Comparison of immune responses induced by Bacillus calmette-Guerin when given at birth or at 6 weeks of age in Ugandan Infants

Includes bibliographical references.In Uganda, infants delivered at a health care facility receive the tuberculosis vaccine Bacillus Calmette-Guerin (BCG) on the first day of life. Infants delivered at home receive BCG at their first health care facility visit at 6 weeks of age. Our aim was to determine the effect of this delay in BCG administration on the induced immune response. Our hypothesis was that infants who received BCG at 6 weeks of age would show an enhanced BCG-induced T cell immunity compared to infants vaccinated at birth. We optimised several polychromatic flow cytometry reagent panels to compare BCG-specific immunity in 9 months-old infants who had received the vaccine either at birth or at 6 weeks of age. We used a 12-hour whole blood intracellular cytokine/cytotoxic marker assay to measure T cell-associated cytokine expression and memory phenotypes. We also compared the capacity of BCGspecific T cells to proliferate and produce cytokines upon antigenic stimulation with a 6-day proliferation assay. Finally, we measured plasma soluble cytokines levels in the two groups of infant using multiplex assay. We enrolled 92 infants: 50 had received BCG at birth and 42 at 6 weeks of age. BCG induced predominantly CD4⁺ T cell responses, and lesser CD8⁺ T cell responses, in both groups. Birth vaccination was associated with greater induction of CD4⁺ and CD8⁺ T cells expressing either IFN-γ alone, or IFN-γ together with perforin, compared with delayed vaccination. Further, birth vaccination induced proliferating cells that had greater capacity to produce IFN-γ, TNF-α and IL-2 together, compared with delayed vaccination. In conclusion, distinct patterns of T cell induction occurred when BCG was given at birth and at 6 weeks of age. We propose that this diversity might impact protection against tuberculosis. Our results differ from those of delayed BCG vaccination studies in South Africa and the Gambia, suggesting geographical and population heterogeneity may affect BCG-induced T cell response

Vδ2+ T cell response to malaria correlates with protection from infection but is attenuated with repeated exposure.

Vδ2+ γδ T cells are semi-innate T cells that expand markedly following P. falciparum (Pf) infection in naïve adults, but are lost and become dysfunctional among children repeatedly exposed to malaria. The role of these cells in mediating clinical immunity (i.e. protection against symptoms) to malaria remains unclear. We measured Vδ2+ T cell absolute counts at acute and convalescent malaria timepoints (n = 43), and Vδ2+ counts, cellular phenotype, and cytokine production following in vitro stimulation at asymptomatic visits (n = 377), among children aged 6 months to 10 years living in Uganda. Increasing age was associated with diminished in vivo expansion following malaria, and lower Vδ2 absolute counts overall, among children living in a high transmission setting. Microscopic parasitemia and expression of the immunoregulatory markers Tim-3 and CD57 were associated with diminished Vδ2+ T cell pro-inflammatory cytokine production. Higher Vδ2 pro-inflammatory cytokine production was associated with protection from subsequent Pf infection, but also with an increased odds of symptoms once infected. Vδ2+ T cells may play a role in preventing malaria infection in children living in endemic settings; progressive loss and dysfunction of these cells may represent a disease tolerance mechanism that contributes to the development of clinical immunity to malaria

Rapid gene fusion testing using the NanoString nCounter platform to improve pediatric leukemia diagnoses in Sub-Saharan Africa

Risk stratification and molecular targeting have been key to increasing cure rates for pediatric cancers in high-income countries. In contrast, precise diagnosis in low-resource settings is hindered by insufficient pathology infrastructure. The Global HOPE program aims to improve outcomes for pediatric cancer in Sub-Saharan Africa (SSA) by building local clinical care and diagnostic capacity. This study aimed to assess the feasibility of implementing molecular assays to improve leukemia diagnoses in SSA. Custom NanoString nCounter gene fusion assays, previously validated in the US, were used to test samples from suspected leukemia patients. The NanoString platform was chosen due to relatively low cost, minimal technical and bioinformatics expertise required, ability to test sub-optimal RNA, and rapid turnaround time. Fusion results were analyzed blindly, then compared to morphology and flow cytometry results. Of 117 leukemia samples, 74 were fusion-positive, 30 were negative, 7 were not interpretable, and 6 failed RNA quality. Nine additional samples were negative for leukemia by flow cytometry and negative for gene fusions. All 74 gene fusions aligned with the immunophenotype determined by flow cytometry. Fourteen samples had additional information available to further confirm the accuracy of the gene fusion results. The testing provided a more precise diagnosis in >60% of cases, and 9 cases were identified that could be treated with an available tyrosine kinase inhibitor, if detected at diagnosis. As risk-stratified and targeted therapies become more available in SSA, implementing this testing in real-time will enable the treatment of pediatric cancer to move toward incorporating risk stratification for optimized therapy

Re-testing as a method of implementing external quality assessment program for COVID-19 real time PCR testing in Uganda.

BackgroundSignificant milestones have been made in the development of COVID19 diagnostics Technologies. Government of the republic of Uganda and the line Ministry of Health mandated Uganda Virus Research Institute to ensure quality of COVID19 diagnostics. Re-testing was one of the methods initiated by the UVRI to implement External Quality assessment of COVID19 molecular diagnostics.Methodparticipating laboratories were required by UVRI to submit their already tested and archived nasopharyngeal samples and corresponding meta data. These were then re-tested at UVRI using the WHO Berlin protocol, the UVRI results were compared to those of the primary testing laboratories in order to ascertain performance agreement for the qualitative & quantitative results obtained. Ms Excel window 12 and GraphPad prism ver 15 was used in the analysis. Bar graphs, pie charts and line graphs were used to compare performance agreement between the reference Laboratory and primary testing Laboratories.ResultsEleven (11) Ministry of Health/Uganda Virus Research Institute COVID19 accredited laboratories participated in the re-testing of quality control samples. 5/11 (45%) of the primary testing laboratories had 100% performance agreement with that of the National Reference Laboratory for the final test result. Even where there was concordance in the final test outcome (negative or positive) between UVRI and primary testing laboratories, there were still differences in CT values. The differences in the Cycle Threshold (CT) values were insignificant except for Tenna & Pharma Laboratory and the UVRI(p = 0.0296). The difference in the CT values were not skewed to either the National reference Laboratory(UVRI) or the primary testing laboratory but varied from one laboratory to another. In the remaining 6/11 (55%) laboratories where there were discrepancies in the aggregate test results, only samples initially tested and reported as positive by the primary laboratories were tested and found to be false positives by the UVRI COVID19 National Reference Laboratory.ConclusionFalse positives were detected from public, private not for profit and private testing laboratories in almost equal proportion. There is need for standardization of molecular testing platforms in Uganda. There is also urgent need to improve on the Laboratory quality management systems of the molecular testing laboratories in order to minimize such discrepancies

Line graphs comparing CT values of N genes of quality control samples from different facilities and the UVRI COVID19 National Reference Laboratory.

3A)CT value of Qc samples from Test & fly compared to UVRI, B)CT values of QC samples from Bwindi Community Hospital compared to UVRI, C) CT value of QC samples from MAIA compared to UVRI, D) CT values of QC samples from Mulago NRH compared to UVRI, E)CT values of QC samples from Safari Laboratory compared to that of UVRI, F)CT values of QC samples from Gulu University Laboratory compared to that of UVRI, G)CT values of QC samples from Tenna & Pharma Laboratory compared to that of UVRI.</p

Comparison of CT values of samples with discrepant results between the UVRI COVID19 National Reference Laboratory and the primary testing laboratories.

Comparison of CT values of samples with discrepant results between the UVRI COVID19 National Reference Laboratory and the primary testing laboratories.</p

Performance agreement between UVRI and initial testing laboratories.

Key: UVRI:Uganda Virus Research Institute. Examina: Examina Medical Laboratory. MAIA: MAIA medicals. T & F: Test and Fly Laboratory. KRRH: Kabale Regional Referral Hospital. S.Day: Same Day Laboratory. BCH: Bwindi Community Hospital. M.SAFE: Medsafe Hospital Limited. GUV: Gulu University multifunctional Laboratory.</p

S1 Data -

BackgroundSignificant milestones have been made in the development of COVID19 diagnostics Technologies. Government of the republic of Uganda and the line Ministry of Health mandated Uganda Virus Research Institute to ensure quality of COVID19 diagnostics. Re-testing was one of the methods initiated by the UVRI to implement External Quality assessment of COVID19 molecular diagnostics.Methodparticipating laboratories were required by UVRI to submit their already tested and archived nasopharyngeal samples and corresponding meta data. These were then re-tested at UVRI using the WHO Berlin protocol, the UVRI results were compared to those of the primary testing laboratories in order to ascertain performance agreement for the qualitative & quantitative results obtained. Ms Excel window 12 and GraphPad prism ver 15 was used in the analysis. Bar graphs, pie charts and line graphs were used to compare performance agreement between the reference Laboratory and primary testing Laboratories.ResultsEleven (11) Ministry of Health/Uganda Virus Research Institute COVID19 accredited laboratories participated in the re-testing of quality control samples. 5/11 (45%) of the primary testing laboratories had 100% performance agreement with that of the National Reference Laboratory for the final test result. Even where there was concordance in the final test outcome (negative or positive) between UVRI and primary testing laboratories, there were still differences in CT values. The differences in the Cycle Threshold (CT) values were insignificant except for Tenna & Pharma Laboratory and the UVRI(p = 0.0296). The difference in the CT values were not skewed to either the National reference Laboratory(UVRI) or the primary testing laboratory but varied from one laboratory to another. In the remaining 6/11 (55%) laboratories where there were discrepancies in the aggregate test results, only samples initially tested and reported as positive by the primary laboratories were tested and found to be false positives by the UVRI COVID19 National Reference Laboratory.ConclusionFalse positives were detected from public, private not for profit and private testing laboratories in almost equal proportion. There is need for standardization of molecular testing platforms in Uganda. There is also urgent need to improve on the Laboratory quality management systems of the molecular testing laboratories in order to minimize such discrepancies.</div

Targets and genes reported by platforms of various testing laboratories in Uganda.

Key: ORF1 = open reading frame one. E-gene = Envelope gene. N-gene = Nucleocapsid gene. IC = Internal Control. ORF1ab = Open Reading Frame 1 ab.</p