Alternative pre-analytic sample handling techniques for glucose measurement in the absence of fluoride tubes in low resource settings.

INTRODUCTION: Sodium fluoride (NaF) tubes are the recommended tubes for glucose measurements, but these are expensive, have limited number of uses, and are not always available in low resource settings. Alternative sample handling techniques are thus needed. We compared glucose stability in samples collected in various tubes exposed to different pre-analytical conditions in Uganda. METHODS: Random (non-fasted) blood samples were drawn from nine healthy participants into NaF, Ethylenediaminetetraacetic acid (EDTA), and plain serum tubes. The samples were kept un-centrifuged or centrifuged with plasma or serum pipetted into aliquots, placed in cool box with ice or at room temperature and were stored in a permanent freezer after 0, 2, 6, 12 and 24 hours post blood draw before glucose analysis. RESULTS: Rapid decline in glucose concentrations was observed when compared to baseline in serum (declined to 64%) and EDTA-plasma (declined to 77%) after 6 hours when samples were un-centrifuged at room temperature whilst NaF-plasma was stable after 24 hours in the same condition. Un-centrifuged EDTA-plasma kept on ice was stable for up to 6 hours but serum was not stable (degraded to 92%) in the same conditions. Early centrifugation prevented glucose decline even at room temperature regardless of the primary tube used with serum, EDTA-plasma and NaF-plasma after 24 hours. CONCLUSION: In low resource settings we recommend use of EDTA tubes placed in cool box with ice and analysed within 6 hours as an alternative to NaF tubes. Alternatively, immediate separation of blood with manual hand centrifuges will allow any tube to be used even in remote settings with no electricity

Schistosoma mansoni infection alters the host pre-vaccination environment resulting in blunted Hepatitis B vaccination immune responses

Schistosomiasis is a disease caused by parasitic flatworms of the Schistosoma spp., and is increasingly recognized to alter the immune system, and the potential to respond to vaccines. The impact of endemic infections on protective immunity is critical to inform vaccination strategies globally. We assessed the influence of Schistosoma mansoni worm burden on multiple host vaccine-related immune parameters in a Ugandan fishing cohort (n = 75) given three doses of a Hepatitis B (HepB) vaccine at baseline and multiple timepoints post-vaccination. We observed distinct differences in immune responses in instances of higher worm burden, compared to low worm burden or non-infected. Concentrations of pre-vaccination serum schistosome-specific circulating anodic antigen (CAA), linked to worm burden, showed a significant bimodal distribution associated with HepB titers, which was lower in individuals with higher CAA values at month 7 post-vaccination (M7). Comparative chemokine/cytokine responses revealed significant upregulation of CCL19, CXCL9 and CCL17 known to be involved in T cell activation and recruitment, in higher CAA individuals, and CCL17 correlated negatively with HepB titers at month 12 post-vaccination. We show that HepB-specific CD4(+) T cell memory responses correlated positively with HepB titers at M7. We further established that those participants with high CAA had significantly lower frequencies of circulating T follicular helper (cTfh) subpopulations pre- and post-vaccination, but higher regulatory T cells (Tregs) post-vaccination, suggesting changes in the immune microenvironment in high CAA could favor Treg recruitment and activation. Additionally, we found that changes in the levels of innate-related cytokines/chemokines CXCL10, IL-1 & beta;, and CCL26, involved in driving T helper responses, were associated with increasing CAA concentration. This study provides further insight on pre-vaccination host responses to Schistosoma worm burden which will support our understanding of vaccine responses altered by pathogenic host immune mechanisms and memory function and explain abrogated vaccine responses in communities with endemic infections.Author summarySchistosomiasis drives host immune responses for optimal pathogen survival, potentially altering host responses to vaccine-related antigen. Chronic schistosomiasis and co-infection with hepatotropic viruses are common in countries where schistosomiasis is endemic. We explored the impact of Schistosoma mansoni (S. mansoni) worm burden on Hepatitis B (HepB) vaccination of individuals from a fishing community in Uganda. We demonstrate that higher schistosome-specific antigen (circulating anodic antigen, CAA) concentration pre-vaccination, is associated with lower HepB antibody titers post-vaccination at month 7. We show higher pre-vaccination levels of CCL17 in instances of high CAA that negatively associate with HepB antibody titers month 12 post-vaccination and coincided with lower frequencies of circulating T follicular helper cell populations (cTfh), proliferating antibody secreting cells (ASCs), and higher frequencies of regulatory T cells (Tregs). We also show that monocyte function is important in HepB vaccine responses, and high CAA is associated with alterations in the early innate cytokine/chemokine microenvironment. Our findings suggest that in individuals with high CAA and likely high worm burden, schistosomiasis can create an environment that is polarized against optimal host immune responses to the vaccine, which puts many endemic communities at risk for infection against HepB and other diseases that are preventable by vaccines.Cancer Signaling networks and Molecular Therapeutic

Developing and improving laboratory methods, for diabetes diagnostics for sub-Saharan Africa

Background: Diabetes mellitus is a metabolic disorder associated with chronic hyperglycaemia resulting from defects in beta cell insulin secretion or insulin action. Diabetes is a growing problem in sub-Saharan Africa (SSA), but the availability of biochemical and immunological tests to help with the diagnosis and management of diabetes has been limited. In this thesis, we carried out two projects to improve laboratory diagnostic methods for diabetes in an African population. Project 1: HbA1c assays: HbA1c is commonly used for diagnosis and monitoring of diabetes. However, it is affected by a number of pre-analytical factors like haemoglobinopathies, which are highly prevalent in SSA. We aimed to: 1) determine the performance and comparability of four major HbA1c methodologies; immunoassay, capillary electrophoresis, boronate affinity, and anion exchange high performance liquid chromatography, and 2) determine the impact of haemoglobinopathies on HbA1c measurement by comparing to mean continuous glucose monitoring measurements. We found good agreement between all four HbA1c methodologies, suggesting whichever methodology a laboratory uses results are comparable. There was a good correlation (>0.80) between all HbA1c methodologies and mean glucose values from continuous glucose monitoring did not differ in those with haemoglobinopathies. Project 2: Islet autoantibodies: Glutamic acid decarboxylase autoantibody (GAD65Ab), tyrosine phosphatase autoantibody (IA-2A) and zinc transporter 8 autoantibody (ZNT8A) are autoimmune biomarkers in diabetes. Studies in SSA have shown associations between these islet autoantibodies and type 1 diabetes development, however presence / prevalence of autoantibodies was defined based on White Caucasian population-based cut offs, which may not be appropriate for the African population. We aimed to determine population based cut offs for positivity at the 97.5th and 99th percentile in the Ugandan population. The 97.5th and 99th percentile cut-offs were; GAD65Ab; ≥44.8, ≥144 U/ml; IA-2A ≥57, ≥107 U/ml; ZNT8A ≥97.6, ≥351 U/ml, respectively. Applying the manufacturer cut offs (GAD65Ab ≥5, IA-2A ≥7.5, ZNT8A ≥15) to the Ugandan non-diabetic controls, led to higher proportions classed as positive for a single autoantibody compared with using our cut offs: (5% GAD65Ab, 12% IA-2A, 8.4% ZNT8A). This implies that using the manufacturer’s cut-offs overestimates the number of positives in our population.National Institute for Health Research (NIHR

Identification and characterisation of diabetes in Uganda: protocol for the nested, population-based ‘Diabetes in low-resource Populations’ (DOP) Study

Introduction Sub-Saharan Africa is experiencing an increasing burden of diabetes, but there are little reliable data, particularly at the community level, on the true prevalence or why this condition affects young and relatively lean individuals. Moreover, the detection of diabetes in Africa remains poor, not only due to a lack of resources but because the performance of available diagnostic tests is unclear.Methods This research aims to (1) determine the prevalence and risk factors of diabetes in a rural Ugandan population, (2) use clinical and biochemical markers to define different diabetes phenotypes and (3) study the progression of diabetes in this population. We will also assess the utility of the widely used tests (glycated haemoglobin (HbA1c), oral glucose tolerance test (OGTT) and fasting glucose) in diagnosing diabetes.Design This is a population-based study nested within the longstanding general population cohort in southwestern Uganda. We will undertake a population survey to identify individuals with diabetes based on fasting glucose, HbA1c, OGTT results or history of pre-existing diabetes.Participants The study intends to enrol up to 11 700 individuals aged 18 years and above, residing within the study area and not pregnant or within 6 months post-delivery date. All participants will have detailed biophysical and biochemical/metabolic measurements. Individuals identified to have diabetes and a random selection of controls will have repeat tests to test reproducibility before referral and enrolment into a diabetic clinic. Participants will then be followed up for 1 year to assess the course of the disease, including response to therapy and diabetes-related complications.Conclusions These data will improve our understanding of the burden of diabetes in Uganda, the risk factors that drive it and underlying pathophysiological mechanisms, as well as better ways to detect this condition. This will inform new approaches to improve the prevention and management of diabetes.Ethics and dissemination This study protocol was approved by the Uganda Virus Research Institute Research Ethics Committee (REC) (number: G.C./127/21/09/858), the London School of Hygiene and Tropical Medicine REC (number: 26638) and the Uganda National Council for Science and Technology (protocol number: HS1791ES). Written informed consent will be obtained from all participants before being enrolled on to the study and conducting study-related procedures. Research findings will be disseminated in policy briefs, seminars, local and international conferences and publications in peer-reviewed open-access journals. As part of the dissemination plans, findings will also be disseminated to patient care groups and to clinicians.Trial registration number NCT05487079