No evidence of frequent human immunodeficiency virus type 1 infection in seronegative at-risk individuals

The possible existence of human immunodeficiency virus type 1 (HIV-1) infection in asymptomatic seronegative at-risk individuals was investigated in a prospective study of 55 seronegative high-risk individuals (42 homosexual men and 13 heterosexual individuals) and 32 seronegative hemophiliacs treated with factor VIII or IX concentrates before viral inactivation by heat treatment and systematic screening of blood donations. Tests used include the polymerase chain reaction assay with three primer pairs (one in the gag region and two in the pol region) and tests for serum p24 antigen, anti-nef serology (Western blot), and five biologic markers frequently altered by HIV infection (CD4 lymphocyte count, serum beta 2-microglobulin and neopterin concentration, and serum IgG and IgA concentration). Although 91 of 92 HIV-1-seropositive persons were positive in testing with at least one primer pair, no positive result was observed in seronegative at-risk individuals or in 117 seronegative low-risk controls. No nef antibody was found in seronegative at-risk individuals or seronegative controls, but 44 (47%) of 92 HIV-1-seropositive persons had nef antibodies. These findings do not support the existence of frequent HIV-1 infection in seronegative at-risk individuals.     

Lack of detection of human immunodeficiency virus type 1 DNA by polymerase chain reaction in the plasma and lymphocytes of seronegative exposed hemophiliacs

Direct detection of human immunodeficiency virus type 1 (HIV-1) DNA in serum or plasma samples has been reported in seronegative as well as seropositive individuals. An alkaline lysis procedure was adapted for polymerase chain reaction (PCR) analysis of plasma specimens. Eighty- five seronegative hemophiliacs, 52 of whom had been exposed to HIV- contaminated blood components, and 19 seronegative at-risk individuals were studied. Each sample was extracted and amplified with SK38/39 gag primers at least three times. Seventy-six samples (72%) were consistently negative for HIV-1 DNA, 24 (22%) were positive only once, and 4 (3%) were positive twice. Genomic DNA from peripheral mononuclear cells was prepared from 12 of 76 negative samples, 18 of 24 samples that were positive once, and 2 of 4 samples that were positive twice and analyzed with both gag and long terminal repeat primers. None (0/32) of these cellular DNAs were positive for HIV-1, which suggests that these seronegative exposed hemophiliacs were not latently infected with HIV-1. In contrast, all (10/10) control cells from seropositive patients were positive with both primer pairs. The detection of HIV-1 DNA in serum or plasma may be prone to a high level of false-positive PCR signals and should be interpreted with caution.     

Detection of human immunodeficiency virus type 1 by a highly sensitive and specific polymerase chain reaction method

A sensitive and specific polymerase chain reaction (PCR)-based assay was developed for detection of a single copy of human immunodeficiency virus type 1 (HIV-1) sequence. The different methodologies for preparation of clinical DNA samples were evaluated. The DNA extracted by the Ficoll-Histopaque method gave the best quality for PCR. DNA samples equivalent to 18 μl or less of blood from HIV-1 seropositive individuals were positive by this assay. This procedure should be suitable for early diagnosis of HIV-1 infection in many clinical situations.     

A comparison of polymerase chain reaction and an infectivity assay for human immunodeficiency virus type 1 titration during virus inactivation of blood components

Three examples of human plasma-derived concentrates, intermediate- purity factors VIII and IX, and fibrinogen were spiked with tissue culture-grown human immunodeficiency virus type 1 (HIV-1) strain RF. All examples were freeze-dried and heated at 80 degrees C for 72 hours by using validated production process models. HIV-1 infectivity was measured by a syncytial infectivity assay in C8166 cells and then compared with levels determined by nested HIV polymerase chain reaction (PCR). The infectivity assay demonstrated a reduction index of at least 4.5 log10, while PCR showed an average 1.7 log10. Large amounts of HIV- 1 RNA (10(5)) were still detectable by PCR in samples in which infectivity assays failed to detect any HIV-1. These data suggest that HIV-1 PCR levels do not parallel HIV-1 infectivity levels during virus- inactivation procedures involved in coagulation factor concentrate production. PCR was able to detect the RNA associated with inactivated HIV-1 particles in the factor concentrates, which allows the conclusion that PCR is not a useful test with which to monitor virus-inactivation procedures such as heating at 80 degrees C for 72 hours. This judgment contrasts with the more definite and sensitive role of PCR in diagnosing HIV-1 infection in patients in whom a positive HIV-1 PCR result correlates with active HIV-1 infection and with PCR's usefulness in monitoring virus removal.     

Failure to detect evidence of human immunodeficiency virus type 1 (HIV- 1) infection by polymerase chain reaction assay in blood donors with isolated core antibodies (anti-p24 or -p17) to HIV-1

Individuals with isolated and persistent core antibodies (anti-p24 or -p17) to HIV-1 are sometimes diagnosed through the systematic screening of blood donations. The significance of such serum reactivities remains unknown. Polymerase chain reaction (PCR) is a new technique allowing the direct detection of HIV-1 DNA in blood samples. In this study, PCR was used to detect HIV-1 DNA in 20 individuals with isolated and persistent core antibodies (14 anti-p24 and 6 -p17), in seven sexual partners of these individuals, in 55 HIV-1-seropositive individuals (positive controls), and in 105 HIV-1-seronegative blood donors at low risk of HIV-1 infection (negative controls). No HIV-1 DNA was detected in individuals with isolated and persistent core antibodies, in their sexual partners, or in negative control individuals, although PCR was positive in 54 of 55 seropositive control individuals. These results strongly suggest that individuals with isolated and persistent core antibodies are not infected with HIV-1.     

Rare detection of hepatitis B and hepatitis C virus genomes by polymerase chain reaction in seronegative donors with elevated alanine aminotransferase

BACKGROUND: Since screening for antibody to hepatitis C virus (HCV) was introduced in 1990, posttransfusion hepatitis has been reduced to nearly background levels. This has led to reconsideration of the value of testing donated blood for elevated alanine aminotransferase (ALT). The contribution of ALT testing in detecting seronegative infection was evaluated by the performance of polymerase chain reaction (PCR) for hepatitis B virus (HBV) or HCV in plasma from ALT-elevated blood units. STUDY DESIGN AND METHODS: Testing was performed on 375 units of plasma, derived from an equivalent of 47,500 blood donations, with a highly sensitive hemi-nested PCR procedure. Using a triplet of primers directed at the conserved regions of HBV DNA and 5'-noncoding regions of HCV RNA, the hemi-nested PCR assay can reliably amplify 10 viral molecules to levels detectable in ethidium bromide-stained agarose gels. Pools of plasma from groups of four donors were screened with hemi-nested PCR. For any reactive pools, the plasma from individual donors was retested twice on different aliquots. RESULTS: Two of 375 units, both with midrange ALT elevation, were repeatedly reactive in hemi-nested PCR (one each for HBV DNA and HCV RNA). However, samples from the two suspect donors tested 9 and 5 months later revealed no seroconversion, elevated ALT, or viral genomes in hemi-nested PCR. CONCLUSION: The lack of confirmed HBV or HCV infection in this study representing an estimated 47,500 voluntary blood donations suggests that routine ALT testing for further prevention of posttransfusion hepatitis after exclusion of HBV- and/or HCV-seropositive blood may be superfluous.     

Indeterminate human immunodeficiency virus type 1 western blot may indicate an abortive infection in some low-risk blood donors

BACKGROUND: The infectious status of persons with an indeterminate human immunodeficiency virus type 1 (HIV-1) Western blot must be established. STUDY DESIGN AND METHODS: Evaluation of the CD4 and CD8 T- cell subsets and the expression of HIV-1-integrated sequences by Southern blot and polymerase chain reaction were studied in a group of low-risk subjects with an indeterminate Western blot. RESULTS: From a total of 45,000 blood donors and 50 patients with chronic renal failure on hemodialysis who were tested during the period of 1985 through 1990, 50 sera (0.1%) had an indeterminate Western blot. A low CD4:CD8 ratio (0.7-1.2) was detected in 14 of 24 tested subjects, whereas the unfractionated and adherence-enriched cells of 7 (32%) and 5 (23%) of 22 patients, respectively, could be stained with a p24 monoclonal antibody. A transient positive culture was detected in 3 of 20 subjects, but these viral isolates could not be transmitted to CEM-A310 cells. Ultracentrifuged culture supernatants hybridized under high- stringency conditions with genomic gag-pol (4 cases), env (3 cases), and tat (1 case) cDNA fragments of the HXB2 HIV-1 clone. In one case, DNA obtained from adherent but not unfractionated mononuclear cells contained 3.3- and 3.9-kb env- and gag-pol-related HIV-1 sequences, respectively; these sequences were heavier than expected. Polymerase chain reaction analysis for gag and pol but not env sequences was positive in 1 and 2 of 7 cases, respectively. A female patient with a positive viral culture and who was positive for pol in polymerase chain reaction demonstrated a full seroconversion 19 months later. CONCLUSION: The results strongly suggest that, rarely, some low-risk subjects with indeterminate Western blot results might be infected with low-level replicative strains or HIV-related viruses; thus, an exhaustive immunologic and virologic workup is needed for the investigation of these subjects.     

Detection of the human immunodeficiency virus genome with a biotinylated DNA probe generated by polymerase chain reaction

A non-radiolabelled DNA probe was developed for detection of the human immunodeficiency virus type 1 (HIV-1) genome using the polymerase chain reaction (PCR) technology. Primers amplifying a 395 base pair segment of a portion of the polymerase region of the HIV-1 genome were used both to amplify sample target DNA and to generate a biotinylated DNA probe used in Southern blot hybridization. This probe performed as well as one produced by nick translation using biotinylated nucleotides or an enzyme labelled oligonucleotide probe.     

Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes

Human immunodeficiency virus type 1 (HIV-1) infection of T lymphocytes requires cellular proliferation and DNA synthesis. Human monocytes were shown to have low DNA synthesis rates, yet the monocytotropic BaL isolate of HIV-1 was able to infect these cells efficiently. Monocytes that were irradiated to assure no DNA synthesis could also be readily infected with HIV-1BaL. Such infections were associated with the integration of HIV-1BaL DNA into the high molecular weight, chromosomal DNA of monocytes. Thus, normal, nonproliferating monocytes differ from T lymphocytes in that a productive HIV-1 infection can occur independently of cellular DNA synthesis. These results suggest that normal nonproliferating mononuclear phagocytes, which are relatively resistant to the destructive effects of this virus, may serve as persistent and productive reservoirs for HIV-1 in vivo.     

Quantitative estimation of human immunodeficiency virus type-1 provirus in CD4+ T lymphocytes using the polymerase chain reaction.

The proviral burden of peripheral blood lymphocytes in 29 patients infected with human immunodeficiency virus type-1 (HIV-1) was estimated using a polymerase chain reaction method which we recently refined. The mean numbers of HIV-1 provirus in eight patients with AIDS and AIDS-related complex treated with zidovudine (AZT), in 10 asymptomatic patients treated with AZT, and in 11 asymptomatic untreated patients were 892, 436, and 406 copies in 1 x 10(5) CD4- T lymphocytes, respectively. These results demonstrate that patients may have more HIV-1 provirus copies in an advanced than early stage of disease, and that the fraction of the infected lymphocytes is much higher than previously thought, especially in asymptomatic patients. There was no difference in the viral burden in CD4+ T lymphocytes regardless of whether AZT had been administered or not. These findings validate the method used here to quantitate HIV-1 provirus DNA and confirm that AZT is not effective in reducing the amount of provirus DNA in lymphocytes.     

The use of polymerase chain reaction in plasma pools for the concomitant detection of hepatitis C virus and HIV type 1 RNA

BACKGROUND: Detection of viral nucleic acids might increase blood transfusion safety through the detection of recently infected blood donors during the preseroconversion window period. Individual screening is difficult to apply, because of technical and financial constraints. STUDY DESIGN AND METHODS: A polymerase chain reaction (PCR)-based assay including a polyethylene glycol precipitation step was developed for the concomitant detection of hepatitis C virus (HCV) and HIV type 1 (HIV-1) RNA in plasma pools corresponding to 50 blood donations by the use of commercial assays. RESULTS: The assay had a sensitivity of less than 33 copies per mL for HCV RNA and 1000 copies per mL for HIV-1 RNA for each individual sample included in the pool. The eight preseroconversion samples with HCV RNA between 1,250 and 762,000 copies per mL were all detected when 100-microL aliquots from the samples were introduced into 5-mL pools of 50 blood donations. CONCLUSIONS: A PCR- based pooling assay associating a prepurification step with polyethylene glycol allows for the screening of blood donations for HCV and HIV-1 RNA without marked loss of sensitivity from that seen with commercially available assays. This procedure might increase blood safety through systematic screening of blood donations at relatively low cost.     

Long-term follow-up of blood donors with indeterminate human immunodeficiency virus type 1 results on Western blot

BACKGROUND: At present, tens of thousands of United States blood donors who are at low risk for human immunodeficiency virus type 1 (HIV-1) infection are indefinitely deferred. These persons are repeatably reactive for HIV-1 antibody in enzyme immunoassay (EIA) and are indeterminate in Western blot. STUDY DESIGN AND METHODS: To determine the significance and persistence of anti-HIV-1 reactivity in plasma from volunteer blood donors with HIV-1-indeterminate Western blots, 66 donors were retested for HIV-1 antibody by the same manufacturers' EIA and Western blot 5 to 7 years after the initial Western blot. In addition, donors' peripheral blood mononuclear cells were tested by polymerase chain reaction (PCR) for HIV-1 DNA gag sequences. RESULTS: Thirty-five (53%) of 66 donors were still repeatedly reactive for HIV-1 on EIA and indeterminate on Western blot, 23 (35%) were negative on EIA and indeterminate on Western blot, 7 (11%) were negative in EIA and Western blot, and 1 (2%) was repeatedly reactive on EIA and negative on Western blot. Donors with persistently indeterminate Western blots had a band pattern nearly identical to that on the original Western blot. No donor was positive in Western blot, p24 antigen, or PCR testing. No donor had signs or symptoms of HIV-1 infection. CONCLUSION: Long-term follow-up of Western blot-indeterminate blood donors does not reveal evidence of HIV-infection. A mechanism to return these donors to the donor pool should be considered.     

Novel polysulfated galactose-derivatized dendrimers as binding antagonists of human immunodeficiency virus type 1 infection

Evidence indicates that galactosyl ceramide (GalCer) and its 3'-sulfated derivative, sulfatide (SGalCer), may act as alternate coreceptors for human immunodeficiency virus type 1 (HIV-1) in CD4(-) cells. Glycosphingolipids (GSLs) may also be necessary for fusion of HIV-1 and host cell membranes. Using an enzyme-linked immunosorbent assay to determine which GSL was the best ligand for both recombinant and virus-associated gp120, we found that SGalCer was the best ligand for each rgp120 and HIV-1 isolate tested. Therefore, novel multivalent glycodendrimers, which mimic the carbohydrate clustering reportedly found in lipid rafts, were synthesized based on the carbohydrate moiety of SGalCer. Here we describe the synthesis of a polysulfated galactose functionalized, fifth generation DAB dendrimer (PS Gal 64mer), containing on average two sulfate groups per galactose residue. Its ability to inhibit HIV-1 infection of cultured indicator cells was compared to that of dextran sulfate (DxS), a known, potent, binding inhibitor of HIV-1. The results indicate that the PS Gal 64mer inhibited infection by the HIV-1 isolates tested as well as DxS.     

Serum factors in the progression of human immunodeficiency virus type 1 infection to AIDS

We studied the prevalence of four serum factors in individuals at different stages of human immunodeficiency virus-1 (HIV-1) infection. Soluble interleukin-2 receptors (sIL-2R) were elevated in all antibody-positive groups compared with high-risk, antibody-negative controls. Paraproteins, usually of the IgG-kappa isotype, were found in the sera of a significant number of HIV-1-infected individuals as were antibodies to lymphocytes (ALAs). Serum factors that inhibit proliferation of peripheral blood mononuclear cells from healthy donors appear late in the course of infection and were associated with increasing clinical severity. Measurement of these factors may prove to be useful in defining the stages of infection and in predicting the appearance or exacerbation of symptoms. They may also play a role in the development of the HIV-1-induced immune defects that lead to the expression of clinical acquired immunodeficiency syndrome.     

Cytomegalovirus as a co-factor of disease progression in human immunodeficiency virus type 1 infection

Summary Cytomegalovirus has been suggested as a co-factor of disease progression in patients with human immunodeficiency virus type 1 infection. Cytomegalovirus infection is highly prevalent among populations at risk of human immunodeficiency virus 1 infection, and has been associated with both an increased susceptibility to infection and a more rapid course of the disease towards immunodeficiency. Cytomegalovirus can have a direct immunosuppressive effect (through infection of immune cells) and can enhance the replication of human immunodeficiency virus (through the transactivation of the genic immunodeficiency virus expression, the stimulation of cytokine production, and the increase in Fc receptor expression on target cells). The role of cytomegalovirus as a co-factor of the progression towards immunodeficiency in subjects infected with the human immunodeficiency virus type 1 needs to be elucidated with more extensive clinical studies and the application of new molecular biology techniques.     

Topographic and quantitative display of integrated human immunodeficiency virus-1 provirus DNA in human lymph nodes by real-time polymerase chain reaction

In situ polymerase chain reaction (isPCR) has been applied in many fields that require detection of a genomic marker in combination with its topographic localization in tissue. We describe here a novel approach that circumvents the major drawbacks of in situ PCR, ie, low sensitivity, leakage of DNA from cells, and inability to quantify the DNA input. Frozen sections of a lymph node from a human immunodeficiency virus (HIV)-1-infected patient were fixed on glass microscope slides, and the glass was scored into square fragments of 0.5-mm edge length using a diamond cutting device. Slides were then attached to adhesive, elastic plastic foil and finally broken, and the foil was extended to allow sorting of fragments into PCR microtiter plates. The material was tested for HIV-1 proviral DNA by a sensitive real-time PCR protocol. Subjacent sections were stained for follicular dendritic cells to identify follicles. The fragmentation process prevented leakage of amplified DNA to neighboring areas as often experienced with in situ PCR. Provirus was clearly associated with follicular areas, in which provirus-carrying cells represented an average of 0.8% of the total cell population (peak density, 3.1% of all follicular cells). The results of this method suggest that the high density of provirus-containing cells in follicles may be important for the persistence of proviral DNA in infected persons.