Hydroxyurea with dose escalation for primary stroke risk reduction in children with sickle cell anaemia in Tanzania (SPHERE): an open-label, phase 2 trial.

BACKGROUND: Transcranial Doppler screening with chronic transfusions reduces stroke risk in children with sickle cell anaemia but is not feasible in low-resource settings. Hydroxyurea is an alternative treatment to decrease stroke risk. We aimed to estimate stroke risk in children with sickle cell anaemia in Tanzania and to determine the efficacy of hydroxyurea to decrease and prevent stroke. METHODS: We did an open-label, phase 2 trial (SPHERE) at Bugando Medical Centre, Mwanza, Tanzania. Children aged 2-16 years with a diagnosis of sickle cell anaemia confirmed by haemoglobin electrophoresis were eligible for enrolment. Participants had transcranial Doppler ultrasound screening by a local examiner. Participants with elevated Doppler velocities, either conditional (170-199 cm/s) or abnormal (≥200 cm/s), received oral hydroxyurea starting at 20 mg/kg once daily and escalated every 8 weeks by 5 mg/kg per day to the maximum tolerated dose. Participants with normal Doppler velocities (<170 cm/s) received usual care from the sickle cell anaemia clinic and were rescreened after 12 months to determine whether they qualified for treatment on trial. The primary endpoint was change in transcranial Doppler velocity from the baseline visit to after 12 months of hydroxyurea treatment, analysed in all patients who had paired baseline and follow-up measurements collected after 12 months of treatment. Safety was analysed in the per-protocol population (all participants who received study treatment). This study is registered with ClinicalTrials.gov, NCT03948867. FINDINGS: Between April 24, 2019, and April 9, 2020, 202 children were enrolled and had transcranial Doppler screening. Sickle cell anaemia was confirmed by DNA-based testing in 196 participants (mean age 6·8 years [SD 3·5], 103 [53%] were female, and 93 [47%] were male). At the baseline screening, 47 (24%) of 196 participants had elevated transcranial Doppler velocities (43 [22%] conditional, four [2%] abnormal); 45 initiated hydroxyurea at a mean dose of 20·2 mg/kg per day (SD 1·4) with escalation to a mean dose of 27·4 mg/kg per day (5·1) after 12 months. Treatment response was analysed after 12 months (± 1 month; median 11 months, IQR 11-12) and 24 months (±3 months; median 22 months, 22-22). Transcranial Doppler velocities decreased to a mean of 149 cm/s (SD 27) compared with 182 cm/s (12) at baseline, which was significantly lower than baseline (p<0·0001), with an average decline of 35 cm/s (SD 23) after 12 months of treatment in 42 participants with paired results available at baseline and 12 months. No clinical strokes occurred, and 35 (83%) of 42 participants reverted to normal transcranial Doppler velocities. Clinical adverse events were mild, and dose-limiting toxicities were uncommon. The most common grade 3 adverse events were malaria (12 [29%] episodes in 45 patients) and sepsis (13 [32%] episodes). There were three serious adverse events, none of which were treatment-related, and no treatment-related deaths occurred. INTERPRETATION: Children with sickle cell anaemia in Tanzania have a high baseline stroke risk. Hydroxyurea at the maximum tolerated dose significantly lowers transcranial Doppler velocities and reduces primary stroke risk. Transcranial Doppler screening plus hydroxyurea at the maximum tolerated dose is an effective stroke prevention strategy, supporting wider hydroxyurea access for patients with sickle cell anaemia across sub-Saharan Africa. FUNDING: American Society of Hematology, National Institutes of Health, Cincinnati Children's Research Foundation.

Stroke Prevention with Hydroxyurea Enabled through Research and Education: A Phase 2 Primary Stroke Prevention Trial in Sub-Saharan Africa.

INTRODUCTION: Stroke is a severe complication of sickle cell anemia (SCA), with devastating sequelae. Transcranial Doppler (TCD) ultrasonography predicts stroke risk, but implementing TCD screening with suitable treatment for primary stroke prevention in low-resource environments remains challenging. SPHERE (NCT03948867) is a prospective phase 2 open-label hydroxyurea trial for SCA in Tanzania. METHODS: After formal training and certification, local personnel screened children 2-16 years old; those with conditional (170-199 cm/s) or abnormal (≥200 cm/s) time-averaged mean velocities (TAMVs) received hydroxyurea at 20 mg/kg/day with dose escalation to maximum tolerated dose (MTD). The primary study endpoint is change in TAMV after 12 months of hydroxyurea; secondary endpoints include SCA-related clinical events, splenic volume and function, renal function, infections, hydroxyurea pharmacokinetics, and genetic modifiers. RESULTS: Between April 2019 and April 2020, 202 children (average 6.8 ± 3.5 years, 53% female) enrolled and underwent TCD screening; 196 were deemed eligible by DNA testing. Most had numerous previous hospitalizations and transfusions, with low baseline hemoglobin (7.7 ± 1.1 g/dL) and %HbF (9.3 ± 5.4%). Palpable splenomegaly was present at enrollment in 49 (25%); average sonographic splenic volume was 103 mL (range 8-1,045 mL). TCD screening identified 22% conditional and 2% abnormal velocities, with hydroxyurea treatment initiated in 96% (45/47) eligible children. CONCLUSION: SPHERE has built local capacity with high-quality research infrastructure and TCD screening for SCA in Tanzania. Fully enrolled participants have a high prevalence of elevated baseline TCD velocities and splenomegaly. SPHERE will prospectively determine the benefits of hydroxyurea at MTD for primary stroke prevention, anticipating expanded access to hydroxyurea treatment across Tanzania.

Primary resistance against integrase strand transfer inhibitors in integrase strand transfer inhibitor-naive patients failing first- and second-line ART in Tanzania.

INTRODUCTION: Sub-Saharan African countries are introducing integrase strand transfer inhibitors (INSTIs) in their ART programmes as the preferred first-line regimen, and dolutegravir is the INSTI of choice due to its potency, tolerability and high genetic barrier to resistance. Dolutegravir was introduced into the first-line ART regimen in Tanzania in 2019. However, there is a paucity of data on the occurrence of mutations in HIV lineages circulating in Tanzania. This study aimed to determine the prevalence of INSTI primary resistance mutations in Tanzanian patients exposed to ART but not INSTIs. METHODS: Plasma samples from 50 INSTI-naive patients failing first- or second-line ART [median (IQR) age: 40 (21.93-46.41) years; 68% women] were subjected to Sanger sequencing of the HIV integrase gene. Participants had been on ART for a median (IQR) duration of 7.32 (4.73-9.29) years, with 80% and 20% failing first- and second-line ART, respectively. RESULTS: No major INSTI mutations were found, but 2 (4%) participants had the accessory mutation T97A. Using the REGA HIV-1 subtyping tool, HIV subtype A1 (53.1%) was found to be dominant, followed by subtypes C (30.6%) and D (16.3%). CONCLUSIONS: This study found no current evidence for transmitted resistance against INSTIs among unexposed patients failing ART and supports the scale-up of INSTI-based regimens. However, the presence of accessory mutations calls for the surveillance of INSTI resistance mutations to ensure that the anticipated long-term desired outcomes are achieved.

Whole-genome sequencing of SARS-CoV-2 isolates from symptomatic and asymptomatic individuals in Tanzania.

Background: Coronavirus Disease-2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accounts for considerable morbidity and mortality globally. Paucity of SARS-CoV-2 genetic data from Tanzania challenges in-country tracking of the pandemic. We sequenced SARS-CoV-2 isolated in the country to determine circulating strains, mutations and phylogenies and finally enrich international genetic databases especially with sequences from Africa. Methods: This cross-sectional study utilized nasopharyngeal swabs of symptomatic and asymptomatic adults with positive polymerase chain reaction tests for COVID-19 from January to May 2021. Viral genomic libraries were prepared using ARTIC nCoV-2019 sequencing protocol version three. Whole-genome sequencing (WGS) was performed using Oxford Nanopore Technologies MinION device. In silico genomic data analysis was done on ARTIC pipeline version 1.2.1 using ARTIC nCoV-2019 bioinformatics protocol version 1.1.0. Results: Twenty-nine (42%) out of 69 samples qualified for sequencing based on gel electrophoretic band intensity of multiplex PCR amplicons. Out of 29 isolates, 26 were variants of concern [Beta (n = 22); and Delta (n = 4)]. Other variants included Eta (n = 2) and B.1.530 (n = 1). We found combination of mutations (S: D80A, S: D215G, S: K417N, ORF3a: Q57H, E: P71L) in all Beta variants and absent in other lineages. The B.1.530 lineage carried mutations with very low cumulative global prevalence, these were nsp13:M233I, nsp14:S434G, ORF3a:A99S, S: T22I and S: N164H. The B.1.530 lineage clustered phylogenetically with isolates first reported in south-east Kenya, suggesting regional evolution of SARS-CoV-2. Conclusion: We provide evidence of existence of Beta, Delta, Eta variants and a locally evolving lineage (B.1.530) from samples collected in early 2021 in Tanzania. This work provides a model for ongoing WGS surveillance that will be required to inform on emerging and circulating SARS-CoV-2 diversity in Tanzania and East Africa.

Audit of clinical-laboratory practices in haematology and blood transfusion at Muhimbili National Hospital in Tanzania.

In Tanzania, there is paucity of data for monitoring laboratory medicine including haematology. This therefore calls for audits of practices in haematology and blood transfusion in order to provide appraise practice and devise strategies that would result in improved quality of health care services. This descriptive cross-sectional study which audited laboratory practice in haematology and blood transfusion at Muhimbili National Hospital (MNH) aimed at assessing the pre-analytical stage of laboratory investigations including laboratory request forms and handling specimen processing in the haematology laboratory and assessing the chain from donor selection, blood component processing to administration of blood during transfusion. A national standard checklist was used to audit the laboratory request forms (LRF), phlebotomists' practices on handling and assessing the from donor selection to administration 6f blood during transfusion. Both interview and observations were used. A total of 195 LRF were audited and 100% of had incomplete information such as patients' identification numbers, time sample ordered, reason for request, summary of clinical assessment and differential diagnoses. The labelling of specimens was poorly done by phlebotomists/clinicians in 82% of the specimens. Also 65% (132/202) of the blood samples delivered in the haematology laboratory did not contain the recommended volume of blood. There was no laboratory request form specific for ordering blood and there were no guidelines for indication of blood transfusion in the wards/ clinics. The blood transfusion laboratory section was not participating in external quality assessment and the hospital transfusion committee was not in operation. It is recommended that a referral hospital like MNH should have a transfusion committee to provide an active forum to facilitate communication between those involved with transfusion, monitor, coordinate and audit blood transfusion practices as per national guidelines.

Risk factors for anaemia among HIV infected children attending HIV care and treatment clinic at Muhimbili National Hospital in Dar es Salaam, Tanzania.

There is paucity of data describing the risk factors for anaemia among HIV infected children in Tanzania. This cross sectional study aimed at determining the contributing factors for anaemia among HIV-infected children attending Muhimbili National Hospital in Dar es Salaam. Both univariate and multivariate logistic regression analyses were performed to identify possible factors associated with anaemia in HIV-infected children. In this study a total of 75 (44%) patients among 167 recruited HIV children aged 6 months to 59 months were found to be anaemic (Hg<11 g/dl). Multivariate logistic regression demonstrated that not being on HAART (OR 3.40, 95%CI (1.20-9.60), having CD4% <25% (OR 2.30, 95%CI (1.20-34.60), having a history of tuberculosis (TB) (OR 3.23, 95%CI (1.10-9.70) and having hookworm infestation (OR 5.97, 95%CI (1.92-18.4) were independent risk factors for anaemia among HIV infected children. The analyses also showed that being HIV positive for ≥ 2.5 years resulted into a low risk of severe anaemia compared to being HIV positive for < 2.5 years. Taking multivitamins (OR 0.07, 95%, CI (0.020-0.30) and antihelminthics (OR 0.27, 95%CI (0.10-0.74) were also protective against anaemia in children. Similar factors (with exception of using antihelmintics) were associated with severe anaemia. In conclusion the factors associated with anaemia in HIV infected children were multifactorial in nature. Efforts to correct anaemia in HIV infected children should include use of HAART and treatment of infections such as TB and hookworms.