The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4 ؉ and CD8 ؉ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at ؊70°C for <3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at ؊70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.

Eleven laboratories evaluated the use of dried blood and plasma spots for quantitation of human immunodeficiency virus (HIV) RNA by two commercially available RNA assays, the Roche Amplicor HIV-1 Monitor and the bioMerieux NucliSens HIV-1 QT assays. The recovery of HIV RNA was linear over a dynamic range extending from 4,000 to 500,000 HIV type 1 RNA copies/ml. The Monitor assay appeared to have a broader dynamic range and seemed more sensitive at lower concentrations. However, the NucliSens assay gave more consistent results and could be performed without modification of the kit. HIV RNA was stable in dried whole blood or plasma stored at room temperature or at ؊70°C for up to 1 year. Dried blood and dried plasma spots can be used as an easy and inexpensive means for the collection and storage of specimens under field conditions for the diagnosis of HIV infection and the monitoring of antiretroviral therapy.Human immunodeficiency virus (HIV) RNA levels in blood plasma are used to monitor the response to antiretroviral drug therapy in developed countries. Since the International AIDS Conference held in Durban, South Africa, in 2000, there has been a global effort to provide access to antiretroviral drugs to all infected individuals, including those in the resource-poor countries which have been hit the hardest by the AIDS pandemic. Studies have demonstrated that the provision of treatment is not enough, however. In order for the drugs to be most effective, both for an individual and for public health reasons, it will be necessary to monitor the responses to the therapy. HIV RNA assays typically use plasma, which implies access to laboratory equipment that may not be readily available in all field settings. Therefore, alternative specimens must be considered for use in these situations.Whole blood dried on filter paper (dried blood spots [DBSs]) has been used to qualitatively detect HIV antibodies (13, 15), HIV DNA (1, 3, 5-7, 9, 10, 11, 12, 16, 18) and HIV RNA (4, 14, 17; J. L. Gerstel and A. M. Comeau, Proc. 10th Natl. Neonatal Screening Symp., abstr. P-19, p. 64, 1994). In addition, dried plasma spots (DPSs) have also been used to quantitate the HIV load (8). Although the stabilities of antibodies and DNA in DBSs have been determined (2, 6, 13), there has been some question regarding the stability of the HIV RNA when it is dried and stored at room temperature (14, 17).The purpose of this study was to compare two different methods for quantitatively measuring HIV RNA in DBSs and DPSs and to assess their long-term stability at room temperature and at Ϫ70C.(This study was presented in part at the 12th World AIDS Conference, Geneva, Switzerland, 28 June to 3 July 1998 [S. Cassol, A. Comeau, S. Fiscus, G. Aldrovandi, J. Sullivan, J. Bremer, and B. Jackson, Abstr. 12th World AIDS Conf., abstr. 33166, 1998].) MATERIALS AND METHODS Specimens.Three different panels were prepared by the Virus Quality Assurance Laboratory by using filter paper (903; Schleicher & Schuell, Keene, N.H.) spotted with 50 l of plasma or whol...

Background The safety and efficacy of adding antiretroviral drugs to standard zidovudine prophylaxis in infants of mothers with human immunodeficiency virus (HIV) infection who did not receive antenatal antiretroviral therapy (ART) because of late identification are unclear. We evaluated three ART regimens in such infants. Methods Within 48 hours after their birth, we randomly assigned formula-fed infants born to women with a peripartum diagnosis of HIV type 1 (HIV-1) infection to one of three regimens: zidovudine for 6 weeks (zidovudine-alone group), zidovudine for 6 weeks plus three doses of nevirapine during the first 8 days of life (two-drug group), or zidovudine for 6 weeks plus nelfinavir and lamivudine for 2 weeks (three-drug group). The primary outcome was HIV-1 infection at 3 months in infants uninfected at birth. Results A total of 1684 infants were enrolled in the Americas and South Africa (566 in the zidovudine-alone group, 562 in the two-drug group, and 556 in the three-drug group). The overall rate of in utero transmission of HIV-1 on the basis of Kaplan–Meier estimates was 5.7% (93 infants), with no significant differences among the groups. Intra-partum transmission occurred in 24 infants in the zidovudine-alone group (4.8%; 95% confidence interval [CI], 3.2 to 7.1), as compared with 11 infants in the two-drug group (2.2%; 95% CI, 1.2 to 3.9; P = 0.046) and 12 in the three-drug group (2.4%; 95% CI, 1.4 to 4.3; P = 0.046). The overall transmission rate was 8.5% (140 infants), with an increased rate in the zidovudine-alone group (P = 0.03 for the comparisons with the two- and three-drug groups). On multivariate analysis, zidovudine monotherapy, a higher maternal viral load, and maternal use of illegal substances were significantly associated with transmission. The rate of neutropenia was significantly increased in the three-drug group (P<0.001 for both comparisons with the other groups). Conclusions In neonates whose mothers did not receive ART during pregnancy, prophylaxis with a two- or three-drug ART regimen is superior to zidovudine alone for the prevention of intrapartum HIV transmission; the two-drug regimen has less toxicity than the three-drug regimen. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development [NICHD] and others; ClinicalTrials.gov number, NCT00099359.)

Perinatal human immunodeficiency virus type 1 (HIV-1) transmission is characterized by acquisition of a homogeneous viral quasispecies, yet the selective factors responsible for this genetic bottleneck are unclear. We examined the role of maternal autologous neutralizing antibody (aNAB) in selective transmission of HIV-1 escape variants to infants. Maternal sera from 38 infected mothers at the time of delivery were assayed for autologous neutralizing antibody activity against maternal time-of-delivery HIV-1 isolates in vitro. Maternal sera were also tested for cross-neutralization of infected-infant-first-positive-time-point viral isolates. Heteroduplex and DNA sequence analyses were then performed to identify the initial infecting virus as a neutralization-sensitive or escape HIV-1 variant. In utero transmitters (n ‫؍‬ 14) were significantly less likely to have aNAB to their own HIV-1 strains at delivery than nontransmitting mothers (n ‫؍‬ 17, 14.3% versus 76.5%, P ‫؍‬ 0.003). Cross-neutralization assays of infected-infant-first-positive-time-point HIV-1 isolates indicated that while 14/21 HIV-1-infected infant first positive time point isolates were resistant to their own mother's aNAB, no infant isolate was inherently resistant to antibody neutralization by all sera tested. Furthermore, both heteroduplex (n ‫؍‬ 21) and phylogenetic (n ‫؍‬ 9) analyses showed that selective perinatal transmission and/or outgrowth of maternal autologous neutralization escape HIV-1 variants occurs in utero and intrapartum. These data indicate that maternal autologous neutralizing antibody can exert powerful protective and selective effects in perinatal HIV-1 transmission and therefore has important implications for vaccine development.

No abstract