis of such sequences would address whether natural variation of HIV-1 IN contributes to the pathogenesis of AIDS and might also identify amino acid residues important for IN function. Sequences encoding HIV-1 IN were amplified from cryopreserved lymphocytes or plasma obtained at different times from 10 hemophilia patients who had been observed for up to 17 years. The region of the HIV-1 genome that encodes the 288-amino acid IN protein was sequenced from a total of 102 clones; information was obtained for 99.97% of 29,478 amino acid positions. Phylogenetic analysis indicated that patient samples were unique. Interpatient nucleic acid distances ranged from 0.8% to 4.9%, highlighting the tight conservation of this genomic region. No major differences were found between DNA and RNA or between early and late time points from the same patient. Significantly, no amino acid changes that might account for the variable rate of disease progression between patients were evident. Only one amino acid substitution involved a highly conserved residue known to be important for enzymatic activity. However, several interesting amino acid substitutions were noted, including residues within the C-terminal region of the protein for which sequence comparisons between animal retroviruses have not been very informative. These results should encourage the pursuit of anti-integrase therapies, especially inasmuch as the apparent biologic constraints on the IN sequence may deter the development of drug resistance.

Objective: To characterize the natural history of viremia with human immunodeficiency virus type 1 (HIV-1) and its association with disease progression from infection to acquired immunodeficiency syndrome (AIDS)., Design: Prospective cohort study. Annual specimens were tested for quantitative virion-associated HIV-1 RNA, p24 antigen, and CD4+ lymphocyte levels., Participants: A total of 42 homosexual men who seroconverted to HIV-1 between 1982 and 1985., Main Outcome Measures: Trends over time in serum HIV-1 RNA level, correlations between serum HIV-1 RNA and other markers, and prediction of AIDS using these markers., Results: HIV-1 RNA levels were stable over time, increasing by 10-fold or more in only six (14%) of the 42 subjects during 3 to 11 years of follow-up. Mean HIV-1 RNA levels were 10(3.8) copies/mL if AIDS occurred in less than 4 years, 10(3.07) copies/mL if AIDS developed within 4 through 9 years, and 10(2.27) copies/mL if AIDS did not develop within 6 through 11 years. In both univariate and multivariate models, initial and subsequent HIV-1 RNA levels, p24 antigenemia, and percentage of CD4+ lymphocytes were independently predictive of AIDS., Conclusions: The stability of virion-associated HIV-1 RNA levels suggests that an equilibrium between HIV-1 replication rate and efficacy of immunologic response is established shortly after infection and persists throughout the asymptomatic period of the disease. Thus, defective immunologic control of HIV-1 infection may be as important as the viral replication rate for determining AIDS-free survival. Because individual steady-state levels of viremia were established soon after infection, HIV-1 RNA levels may be useful markers for predicting clinical outcome.

Blood samples were obtained from 16 hemophiliacs who had a 50%-94% defined risk of human immunodeficiency virus (HIV) type 1 infection on the basis of treatment history and from 14 controls not at risk for HIV infection. HIV-1 was not detected in any of 12 patient samples by cocultivation nor in 14 patient samples by the polymerase chain reaction. Peripheral blood cells from 7 seronegative hemophiliacs at highest risk of seroconversion (94%) were less susceptible to HIV-1 infection in vitro than were cells from healthy controls (P < .025, one-tailed Wilcoxon rank sum test). In contrast, the susceptibility to HIV-1 infection of lymphocytes from 6 seronegative hemophiliacs at moderate risk (50%-56%) of seroconversion did not differ from that of cells from controls or from high-risk hemophiliacs. Therefore, prolonged periods of seronegative HIV-1 infection are not common in this high-risk population. In addition, among hemophiliacs there may exist heterogeneity in susceptibility to HIV-1 infection in vitro and in vivo.

To evaluate the effects of human immunodeficiency virus type 1 (HIV-1) infection on the loss of factor VIII alloantibodies, we identified 77 patients with a history of inhibitors from among a large cohort of HIV-1-infected participants enrolled in a natural history study of HIV-1 infection in hemophilia. Fifty-six patients were high responders with inhibitors titers greater than 5 Bethesda Units (BU) measured on at least one occasion. From May 1985 to December 1989, 13 of the high-responder patients were rechallenged with factor VIII concentrates after several years of treatment with other plasma products. All exhibited excellent hemostasis upon reinstitution of factor VIII. Seven of the 13 patients (11.3-46.3 years of age) were in the advanced stages of HIV-1 infection at the time of rechallenge. Inhibitor titers measured subsequent to the reinstitution of factor VIII were consistently less than 1 BU in five of these seven patients. The remaining six patients (6.1-57.5 years of age) had mild to moderate CD4+ lymphocyte depletion (absolute CD4+ cells: 262-935/mm3) at the time of factor VIII rechallenge. Follow-up inhibitor titers were negative 7-42 months after consistent factor VIII use in these six patients. The lack of anamnestic response to factor VIII in all 13 patients who were rechallenged indicates that HIV-1-infected patients who have a history of high-responder inhibitors frequently benefit from the reintroduction of factor VIII use for the control of bleeding, regardless of their stage of HIV-1 disease.

We used the polymerase chain reaction (PCR) to determine the frequency of silent human immunodeficiency virus type 1 (HIV-1) infections in seronegative high-risk individuals with hemophilia who had been exposed to contaminated blood products more than 3 years previously. In a cross-sectional study of a cohort of 57 prospectively followed seronegative hemophiliacs who received multiple transfusions before 1986, HIV-1 proviral DNA was found transiently in only one patient. These data suggest that the rate of HIV infection among high-risk antibody negative individuals with hemophilia is very low to absent, in the range of 0% to 2%. These findings should provide considerable reassurance to seronegative persons with hemophilia and their sexual partners.

In order to optimize detection of human immunodeficiency virus-1 (HIV-1)-infected cells, the temporal appearance of virus antigens in newly infected H9 cell cultures was examined. Analyses were accomplished by indirect immunofluorescence labeling with each of 10 monoclonal antibodies and evaluation by flow cytometry. Of the antibodies examined, those specific for HIV-1 capsid protein p24, matrix protein p17, or their precursor molecule p55 allowed the earliest and most sensitive detection in infected cells fixed to allow detection of intracellular antigen. Discrimination of infected cells from uninfected cells was much less sensitive when three antibodies specific for HIV-1 glycoproteins were used to detect intracellular or cell surface antigen. In several experiments involving the time course of infection, we observed no differences in cell numbers between infected and uninfected H9 cultures initiated at identical cell concentrations. We hypothesized that it might be possible to quantitate infectious HIV-1 virions from the kinetics of infected cell appearance. Straight-line relationships between the log p24-positive cells and the time after infection were observed. These quantitative observations were employed to calculate the number of infectious units originally added to the culture that were capable of infecting H9 cells. The production of infectious virus, but not of cytopathic effects, was required. The results of this novel approach to the titration of infectious HIV-1 particles agreed well with those from median cell culture infective dose determination. This method could be employed with other infectious agents for which detection of cell-associated antigens is possible in cell cultures not destroyed by infection.