Luminex-based virtual crossmatching for renal transplantation in South Africa

Background. Current practice in the Johannesburg renal transplantation programme is to perform a transplant when the patient’s complement-dependent cytotoxicity and flow cytometric crossmatches are negative. However, even in patients with negative crossmatches early graft rejections have occurred. We retrospectively evaluated the use of Luminex anti-human leukocyte antigen (HLA) antibody detection technology, often termed ‘virtual crossmatching’, compared with the flow cytometric crossmatch, for predicting graft outcome in renal transplant patients. Methods. Sixty-four recipients were crossmatched against multiple donors during their routine work-up for transplant (111 crossmatches); 17 of these patients received transplants during the study period. Anti-HLA antibody detection was performed using Luminex technology and the results were compared with the flow cytometric crossmatch results and with short-term graft success. Results. Compared with flow cytometric crossmatch results, the sensitivity and specificity of Luminex virtual crossmatching was 85.7% and 90.7% for the T-cell crossmatch and 100% and 87.2% for the B-cell crossmatch. Both the sensitivity and specificity of Luminex for predicting short-term graft success were 100%. Conclusions. Strong evidence is provided that single-antigen assays provide improved sensitivity to detect clinically relevant anti-HLA antibodies and can reliably be used to predict shortterm graft success. We recommend incorporation of single-antigen Luminex methodology into the routine work-up algorithm of renal transplant recipients in South Africa.S Afr Med J, 2012;102:40-4

Multi-analyte profiling of ten cytokines in South African HIV-infected patients with Immune Reconstitution Inflammatory Syndrome (IRIS)

Abstract Background Immune reconstitution inflammatory syndrome (IRIS) is an important complication of HAART in sub-Saharan Africa, where opportunistic infections (OIs) including mycobacteria and cryptococcus are common. The immune system's role in HIV infected patients is complex with cytokine expression strongly influencing HIV infection and replication. Methods We determined the expression patterns of 10 cytokines by Luminex multi-analyte profiling in 17 IRIS nested case-control pairs participating in a prospective South African cohort initiating anti-retroviral therapy. Results Interferon-gamma (IFN-γ) expression was significantly elevated in IRIS cases compared to controls (median 9.88 pg/ml versus 2.68 pg/ml, respectively, P = 0.0057), while other cytokines displayed non-significant differences in expression. Significant correlation was observed between IL-6, IL-10, and IFN-γ expression in the IRIS patients. Conclusions Significantly increased expression levels of IFN-γ suggest that this cytokine possibly plays a role in IRIS pathology and is a potential diagnostic marker

Polychromatic immunophenotypic characterization of T cell profiles among HIV-infected patients experiencing immune reconstitution inflammatory syndrome (IRIS)

Abstract Objective To immunophenotype CD4+ and CD8+ T cell sub-populations in HIV-associated immune reconstitution inflammatory syndrome (IRIS). Design Nested case-control immunological study. Methods ART-naïve HIV-infected patients were prospectively observed for IRIS during the first 6 months of ART. Twenty-two IRIS cases and 22 ART-duration matched controls were sampled for T cell immunophenotyping. Results IRIS cases demonstrated significantly lower CD4 cell counts compared to controls (baseline: 79 versus 142, p = 0.02; enrollment: 183 versus 263, p = 0.05, respectively) with no differences in HIV RNA levels. Within CD4+T cells, cases exhibited more of an effector memory phenotype compared to controls (40.8 versus 27.0%, p = 0.20), while controls trended towards a central memory phenotype (43.8 versus 30.8%, p = 0.07). Within CD8+ T cells, controls exhibited more central memory (13.9 versus 7.81%, p = 0.01, respectively) and effector (13.2 versus 8.8%, p = 0.04, respectively) phenotypes compared to cases, whereas cases demonstrated more terminal effectors than controls (28.8 versus 15.1%, p = 0.05). Cases demonstrated increased activation of CD8+ T cell effector memory, terminal effector, and effector subsets than controls (p = 0.04, 0.02, and 0.02, respectively). Conclusion CD4+ and CD8+ T cell subset maturational phenotypes were heterogeneous among IRIS cases and controls. However, IRIS cases demonstrated significant increases in activation of CD8+ T cell effector subpopulations

FOXP3 Expression Is Upregulated in CD4+T Cells in Progressive HIV-1 Infection and Is a Marker of Disease Severity

Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection.FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution.HIV infected individuals had significantly higher frequencies of CD4(+)FOXP3(+) T cells (median of 8.11%; range 1.33%-26.27%) than healthy controls (median 3.72%; range 1.3-7.5%; P = 0.002), despite having lower absolute counts of CD4(+)FOXP3(+) T cells. There was a significant positive correlation between the frequency of CD4(+)FOXP3(+) T cells and viral load (rho = 0.593 P = 0.003) and a significant negative correlation with CD4 count (rho = -0.423 P = 0.044). 48% of our patients had CD4 counts below 200 cells/microl and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%-26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4(+) T cells following antigenic or other stimulation.FOXP3 expression in the CD4(+) T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression

Luminex-based virtual crossmatching for renal transplantation in South Africa

Background. Current practice in the Johannesburg renal transplantation programme is to perform a transplant when the patient’s complement-dependent cytotoxicity and flow cytometric crossmatches are negative. However, even in patients with negative crossmatches early graft rejections have occurred. We retrospectively evaluated the use of Luminex anti-human leukocyte antigen (HLA) antibody detection technology, often termed ‘virtual crossmatching’, compared with the flow cytometric crossmatch, for predicting graft outcome in renal transplant patients. Methods. Sixty-four recipients were crossmatched against multiple donors during their routine work-up for transplant (111 crossmatches); 17 of these patients received transplants during the study period. Anti-HLA antibody detection was performed using Luminex technology and the results were compared with the flow cytometric crossmatch results and with short-term graft success. Results. Compared with flow cytometric crossmatch results, the sensitivity and specificity of Luminex virtual crossmatching was 85.7% and 90.7% for the T-cell crossmatch and 100% and 87.2% for the B-cell crossmatch. Both the sensitivity and specificity of Luminex for predicting short-term graft success were 100%. Conclusions. Strong evidence is provided that single-antigen assays provide improved sensitivity to detect clinically relevant anti-HLA antibodies and can reliably be used to predict short-term graft success. We recommend incorporation of single-antigen Luminex methodology into the routine work-up algorithm of renal transplant recipients in South Africa

Immunogenicity of a combined schedule of trivalent oral and inactivated polio vaccines in South African infants

Background: South Africa transitioned from using live-attenuated trivalent oral polio vaccine (tOPV), to a combination of tOPV and inactivated polio vaccine (IPV) in April 2009. We evaluated the immunogenicity of the South African combined tOPV-IPV schedule versus the tOPV-only schedule in South African infants. Methods: Serum samples of HIV-unexposed infants were analysed retrospectively from two cohorts; infants enrolled from April 2005 through June 2006 and infants enrolled from December 2009 to April 2010. The primary vaccination series of the tOPV-only schedule included doses at birth, 6, 10 and 14 weeks, and the tOPV-IPV schedule included tOPV at birth and 6 weeks and IPV at 6, 10 and 14 weeks. Serum polio neutralising antibody titres to serotype-1, serotype-2 and serotype-3 were evaluated in infants at 18 weeks of age. Results: Infants who received the tOPV-IPV schedule had higher GMTs than infants who received tOPV-only for serotype-2 (9.63 vs. 8.80, P < 0.001) and serotype-3 (10.01 vs. 8.53, P < 0.001), as well as higher sero-protective titres for serotype-1 (100% vs. 96%, P = 0.014). Conclusion: Our data support the option of the South African combined polio vaccination schedule as an immunogenic option for a combined schedule

Multi-analyte profiling of ten cytokines in South African HIV-infected patients with Immune Reconstitution Inflammatory Syndrome (IRIS)

Abstract Background Immune reconstitution inflammatory syndrome (IRIS) is an important complication of HAART in sub-Saharan Africa, where opportunistic infections (OIs) including mycobacteria and cryptococcus are common. The immune system's role in HIV infected patients is complex with cytokine expression strongly influencing HIV infection and replication. Methods We determined the expression patterns of 10 cytokines by Luminex multi-analyte profiling in 17 IRIS nested case-control pairs participating in a prospective South African cohort initiating anti-retroviral therapy. Results Interferon-gamma (IFN-γ) expression was significantly elevated in IRIS cases compared to controls (median 9.88 pg/ml versus 2.68 pg/ml, respectively, P = 0.0057), while other cytokines displayed non-significant differences in expression. Significant correlation was observed between IL-6, IL-10, and IFN-γ expression in the IRIS patients. Conclusions Significantly increased expression levels of IFN-γ suggest that this cytokine possibly plays a role in IRIS pathology and is a potential diagnostic marker.</p