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Performance assessment of neonatal dried blood spot testing for HIV antibody.

A study was undertaken to assess the performance of laboratories participating in the UK unlinked anonymised neonatal screening programme for the detection of HIV antibody. A coded panel of 100 contrived dried blood spots prepared form well characterised anti-HIV-1 and anti-HIV-2 positive sera and an anti-HIV negative serum was distributed to eight testing centres. Fifty-one out of 52 anti-HIV-1 positive samples were identified by all eight laboratories: five laboratories identified all 52 specimens. Only one laboratory detected all four anti-HIV-2 positive spots. None of the laboratories reported a seronegative spot as reactive. The sensitivity of anonymised neonatal anti-HIV-1 screening was deemed satisfactory in all participating laboratories but the margin of sensitivity was insufficient to allow pooling of dried blood spot eluates before testing. Technical modifications would be necessary to increase sensitivity for anti-HIV-2 were HIV-2 infection to be reported more frequently in the UK. Performance assessment of the screening programme should be repeated at regular intervals.

AIDS Serodiagnosis↗

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.

Amino Acid Sequence↗

Salivary and urinary diagnosis of human immunodeficiency viruses 1 and 2 infection in Côte d'Ivoire, using two assays.

This investigation, done at the Institut Pasteur de Cote d'Ivoire 'blind' of the previous serological findings, suggests that GACELISA, a commercial immunoglobulin G capture enzyme immunoassay for anti-human immunodeficiency virus antibody, can be successfully applied to unprocessed saliva and urine specimens. Its accuracy may be as high as that of conventional enzyme assays on serum tested under similar conditions. However, the role of GACPAT, a similar assay, as a cheap alternative screening test for urine remains in doubt unless its non-specificity can be controlled.

HIV Antibodies↗

A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates.

We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridine-triphosphate (bio-dUTP) using a poly(rA) template hybridized with oligo(dT) primer that is immobilized on the surface of a 96-well microtiter plate. This assay is thus semi-automated by adapting it to an ELISA testing format. The incorporation of bio-dUTP was enhanced by adding cold dTTP to the reaction mixture, optimally in a molar ratio 4:1 (dTTP:bio-dUTP). This non-RI RTA is more sensitive than the conventional RI assay for the detection of purified Rous-associated virus 2 (RAV-2) and of human immunodeficiency virus type 1 (HIV-1) lysate. Because of its simple procedure, higher sensitivity and non-use of RI materials, the assay can be utilized not only for virological studies but also for routine safety screening of biological products for retroviral contamination.

Avian Leukosis Virus↗

A new method for measuring reverse transcriptase activity by ELISA.

A new and sensitive assay of reverse transcriptase (RT) activity of retroviruses measures the incorporation of digoxigenin-labelled dUTP in newly synthesized DNA instead of radioactively labelled (3H- or 32P-)dTTP. To avoid difficulties associated with separation of non-incorporated nucleotides from the newly synthesized DNA, biotin-labelled dUTP is added to the reaction mixture in very low concentrations. After reverse transcription, the newly synthesized, doubly labelled DNA is immobilized on streptavidin-coated ELISA wells and evaluated photometrically by binding of peroxidase-conjugated anti-digoxigenin-antibodies (sheep) and subsequent colour development with 2,2'-azino-di[3-ethylbenzthiazolin-sulfonate(6)] (ABTSR) as substrate. For better standardization, it is suggested that RT activity is given in units (one unit of RT is the amount of enzyme incorporating one nanomole of labelled dNTP in 10 min at 37 degrees C into an acid precipitable DNA) rather than in cpm (counts per minute). The method is specific and easy to perform.

Avian Myeloblastosis Virus↗

HIV-1 ancestry primordial expansions of RRE and RRE related sequences.

The human T-lymphotropic retroviruses HTLV-I and HIV-1/-2 share a complementary patchwork homology in which the RRE regions of HIV-1 and -2 cover 78.1% of a 169 nucleotide (nt) region (PX1,2) positioned precisely between open reading frames PX I and PX II of ATLV (HTLV-I). The sequence character of the PX1,2 region is shown to be influenced by a primordial expansion, CTC2T, originating several hundred nucleotides upstream. A second primordial expansion, AGCU(U/C), is identified and shown to represent 52.1% of the HIV-1 RRE region. It is argued that prior to the present AIDS pandemic the efficiency of the rev receptor was enhanced by an ancestral recombination event.

Base Sequence↗

(+-)-carbocyclic 5'-nor-2'-deoxyguanosine and related purine derivatives: synthesis and antiviral properties.

Beginning with 3-cyclopenten-1-ylamine hydrochloride, the 5'-nor derivatives of carbocyclic 2'-deoxyguanosine (2), 2'-deoxyadenosine (3), and 2,6-diaminopurine 2'-deoxyribofuranoside (4) have been prepared. These compounds were evaluated for antiviral potential versus herpes simplex virus, varicella-zoster virus, cytomegalovirus, vaccinia virus, vesicular stomatitis virus, and human immunodeficiency virus and found to lack activity. Also, compounds 2-4 were virtually nontoxic toward the host (human diploid fibroblast ESM and HEL) cells. These biological properties may be due to the inability of 2-4 to be phosphorylated to the requisite nucleotide level that is likely to be necessary for biological activity by correlation to carbocyclic 2'-deoxyguanosine (1), which possesses significant antiviral properties as a result of conversion to its 5'-triphosphate derivative.

2-Aminopurine↗

HIV infection and primary resistance to antituberculosis drugs in Abidjan, Côte d'Ivoire.

OBJECTIVE: To determine the prevalence of Mycobacterium tuberculosis resistance to antituberculosis drugs, and to relate this resistance to HIV serologic status. DESIGN: Cross-sectional prevalence study. SETTING: The two major outpatient tuberculosis clinics in Abidjan, Côte d'Ivoire, West Africa. PATIENTS: Sixty individuals with newly diagnosed pulmonary tuberculosis and sputum smears positive for acid-fast bacilli. MAIN OUTCOME MEASURES: HIV serologic status and in vitro testing for susceptibility of M. tuberculosis isolates to antituberculosis drugs. RESULTS: M. tuberculosis was isolated from 82% (49 out of 60) of sputum specimens. Thirty-five per cent (17 out of 49) were obtained from HIV-seropositive and 65% (32 out of 49) from HIV-seronegative patients. There was no statistically significant difference in the proportion of resistant isolates from HIV-seropositive versus HIV-seronegative patients, although the relatively small sample size limited power. Of the total number of isolates, 17% were resistant to isoniazid; resistance was less to streptomycin (7%), rifampin (2%), pyrazinamide (0%), and ethambutol (0%). Eighteen and 21% of mycobacterial isolates from HIV-seropositive and HIV-seronegative individuals, respectively, were resistant to one or more of these drugs. CONCLUSIONS: Surveys of this type are useful in planning and evaluating tuberculosis preventive therapy in individuals with dual infection.

AIDS-Related Opportunistic Infections↗

Prevalence of hepatitis B and C and HIV antibodies in children in a Romanian orphanage.

Two hundred and fifteen children in an orphanage in Romania were examined for serum markers of present or past hepatitis B and C virus and HIV infection. In total, 183 children (85.1%) had at least one marker (HBsAg, anti-HBs or anti-HBc) of hepatitis B virus infection. An HBsAg carrier state was diagnosed in 38 (20.8%) of the infected children. Among the carriers 24.3% were HBeAg carriers, 51.4% had anti-HBe and 24.3% had neither HBe antigen nor antibody. Nine children (4.2%) had antibodies to hepatitis C virus. All sera were negative in tests for HIV antibodies. False-positive reactions represented a considerable problem with these sera. Six percent of the sera gave false-positive reactions in indirect ELISA tests for hepatitis C and HIV. Sera giving false-positive reactions had rather high serum IgG levels. The results of this study indicate that these children have been heavily exposed to hepatitis B virus and to a certain degree to hepatitis C virus, while there were no cases of HIV infection in this orphanage.

Child↗

Selective inhibition of human immunodeficiency viruses by racemates and enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine.

2',3'-Dideoxy-5-fluoro-3'-thiacytidine (FTC) has been shown to be a potent and selective compound against human immunodeficiency virus type 1 in acutely infected primary human lymphocytes. FTC is also active against human immunodeficiency virus type 2, simian immunodeficiency virus, and feline immunodeficiency virus in various cell culture systems, including human monocytes. The antiviral activity can be prevented by 2'-deoxycytidine, but not by other natural nucleosides, suggesting that FTC must be phosphorylated to be active and 2'-deoxycytidine kinase is responsible for the phosphorylation. By using chiral columns or enzymatic techniques, the two enantiomers of FTC were separated. The (-)-beta-enantiomer of FTC was about 20-fold more potent than the (+)-beta-enantiomer against human immunodeficiency virus type 1 in peripheral blood mononuclear cells and was also effective in thymidine kinase-deficient CEM cells. Racemic FTC and its enantiomers were nontoxic to human lymphocytes and other cell lines at concentrations of up to 100 microM. Studies with human bone marrow cells indicated that racemic FTC and its (-)-enantiomer had a median inhibitory concentration of > 30 microM. The (+)-enantiomer was significantly more toxic than the (-)-enantiomer to myeloid progenitor cells. The susceptibilities to FTC of pretherapy isolates in comparison with those of posttherapy 3'-azido-3'-deoxythymidine-resistant viruses in human lymphocytes were not substantially different. Similar results were obtained with well-defined 2',3'-dideoxyinosine- and nevirapine-resistant viruses. (-)-FTC-5'-triphosphate competitively inhibited human immunodeficiency virus type 1 reverse transcriptase, with an inhibition constant of 2.9 microM, when a poly(I)n.oligo(dC)19-24 template primer was used. These results suggest that further development of the (-)-Beta-enantiomer of FTC is warranted as an antiviral agent for infections caused by human immunodeficiency viruses.

Antiviral Agents↗

Risk of HIV infection from transfusion with blood negative for HIV antibody in a west African city.

OBJECTIVE: To estimate the risk of infection with HIV (HIV 1 or HIV 2, or both) from transfusion of a screened unit of blood in a high prevalence area in west Africa. DESIGN: Retrospective cohort study for January-July 1991. SETTING: National Blood Transfusion Centre, Abidjan, Côte d'Ivoire. SUBJECTS: Repeat donors (5831 units of blood) and first time donors (5076 units) in the first five months of 1991. MAIN OUTCOME MEASURES: Prevalence and estimated incidence of HIV infection in repeat and first time donors; estimated rate of potentially infected, HIV antibody negative units; and rate of (false negative) potentially infected units assuming a laboratory test sensitivity of 99%. RESULTS: Overall HIV prevalence was 11.0% in first time donors and 2.1% in repeat donors. In the first seven months of 1991, 29 HIV antibody positive (27 HIV 1, 1 HIV 2, 1 dually reactive) donors with a seronegative unit of blood earlier in the year were identified; 26 had donated blood eight weeks or less before their estimated dates of seroconversion and may have been infectious (minimum rate 26/5831 (4.5/1000 potentially infected units)). Estimated incidence of infection in repeat donors was 1.2-2.5%. Laboratory test insensitivity would result in an estimated 1.1/1000 false negative units from first time donors and 0.2/1000 units from regular donors. The overall rate of potentially infected units (all donors, seroconversions, and errors) was estimated at 5.4-10.6/1000. CONCLUSIONS: The risk of HIV infection from a single unit of blood remains substantial (5.4-10.6/1000 units). To prevent infection from blood transfusion in areas of high incidence and prevalence of HIV all but absolutely essential transfusions should be avoided, and donors with low incidence of HIV infection should be selected.

Blood Donors↗

Recombinant antigens in viral diagnosis.

DNA fragments coding for a variety of different viral antigens have been cloned and expressed in Escherichia coli. Selected purified recombinant antigens were used for detection of specific antibodies by means of the ELISA technique. This approach has been used for the development of four different ELISAs for the detection of HIV- and EBV-specific antibodies.

Antibodies, Viral↗

[Follow-up of blood donors with indeterminate reaction to anti-HIV 1/2 EIA or isolated detection of antibodies to HIV-1 core antigens (anti-p24)].

Over a period of 20 months we identified 10 voluntary blood donors with indeterminate reactions in anti-HIV 1/2 EIA tests. Western blot (WB) patterns of 6 of these 10 donors showed isolated core antibodies (anti-p24), in 4 cases WB patterns were negative. After a median follow-up period of 58 weeks (range 12-93) we have not seen a case of typical seroconversion.

AIDS Serodiagnosis↗

[Analysis of new criteria for increasing the specificity of commercial Western blots].

We examined the frequency of serum cross-reactivity on Western blot for HIV1 and HIV2. 661 patients with tuberculosis in Abidjan, and 4,899 asymptomatic persons for HIV1 and HIV2 infections were tested. All specimens positive on ELISA for HIV1 or HIV2 were further characterized by synthetic peptide based tests. Confirmed positive samples were tested by HIV1 and HIV2 specific Western blot criteres utilisis. Dual serologic reactivity on synthetic peptide tests was significantly more frequent in HIV positive patients with tuberculosis than asymptomatic subjects. Positive HIV1 Western blots were seen in 61%-86% of specimens positive for HIV2 only on synthetic peptide tests. [Cross-reactivity, to HIV2 Western blots by HIV1 positive specimens was significantly more frequent in patients with tuberculosis than in asymptomatic subjects.] Using recently recommended criteria for HIV1 and HIV2 Western blot interpretation (presence of 2 env bands) reduced the overall proportion of HIV1 positive specimens having a positive HIV2 Western blot from 39% to 14% and HIV2 positive specimens having a positive HIV1 Western blot from 31% to 8%.

Acquired Immunodeficiency Syndrome↗