Effects of Hydroxyurea Treatment on Haemolysis in Patients with Sickle Cell Disease at Muhimbili National Hospital, Tanzania

Tanzania is one of the countries with a high burden of sickle cell disease (SCD). Haemolytic anaemia is a clinical feature of SCD, and has been linked to major complications leading to morbidity and mortality. Treatment with hydroxyurea (HU) has shown to induce foetal haemoglobin (HbF) which in turn decreases haemolysis in patients. This study aimed to investigate the effects of HU on haemolysis in SCD patients attending Muhimbili National Hospital, Tanzania by comparing their haemolytic parameters before and after therapy. Patients meeting the criteria were initiated on HU therapy for 3 months. Two haemolytic biomarkers: unconjugated plasma bilirubin levels and absolute reticulocyte counts were measured from patients’ blood samples at baseline and after 3 months of HU therapy and compared. Both absolute reticulocyte counts and indirect plasma bilirubin levels significantly declined after HU therapy. Median (IQR) plasma unconjugated bilirubin levels dropped significantly from 20.3 (12.7–34.4) μmol/L to 14.5 (9.6–24.1) μmol/L (p < 0.001) and mean (SD) absolute reticulocyte counts dropped significantly from 0.29 (0.1) x 109/L to 0.17 (0.1) x 109/L (p < 0.001) after therapy, thus, a decline in both haemolytic biomarkers after treatment was observed. This study found a potential for use of HU therapy in managing SCD patients in our settings evidenced by improvements in their haemolytic parameters. Clinical trials with a lager sample size conducted for a longer time period would be beneficial in guiding towards the inclusion of HU in treatment protocols for the Tanzanian population. Keywords: Sickle cell disease; hydroxyurea; haemolysis; foetal haemoglobin &nbsp

Identifying genetic variants and pathways associated with extreme levels of fetal hemoglobin in sickle cell disease in Tanzania

Background Sickle cell disease (SCD) is a blood disorder caused by a point mutation on the beta globin gene resulting in the synthesis of abnormal hemoglobin. Fetal hemoglobin (HbF) reduces disease severity, but the levels vary from one individual to another. Most research has focused on common genetic variants which differ across populations and hence do not fully account for HbF variation. Methods We investigated rare and common genetic variants that influence HbF levels in 14 SCD patients to elucidate variants and pathways in SCD patients with extreme HbF levels (≥7.7% for high HbF) and (≤2.5% for low HbF) in Tanzania. We performed targeted next generation sequencing (Illumina_Miseq) covering exonic and other significant fetal hemoglobin-associated loci, including BCL11A, MYB, HOXA9, HBB, HBG1, HBG2, CHD4, KLF1, MBD3, ZBTB7A and PGLYRP1. Results Results revealed a range of genetic variants, including bi-allelic and multi-allelic SNPs, frameshift insertions and deletions, some of which have functional importance. Notably, there were significantly more deletions in individuals with high HbF levels (11% vs 0.9%). We identified frameshift deletions in individuals with high HbF levels and frameshift insertions in individuals with low HbF. CHD4 and MBD3 genes, interacting in the same sub-network, were identified to have a significant number of pathogenic or non-synonymous mutations in individuals with low HbF levels, suggesting an important role of epigenetic pathways in the regulation of HbF synthesis. Conclusions This study provides new insights in selecting essential variants and identifying potential biological pathways associated with extreme HbF levels in SCD interrogating multiple genomic variants associated with HbF in SCD

Fetal Hemoglobin is Associated with Peripheral Oxygen Saturation in Sickle Cell Disease in Tanzania.

Fetal hemoglobin (HbF) and peripheral hemoglobin oxygen saturation (SpO2) both predict clinical severity in sickle cell disease (SCD), while reticulocytosis is associated with vasculopathy, but there are few data on mechanisms. HbF, SpO2 and routine clinical and laboratory measures were available in a Tanzanian cohort of 1175 SCD individuals aged≥5years and the association with SpO2 (as response variable transformed to a Poisson distribution) was assessed by negative binomial model with age and sex as covariates. Increase in HbF was associated with increased SpO2 (rate ratio, RR=1.19; 95% confidence intervals [CI] 1.04, 1.37 per natural log unit of HbF; p=0.0004). In univariable analysis, SpO2 was inversely associated with age, reticulocyte count, and log (total bilirubin) and directly with pulse, SBP, hemoglobin, and log(HbF). In multivariable regression log(HbF) (RR 1.191; 95%CI 1.04, 1.37; p=0.013), pulse (RR 1.01; 95%CI 1.00, 1.01; p=0.026), SBP (RR 1.008; 95%CI 1.00, 1.02; p=0.014), and hemoglobin (1.120; 95%CI 1.05, 1.19; p=0.001) were positively and independently associated with SpO2 while reticulocyte count (RR 0.985; 95%CI 0.97, 0.99; p=0.019) was independently inversely associated with SpO2. In SCD, improving SpO2, in part through cardiovascular compensation and associated with reduced reticulocytosis, may be a mechanism by which HbF reduces disease severity

Genome wide association study of fetal hemoglobin in sickle cell anemia in Tanzania.

BACKGROUND: Fetal hemoglobin (HbF) is an important modulator of sickle cell disease (SCD). HbF has previously been shown to be affected by variants at three loci on chromosomes 2, 6 and 11, but it is likely that additional loci remain to be discovered. METHODS AND FINDINGS: We conducted a genome-wide association study (GWAS) in 1,213 SCA (HbSS/HbSβ0) patients in Tanzania. Genotyping was done with Illumina Omni2.5 array and imputation using 1000 Genomes Phase I release data. Association with HbF was analysed using a linear mixed model to control for complex population structure within our study. We successfully replicated known associations for HbF near BCL11A and the HBS1L-MYB intergenic polymorphisms (HMIP), including multiple independent effects near BCL11A, consistent with previous reports. We observed eight additional associations with P<10(-6). These associations could not be replicated in a SCA population in the UK. CONCLUSIONS: This is the largest GWAS study in SCA in Africa. We have confirmed known associations and identified new genetic associations with HbF that require further replication in SCA populations in Africa

A ten year review of the sickle cell program in Muhimbili National Hospital, Tanzania.

BACKGROUND: Africa has the highest burden of Sickle cell disease (SCD) but there are few large, systematic studies providing reliable descriptions of the disease spectrum. Tanzania, with 11,000 SCD births annually, established the Muhimbili Sickle Cell program aiming to improve understanding of SCD in Africa. We report the profile of SCD seen in the first 10 years at Muhimbili National Hospital (MNH). METHODS: Individuals seen at MNH known or suspected to have SCD were enrolled at clinic and laboratory testing for SCD, haematological and biochemical analyses done. Ethnicity was self-reported. Clinical and laboratory features of SCD were documented. Comparison was made with non-SCD population as well as within 3 different age groups (< 5, 5-17 and ≥ 18 years) within the SCD population. RESULTS: From 2004 to 2013, 6397 individuals, 3751 (58.6%) SCD patients, were enrolled, the majority (47.4%) in age group 5-17 years. There was variation in the geographical distribution of SCD. Individuals with SCD compared to non-SCD, had significantly lower blood pressure and peripheral oxygen saturation (SpO2). SCD patients had higher prevalence of severe anemia, jaundice and desaturation (SpO2 < 95%) as well as higher levels of reticulocytes, white blood cells, platelets and fetal hemoglobin. The main causes of hospitalization for SCD within a 12-month period preceding enrolment were pain (adults), and fever and severe anemia (children). When clinical and laboratory features were compared in SCD within 3 age groups, there was a progressive decrease in the prevalence of splenic enlargement and an increase in prevalence of jaundice. Furthermore, there were significant differences with monotonic trends across age groups in SpO2, hematological and biochemical parameters. CONCLUSION: This report confirms that the wide spectrum of clinical expression of SCD observed elsewhere is also present in Tanzania, with non-uniform geographical distribution across the country. Age-specific analysis is consistent with different disease-patterns across the lifespan

Specific Receptor Usage in Plasmodium falciparum Cytoadherence Is Associated with Disease Outcome

Our understanding of the basis of severe disease in malaria is incomplete. It is clear that pathology is in part related to the pro-inflammatory nature of the host response but a number of other factors are also thought to be involved, including the interaction between infected erythrocytes and endothelium. This is a complex system involving several host receptors and a major parasite-derived variant antigen (PfEMP1) expressed on the surface of the infected erythrocyte membrane. Previous studies have suggested a role for ICAM-1 in the pathology of cerebral malaria, although these have been inconclusive. In this study we have examined the cytoadherence patterns of 101 patient isolates from varying clinical syndromes to CD36 and ICAM-1, and have used variant ICAM-1 proteins to further characterise this adhesive phenotype. Our results show that increased binding to CD36 is associated with uncomplicated malaria while ICAM-1 adhesion is raised in parasites from cerebral malaria cases

Perspectives on Building Sustainable Newborn Screening Programs for Sickle Cell Disease: Experience from Tanzania

The prevalence of sickle cell disease is high in Africa, with significant public health effects on the affected countries. Many of the countries with the highest prevalence of the disease also have poor health care systems and a high burden of infectious diseases with many other competing health care priorities. Although considerable efforts have been made to implement newborn screening for sickle cell disease programs in Africa, coverage is still low. Tanzania has one of the highest birth prevalence of children with sickle cell disease in Africa. In 2015, the country implemented a pilot project for Newborn Screening for Sickle Cell Disease to assess feasibility. Several efforts have been made afterwards to continue providing the screening services as well as related comprehensive care services. Using qualitative methods, we conducted in-depth interviews and focus group discussions with policy makers (n = 4), health care providers (n = 21) and families (n = 15) to provide an analysis of their experiences and perspectives on efforts to expand and sustain newborn screening for sickle cell disease and related comprehensive care services in the country. Thematic content analysis was used to analyze the data through the framework analysis method. The findings have demonstrated both the opportunities and areas that need addressing in the implementation and sustainability of the services in low resource settings. A key area of strengthening is full integration of the services in countries’ health care systems to facilitate the coverage, accessibility and affordability of the services. Although the coverage of newborn screening services for sickle cell disease is still low, efforts at the local level to sustain the implementation of the programs and related comprehensive care services are encouraging and can be used as a model for other programs implemented in low resources settings

Genetic variants at HbF-modifier loci moderate anemia and leukocytosis in sickle cell disease in Tanzania

Fetal hemoglobin (HbF) is a recognized modulator of sickle cell disease (SCD) severity. HbF levels are strongly influenced by genetic variants at three major genetic loci, Xmn1-HBG2, HMIP-2, and BCL11A, but the effect of these loci on the hematological phenotype in SCD, has so far not been investigated. In a cohort of individuals with SCD in Tanzania (HbSS and HbS/β° thalassemia, n = 726, aged 5 or older), HbF levels were positively correlated with hemoglobin, red blood cell (RBC) indices, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH), and negatively with white blood cell (WBC) and platelet counts (all P &lt;0.0001). We subsequently assessed the contribution of the three HbF modifier loci and detected diverse effects, including a reduction in anemia, leukocytosis, and thrombocytosis associated with certain HbF-promoting alleles. The presence of the 'T' allele at Xmn1-HBG2 led to a significant increase in hemoglobin (P = 9.8 × 10-3) but no changes in cellular hemoglobin content. Xmn1-HBG2 'T' also has a weak effect decreasing WBC (P = 0.06) and platelet (P = 0.06) counts. The BCL11A variant (rs11886868-'C') increases hemoglobin (P = 2 × 10-3) and one of the HBS1L-MYB variants decreases WBC values selectively (P = 2.3 × 10-4). The distinct pattern of effects of each variant suggests that both, disease alleviation through increased HbF production, and 'pleiotropic' effects on blood cells, are involved, affecting a variety of pathways.</p