Evaluating the performance of the GeneXpert HIV-1 qualitative assay as a consecutive test for a new early infant diagnosis algorithm in South Africa.

BACKGROUND: The proportion of HIV-exposed infants and young children infected with HIV in South Africa (SA) has declined markedly over the past decade as a result of the country's comprehensive prevention of mother-to-child transmission programme. This decrease has in turn reduced the positive predictive value (PPV) of diagnostic assays, necessitating review of early infant diagnosis (EID) algorithms to ensure improved accuracy. OBJECTIVES: To evaluate the performance of the GeneXpert HIV-1 qualitative assay (Xpert EID) as a consecutive test for infants with an 'HIV-detected' polymerase chain reaction screening test at birth. METHODS: We retrospectively analysed a longitudinal cohort of HIV-exposed infants on whom birth testing was performed, using whole-blood ethylenediaminetetra-acetic acid samples, from four tertiary sites in Gauteng Province between June 2014 and December 2019. Birth samples from all infants with a Cobas AmpliPrep/Cobas TaqMan HIV-1 Qualitative Test v2.0 (CAP/CTM v2.0) HIV-detected screening test, a concurrent Xpert EID test and a subsequent confirmatory CAP/CTM v2.0 test on a separate specimen were included. Performance of the Xpert EID in predicting final HIV status was determined as proportions with 95% confidence intervals (CIs). A comparison of indeterminate CAP/CTM v2.0 results, as per National Health Laboratory Service resulting practice, with discordant CAP/CTM v2.0 v. Xpert EID results was performed. RESULTS: Of 150 infants who met the inclusion criteria, 6 (3.9%) had an Xpert EID result discordant with final HIV status: 5 (3.3%) were false negatives and 1 (0.7%) was false positive. As a consecutive test, the Xpert EID yielded a sensitivity of 96.5% (95% CI 92 - 98.9), specificity of 85.7% (95% CI 42.1 - 99.6), PPV of 99.3% (95% CI 95.7 - 99.9), negative predictive value of 54.5% (95% CI 32.5 - 74.9) and overall accuracy of 96.1% (95% CI 91.5 - 98.5). Using discordant CAP/CTM v2.0/Xpert EID results as criteria to verify indeterminate results instead of current practice would have reduced the number of indeterminate screening results by 42.1%, from 18 (12.6%) to 11 (7.2%), without increasing the false-positive rate. CONCLUSIONS: Addition of the Xpert EID as a consecutive test for specimens with an HIV-detected PCR screening result has the potential to improve the PPV and reduce the indeterminate rate, thereby reducing diagnostic challenges and time to final status, in SA's EID programme.

Detection and molecular characterization of urinary tract HIV-1 populations.

BACKGROUND: Identification of all possible HIV reservoirs is an important aspect in HIV eradication efforts. The urinary tract has however not been well studied as a potential HIV reservoir. In this pilot study we molecularly characterized HIV-1 viruses in urine and plasma samples to investigate HIV-1 replication, compartmentalization and persistence in the urinary tract. METHODS: Prospectively collected urine and blood samples collected over 12-36 months from 20 HIV-1 infected individuals were analysed including sampling points from prior to and after ART initiation. HIV-1 pol gene RNA and DNA from urine supernatant and urine pellets respectively were analysed and compared to plasma RNA viruses from the same individual. RESULTS: HIV-1 nucleic acid was detected in urine samples from at least one time point in 8/20 (40%) treatment-naïve subjects compared to 1/13 (7.7%) individuals on antiretroviral treatment (ART) during periods of plasma viral suppression and 1/7 (14.3%) individuals with virological failure. HIV-1 RNA was undetectable in urine samples after ART initiation but HIV-1 DNA was detectable in one patient more than 6 months after treatment initiation. There was co-clustering of urine-derived pol sequences but some urine-derived sequences were interspersed among the plasma-derived sequences. CONCLUSIONS: Suppressive ART reduces HIV-1 replication in the urinary tract but HIV-1 DNA may persist in these cells despite treatment. A larger number of sequences would be required to confirm HIV compartmentalization in the urinary tract.

Columbus® computer navigated total knee arthroplasty: Gap balancing versus measured resection.

BACKGROUND: The aim of this study was to compare the medium term functional outcome and patient satisfaction of gap balanced (GB) with measured resection (MR) total knee arthroplasty (TKA) using computer navigation. METHODS: A cohort of 144 consecutive computer navigated TKA were retrospectively identified from an arthroplasty database. Functional assessment using the Oxford Knee Score (OKS) and patient satisfaction were obtained from 113 patients at a mean follow-up of 5.4 (range four to seven) years. There were 44 patients in the GB group and 69 patients in the MR group. RESULTS: The mean OKS for the GB group was 36.9 (SD 9.2) and for the MR was 33.6 (SD 9.8), with a difference of 3.3 (95% CI 0.3 to 6.3) points, which was statistically significant (p=0.01). Linear regression analysis confirmed the independent effect of surgical technique when adjusting for confounding factors and surgeon, with the GB group achieving a greater post-operative OKS (R2=0.39, 3.0 points, 95% CI 1.2 to 4.8, p=0.001). There was a greater rate of patient satisfaction in the GB group (88.6%, n=39/44) compared to the MR group (81.1%, n=56/69), but this was not statistically significant (odds ratio 1.8, 95% CI 0.6 to 5.5, p=0.31). CONCLUSION: Computer navigated Columbus® TKA using a GB technique results in a statistically significantly greater functional outcome but no significant difference in patient satisfaction in the medium term compared to patients undergoing a MR technique.

Pre-treatment minority HIV-1 drug resistance mutations and long term virological outcomes: is prediction possible?

BACKGROUND: Although the use of highly active antiretroviral therapy in HIV positive individuals has proved to be effective in suppressing the virus to below detection limits of commonly used assays, virological failure associated with drug resistance is still a major challenge in some settings. The prevalence and effect of pre-treatment resistance associated variants on virological outcomes may also be underestimated because of reliance on conventional population sequencing data which excludes minority species. We investigated long term virological outcomes and the prevalence and pattern of pre-treatment minority drug resistance mutations in individuals initiating HAART at a local HIV clinic. METHODS: Patient's records of viral load results and CD4 cell counts from routine treatment monitoring were used and additional pre-treatment blood samples for Sanger sequencing were obtained. A selection of pre-treatment samples from individuals who experienced virological failure were evaluated for minority resistance associated mutations to 1 % prevalence and compared to individuals who achieved viral suppression. RESULTS: At least one viral load result after 6 months or more of treatment was available for 65 out of 78 individuals followed for up to 33 months. Twenty (30.8 %) of the 65 individuals had detectable viremia and eight (12.3 %) of them had virological failure (viral load > 1000 RNA copies/ml) after at least 6 months of HAART. Viral suppression, achieved by month 8 to month 13, was followed by low level viremia in 10.8 % of patients and virological failure in one patient after month 20. There was potentially reduced activity to Emtricitabine or Tenofovir in three out of the eight cases in which minority drug resistance associated variants were investigated but detectable viremia occurred in one of these cases while the activity of Efavirenz was generally reduced in all the eight cases. CONCLUSIONS: Early viral suppression was followed by low level viremia for some patients which may be an indication of failure to sustain viral suppression over time. The low level viremia may also be representing early stages of resistance development. The mutation patterns detected in the minority variants showed potential reduced drug sensitivity which highlights their potential to dominate after treatment initiation. TRIAL REGISTRATION: Not applicable.

Variability at the FCGR locus: characterization in Black South Africans and evidence for ethnic variation in and out of Africa.

This study set out to comprehensively investigate all known functional FcγR variants in South African Black and Caucasian individuals. Population diversity was further assessed using data from the 1000 Genomes Project. In our cohort, Black South Africans neither possessed the haplotypes previously associated with increased surface densities of FcγRIIb and FcγRIIIa nor the FCGR2C haplotype recently associated with increased vaccine efficacy in the RV144 HIV-1 vaccine trial (despite 48.7% bearing the c.134-96T tag allele). Moreover, Africans (South Africans, Luhya Kenyans and Yoruba Nigerians) lack the FCGR2C c.798+1G splice-site allele required for the expression of functional FcγRIIc. Although the presence or absence of surface FcγRIIc did not affect natural killer cell-mediated antibody-dependent cellular cytotoxicity capability, this may be significant for other FcγRIIc-mediated functions. Overall, allele distribution and linkage disequilibrium in Africans and Caucasians differed in a manner that would suggest a differentially maintained balance of FcγR-mediated cell activation in these populations. Finally, significant variation observed among different African populations precludes the use of any one African population as a proxy for FcγR diversity in Africans. In conclusion, the findings of this study highlight further ethnic variation at the FCGR gene locus, in particular for FCGR2C, a gene with increasingly recognized clinical significance.

Frequencies of immune hypersensitivity reaction-associated HLA class I alleles in healthy South African Indian and mixed ancestry populations determined by a novel real-time PCR assay.

We have determined the frequencies of human leucocyte antigen (HLA)-B*57:01, HLA-B*35:05, HLA-C*04 and HLA-C*08 in healthy individuals of South African Indian (SAI) ethnicity (n = 50) and South African mixed (SAM) ancestry (n = 50) using real-time allele-specific polymerase chain reaction (AS-PCR) assay. HLA-B*57:01 associates with immune hypersensitivity reaction (IHR) in individuals exposed to abacavir (ABC), while nevirapine (NVP) IHR associates with HLA-B*35:05, HLA-C*04 and HLA-C*08. Real-time AS-PCR assays typically use less DNA, are more cost-effective and rapid compared with conventional genotyping methods, such as sequence-based typing (SBT). The assay was developed using samples of known HLA class I genotype and subsequently applied to the SAI and SAM samples. HLA-B*57:01 was detected in SAM and SAI populations at frequencies of 8.0% and 12.0%, respectively, while HLA-B*35:05 was not found in SAI individuals, but was present in 6.0% of SAM individuals. HLA-C*04 was detected in 22.0% and 24.0% of SAM and SAI individuals, respectively, while 10.0% and 8.0% of SAM and SAI individuals, respectively, were HLA-C*08 positive. This study reports the development of a novel real-time AS-PCR assay to identify HLA class I alleles associated with ABC and NVP IHR and has established the frequencies of these alleles present in healthy SAI and SAM populations. Using South African demographic data, our hypothetical analysis suggests that a substantial number of individuals would benefit from the assay.

Influence of intragenic CCL3 haplotypes and CCL3L copy number in HIV-1 infection in a sub-Saharan African population.

Two CCL3 haplotypes (HapA1 and Hap-A3) and two polymorphic positions shared by the haplotypes (Hap-2SNP (single nucleotide polymorphism)) were investigated together with CCL3L copy number (CN), for their role in HIV-1 disease. Hap-A1 was associated with protection from in utero HIV-1 infection: exposed uninfected (EU) infants had higher representation of wild type (WT)/Hap-A1 than infected infants (excluding intrapartum (IP)-infected infants), which maintained significance post maternal Nevirapine (mNVP) and viral load (MVL) correction (P=0.04; odds ratio (OR)=0.33). Mother-infant pair analyses showed the protective effect of Hap-A1 is dependent on its presence in the infant. Hap-A3 was associated with increased IP transmission: WT/Hap-A3 was increased in IP-transmitting vs non-transmitting (NT) mothers, and remained significant post mNVP and MVL correction (P=0.02; OR=3.50). This deleterious effect of Hap-A3 seemed dependent on its presence in the mother. Hap-2SNP was associated with lower CD4 count in the NT mothers (P=0.03). CCL3 Hap-A1 was associated with high CCL3L CN in total (P=0.001) and EU infants (P=0.006); the effect was not additive, however, having either Hap-A1 or high CCL3L CN was more significantly (P=0.0008) associated with protection from in utero infection than Hap-A1 (P=0.028) or high CCL3L CN (P=0.002) alone. Linkage disequilibrium between Hap-A1 and high CCL3L CN appears unlikely given that a Nigerian population showed an opposite relationship.

Killer-cell immunoglobulin-like receptor genotyping and HLA killer-cell immunoglobulin-like receptor-ligand identification by real-time polymerase chain reaction.

The effector function of natural killer (NK) cells is modulated by surface expression of a range of killer-cell immunoglobulin-like receptors (KIRs) that interact with human leukocyte antigen (HLA) class I ligands. We describe the use of real-time polymerase chain reaction (PCR) assays that allow easy and quick detection of 16 KIR genes and the presence/absence of KIR-ligands based on allelic discrimination at codon 80 in the HLA-A/B Bw4 and HLA-C C1/C2 genes. These methods overcome the tedious and expensive nature of conventional KIR genotyping and HLA class I typing using sequence-specific primer (SSP) PCR, sequence-specific oligonucleotide (SSO) hybridization or sequence-based typing (SBT). Using these two cost-effective assays, we measured the frequencies of KIRs, KIR-ligands and KIR/KIR-ligand pairs in a cohort of Black women recruited in South Africa.

Viral genetic determinants of nonprogressive HIV type 1 subtype C infection in antiretroviral drug-naive children.

Characterization of HIV-1 from slow progressors is important to facilitate vaccine and antiviral drug development. To identify virus attenuations that may contribute to slower rates of disease progression, the full-length viral genomes from primary isolates of six slow progressing HIV-positive children were sequenced. Proviral DNA was extracted from cocultured peripheral blood mononuclear cells and used to PCR amplify, sequence, and extensively analyze the near full-length genomes and LTR regions. All primary HIV-1 isolates were HIV-1 subtype C throughout their genome, and amino acid (AA) sequence analysis revealed open reading frames for all genes. However, all isolates had at least one unusual gene/protein. For example, isolate LT5 had a 2AA insertion in the Vpr mitochondriotoxic domain. Isolate LT21 contained an additional 5AA in the C-terminus of tat exon 2, while integrase in isolate LT39 had an additional 4AA at the C-terminus. Rev from isolates LT45 and LT46 did not have the characteristic subtype C 16AA truncation, and in addition, had a further 3AA. Furthermore, altered functional domains were noted in several isolates, such as the cAMP-dependent kinase PKA phosphorylation site in Nef (LT5), a Vpr mutation involved in decreased proapoptotic activity (all isolates), and the Nef ExxxLL motif involved in the interaction with AP-1 and AP-2 (LT46). The slower HIV-1 disease progression in these six children may be attributed to altered protein functions. For example, LT46 Nef is unable to bind AP-1 and AP-2 and therefore is inactive on CD4 endocytosis. The biological relevance of these findings requires further investigation.

Identification of new variants within the two functional genes CCL3 and CCL3L encoding the CCL3 (MIP-1alpha) chemokine: implications for HIV-1 infection.

The CC chemokine CCL3 is encoded by two functional genes, namely CCL3 and CCL3L, and has been identified as a key chemokine in HIV-1 susceptibility and disease progression. The complete CCL3 and CCL3L genes and core promoters of 43 African mother-infant pairs (86 samples) and 28 Caucasian adults in South Africa were sequenced and extensively analysed for genetic variations. Africans were found to be more polymorphic in both genes with 25 single nucleotide polymorphisms (SNPs) in the CCL3 gene and 14 gene copy number single nucleotide polymorphisms (gcnSNPs) in the CCL3L gene, compared to nine CCL3 SNPs and eight CCL3L gcnSNPs in Caucasians. A total of 14 polymorphisms across the two genes were newly identified in this study, most (12/14) of which were exclusive to the African population. In addition, two indels were identified and characterized in the CCL3 and CCL3L genes of a small number of individuals. Of the numerous unique intragenic haplotypes found in the two genes, none were shared by the two population groups. A newly identified five-SNP CCL3 haplotype (Hap-C1) found in a high frequency in Caucasians, however, seems to be evolutionarily related to the most prevalent newly identified African seven-SNP CCL3 haplotype (Hap-A1). Hap-A1 also includes an SNP in the core promoter region and previous CCL3 haplotypes that have been reported to be associated with HIV-1 infection appear to be smaller haplotypes within Hap-A1. We thus propose Hap-A1 as a likely candidate for influencing levels of CCL3 production and in turn outcomes of HIV-1 infection.

Intra-articular hip viscosupplementation with synthetic hyaluronic acid for osteoarthritis: efficacy, safety and relation to pre-injection radiographs.

OBJECTIVE: The aim of this study was to determine the efficacy and safety of viscosupplementation with synthetic hyaluronic acid to the hip joint and to determine if there was any relation to pre-injection radiographic changes of osteoarthritis (OA). METHODS: Three Suplasyn injections were performed each to 15 hips with OA. Standing antero-posterior radiographs of the pelvis were performed prior to injection and scored according to Kelgren and Lawrence grades along with recordings of the minimum joint space width. Harris Hip Scores (HHS) which contain a component for pain, function, activities, absence of deformity and range of motion were recorded pre-injection and at 3 and 6 months. RESULTS: We established that at 3 months the HHS is significantly higher (P < 0.05). At 6 months, four hips had been excluded as they has went on to total hip arthroplasty (these hips showed a lower HHS at 3 months). For the remaining hips the HHS was highly significantly increased (P < 0.001). No side effects or complications were observed. Analysis of the pre-injection radiographs showed a trend towards a bigger increase in HHS with less radiographic OA changes. CONCLUSIONS: Viscosupplementation performed under fluoroscopic guidance is an effective and safe method of treating hip OA and appears to be more efficacious in those with less radiographic changes of OA.

Human immunodeficiency virus type 1 envelope-stimulated interleukin-2 production and survival of infected children with severe and mild clinical disease.

Interleukin (IL)-2 production after stimulation with human immunodeficiency virus type 1 (HIV-1) envelope (Env) peptides, tetanus toxoid, and phytohemagglutinin was measured in peripheral blood mononuclear cells (PBMC) from 25 HIV-1-infected children with mild and 24 with severe clinical disease and from 15 uninfected children. Env-specific IL-2 production was detected in PBMC of 26.5% of HIV-1-infected children but in none of the uninfected. The absence of Env-specific responses at enrollment among infected children was associated with a 6-fold increased risk of mortality within a year, adjusting for clinical severity (P=.04). Among those with severe clinical disease, Env-stimulated IL-2 reactivity in PBMC was negatively correlated with HIV-1 RNA copy numbers in plasma at enrollment and was positively correlated with CD4 T cell percentages 1 year later. HIV-specific cellular immune responses may play a role in containing progression of HIV-1 infection in children, despite early deficits in cell-mediated immunity.

Altered expression of L-selectin (CD62L) on polymorphonuclear neutrophils of children vertically infected with human immunodeficiency virus type 1.

The expression of the leukocyte adhesion molecule L-selectin (CD62L) on polymorphonuclear neutrophils (PMN) and circulating levels of the soluble form of the receptor (sCD62L) were determined for a group of HIV-1-infected children, categorized as having mild or severe disease, and a group of uninfected control children. The fluorescence intensity of CD62L on PMN was significantly reduced in the HIV-1-infected children with mild disease compared to the uninfected controls. The proportion of lymphocytes expressing CD62L, as well as their corresponding fluorescence intensities, was significantly reduced in both the mild and the severe disease groups compared to the uninfected children, while peripheral levels of sCD62L were significantly elevated in the HIV-1-infected children with mild and severe disease compared to the controls. Altered cell migration resulting from reduced expression of CD62L may be an important contributor to the increased susceptibility to secondary microbial infections seen in HIV-1-infected children.

Reduced HIV-stimulated T-helper cell reactivity in cord blood with short-course antiretroviral treatment for prevention of maternal-infant transmission.

T-helper cell responses to HIV have been associated with protection against maternal-infant HIV transmission in the absence of antiretroviral treatment, but the effects of antiretroviral treatment, now widely used for prevention, on development of these cell-mediated responses is unknown. We tested whether development of T-helper cell responses to HIV and other antigens would be affected by exposure to short-course regimens of zidovudine-lamivudine (ZDV-3TC) given to prevent maternal-infant HIV transmission. Cord blood samples were collected from 41 infants of HIV-infected mothers enrolled in a clinical trial in which they were treated with regimens of ZDV-3TC and from 29 infants whose HIV-infected mothers were not treated with any antiretroviral drugs. T-helper cell reactivity to HIV envelope peptides and other antigens was measured in vitro using a sensitive culture supernatant titration assay based on IL-2-dependent proliferation. Infants in the clinical trial were followed to 18 months to determine their HIV infection status, and venous blood samples were re-tested at 4.5 and 9 months for T-cell reactivity to HIV. HIV-stimulated T-helper cell reactivity in cord blood was detected 10-fold less frequently among those exposed to antiretroviral prophylaxis (2.4%) than among those unexposed (24.1%) (P = 0.007). Reductions in HIV-stimulated responses in cord blood occurred despite detectable HIV RNA (mean 3.38 standard deviation 0.76 log(10) copies per ml) at delivery among treated women and occurred independent of treatment duration. Our results suggest that short-course antiretroviral treatment given to prevent maternal-infant HIV transmission may attenuate HIV-stimulated T-cell memory responses in the neonate.

Duration of sample storage dramatically alters expression of the human immunodeficiency virus coreceptors CXCR4 and CCR5.

Expression of the chemokine receptors CXCR4 and CCR5 was monitored using EDTA-anticoagulated whole blood held for different time periods prior to fluorescent-antibody staining. When left overnight CXCR4 expression on leukocytes was substantially increased, whereas CCR5 expression was reduced. The results were similar when heparin and acid-citrate-dextrose were used as anticoagulants.

Altered expression of CD88 and associated impairment of complement 5a-induced neutrophil responses in human immunodeficiency virus type 1-infected patients with and without pulmonary tuberculosis.

The effect of infection with human immunodeficiency virus type 1 (HIV patient group), infection with Mycobacterium tuberculosis (TB patient group), and coinfection with both of these organisms (HIV/TB patient group) on the expression of CD88 on polymorphonuclear leukocytes (PMNL) was determined by using a receptor-specific monoclonal antibody and flow cytometry. A significant reduction in the fluorescence intensity of CD88 on PMNL was observed in the HIV and HIV/TB groups, compared with both the healthy donor (HD) and TB groups. Furthermore, when degranulation of PMNL was induced by ligation of CD88 by complement 5a (C5a), a large proportion of patients in the HIV and the HIV/TB groups was found to have reciprocal degranulation responses. Patients in the 2 HIV groups also were found to have significantly reduced C5a-induced chemotactic responses and significantly elevated peripheral levels of C5a des Arg, compared with the HD and TB groups. These differences may contribute to the increased susceptibility of HIV-1-infected individuals to secondary microbial infections.

Defective neutrophil degranulation induced by interleukin-8 and complement 5a and down-regulation of associated receptors in children vertically infected with human immunodeficiency virus type 1.

The polymorphonuclear neutrophils (PMNs) of patients infected with human immunodeficiency virus type 1 (HIV-1) show impaired microbicidal responses. The present study assessed the functional integrity of PMN degranulation responses and the expression of specific receptors that mediate these responses in a group of children vertically infected with HIV-1. PMN degranulation in response to interleukin-8 (IL-8) and complement 5a (C5a) was measured in a group of HIV-1-infected children with mild and severe clinical disease and in an uninfected control group. In addition, the expression of CXCR1, CXCR2, and CD88 on whole-blood PMNs was quantified by flow cytometry. Although CXCR1 expression was found to be largely unaltered in the HIV-1-infected children relative to that in the control children, the intensity of CXCR2 expression was significantly reduced in those with severe disease. Furthermore, there was a significant reduction in the percentage of cells expressing CD88 and in the intensity of CD88 fluorescence in the HIV-1-infected children compared to that in control children, with CD88 fluorescence intensity more significantly reduced in the presence of severe disease. PMNs from a large proportion of the HIV-1-infected children either showed reciprocal degranulation responses or were unresponsive to IL-8 and C5a, whereas the PMNs from the uninfected children showed positive responses. Inefficient agonist-induced degranulation may contribute to the increased susceptibility of HIV-1-infected children to secondary microbial infections. Furthermore, reduced expression of CXCR2 and CD88 may be suggestive of defects in other functions of PMNs from HIV-1-infected children.

Distribution of the human immunodeficiency virus coreceptors CXCR4 and CCR5 on leukocytes of persons with human immunodeficiency virus type 1 infection and pulmonary tuberculosis: implications for pathogenesis.

Expression of CXCR4 was significantly reduced from normal on all cell subsets of persons with pulmonary tuberculosis (TB group), with HIV-1 infection (HIV group), and those with both infections (HIV/TB group), except for on monocytes in the HIV group. The reductions were most notable in the two TB groups. Interestingly, the duration of antituberculosis treatment was significantly negatively correlated with the expression of CXCR4 on CD4+ and CD8+CD45RO+ cells, monocytes and NK cells, viral load, and proportions of CD38-expressing CD8+ lymphocytes, in HIV/TB patients. By contrast, CCR5 expression on most cell subsets analyzed was increased in all the disease groups, except for on monocytes in the two TB groups. There was no change in CCR5 expression on CD4+ cells when based on the disease groupings. However, higher proportions of CD4+CD45RA+ and CD8+ lymphocytes as well as B cells expressing CCR5 correlated with advancing HIV-1 disease, as did decreased proportions of CXCR4-expressing CD4+CD45RA+ cells.