Deborah K. Glencross

Providing immediate CD4 count results at HIV testing improves ART initiation.

BackgroundIn South Africa, CD4 count results are typically available within a week of testing. However, 35%–55% of newly diagnosed HIV-positive patients do not return for their CD4 results and therefore, do not access further care.12 We evaluated the impact of a CD4 count result and patient written information provided immediately after diagnosis on retention in care. MethodsHIV-infected subjects were randomized to 3 arms; receipt of a CD4 result at time of HIV diagnosis, receipt of written information, and standard of care (CD4 collection after 1 week) or standard of care alone. The outcome of interest was enrollment for further care within 1 month for pre-antiretroviral therapy (ART) care or within 3 months for ART initiation. Secondary outcome was time taken from diagnosis to each stage of care pathway. Independent predictors of retention were assessed with multivariate analysis. ResultsThree hundred forty-four patients recruited, of which 64.5% were females with a median age of 30 years (interquartile range: 27–35). Subjects were similar in age, gender, CD4 count, education, and employment status. Providing CD4 results at HIV diagnosis increases the likelihood of reporting for ART initiation (risk ratio = 2.1; 95% confidence interval = 1.39 to 3.17) compared with standard of care. Written information only reduced the time to presentation for pre-ART care although increasing age was associated with retention. There was 49% attrition in the standard of care arms. ConclusionsReceipt of a CD4 count at the time of HIV testing increases ART initiation rates. Point-of-care diagnostics can be used to improve retention, but losses to pre-ART care remain high.

Multiple Method Comparison: Statistical Model Using Percentage Similarity

Method comparison typically determines how well two methods agree. This is usually performed using the difference plot model, which measures absolute differences between two methods. This is often not applicable to data with wide ranges of absolute values. An alternative model is introduced that simplifies comparisons specifically for multiple methods compared to a gold standard.

Performance evaluation of the Pima™ point-of-care CD4 analyser using capillary blood sampling in field tests in South Africa

BackgroundPoint-of-care CD4 testing can provide immediate CD4 reporting at HIV-testing sites. This study evaluated performance of capillary blood sampling using the point-of-care Pima™ CD4 device in representative primary health care clinics doing HIV testing.MethodsPrior to testing, prescribed capillary-sampling and instrument training was undertaken by suppliers across all sites. Matching venous EDTA samples were drawn throughout for comparison to laboratory predicate methodology (PLG/CD4). In Phase I, Pima™ cartridges were pipette-filled with EDTA venous blood in the laboratory (N = 100). In Phase II (N = 77), Pima™ CD4 with capillary sampling was performed by a single operator in a hospital-based antenatal clinic. During subsequent field testing, Pima™ CD4 with capillary sampling was performed in primary health care clinics on HIV-positive patients by multiple attending nursing personnel in a rural clinic (Phase-IIIA, N = 96) and an inner-city clinic (Phase-IIIB, N = 139).ResultsPima™ CD4 compared favourably to predicate/CD4 when cartridges were pipette-filled with venous blood (bias -17.3 ± STDev = 36.7 cells/mm3; precision-to-predicate %CV < 6%). Decreased precision of Pima™ CD4 to predicate/CD4 (varying from 17.6 to 28.8%SIM CV; mean bias = 37.9 ± STDev = 179.5 cells/mm3) was noted during field testing in the hospital antenatal clinic. In the rural clinic field-studies, unacceptable precision-to-predicate and positive bias was noted (mean 28.4%SIM CV; mean bias = +105.7 ± STDev = 225.4 cells/mm3). With additional proactive manufacturer support, reliable performance was noted in the subsequent inner-city clinic field study where acceptable precision-to-predicate (11%SIM CV) and less bias of Pima™ to predicate was shown (BA bias ~11 ± STDev = 69 cells/mm3).ConclusionsVariable precision of Pima™ to predicate CD4 across study sites was attributable to variable capillary sampling. Poor precision was noted in the outlying primary health care clinic where the system is most likely to be used. Stringent attention to capillary blood collection technique is therefore imperative if technologies like Pima™ are used with capillary sampling at the POC. Pima™ CD4 analysis with venous blood was shown to be reproducible, but testing at the point of care exposes operators to biohazard risk related to uncapping vacutainer samples and pipetting of blood, and is best placed in smaller laboratories using established principles of Good Clinical Laboratory Practice. The development of capillary sampling quality control methods that assure reliable CD4 counts at the point of care are awaited.

Large‐scale affordable Panleucogated CD4+ testing with proactive internal and external quality assessment: In support of the South African national comprehensive care, treatment and management programme for HIV and AIDS

In order to expand the treatment of human immunodeficiency virus‐1 (HIV) infected patients in Africa, millions will require cost‐effective CD4 counts. Supporting laboratories therefore, need to move away from crisis management and haphazard practices to organized pathology services. The authors reviewed the performance of the simplified single platform (SP) PanLeucogated (PLG) CD4 methodology, introduced into 52 laboratories across the South African National Health Laboratory Service (SA‐NHLS), with a proactive approach to training, internal quality control (IQC), and external quality assessment (EQA).

CD4+ T cell enumeration in HIV infection with limited resources

The incidence of human immunodeficiency virus (HIV) infection continues to increase in South Africa. Limited resources are available for diagnosis and management of the disease and the development of affordable strategies is required. Absolute CD4 counts are used locally predominantly to monitor disease progression and institute prophylaxis against opportunistic infections. A dramatic increase in demand for CD4 counts prompted an investigation for a more cost-effective flow cytometry method than those currently recommended by the Centers for Disease Control (CDC). CD4 counts generated by two different single tube methods using CD3/CD4/CD8 [1(3)] and CD4 [1(1)] antibodies, respectively, were compared to the CDC recommended 6 tube 2 colour panel [6(2)]. Whole blood analysis using the Coulter Multi-Q-Prep system and an Epics XL Flow Cytometer (Coulter, Hialeah, FL) was performed for each of the three methods. Random samples from HIV positive adult patients were compared. A mean difference in the absolute CD4 counts of less than 10x10(6)/l was generated by both of the alternative panels when compared with the 6(2) panel. The precision of the three methods is comparable. In reagents alone, the 1(3) and 1(1) methods represent a cost saving of 76% and 93%, respectively, over the 6(2) method. The 1(3) and 1(1) panels would permit more affordable CD4 counts to be determined by the gold standard methodology of flow cytometry with no clinically significant sacrifices in accuracy or precision.

African regional external quality assessment for CD4 T‐cell enumeration: Development, outcomes, and performance of laboratories

An independent African Regional External Quality Assessment Scheme (AFREQAS) was implemented from Johannesburg. The aim was to establish a network of CD4 laboratories supporting HIV/AIDS anti‐retroviral therapy programs and improve the quality of regional CD4 testing with EQA assessment, feedback, remedial action, and technical training. The overall performance from 2002 to 2006 (Trials 1–20) is reported, together with cumulative longitudinal performance of the different CD4 methods used.

Melatonin has no effect on the growth, morphology or cell cycle of human breast cancer (MCF-7), cervical cancer (HeLa), osteosarcoma (MG-63) or lymphoblastoid (TK6) cells

Melatonin was previously shown to inhibit proliferation of MCF-7 human breast cancer cells. In this study the effect of melatonin on MCF-7 cells was further examined, while human cervical carcinoma (HeLa), osteosarcoma (MG-63) and lymphoblastoid (TK6) cells were tested for the first time. Haemocytometer counts, DNA content, flow cytometry and indirect immunofluorescence for nucleolar proteins, actin and beta-tubulin showed no differences in the growth, cell cycle or morphology between melatonin-exposed and control cells. The direct antiproliferative effect of melatonin thus seems to be confined to a melatonin-responsive subclone of MCF-7 cells and not applicable to the majority of cancer cells.

CD8/CD38 activation yields important clinical information of effective antiretroviral therapy: Findings from the first year of the CIPRA‐SA cohort

The affordable, reliable, and simplified flow cytometric method on single platform with 2‐color (CD45+, CD4+) Panleucogating (PLG‐CD4) is used to monitor HIV+ patients on antiretroviral therapy (ART) in South Africa (SA). Viral load (VL) assays are also used to monitor response to ART but are labor‐intensive with costs that, in the long run, may remain unsustainable. Cheaper and quicker alternatives to VL such as CD38 tests on CD8+ T cells may therefore play a role in securing the continuation of ART programs in high‐volume resource‐restricted settings.

Natural killer cell activation distinguishes Mycobacterium tuberculosis-mediated immune reconstitution syndrome from chronic HIV and HIV/MTB coinfection.

BackgroundWith increased access to antiretroviral treatment (ART), immune reconstitution inflammatory syndrome (IRIS) in Mycobacterium tuberculosis (MTB)–infected populations remains a clinical challenge. We studied a cross-sectional cohort of HIV-infected subjects in Johannesburg (South Africa) to help define the immune correlates that best distinguish IRIS from ongoing MTB cases. MethodsWe studied HIV+ subjects developing MTB-related unmasking tuberculosis-related immune reconstitution inflammatory syndrome (uTB-IRIS) after ART initiation; control groups were subjects with HIV and HIV/tuberculosis-coinfected subjects with comparable ART treatment. Testing was conducted with whole blood–based 4-color flow cytometry and plasma-based Luminex cytokine assessment. ResultsNatural killer cell activation, C-reactive protein, and interleukin 8 serum concentration were significantly higher in uTB-IRIS subjects compared with both control groups. In addition, all MTB-coinfected subjects, independent of clinical presentation, had higher neutrophils and T-cell activation, together with lower lymphocytes, CD4+ T-cell, and myeloid dendritic cell counts. Using conditional inference tree analysis, we show that elevated natural killer cell activation in combination with lymphocyte count characterizes the immunological profile of uTB-IRIS. ConclusionOur results support a role for innate immune effectors in the immunopathogenesis of unmasking MTB-related IRIS and identify new immune parameters defining this pathology.

Increased microbial translocation in ≤180 days old perinatally human immunodeficiency virus-positive infants as compared with human immunodeficiency virus-exposed uninfected infants of similar age

Background: The effect of early versus deferred antiretroviral treatment (ART) on plasma concentration of lipopolysaccharide (LPS) and host LPS-binding molecules in human immunodeficiency virus (HIV)-infected infants up to 1 year of age was investigated. Methods: We evaluated 54 perinatally HIV-infected and 22 HIV-exposed uninfected infants (controls) at the first and second semester of life. All HIV-infected infants had a baseline CD4 of ≥25%, participated in the Comprehensive International Program of Research on AIDS Children with HIV Early Antiretroviral Therapy trial in South Africa, and were randomized in the following groups: group 1 (n = 20), ART deferred until CD4 <25% or severe HIV disease; and group 2 (n = 34), ART initiation within 6 to 12 weeks of age. LPS, endotoxin-core antibodies, soluble CD14 (sCD14), and LPS-binding protein (LBP) were measured in cryopreserved plasma. T-cell activation was measured in fresh whole blood. Results: At the first semester, LPS concentration was higher in HIV-infected infants than in controls; sCD14, LBP, and T-cell activation were higher in group 1 than in group 2 and controls. Although LPS was not correlated with study variables, viral load was positively associated with sCD14, LBP, or endotoxin-core antibodies. At the second semester, LPS was not detectable and elevated host LPS-control molecules values were sustained in all groups and in conjunction with ART in all HIV-infected infants. Conclusions: Although plasma concentration of LPS was higher in perinatally HIV-infected infants 0 to 6 months of age than in controls independent of ART initiation strategy, concentration of LPS-control molecules was higher in infants with deferred ART, suggesting the presence of increased microbial translocation in HIV-infected infants with sustained early viral replication.