Biomedical subjects

S Pahwa

Publications and source records attributed to S Pahwa.

At least 19 recordsLinked to original sources

PubMed2001

Human immunodeficiency virus gag and pol-specific CD8 T cells in perinatal HIV infection.

BACKGROUND: Binding of fluorochrome-conjugated MHC class I tetramers is a powerful means to detect antigen-specific CD8 T lymphocytes. In human immunodeficiency virus (HIV) infection, cellular immune response is essential in curtailing HIV disease progression but gaps persist in our understanding of HIV-specific cells during the disease course. In this study, we evaluated tetramer binding HIV-specific CD8 T cells in HIV-infected children. METHODS: Fluorescently labeled tetramers for HIV gag and pol were utilized to quantify antigen-specific cells by flow cytometry using a whole blood labeling method in a cohort of 19 HLA-A2+ HIV-infected children (age range 1 month to 17 years). RESULTS: Fourteen children had detectable gag (median 0.4%) and pol (median 0.1%) binding CD8 T cells, three children had gag binding cells only, and two had neither. Numbers of gag and pol binding cells correlated with each other and each correlated independently with total CD8 T cells and total CD4 T cells. CONCLUSIONS: HIV gag and pol-specific CD8 T cells are maintained during the chronic phase of HIV infection in children and CD4 lymphocytes appear to be important for sustaining their levels.

Adolescent↗

PubMed2001

The HIV protease inhibitor Indinavir inhibits cell-cycle progression in vitro in lymphocytes of HIV-infected and uninfected individuals.

Indinavir (IDV) is a potent and selective human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) widely used in antiretroviral therapy for suppression of HIV, but its effects on the immune system are relatively unknown. Recently, it has been reported that PIs inhibit lymphocyte apoptosis. In the present study we have investigated the effects of ex vivo addition of IDV on lymphocyte activation and apoptosis in cells from HIV-infected children (n = 18) and from healthy uninfected individuals (controls, n = 5) as well as in Jurkat and PM1 T-cell lines. Pretreatment of control peripheral blood mononuclear cell (PBMC) cultures with IDV resulted in a dose-dependent inhibition of lymphoproliferative responses to different activation stimuli. Additionally, this treatment led to cell-cycle arrest in G0/G1 phase in anti-CD3 monoclonal antibody-stimulated PBMC cultures in controls and in 15 of 18 HIV-infected children. Spontaneous- or activation-induced apoptosis of PBMCs from HIV-infected or uninfected individuals or of Fas-induced apoptosis in Jurkat and PM1 T cell lines were not inhibited by IDV. Moreover, IDV did not inhibit activation of caspases-1, -3, -4, -5, -9, and -8 in lysates of Jurkat T cells undergoing Fas-induced apoptosis. The findings indicate that IDV interferes with cell-cycle progression in primary cells but does not directly affect apoptosis. It is concluded that IDV may prolong cell survival indirectly by inhibiting their entry into cell cycle. In individuals on PI therapy, PI-mediated effects could potentially modulate immunologic responses independently of antiviral activity against HIV.

Adolescent↗

PubMed2001

Immunophenotypic analysis of HIV-infected children: alterations within the first year of life, changes with disease progression, and longitudinal analyses of lymphocyte subsets.

Perinatal infection with human immunodeficiency virus (HIV) results in tremendous activation of the pediatric immune system. An important component of understanding the pathogenesis of this disease is to characterize and quantify antigenic indicators of activation within the peripheral lymphocyte population. We measured T-lymphocyte activation and maturation antigens in a cohort of 112 HIV-infected children treated with antiretroviral therapy according to the current standard of care. Changes in expression of CD95, HLA-DR, and CD45RO were evident in 22 HIV-infected children younger than 1 year of age. A comparison of phenotypic profiles of children in mild, moderate, and severe immune categories revealed perturbations of CD28, CD38, CD45RA, CD45RO, CD95, and HLA-DR. Finally, a novel analysis of 56 HIV-infected children based on the repeated collection of data over time (median of seven observations over 33 months) demonstrated a strong negative correlation between the percentage CD4 and the percentage of CD45RO, CD95, and HLA-DR on both CD4 and CD8 cells. Our data implicate persistent immune activation, beginning within the first year of life, as a major driving force in the pathogenesis of perinatally acquired HIV disease.

ADP-ribosyl Cyclase↗

PubMed2001

Evaluation of T cell receptor gene rearrangement excision circles after antiretroviral therapy in children infected with human immunodeficiency virus.

Immune reconstitution after antiretroviral therapy in human immunodeficiency virus (HIV)infected patients may result from the recovery of thymus function, peripheral redistribution, or decreased T cell destruction. This study investigated levels of T cell receptor gene rearrangement excision circles (TRECs) as a measure of recent thymic emigrant cells in peripheral blood lymphocytes of 50 HIV-infected infants and children who were followed-up for 40 months after the start or change of antiretroviral therapy. At baseline, patients exhibited fewer TRECs than did uninfected control subjects. The increase in TRECs after antiretroviral therapy was greater in infants than in older HIV-infected children. Of interest, patients who demonstrated discordant responses (i.e., increased CD4 T cell counts without significant virologic suppression) also had substantial gains in TRECs. Furthermore, TRECs correlated positively with the number of CD4 and naive T cells and negatively with age and virus load. Measurement of TRECs may serve as a useful tool for evaluating immune reconstitution in HIV-infected children receiving antiretroviral therapy.

Adolescent↗

PubMed2001

Possible linkage of amprenavir with intracranial bleeding in an HIV-infected hemophiliac.

The use of protease Inhibitors (PI) has been associated with many adverse effects including increased tendency to bleed, which is particularly problematic in individuals with congenital coagulation disorders. We report the occurrence of spontaneous intracranial bleeding in an human immunodeficiency virus (HIV)-infected adolescent with hemophilia A who was receiving amprenavir (APV). The bleeding resolved on discontinuation of APV. This case report highlights a need for awareness of increased bleeding as a potentially serious complication associated with the use of all currently licensed PIs in individuals with hemophilia.

Adult↗

PubMed2001

Apoptosis in T-lymphocyte subsets in human immunodeficiency virus-infected children measured immediately ex vivo and following in vitro activation.

Phosphatidylserine molecules are translocated to the outer plasma membrane of lymphocytes undergoing apoptosis and can be detected by the binding of fluorochrome-conjugated annexin V. Using the annexin V assay, we examined CD4 and CD8 T cells from human immunodeficiency virus (HIV)-infected children for apoptosis upon isolation or following in vitro culture. Immediate ex vivo analysis or overnight culture showed significantly higher levels of apoptosis in CD8 cells than in CD4 cells. Following culture with the activating stimulus phytohemagglutinin or anti-CD3 monoclonal antibody, we observed an increase in the percentage of apoptotic CD4 cells, whereas there was no change in the rate of CD8 cell death. These results demonstrate that in HIV-infected children, CD8 apoptosis may occur at a greater rate than CD4 apoptosis in vivo; greater CD4 depletion may be observed due to more efficient mechanisms for peripheral lymphocyte replacement in the CD8 compartment. Furthermore, our data suggest that CD8 lymphocytes may be maximally activated in vivo, a condition which may lead to the exhaustion of CD8-mediated immunity. These findings clarify the differences between the CD4 and CD8 apoptotic responses to HIV.

Annexin A5↗

PubMed2000

Patterns of CD8 T cell clonal dominance in response to change in antiretroviral therapy in HIV-infected children.

OBJECTIVE: To examine the influence of change in antiretroviral therapy (ART) on patterns of CD8 T cell clonal dominance in HIV-infected children. DESIGN: Seventeen HIV-infected children with plasma virus loads between 3.1 and 5.7 log10 were investigated before and after changes in ART. METHODS: CDR3 spectratyping was performed in 22 T cell receptor (TCR) Vbeta subfamilies by multiplex polymerase chain reaction (PCR) in purified peripheral blood CD8 T cells in conjunction with CD4 cell counts, plasma HIV-RNA copies and lymphoproliferative assays (LPA). RESULTS: CD8 T cell clonal dominance in two or more Vbeta families was present in eight out of 17 children. After a change in therapy, 13 patients (76%) acquired new clones whereas three patients (17.6%) showed a loss in CD8 cell clones. An increase in the numbers of dominant clones correlated with an increase in percentage CD4 cell counts (P < 0.001) and with improved LPA responses to tetanus (P < 0.05) and alloantigens (P < 0.01). CD4 cell increase was associated with an initial mean gain of 3.1+/-2.1 CD8 cell clones, independent of a virological response. A loss of CD8 cell clones or failure to achieve CD4 T cell increase was associated with failure to achieve virological suppression. CONCLUSION: Children with chronic HIV infection manifest CD8 T cell clonal dominance, which appears to be dependent upon the adequacy of the CD4 cells. With optimization of therapy, a gain in clonal dominance is the predominant response, except in situations of failure to contain viral replication.

Adolescent↗

PubMed2000

CD4 T lymphocytes are primed to express Fas ligand by CD4 cross-linking and to contribute to CD8 T-cell apoptosis via Fas/FasL death signaling pathway.

CD4 molecules serve as coreceptors for the T-cell receptor (TCR)/CD3 complex that are engaged coordinately with TCR and facilitate antigen-specific T-cell activation leading to interleukin 2 (IL-2) production and proliferation. However, cross-ligation of CD4 molecules prior to TCR stimulation has been shown to prime CD4 T cells to undergo apoptosis. Although in vivo and in vitro experiments have implicated the involvement of Fas/FasL interaction in this CD4 cross-linking (CD4XL)-induced apoptosis, detailed mechanisms to account for cell death induction have not been elucidated. In the present study, we demonstrate that CD4XL in purified T cells not only led to Fas up-regulation but also primed CD4 T cells to express FasL upon CD3 stimulation and rendered the T cells susceptible to Fas-mediated apoptosis. Notably, in addition to CD4(+) T cells, CD4XL-induced sensitization for apoptosis was observed in CD8(+) T cells as well and was associated with Bcl-x down-modulation. Both CD4 and CD8 T-cell subsets underwent apoptosis following cell-cell contact with FasL(+) CD4 T cells. CD28 costimulation abrogated CD4XL/CD3-induced apoptosis with restoration of IL-2 production and prevented Bcl-x down-modulation. As CD4 molecules are the primary receptors for human immunodeficiency virus 1 (HIV-1), we conclude that HIV-1 envelope mediated CD4XL can lead to the generation of FasL-expressing CD4(+) T cells that can lead to apoptosis of CD4 as well as CD8 T cells. These findings implicate a novel mechanism for CD8 T-cell depletion in HIV disease.

Antigens, CD↗

PubMed2000

Correlation of loss of CD4 T cells with plasma levels of both soluble form Fas (CD95) Fas ligand (FasL) in HIV-infected infants.

Fas and Fas ligand (FasL), members of the TNFR and TNF families of molecules involved in apoptosis, respectively, are expressed in membrane-associated as well as soluble forms. Soluble Fas (sFas) and sFasL were evaluated in sequential samples from 16 HIV-infected and 11 HIV-exposed uninfected infants at ages 0-13 months. Regardless of the state of infection, age-dependent decreases in peripheral CD4 T cell counts and increases in sFas and sFasL were noted. However, decreases of the percentage CD4 T cells were more prominent in HIV-infected infants, and this was correlated significantly with increased plasma levels of sFas and sFasL (P = 0.002 and 0.004, respectively). Moreover, the levels of sFas in HIV-infected infants were found to be directly correlated with plasma HIV RNA (P = 0.03) and were significantly increased as early as age <1 month and prior to the onset of CD4 T cell decline. In uninfected infants, there was no such correlation between CD4 counts and the levels of sFas/sFasL. Plasma levels of sFas and sFasL may thus be important indicators of disease progression in perinatal HIV infection.

Age Factors↗

PubMed2000

Alterations in apoptosis of cord and adult peripheral blood mononuclear cells induced by in vitro infection with respiratory syncytial virus.

Respiratory syncytial virus (RSV), a major cause of morbidity in children, results in severe lower respiratory tract infections. With an in vitro infection system of isolated cord or adult peripheral blood mononuclear cells, addition of virus to cell cultures resulted in significant reductions in cell deaths, as measured by 2 independent assays: quantitation of cells with subdiploid levels of DNA and cells with DNA strand breaks. Decreased cell death was observed in lymphocytes and monocytes of cord and adult samples, with more dramatic effects evident in cells from cord blood. This may be linked to the increased virulence observed in infants with RSV infection. These data suggest that RSV may be equipped with some mechanism to prevent apoptosis, which is a major component of the host defense system used to eliminate virally infected cells.

Adult↗

PubMed1999

Chemokine pattern in relation to disease state in human immunodeficiency virus-infected children.

Although beta chemokines can block human immunodeficiency virus (HIV) entry into target cells, their role in HIV disease progression is controversial. To determine the association of RANTES with HIV disease state, we examined constitutive mRNA expression by reverse-transcribed polymerase chain reaction (RT-PCR) in peripheral blood mononuclear cells (PBMCs) and induction of RANTES secretion by enzyme-linked immunosorbent assay (ELISA) in anti-CD3 monoclonal antibody (MAb)-stimulated cultures of PBMCs, and in CD4+ and CD8+ T cell subsets of 17 HIV-infected children. In comparison with uninfected subjects, PBMCs of HIV-infected children were deficient in both constitutive RANTES mRNA expression as well as in stimulus-induced RANTES production. Children in clinical category C were found to be more deficient than children in clinical category A. Expression of RANTES mRNA in PMBCs was inversely correlated with plasma virus load and correlated directly with CD4+ T cell counts. In T cell subsets, RANTES production was equivalent between CD4+ and CD8+ T cells in patients and controls but CD8+ T cells of children in clinical category A produced higher RANTES levels than those of children in clinical category C. The beta-chemokine RANTES may play an important role in slowing clinical disease progression in HIV-infected children.

Adolescent↗

PubMed1999

CD4-mediated signals induce T cell dysfunction in vivo.

Triggering of CD4 coreceptors on both human and murine T cells can suppress TCR/CD3-induced secretion of IL-2. We show here that pretreatment of murine CD4+ T cells with the CD4-specific mAb YTS177 inhibits the CD3-mediated activation of the IL-2 promoter factors NF-AT and AP-1. Ligation of CD4 molecules on T cells leads to a transient stimulation of extracellular signal-regulated kinase (Erk) 2, but not c-Jun N-terminal kinase (JNK) activity. Pretreatment with anti-CD4 mAb impaired anti-CD3-induced Erk2 activation. Costimulation with anti-CD28 overcame the inhibitory effect of anti-CD4 Abs, by induction of JNK activation. The in vivo relevance of these studies was demonstrated by the observation that CD4+ T cells from BALB/c mice injected with nondepleting anti-CD4 mAb were inhibited in their ability to respond to OVA Ag-induced proliferation and IL-2 secretion. Interestingly, in vivo stimulation with anti-CD28 mAb restored IL-2 secretion. Furthermore, animals pretreated with anti-CD4 elicited enhanced IL-4 secretion induced by OVA and CD28. These observations suggest that CD4-specific Abs can inhibit T cell activation by interfering with signal 1 transduced through the TCR, but potentiate those delivered through the costimulatory molecule CD28. These studies have relevance to understanding the mechanism of tolerance induced by nondepleting anti-CD4 mAb used in animal models for allograft studies, autoimmune pathologies, and for immunosuppressive therapies in humans.

Animals↗

PubMed1999

Clonal dominance patterns of CD8 T cells in relation to disease progression in HIV-infected children.

CD8 T cells are important mediators of cellular immune responses as evidenced by clonal expansions in the CD8 TCR V beta repertoire during primary HIV infection in adults. This study investigated the CD8 TCR V beta repertoire by complementarity-determining region 3 length analysis using multiplex PCR in purified peripheral blood CD8 T cells of 22 HIV-infected children (age range was 0.75-15 yr, mean was 8.2 +/- 4.1 yr). Evidence of clonal dominance in one or more V beta families was obtained in 15 of 22 children. The patterns of clonal dominance were designated as major, minor, single, and none to indicate the involvement of three or more, two, one, or no V beta families, respectively. A pattern of major or minor clonal dominance was observed in 12 children (group 1), whereas 10 children had single or no clonal dominance (group 2). In comparison with group 2, the children in group 1 had a higher percentage of CD4 cells (28.3 +/- 11.6 vs 8.6 +/- 4.8, p < 0.001); a higher stimulation index in lymphoproliferative responses to Candida (92.0 +/- 59.5 vs 12.3 +/- 14.4, p = 0.002), tetanus (76.3 +/- 51.2 vs 11.2 +/- 12.7, p = 0.002), and alloantigens (178.3 +/- 298.9 vs 32.9 +/- 35.2, p < 0.001); and a lower percentage of CD8+HLA-DR+CD38+ cells (37.4 +/- 13.1 vs 54.6 +/- 14.2, p < 0.01). The number of dominant CD8 T cell clones was significantly correlated with the percentage of CD4 T cells (r = 0.669, p < 0.001) but not with plasma HIV RNA. Compared with group 1, patients in group 2 had a 4.8 times greater probability of having < 15% CD4 cells. These findings indicate that CD8 clonal dominance in HIV-infected children reflects robustness of immune responses, regardless of time since infection and virus load.

Adolescent↗

PubMed1999

Serum HIV-1 p24 antibody, HIV-1 RNA copy number and CD4 lymphocyte percentage are independently associated with risk of mortality in HIV-1-infected children. National Institute of Child Health and Human Development Intravenous Immunoglobulin Clinical Trial Study Group.

OBJECTIVE: The role of HIV-1 antibody in modulating disease progression must be assessed in the context of other immune and viral load markers. We evaluated the association between HIV-1 p24 antibody, HIV-1 RNA, immune complex-dissociated (ICD) p24 antigen, CD4 cell percentage, and mortality in a cohort of 218 HIV-infected children enrolled in a trial of intravenous immunoglobulin prophylaxis of bacterial infections. METHODS: CD4 cell percentage was measured and sera collected and stored at baseline and every 3 months on study (1988-1991). Stored sera were assayed for HIV-1 p24 antibody, HIV-1 RNA, and ICD p24 antigen. Mortality was recorded during the trial and updated through 1996 (mean total follow-up, 6.3 years). RESULTS: Eighty-one (37%) children died; probability of mortality for children with baseline HIV-1 p24 antibody concentrations of undetectable (< 1), 1-4, 5-124, and > or = 125 reciprocal titer units (RTU) was 61, 50, 24, and 10%, respectively. A 3.5-fold increase in the relative risk (RR) of death [95% confidence interval (CI), 2.2-5.5] was observed among children with baseline HIV-1 p24 antibody concentration < 5 RTU compared with > or = 5 RTU. In multivariate analyses, p24 antibody, HIV-1 RNA, and CD4 cell percentage but not ICD p24 antigen were independently associated with mortality; the RR of death increased by 1.7 (95% CI, 1.3-2.1) for each log10 decrement in baseline HIV-1 p24 antibody. CONCLUSIONS: HIV-1 p24 antibody, HIV-1 RNA and CD4 cell percentage independently predict mortality amongst infected children. Whereas CD4 cell percentage provides an estimate of the general degree of immune suppression, HIV-1 p24 antibody could provide an easily obtained, inexpensive assessment of CD4 cell function and could augment prognostic information provided by CD4 cell count and viral load for clinical management of infected children.

CD4 Lymphocyte Count↗

PubMed1999

Virologic and immunologic response to nucleoside reverse-transcriptase inhibitor therapy among human immunodeficiency virus-infected infants and children.

Plasma human immunodeficiency virus RNA and CD4 lymphocyte response to nucleoside reverse-transcriptase therapy were evaluated in a large, comparative pediatric trial. Both baseline values and changes in the two laboratory markers over time correlated well with clinical outcome and possessed independent predictive value. In comparison of RNA reduction from baseline between the dideoxyinosine (ddI) and zidovudine+ddI therapeutic arms, marginal superiority of the combination arm was not correlated with an observed clinical benefit. Despite the size of this trial and the significantly higher rate of clinical end points in the zidovudine monotherapy group, attempts to establish surrogacy for plasma RNA were difficult. Nevertheless, plasma RNA and CD4 lymphocyte count together possess strong clinical predictive power and are valuable tools for both the clinician and the evaluation of new therapies.

Adolescent↗

PubMed1999

Serum vitamin A concentrations in a North American cohort of human immunodeficiency virus type 1-infected children. National Institute of Child Health and Human Development Intravenous Immunoglobulin Clinical Trial Study Group.

BACKGROUND: Vitamin A deficiency is associated with increased risks of vertical transmission of HIV-1 (HIV) and of disease progression and mortality among HIV-infected adults. The objectives of the study were to describe serum vitamin A concentrations among HIV-infected children in the National Institute of Child Health and Human Development IVIG Clinical Trial, to examine changes in vitamin A concentrations and to investigate the relationships between vitamin A concentrations and morbidity and mortality. METHODS: Blood was collected from children at baseline and at 3-month intervals throughout the study. Serum samples were stored at -70 degrees C at a central repository until retrieved for vitamin A assay. Samples were hexane-extracted and assayed by high performance liquid chromatography. The rate of change in vitamin A concentrations, calculated by fitting a linear regression model, was expressed as micrograms/dl/year. RESULTS: The median vitamin A concentration at baseline (n = 207 children) was 31.0 microg/dl [range, undetectable (< 10 microg/dl) to 98 microg/dl]. The rate of change in vitamin A concentrations (n = 180 children) did not vary significantly by any factor other than baseline vitamin A concentration. Baseline vitamin A concentration was not associated with morbidity (incidence of infections, growth failure, CD4+ percent decline below 15%, increases in serum HIV RNA concentrations above either 10(5) or 10(6) copies/ml or acute care hospitalization). Neither baseline vitamin A concentration nor the rate of change of vitamin A concentrations was associated with mortality. CONCLUSIONS: Among these North American children with relatively normal vitamin A concentrations, vitamin A was not observed to be associated with morbidity or mortality.

AIDS-Related Opportunistic Infections↗

PubMed1999

Predictive value of CD19 measurements for bacterial infections in children infected with human immunodeficiency virus.

We investigated the predictive value of CD19 cell percentages (CD19%) for times to bacterial infections, using data from six pediatric AIDS Clinical Trials Group protocols and adjusting for other potentially prognostic variables, such as CD4%, CD8%, immunoglobulin (IgA) level, lymphocyte count, prior infections, prior zidovudine treatment, and age. In addition, we explored the combined effects of CD19% and IgG level in predicting time to infection. We found that a low CD19% is associated with a nonsignificant 1.2-fold increase in hazard of bacterial infection (95% confidence interval: 0.97, 1.49). In contrast, a high IgG level is associated with a nonsignificant 0.87-fold decrease in hazard of infection (95% confidence interval: 0.68, 1.12). CD4% was more prognostic of time to bacterial infection than CD19% or IgG level. Low CD19% and high IgG levels together lead to a significant (P < 0. 01) 0.50-fold decrease in hazard (95% confidence interval: 0.35, 0. 73) relative to low CD19% and low IgG levels. Similarly, in a model involving assay result changes (from baseline to 6 months) as well as baseline values, the effect of CD19% by itself is reversed from its effect in conjunction with IgG. In this model, CD19% that are increasing and high are associated with decreases in hazard of infection (P < 0.01), while increasing CD19% and increasing IgG levels are associated with significant (at the P = 0.01 level) fourfold increases in hazard of infection relative to stable CD19% and decreasing, stable, or increasing IgG levels. Our data suggest that CD19%, in conjunction with IgG level, provides a useful prognostic tool for bacterial infections. It is highly likely that T-helper function impacts on B-cell function; thus, inclusion of CD4% in such analyses may greatly enhance the assessment of risk for bacterial infection.

AIDS-Related Opportunistic Infections↗

PubMed1999

Genotyping of the CCR5 chemokine receptor by isothermal NASBA amplification and differential probe hybridization.

The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectability of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.

Alleles↗