CD4-T cell enumeration in human immunodeficiency virus (HIV)-infected patients: A laboratory performance evaluation of Muse Auto CD4/CD4% system by World Health Organization prequalification of in vitro diagnostics.

BACKGROUND: CD4 T-cell counts are still widely used to assess treatment eligibility and follow-up of HIV-infected patients. The World Health Organization (WHO) prequalification of in vitro diagnostics requested a manufacturer independent laboratory evaluation of the analytical performance at the Institute of Tropical Medicine (ITM) Antwerp, Belgium, of the Muse Auto CD4/CD4% system (Millipore), a new small capillary-flow cytometer dedicated to count absolute CD4-T cells and percentages in venous blood samples from HIV-infected patients. METHODS: Two hundred and fifty (250) patients were recruited from the HIV outpatient clinic at ITM. Accuracy and precision of CD4 T cell counting on fresh EDTA anticoagulated venous blood samples were assessed in the laboratory on a Muse Auto CD4/CD4% system. Extensive precision analyses were performed both on fresh blood and on normal and low stabilized whole blood controls. Accuracy ((bias) was assessed by comparing results from Muse CD4/CD4% to the reference (single-platform FACSCalibur). Clinical misclassification was measured at 500, 350, 200 and 100 cells/µL thresholds. RESULTS: Intra-assay precision was < 5%, and inter-assay was < 9%. CD4 T cell counts measured on Muse Auto CD4/CD4% System and on the reference instrument resulted in regression slopes of 0.97 for absolute counts and 1.03 for CD4 T cell percentages and a correlation coefficient of 0.99 for both. The average absolute bias as compared to the reference was negligible (4 cells/µL or 0.5%). The absolute average bias on CD4 T cell percentages was < 1%. Clinical misclassification at different CD4 T cell thresholds was small resulting in sensitivities and specificities equal or >90% at all thresholds except at 100 cells/µL (sensitivity = 87%). All samples could be analyzed as there was no repetitive rejection errors recorded. CONCLUSIONS: The Muse Auto CD4/CD4% System performed very well on fresh venous blood samples and met all WHO acceptance criteria for analytical performance of CD4 technologies.

Additional Screening and Treatment of Malaria During Pregnancy Provides Further Protection Against Malaria and Nonmalarial Fevers During the First Year of Life.

Background: Although consensus exists that malaria in pregnancy (MiP) increases the risk of malaria in infancy, and eventually nonmalarial fevers (NMFs), there is a lack of conclusive evidence of benefits of MiP preventive strategies in infants. Methods: In Burkina Faso, a birth cohort study was nested to a clinical trial assessing the effectiveness of a community-based scheduled screening and treatment of malaria in combination with intermittent preventive treatment with sulfadoxine-pyrimethamine (CSST/IPTp-SP) to prevent placental malaria. Clinical episodes and asymptomatic infections were monitored over 1 year of follow-up to compare the effect of CSST/IPTp-SP and standard IPTp-SP on malaria and NMFs. Results: Infants born during low-transmission season from mothers receiving CSST/IPTp-SP had a 26% decreased risk of experiencing a first clinical episode (hazard ratio, 0.74 [95% confidence interval, .55-0.99]; P = .047). CSST/IPTp-SP interacted with birth season and gravidity to reduce the incidence of NMFs. No significant effects of CSST/IPTp-SP on the incidence of clinical episodes, parasite density, and Plasmodium falciparum infections were observed. Conclusions: Our findings indicate that CSST/IPTp-SP strategy may provide additional protection against both malaria and NMFs in infants during the first year of life, and suggest that malaria control interventions during pregnancy could have long-term benefits in infants.

Serum Levels of Soluble CD40 Ligand and Neopterin in HIV Coinfected Asymptomatic and Symptomatic Visceral Leishmaniasis Patients.

Human Immunodeficiency Virus (HIV) co-infection drastically increases the risk of developing overt visceral leishmaniasis (VL). The asymptomatic Leishmania infection window constitutes an opportunity to identify those HIV patients at highest risk by defining early markers associated with disease susceptibility or resistance. As intracellular parasite killing is essential, we investigated whether serum markers of macrophage activation were notably affected in HIV patients with an asymptomatic Leishmania infection or overt visceral leishmaniasis disease. Serum levels of soluble CD40 ligand and neopterin were assessed in 24 active VL-HIV patients, 35 HIV patients with asymptomatic Leishmania infection and 35 HIV endemic controls. All patients were recruited in L. donovani endemic regions of North-West Ethiopia. The serum levels of sCD40L and neopterin significantly decreased and increased in HIV patients with active VL compared to HIV patients with asymptomatic Leishmania infection, respectively. No statistically significant differences could be detected in neopterin and sCD40L levels between Leishmania asymptomatically infected HIV patients and endemic HIV control patients. However, an inverse trend, between Leishmania antibody positivity or VL development and neopterin levels could be seen. The CD4+ T-cell count was inversely correlated with serum neopterin levels, but not with sCD40L levels. Our results in HIV coinfected patients, correspond with the postulated protective role of sCD40L in VL and underline the importance of the CD40-CD40L pathway in resistance against the parasite. Neopterin levels suggest an increased macrophage activation upon infection and could have a value in clinical algorithms to, although non-specifically, improve prediction of VL development in HIV patients with asymptomatic Leishmania infection.

Performance of FACSPresto Point-of-Care Instrument for CD4-T Cell Enumeration in Human Immunodeficiency Virus (HIV)-Infected Patients Attending Care and Treatment Clinics in Belgium and Tanzania.

BACKGROUND: CD4 T-cell counts are widely used to assess treatment eligibility and to follow-up HIV-infected patients. The World Health Organization prequalification of in vitro diagnostics program conducted a performance evaluation of the FACSPresto (BD Biosciences), a new point-of-care instrument to measure absolute CD4-T cell (CD4) counts and percentages in venous and capillary blood samples from HIV-infected patients. METHODS: Patients were recruited in Belgium (200 patients) and in Tanzania (247 patients). Venous blood samples were analyzed in two nearby reference laboratories. In addition, nurses/technicians collected a capillary blood sample by finger prick directly into a FACSPresto CD4 cartridge. Assay precision was assessed on fresh blood and on external quality control samples. Trueness (bias) was assessed by comparing results from FACSPresto with the reference (single-platform FACSCalibur). Clinical misclassification was measured at 200, 350 and 500 cells/µL thresholds. RESULTS: Intra-assay precision was < 6%, and inter-assay < 8%. CD4 results from FACSPresto and reference method resulted in regression slopes of 0.99-1.11 using either venous or capillary blood. Correlation was better for venous than for capillary blood (minimum 0.97 vs 0.93 respectively). Capillary blood resulted in a larger bias than venous blood, with 24 and 83 cells/µL for absolute CD4 counts on capillary blood in Antwerp and Dar es Salaam respectively, vs 12 and 41 cells/µL on venous blood. Bias on CD4% was < 1% on both venous and capillary blood, and was proportionally better than for absolute CD4 counts. Clinical misclassification was in line with the average overestimation, showing a very good specificity, but sensitivity around 70-90%. The rejection rate was 11% on first reading, leading to 6% of all samples without final result after a second reading. CONCLUSIONS: The FACSPresto performed very well on venous blood samples, and well on capillary blood samples.

CD4 results with a bias larger than hundred cells per microliter can have a significant impact on the clinical decision during treatment initiation of HIV patients.

BACKGROUND: CD4 counts are currently used to assess HIV patients for treatment eligibility and to monitor antiretroviral response to treatment. The emerging point-of-care devices could fill an important gap in resource-limited settings. However, the accuracy of CD4-counting instruments is diverse and data on how CD4 measurement errors have an impact on clinical decisions are lacking. METHODS: Clinicians were queried on the use of CD4 results in their clinical setting. Subsequently, the effect of CD4 measurement errors on treatment initiation was put in a statistical model. Based on clinical CD4 databases from Belgium, Cambodia, and Senegal, the percentage of unchanged clinical decisions was calculated (treatment initiation should start within a 3-month delay [one visit]) for escalating CD4 measurement errors, taking into account the strict or preventive application of CD4 thresholds at 350 or 500 cells/µl used by clinicians. RESULTS: To ensure that the treatment was initiated appropriately for at least 95% of patients, an error of 5 - 10 cells/µl was allowed. This is significantly smaller than the bias of ±50 cells/µl most clinicians considered acceptable. For limits of agreement (LOA, 1.96 x error) of 100 cells/µl, corresponding to most CD4 instrument evaluations, the misclassification rate of patients was found to be 3 - 28% at the threshold of 350 cells/µl (strict or flexible), and 13 - 20% at 500 cells/µl. CONCLUSIONS: The maximum allowed CD4 bias on results from new CD4 technologies should not exceed 50 cells/µl (LOA 100 cells/µl) when applied for treatment initiation, to ensure at least 72% of correct clinical decisions. © 2016 International Clinical Cytometry Society.

Human Immunodeficiency Virus (HIV)-Infected Patients Accept Finger Stick Blood Collection for Point-Of-Care CD4 Testing.

INTRODUCTION: HIV-infected patients require antiretroviral treatment for life. To improve access to care, CD4 enumeration and viral load tests have been redesigned to be used as point-of-care techniques using finger-stick blood. Accurate CD4 counting in capillary blood requires a free flowing blood drop that is achieved by blade incision. The aim of this study was to assess the attitude of the patients toward blade-based finger-stick blood donation. METHODS: Four hundred and ninety-nine patients were included (299 patients from South Africa and 200 from Belgium). They completed a questionnaire to express their preference for finger stick or venipuncture, after undergoing both. The South African patient cohort was divided in two groups, receiving either single or multiple finger stick for CD4 and other HIV-related tests. The Belgian patients received a single finger stick for CD4 testing, and were asked to respond directly and again after two days. RESULTS: The majority of the patients preferred the finger stick to the venipuncture. The perceived pain using the blade was superior to a small needle, but similar to a large needle. They preferred up to three finger sticks over one venipuncture. Up to 30% of the patients changed their mind over two days. The main reason for choosing a finger stick was continued bleeding after venipuncture. The most cited objection to finger stick was pain/soreness. CONCLUSION: Patient perceptions support the implementation of donating capillary blood with blade-based finger stick during CD4 point-of-care testing.

The role of NK cells in HIV-1 protection: autologous, allogeneic or both?

Natural killer (NK) cells specialize in killing virally infected- or tumor cells and are part of the innate immune system. The activational state of NK cells is determined by the balance of incoming activating and inhibitory signals mediated by receptor-ligand binding with the target cell. These receptor-ligand bonds mainly consist of the killer immunoglobulin-like receptors (KIR), which are expressed at the cell surface of NK cells, and their ligands: the highly variable human leukocyte antigen -class I molecules (HLA). Absence of an inhibitory receptor-ligand bond lowers the NK cell activation threshold, whereas an activating receptor-ligand bond stimulates the cell, potentially overcoming this threshold and triggering NK cell activation. NK cells influence the course of infection as well as the acquisition of HIV-1. Several lines of evidence relate the activating NK cell receptor KIR3DS1, in the presence or absence of its putative ligand HLA-Bw4, with slower disease progression as well as resistance to HIV-1 infection. Overall, resistance to HIV-1 infection predominantly correlates with activating KIR/HLA profiles, consisting of e.g. activating KIRs, group B haplotypes, or inhibitory KIRs in absence of their ligands. Such a conclusion is less evident for studies of HIV-1 disease progression, with studies reporting beneficial as well as detrimental effects of activating KIR/HLA genotypes. It is likely that KIR/HLA association studies are complicated by the complexity of the KIR and HLA loci and their mutual interactions, as well as by additional factors like route of HIV exposure, immune activation, presence of co-infections, and the effect of anti-HIV-1 antibodies. One newly discovered NK cell activation pathway associated with resistance to HIV-1 infection involves the presence of an iKIR/HLA mismatch between partners. The absence of such an iKIR/HLA bond renders donor-derived allogeneic HIV-1 infected cells vulnerable to NK cell responses during HIV-1 transmission. Therefore, theoretically, HIV-1 would be eliminated before it has the chance to infect the autologous cells in the recipient. While this "alloreactive" NK cell mechanism is especially relevant to HIV transmission in monogamous couples, it would be interesting to investigate how it could influence resistance to HIV in other settings. The objective of this review is to summarize the knowledge about these autologous and alloreactive NK cell responses with regard to HIV-1 outcome.

Antigen-specific interferon-gamma responses and innate cytokine balance in TB-IRIS.

BACKGROUND: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) remains a poorly understood complication in HIV-TB patients receiving antiretroviral therapy (ART). TB-IRIS could be associated with an exaggerated immune response to TB-antigens. We compared the recovery of IFNγ responses to recall and TB-antigens and explored in vitro innate cytokine production in TB-IRIS patients. METHODS: In a prospective cohort study of HIV-TB co-infected patients treated for TB before ART initiation, we compared 18 patients who developed TB-IRIS with 18 non-IRIS controls matched for age, sex and CD4 count. We analyzed IFNγ ELISpot responses to CMV, influenza, TB and LPS before ART and during TB-IRIS. CMV and LPS stimulated ELISpot supernatants were subsequently evaluated for production of IL-12p70, IL-6, TNFα and IL-10 by Luminex. RESULTS: Before ART, all responses were similar between TB-IRIS patients and non-IRIS controls. During TB-IRIS, IFNγ responses to TB and influenza antigens were comparable between TB-IRIS patients and non-IRIS controls, but responses to CMV and LPS remained significantly lower in TB-IRIS patients. Production of innate cytokines was similar between TB-IRIS patients and non-IRIS controls. However, upon LPS stimulation, IL-6/IL-10 and TNFα/IL-10 ratios were increased in TB-IRIS patients compared to non-IRIS controls. CONCLUSION: TB-IRIS patients did not display excessive IFNγ responses to TB-antigens. In contrast, the reconstitution of CMV and LPS responses was delayed in the TB-IRIS group. For LPS, this was linked with a pro-inflammatory shift in the innate cytokine balance. These data are in support of a prominent role of the innate immune system in TB-IRIS.

LPS-binding protein and IL-6 mark paradoxical tuberculosis immune reconstitution inflammatory syndrome in HIV patients.

BACKGROUND: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) remains a poorly understood complication in HIV-TB co-infected patients initiating antiretroviral therapy (ART). The role of the innate immune system in TB-IRIS is becoming increasingly apparent, however the potential involvement in TB-IRIS of a leaky gut and proteins that interfere with TLR stimulation by binding PAMPs has not been investigated before. Here we aimed to investigate the innate nature of the cytokine response in TB-IRIS and to identify novel potential biomarkers. METHODS: From a large prospective cohort of HIV-TB co-infected patients receiving TB treatment, we compared 40 patients who developed TB-IRIS during the first month of ART with 40 patients matched for age, sex and baseline CD4 count who did not. We analyzed plasma levels of lipopolysaccharide (LPS)-binding protein (LBP), LPS, sCD14, endotoxin-core antibody, intestinal fatty acid-binding protein (I-FABP) and 18 pro-and anti-inflammatory cytokines before and during ART. RESULTS: We observed lower baseline levels of IL-6 (p = 0.041), GCSF (p = 0.036) and LBP (p = 0.016) in TB-IRIS patients. At IRIS event, we detected higher levels of LBP, IL-1RA, IL-4, IL-6, IL-7, IL-8, G-CSF (p ≤ 0.032) and lower I-FABP levels (p = 0.013) compared to HIV-TB co-infected controls. Only IL-6 showed an independent effect in multivariate models containing significant cytokines from pre-ART (p = 0.039) and during TB-IRIS (p = 0.034). CONCLUSION: We report pre-ART IL-6 and LBP levels as well as IL-6, LBP and I-FABP levels during IRIS-event as potential biomarkers in TB-IRIS. Our results show no evidence of the possible contribution of a leaky gut to TB-IRIS and indicate that IL-6 holds a distinct role in the disturbed innate cytokine profile before and during TB-IRIS. Future clinical studies should investigate the importance and clinical relevance of these markers for the diagnosis and treatment of TB-IRIS.

Inhibitory KIR/HLA incompatibility between sexual partners confers protection against HIV-1 transmission.

Killer immunoglobulin-like receptors (KIRs) regulate natural killer (NK) cells in a human leukocyte antigen (HLA)-dependent manner. KIR/HLA mismatched hematopoietic stem cell transplants induce alloreactive NK cells, which prevent leukemia relapse. Certain KIR/HLA combinations protect against HIV-1 infection, but the effect of KIR/HLA mismatches between sexual partners has never been investigated. In this study, we analyzed the effect of allogeneic KIR/HLA combinations on HIV-1 transmission in a West African population of HIV-1-discordant and concordant couples. HIV-1-discordant couples were characterized by recipient partners with homozygous KIR2DL2, and by a mismatched recipient partner KIR2DL1/HLA-C2 with index partner HLA-C1/C1 combination expected to allow licensed missing self NK cell killing of index partners' cells. HIV-1-concordant couples on the other hand were characterized by KIR2DL3 homozygous recipient partners with HLA-C1/C2 bearing index partners, resulting in a matched KIR/HLA combination expected to inhibit NK cell killing. In vitro cocultures of healthy donor-derived NK cells and HIV-1 patient-derived CD4(+) T cells confirmed the involvement of these allogeneic KIR/HLA combinations in NK cell-mediated CD4(+) T-cell killing. Our data suggest that KIR/HLA incompatibility between sexual partners confers protection against HIV-1 transmission and that this may be due to alloreactive NK cell killing of the HIV-1-infected partner's cells.

Expression analysis of LEDGF/p75, APOBEC3G, TRIM5alpha, and tetherin in a Senegalese cohort of HIV-1-exposed seronegative individuals.

BACKGROUND: HIV-1 replication depends on a delicate balance between cellular co-factors and antiviral restriction factors. Lens epithelium-derived growth factor (LEDGF/p75) benefits HIV, whereas apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), tripartite motif 5alpha (TRIM5α), and tetherin exert anti-HIV activity. Expression levels of these proteins possibly contribute to HIV-1 resistance in HIV-1-exposed populations. METHODOLOGY/PRINCIPAL FINDINGS: We used real-time PCR and flow cytometry to study mRNA and protein levels respectively in PBMC and PBMC subsets. We observed significantly reduced LEDGF/p75 protein levels in CD4+ lymphocytes of HIV-1-exposed seronegative subjects relative to healthy controls, whereas we found no differences in APOBEC3G, TRIM5α, or tetherin expression. Untreated HIV-1-infected patients generally expressed higher mRNA and protein levels than healthy controls. Increased tetherin levels, in particular, correlated with markers of disease progression: directly with the viral load and T cell activation and inversely with the CD4 count. CONCLUSIONS/SIGNIFICANCE: Our data suggest that reduced LEDGF/p75 levels may play a role in resistance to HIV-1 infection, while increased tetherin levels could be a marker of advanced HIV disease. Host factors that influence HIV-1 infection and disease could be important targets for new antiviral therapies.

Multisite evaluation of a point-of-care instrument for CD4(+) T-cell enumeration using venous and finger-prick blood: the PIMA CD4.

BACKGROUND: CD4(+) T-cell enumeration (CD4 count) is used as a criterion to initiate antiretroviral therapy (ART) in HIV patients and to monitor treatment efficacy. However, simple, affordable, and reliable point-of-care (POC) instruments adapted to resource-limited settings are still lacking. The PIMA CD4 analyzer is a new POC instrument for CD4 counting that uses disposable cartridges and a battery-powered analyzer. METHODS: Whole blood samples were taken by venipuncture or by finger prick from 300 subjects, including HIV-infected patients and HIV (-) controls. CD4 counts were measured by PIMA (using venous or capillary blood) and by FACSCount (using venous blood) considered as the reference. RESULTS: Similar CD4 counts were obtained by PIMA and FACSCount using either HIV+ venous blood or HIV+ finger-prick blood samples. However, with a concordance coefficient of 0.88 and a Pearson correlation of 0.89, finger-prick blood performed not as good as venous blood (0.97 and 0.98, respectively). For a clinical decision to start ART at 200 CD4 cells per microliter, sensitivity of PIMA was 90%/91% and specificity 98%/96% for venous/finger-prick blood, respectively, and for a treatment threshold of 350 CD4 cells per microliter, the sensitivity was 98%/91% and the specificity was 79%/80% for venous/finger-prick blood, respectively. Repeatability (precision) on venous blood resulted in a coefficient of variation of 4%. Using finger-prick blood, the average instrument error frequency resulting in aborted analyses was 14%. CONCLUSIONS: PIMA is a good POC instrument for screening adult HIV-infected patients in resource-limited settings for treatment eligibility. Its performance on finger-prick blood is not as good as on venous blood. Adequate training for correct use of finger-prick blood samples is mandatory.

Evaluation of a flow cytometry method for CD4 T cell enumeration based on volumetric primary CD4 gating using thermoresistant reagents.

Laboratory follow-up of HIV patients in resource-limited settings requires appropriate instruments for CD4 T cell enumeration. In this study, we evaluated the application of a simplified, mobile and robust flow cytometry system, the Apogee Auto 40 analyzer (Auto40) using thermoresistant reagents, for CD4 T cell enumeration. We measured the absolute CD4 counts in fresh whole blood samples from 170 Senegalese subjects, including 129 HIV-positive (HIV+) patients and 41 HIV-negative (HIV-) controls. Based on volumetric primary CD4 gating, cells were stained with commercially available reagents (Easy MoAb CD4;Bio-D, Valenzano, Italy) and analyzed on the Auto40. The results were compared with those from the FACSCount system (Becton Dickinson, San Jose, USA). Repeatability analysis was performed on duplicate testing of 49 samples on both FACSCount and Auto40. The intra-run precision was measured by 10 replicates using 3 clinical blood samples with low, intermediate and high CD4 concentrations. The results from the two instruments were in good agreement. The percent similarity between the results of both instruments was 99%±relative standard deviation of 12.7%. The concordance correlation coefficient was 0.99. The absolute bias and limits of agreement (LOA) between the two instruments, calculated by Bland-Altman analysis, were clinically acceptable (bias: +4 cells/µl; LOA: -111 to +120 cells/µl). The clinical agreement between the two instruments at a cutoff of 200 CD4 cells/µl was 94%. The repeatability of measurements on the Auto40 was also similar to that observed with FACSCount system (bias +0.1 cells/µl, coefficient of variation 2.5% vs bias -1.1cells/µl, coefficient of variation 2.9% respectively). In conclusion, our results indicate that the Auto 40 system, using thermoresistant reagents, is suitable for CD4 T cell enumeration and will be a helpful tool to improve HIV laboratory monitoring in resource-limited settings.

Intracellular detection of differential APOBEC3G, TRIM5alpha, and LEDGF/p75 protein expression in peripheral blood by flow cytometry.

Expression studies on specific host proteins predominantly use quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based assay to study intracellular expression levels of three important host proteins involved in HIV-1 replication: apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), tripartite motif 5alpha (TRIM5α), and lens epithelium-derived growth factor (LEDGF/p75). An indirect intracellular staining (ICS) method was optimized using antibodies designed for other applications like enzyme-linked immunosorbent assay (ELISA), confocal imaging, and western blotting. The median fluorescence intensity (MFI) value--a measure for the protein expression level--increased upon higher antibody concentration and longer incubation time, and was reduced following preincubation with recombinant proteins. Staining of stably transfected or knock-down cell lines supported the method's specificity. Moreover, confocal microscopy analysis of peripheral blood mononuclear cells (PBMC), when stained according to the ICS method, confirmed the localization of APOBEC3G and TRIM5α in the cytoplasm, and of LEDGF/p75 in the nucleus. Also, stimulation with mitogen, interferon-alpha, or interferon-beta resulted in detectable, albeit weak, increases in intracellular expression of APOBEC3G and TRIM5α. After optimization, the method was applied to healthy control and HIV-1 infected subjects. For all subjects studied, the memory subset of CD4+ T cells showed significantly higher expression levels of APOBEC3G, TRIM5α, and LEDGF/p75, while the CD16+ subset of monocytes was characterized by higher expression levels of LEDGF/p75. In addition, we observed that therapy-naïve HIV-1 patients tended to have lower expression levels of APOBEC3G and TRIM5α than HIV-1 negative controls. In summary, our data provide proof-of-principle for the detection of specific host factors at the level of a single cell, which may prove useful for our further understanding of their role in virus-host interactions.

Antiretroviral treatment-associated tuberculosis in a prospective cohort of HIV-infected patients starting ART.

Commencement of antiretroviral treatment (ART) in severely immunosuppressed HIV-infected persons is associated with unmasking of subclinical disease. The subset of patients that are diagnosed with tuberculosis (TB) disease while on ART have been classified as ART-associated TB. Few studies have reported the incidence of ART-associated TB and unmasking TB-IRIS according to the International Network for the Study of HIV-Associated IRIS (INSHI) consensus definition. To determine the incidence and predictors of ART-associated TB, we screened 219 patients commencing ART at the Infectious Diseases Clinic in Kampala, Uganda for TB by symptoms, sputum microscopy, and chest X-rays and followed them for one year. Fourteen (6.4%) patients were diagnosed with TB during followup. Eight (3.8%) patients had ART-associated TB (incidence rate of 4.3 per 100 person years); of these, three patients fulfilled INSHI criteria for unmasking TB-associated IRIS (incidence rate of 1.6 per 100 person years). A body mass index of less than 18.5 kg/m(2) BMI (HR 5.85 95% CI 1.24-27.46, P = .025) and a C-reactive protein greater than 5 mg/L (HR 8.23 95% CI 1.36-38.33, P = .020) were risk factors for ART-associated TB at multivariate analysis. In conclusion, with systematic TB screening (including culture and chest X-ray), the incidence of ART-associated TB is relatively low in settings with high HIV and TB prevalence.

Low-level CD4+ T cell activation in HIV-exposed seronegative subjects: influence of gender and condom use.

Immune activation has been suggested to increase susceptibility to human immunodeficiency virus type 1 (HIV-1) transmission, while at the same time it could be deemed essential for mounting an effective antiviral immune response. In this study, we compared levels of T cell activation between exposed seronegative (ESN) partners in HIV-1 discordant couples and HIV-unexposed control subjects in Dakar, Senegal. ESN subjects showed lower levels of CD38 expression on CD4(+) T cells than did control subjects. However, this was found to be associated with concurrent differences in the use of condoms: ESN subjects reported a higher degree of condom use than did control subjects, which correlated inversely with CD38 expression. In addition, we observed markedly higher levels of T cell activation in women compared with men, irrespective of sexual behavior. These findings question the relevance of low-level CD4(+) T cell activation in resistance to HIV-1 infection and underscore the need to take gender and sexual behavior characteristics of high-risk populations into account when analyzing correlates of protective immunity.

Proof of principle: an HIV p24 microsphere immunoassay with potential application to HIV clinical diagnosis.

The measurement of CD4 counts and viral loads on a single instrument such as an affordable flow cytometer could considerably reduce the cost related to the follow-up of antiretroviral therapy in resource-poor settings. The aim of this study was to assess whether the HIV-1 p24 antigen could be measured using a microsphere-based flow cytometric (FC) assay and the experimental conditions necessary for processing plasma samples. A commercial anti-p24 antibody pair from Biomaric was used to develop a p24 microsphere immunoassay (MIA) using HIV culture supernatant as the source of antigen. The ultrasensitive Perkin Elmer enzyme immunoassay (EIA) served as a reference assay. Quantification of HIV p24 using the heat-mediated immune complex disruption format described for plasma samples was feasible using the Biomaric MIA and applicable to a broad range of HIV-1 Group M subtypes. The inclusion of a tyramide amplification step was successful and increased the fluorescence signal up to 3 logs as compared with the MIA without amplification. The analytical sensitivity of this ultrasensitive Biomaric assay reached 1 pg/mL, whereas the ultrasensitive Perkin Elmer EIA was sensitive to less than 0.17 pg/mL. Our data indicate, for the first time, that the principle of p24 detection using the heat-denatured ultrasensitive format can be applied to FC.

Higher homologous and lower cross-reactive Gag-specific T-cell responses in human immunodeficiency virus type 2 (HIV-2) than in HIV-1 infection.

Human immunodeficiency virus type 2 (HIV-2) infection results in slower CD4(+) T-cell decline, lower plasma viral load levels, and hence slower progression of the disease than does HIV-1 infection. Although the reasons for this are not clear, it is possible that HIV-2 replication is more effectively controlled by host responses. We used aligned pools of overlapping HIV-1 and HIV-2 Gag peptides in an enhanced gamma interferon enzyme-linked immunospot assay to compare the levels of homologous and cross-reactive Gag-specific T-cell responses between HIV-1- and HIV-2-infected patients. HIV-2-infected patients showed broader and stronger homologous Gag-specific T-cell responses than HIV-1-infected patients. In contrast, the cross-reactive T-cell responses in HIV-2-infected patients were both narrower and weaker than those in HIV-1-infected patients, in line with overall weaker correlations between homologous and heterologous T-cell responses among HIV-2-infected patients than among HIV-1-infected patients. Cross-reactive responses in HIV-2-infected patients tended to correlate directly with HIV-1/HIV-2 Gag sequence similarities; this was not found in HIV-1-infected patients. The CD4(+) T-cell counts of HIV-2-infected patients correlated directly with homologous responses and inversely with cross-reactive responses; this was not found in HIV-1-infected patients. Our data support a model whereby high-level HIV-2-specific T-cell responses control the replication of HIV-2, thus limiting viral diversification and priming of HIV-1 cross-reactive T-cell responses over time. However, we cannot exclude the possibility that HIV-2 replication is controlled by other host factors and that HIV-2-specific T-cell responses are better maintained in the context of slow viral divergence and a less damaged immune system. Understanding the nature of immune control of HIV-2 infection could be crucial for HIV vaccine design.

Production of human immunodeficiency virus type 1 (HIV-1) pseudoviruses using linear HIV-1 envelope expression cassettes.

HIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes.