Hydroxyurea treatment is associated with lower malaria incidence in children with sickle cell anemia in sub-Saharan Africa.

Realizing Effectiveness Across Continents with Hydroxyurea (REACH, NCT01966731) provides hydroxyurea at maximum tolerated dose (MTD) for children with sickle cell anemia (SCA) in sub-Saharan Africa. Beyond reducing SCA-related clinical events, documented treatment benefits include ∼50% reduction in malaria incidence. To identify associations and propose mechanisms by which hydroxyurea could be associated with lower malaria rates, infections were recorded across all clinical sites (Angola, Democratic Republic of Congo, Kenya, and Uganda). Hazard ratios (HR) with 95% confidence intervals (CIs) for baseline demographics, and time-varying laboratory and clinical parameters were estimated in a modified Cox gap-time model for repeated events. Over 3387 patient-years of hydroxyurea treatment, 717 clinical malaria episodes occurred in 336 of 606 study participants; over half were confirmed by blood smear and/or rapid diagnostic testing with 97.8% Plasmodium falciparum. In univariate analysis limited to 4 confirmed infections per child, malaria risk was significantly associated with absolute neutrophil count (ANC), splenomegaly, hemoglobin, and achieving MTD; age, malaria season, MTD dose, fetal hemoglobin, α-thalassemia, and glucose-6-phosphate dehydrogenase deficiency had no effect. In multivariable regression of confirmed infections, ANC was significant (HR, 1.37 per doubled value; 95% CI, 1.10-1.70; P = .0052), and ANC values <3.0 × 109/L were associated with lower malaria incidence. Compared with nonpalpable spleen, 1- to 4-cm splenomegaly also was associated with higher malaria risk (HR, 2.01; 95% CI, 1.41-2.85; P = .0001). Hydroxyurea at MTD is associated with lower malaria incidence in SCA through incompletely defined mechanisms, but treatment-associated mild myelosuppression with ANC <3.0 × 109/L is salutary. Splenomegaly is an unexplained risk factor for malaria infections among children with SCA in Africa.

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.

Trends in sickle cell trait and disease screening in the Republic of Uganda, 2014-2019.

OBJECTIVE: Sickle cell disease is an important public health issue that is increasingly recognised as a substantial contributor to morbidity and early childhood mortality in sub-Saharan Africa. We aimed to provide information from large-scale, long-term sickle cell screening efforts in Africa. METHODS: We used nationally representative data from the centralised public health laboratory database in Uganda to examine epidemiological trends in sickle cell screening over a five-year period, comparing age and geographic adjustments to prevalence among different testing cohorts of children aged 0-24 months, and calculating screening coverage within high-burden districts. RESULTS: A total of 324 356 children aged 0-24 months were screened for sickle cell trait and disease from February 2014 to March 2019. A high national burden of sickle cell disease (0.9%) was confirmed among a cohort of samples co-tested with HIV. In the cohort of samples referred specifically for sickle cell testing, the overall prevalence of sickle cell disease was 9.7% and particularly elevated in high-burden districts where focused screening occurred. The majority of children were screened before age 4 months, but the sickle-specific cohort had a larger proportion of affected children tested between age 5-9 months, coincident with onset of disease signs and symptoms. Successful screening coverage of sickle cell disease births was achieved in several high-burden districts. CONCLUSIONS: Examination and analysis of national sickle cell screening trends in Uganda documents the successes of focused screening strategies as an important step towards universal screening. With this evidence and increased healthcare provider knowledge, Uganda can optimise sickle cell diagnosis and management across the country.

OBJECTIF: La drépanocytose est un problème important de santé publique, de plus en plus reconnu comme un contributeur important à la morbidité et à la mortalité infantile en Afrique subsaharienne. Notre objectif était de fournir des informations sur les efforts de dépistage de la drépanocytose à grande échelle et à long terme en Afrique. MÉTHODES: Nous avons utilisé des données représentatives nationales de la base de données centralisée des laboratoires de santé publique en Ouganda pour examiner les tendances épidémiologiques dans le dépistage de la drépanocytose sur une période de cinq ans, en comparant l'âge et les ajustements géographiques à la prévalence dans différentes cohortes de tests d'enfants âgés de 0 à 24 mois, et en estimant la couverture du dépistage dans les districts à forte charge. RÉSULTATS: Un total de 324.356 enfants âgés de 0 à 24 mois ont été dépistés pour le trait drépanocytaire et la maladie de février 2014 à mars 2019. Une charge nationale élevée de la drépanocytose (0,9%) a été confirmée dans une cohorte d'échantillons co-testés pour le VIH. Dans la cohorte d'échantillons référés spécifiquement pour le dépistage de la drépanocytose, la prévalence globale de la drépanocytose était de 9,7% et particulièrement élevée dans les districts à forte charge où un dépistage ciblé avait eu lieu. La majorité des enfants ont été dépistés avant l'âge de 4 mois, mais la cohorte spécifique pour la drépanocytose avait une plus grande proportion d'enfants affectés testés entre l'âge de 5 à 9 mois, coïncidant avec l'apparition des signes et symptômes de la maladie. Une bonne couverture de dépistage des naissances drépanocytaires a été obtenue dans plusieurs districts à forte charge. CONCLUSIONS: L'examen et l'analyse des tendances nationales du dépistage de la drépanocytose en Ouganda documentent les succès des stratégies de dépistage ciblé comme une étape importante vers le dépistage universel. Grâce à ces données et à une meilleure connaissance des prestataires de soins de santé, l'Ouganda peut optimiser le diagnostic et la prise en charge de la drépanocytose dans tout le pays.

AnemoCheck-LRS: an optimized, color-based point-of-care test to identify severe anemia in limited-resource settings.

BACKGROUND: Severe anemia is common and frequently fatal for hospitalized patients in limited-resource settings. Lack of access to low-cost, accurate, and rapid diagnosis of anemia impedes the delivery of life-saving care and appropriate use of the limited blood supply. The WHO Haemoglobin Colour Scale (HCS) is a simple low-cost test but frequently inaccurate. AnemoCheck-LRS (limited-resource settings) is a rapid, inexpensive, color-based point-of-care (POC) test optimized to diagnose severe anemia. METHODS: Deidentified whole blood samples were diluted with plasma to create variable hemoglobin (Hb) concentrations, with most in the severe (≤ 7 g/dL) or profound (≤ 5 g/dL) anemia range. Each sample was tested with AnemoCheck-LRS and WHO HCS independently by three readers and compared to Hb measured by an electronic POC test (HemoCue 201+) and commercial hematology analyzer. RESULTS: For 570 evaluations within the limits of detection of AnemoCheck-LRS (Hb ≤ 8 g/dL), the average difference between AnemoCheck-LRS and measured Hb was 0.5 ± 0.4 g/dL. In contrast, the WHO HCS overestimated Hb with an absolute difference of 4.9 ± 1.3 g/dL for samples within its detection range (Hb 4-14 g/dL, n = 405). AnemoCheck-LRS was much more sensitive (92%) for the diagnosis of profound anemia than WHO HCS (22%). CONCLUSIONS: AnemoCheck-LRS is a rapid, inexpensive, and accurate POC test for anemia. AnemoCheck-LRS is more accurate than WHO HCS for detection of low Hb levels, severe anemia that may require blood transfusion. AnemoCheck-LRS should be tested prospectively in limited-resource settings where severe anemia is common, to determine its utility as a screening tool to identify patients who may require transfusion.

Surveillance for sickle cell disease, United Republic of Tanzania.

OBJECTIVE: To determine the regional- and district-level newborn prevalence of sickle cell trait and disease, and the prevalence of haemoglobin variants and genetic modifiers of sickle cell disease, in the nine regions of north-western United Republic of Tanzania. METHODS: We repurposed dried blood spot samples from children (aged 0-24 months) born to mothers living with human immunodeficiency virus (HIV), collected as part of the HIV Early Infant Diagnosis programme, for sickle cell diagnosis. We performed isoelectric focusing to determine whether samples had normal haemoglobin, sickle cell trait, sickle cell disease or a rare haemoglobin variant. We shipped samples diagnosed as disease or variant to Cincinnati Children's Hospital in the United States of America for deoxyribonucleic-acid-based analyses to determine the prevalence of α-thalassaemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency or fetal haemoglobin genetic modifiers. FINDINGS: We analysed a total of 17 200 specimens during February 2017-May 2018. We observed a prevalence of sickle cell trait and disease of 20.3% (3492/17 200) and 1.2% (210/17 200), respectively. District-level trait varied from 8.6% (5/58) to 28.1% (77/274). Among confirmed sickle cell disease specimens, we noted 42.7% (61/143) had 1-gene deletion and 14.7% (21/143) had 2-gene deletion α-thalassaemia trait. We documented G6PD A- deficiency in 19.2% (14/73) of males. CONCLUSION: Our calculated prevalence is twice as high as previously reported and reinforces the need for enhanced sickle cell diagnostic services. Our district-level data will inform public health policy, allowing screening and disease-modifying hydroxyurea therapy to be focused on high-prevalence areas, until universal newborn screening is available.

Robust clinical and laboratory response to hydroxyurea using pharmacokinetically guided dosing for young children with sickle cell anemia.

Hydroxyurea is FDA-approved and now increasingly used for children with sickle cell anemia (SCA), but dosing strategies, pharmacokinetic (PK) profiles, and treatment responses for individual patients are highly variable. Typical weight-based dosing with step-wise escalation to maximum tolerated dose (MTD) leads to predictable laboratory and clinical benefits, but often takes 6 to 12 months to achieve. The Therapeutic Response Evaluation and Adherence Trial (TREAT, NCT02286154) was a single-center study designed to prospectively validate a novel personalized PK-guided hydroxyurea dosing strategy with a primary endpoint of time to MTD. Enrolled participants received a single oral 20 mg/kg dose of hydroxyurea, followed by a sparse PK sampling approach with three samples collected over three hours. Analysis of individual PK data into a population PK model generated a starting dose that targets the MTD. The TREAT cohort (n = 50) was young, starting hydroxyurea at a median age of 11 months (IQR 9-26 months), and PK-guided starting doses were high (27.7 ± 4.9 mg/kg/d). Time to MTD was 4.8 months (IQR 3.3-9.3), significantly shorter than comparison studies (p < 0.0001), thus meeting the primary endpoint. More remarkably, the laboratory response for participants starting with a PK-guided dose was quite robust, achieving higher hemoglobin (10.1 ± 1.3 g/dL) and HbF (33.3 ± 9.1%) levels than traditional dosing. Though higher than traditional dosing, PK-guided doses were safe without excess hematologic toxicities. Our data suggest early initiation of hydroxyurea, using a personalized dosing strategy for children with SCA, provides laboratory and clinical response beyond what has been seen historically, with traditional weight-based dosing.

Sickle cell screening in Uganda: High burden, human immunodeficiency virus comorbidity, and genetic modifiers.

BACKGROUND: The Uganda Sickle Surveillance Study provided evidence for a large sickle burden among HIV-exposed infants in Uganda. To date, however, no large scale screening program has been developed for Central or East Africa. METHODS: A 3-year targeted sickle cell screening project in Uganda was designed by the Ministry of Health to (1) determine sickle cell trait and disease prevalence within high-burden districts, (2) document the prevalence among HIV-exposed and nonexposed children, (3) confirm previously suggested HIV comorbidity, and (4) estimate the co-inheritance of known genetic modifiers of sickle cell disease. RESULTS: A total of 163 334 dried blood spot samples collected between April 2015 and March 2018 were analyzed, including 112 352 samples within the HIV Early Infant Diagnosis program. A high burden with >1% sickle cell disease was found within targeted East Central and Mid-Northern districts, in both HIV-exposed and nonexposed children. Based on crude birth-rate data, 236 905 sickle cell trait births and 16 695 sickle cell disease births will occur annually in Uganda. Compared to sickle cell disease without HIV, the odds ratio of having sickle cell disease plus HIV was 0.50 (95% confidence interval = 0.40-0.64, P < .0001). Alpha-thalassemia trait and G6PD deficiency were common with sickle cell disease, but with different geospatial distribution. CONCLUSIONS: High sickle cell burden and potential HIV comorbidity are confirmed in Uganda. Genetic modifiers are common and likely influence laboratory and clinical phenotypes. These prospective data document that targeted sickle cell screening is feasible and effective in Uganda, and support development of district-level comprehensive care programs.

Realizing effectiveness across continents with hydroxyurea: Enrollment and baseline characteristics of the multicenter REACH study in Sub-Saharan Africa.

Despite its well-described safety and efficacy in the treatment of sickle cell anemia (SCA) in high-income settings, hydroxyurea remains largely unavailable in sub-Saharan Africa, where more than 75% of annual SCA births occur and many comorbidities exist. Realizing Effectiveness Across Continents with Hydroxyurea (REACH, ClinicalTrials.gov NCT01966731) is a prospective, Phase I/II open-label trial of hydroxyurea designed to evaluate the feasibility, safety, and benefits of hydroxyurea treatment for children with SCA in four sub-Saharan African countries. Following comprehensive training of local research teams, REACH was approved by local Ethics Committees and achieved full enrollment ahead of projections with 635 participants enrolled over a 30-month period, despite half of families living >12 km from their clinical site. At enrollment, study participants (age 5.4 ± 2.4 years) had substantial morbidity, including a history of vaso-occlusive pain (98%), transfusion (68%), malaria (85%), and stroke (6%). Significant differences in laboratory characteristics were noted across sites, with lower hemoglobin concentrations (P < .01) in Angola (7.2 ± 1.0 g/dL) and the DRC (7.0 ± 0.9 g/dL) compared to Kenya (7.4 ± 1.1 g/dL) and Uganda (7.5 ± 1.1 g/dL). Analysis of known genetic modifiers of SCA demonstrated a high frequency of α-thalassemia (58.4% with at least a single α-globin gene deletion) and G6PD deficiency (19.7% of males and 2.4% of females) across sites. The CAR ß-globin haplotype was present in 99% of participants. The full enrollment to REACH confirms the feasibility of conducting high-quality SCA research in Africa; this study will provide vital information to guide safe and effective dosing of hydroxyurea for children with SCA living in Africa.

Characteristics of a rapid, point-of-care lateral flow immunoassay for the diagnosis of sickle cell disease.

Sickle cell disease (SCD) is a common and life-threatening hematological disorder, affecting approximately 400,000 newborns annually worldwide. Most SCD births occur in low-resource countries, particularly in sub-Saharan Africa, where limited access to accurate diagnostics results in early mortality. We evaluated a prototype immunoassay as a novel, rapid, and low-cost point-of-care (POC) diagnostic device (Sickle SCAN) designed to identify HbA, HbS, and HbC. A total of 139 blood samples were scored by three masked observers and compared to results using capillary zone electrophoresis. The sensitivity (98.3-100%) and specificity (92.5-100%) to detect the presence of HbA, HbS, and HbS were excellent. The test demonstrated 98.4% sensitivity and 98.6% specificity for the diagnosis of HbSS disease and 100% sensitivity and specificity for the diagnosis of HbSC disease. Most variant hemoglobins, including samples with high concentrations of HbF, did not interfere with the ability to detect HbS or HbC. Additionally, HbS and HbC were accurately detected at concentrations as low as 1-2%. Dried blood spot samples yielded clear positive bands, without loss of sensitivity or specificity, and devices stored at 37°C gave reliable results. These analyses indicate that the Sickle SCAN POC device is simple, rapid, and robust with high sensitivity and specificity for the detection of HbA, HbS, and HbC. The ability to obtain rapid and accurate results with both liquid blood and dried blood spots, including those with newborn high-HbF phenotypes, suggests that this POC device is suitable for large-scale screening and potentially for accurate diagnosis of SCD in limited resource settings.

Genetic mapping and exome sequencing identify 2 mutations associated with stroke protection in pediatric patients with sickle cell anemia.

Stroke is a devastating complication of sickle cell anemia (SCA), occurring in 11% of patients before age 20 years. Previous studies of sibling pairs have demonstrated a genetic component to the development of cerebrovascular disease in SCA, but few candidate genetic modifiers have been validated as having a substantial effect on stroke risk. We performed an unbiased whole-genome search for genetic modifiers of stroke risk in SCA. Genome-wide association studies were performed using genotype data from single-nucleotide polymorphism arrays, whereas a pooled DNA approach was used to perform whole-exome sequencing. In combination, 22 nonsynonymous variants were identified and represent key candidates for further in-depth study. To validate the association of these mutations with the risk for stroke, the 22 candidate variants were genotyped in an independent cohort of control patients (n = 231) and patients with stroke (n = 57) with SCA. One mutation in GOLGB1 (Y1212C) and another mutation in ENPP1 (K173Q) were confirmed as having significant associations with a decreased risk for stroke. These mutations were discovered and validated by an unbiased whole-genome approach, and future studies will focus on how these functional mutations may lead to protection from stroke in the context of SCA.

Epigenetic and molecular profiles of erythroid cells after hydroxyurea treatment in sickle cell anemia.

Hydroxyurea has been shown to be efficacious for the treatment of sickle cell anemia (SCA), primarily through the induction of fetal hemoglobin (HbF). However, the exact mechanisms by which hydroxyurea can induce HbF remain incompletely defined, although direct transcriptional effects and altered cell cycle kinetics have been proposed. In this study, we investigated potential epigenetic and alternative molecular mechanisms of hydroxyurea-mediated HbF induction by examining methylation patterns within the (G)γ-globin promoter and miRNA expression within primary CD71(+) erythrocytes of patients with SCA, both at baseline before beginning hydroxyurea therapy and after reaching maximum tolerated dose (MTD). Using both cross-sectional analysis and paired-sample analysis, we found that the highly methylated (G)γ-globin promoter was inversely correlated to baseline HbF levels, but only slightly altered by hydroxyurea treatment. Conversely, expression of several specific miRNAs was significantly increased after hydroxyurea treatment, and expression of miR-26b and miR-151-3p were both associated with HbF levels at MTD. The significant associations identified in these studies suggest that methylation may be important for regulation of baseline HbF, but not after hydroxyurea treatment, whereas changes in miRNA expression may be associated with hydroxyurea-mediated HbF induction. This study was registered at ClinicalTrials.gov (NCT00305175).

Genetic predictors for stroke in children with sickle cell anemia.

Stroke is a devastating complication of sickle cell anemia (SCA), affecting 5% to 10% of patients before adulthood. Several candidate genetic polymorphisms have been proposed to affect stroke risk, but few have been validated, mainly because previous studies were hampered by relatively small sample sizes and the absence of additional patient cohorts for validation testing. To verify the accuracy of proposed genetic modifiers influencing stroke risk in SCA, we performed genotyping for 38 published single nucleotide polymorphisms (SNPs), as well as α-thalassemia, G6PD A(-) variant deficiency, and ß-globin haplotype in 2 cohorts of children with well-defined stroke phenotypes (130 stroke, 103 nonstroke). Five polymorphisms had significant influence (P < .05): SNPs in the ANXA2, TGFBR3, and TEK genes were associated with increased stroke risk, whereas α-thalassemia and a SNP in the ADCY9 gene were linked with decreased stroke risk. Further investigation at these genetic regions may help define mutations that confer stroke risk or protection in children with SCA.

Genetic modifiers of sickle cell anemia in the BABY HUG cohort: influence on laboratory and clinical phenotypes.

The recently completed BABY HUG trial investigated the safety and efficacy of hydroxyurea in infants with sickle cell anemia (SCA). To investigate the effects of known genetic modifiers, genomic DNA on 190 randomized subjects were analyzed for alpha thalassemia, beta-globin haplotype, polymorphisms affecting endogenous fetal hemoglobin (HbF) levels (XmnI, BCL11A, and HBS1L-MYB), UGT1A1 promoter polymorphisms, and the common G6PD A(-) mutation. At study entry, infants with alpha thalassemia trait had significantly lower mean corpuscular volume, total bilirubin, and absolute reticulocyte count. Beta-globin haplotypes associated with milder disease had significantly higher hemoglobin and %HbF. BCL11A and XmnI polymorphisms had significant effects on baseline HbF, while UGT1A1 promoter polymorphisms significantly influenced baseline serum bilirubin. At study exit, subjects randomized to placebo still exhibited laboratory effects of alpha thalassemia and other modifiers, while those assigned hydroxyurea had treatment effects that exceeded most genetic influences. The pain phenotype was influenced by HbF modifiers in both treatment groups. These data document that genetic polymorphisms do modify laboratory and clinical phenotypes even in very young patients with SCA. The hydroxyurea effects are more potent, however, indicating that treatment criteria should not be limited to certain genetic subsets, and supporting the use of hydroxyurea for all young patients with SCA.

Hydroxycarbamide alters erythroid gene expression in children with sickle cell anaemia.

Sickle cell anaemia (SCA) is a severe debilitating haematological disorder associated with a high degree of morbidity and mortality. The level of fetal haemoglobin (HbF) is well-recognized as a critical laboratory parameter: lower HbF is associated with a higher risk of vaso-occlusive complications, organ damage, and early death. Hydroxycarbamide treatment can induce HbF, improve laboratory parameters, and ameliorate clinical complications of SCA but its mechanisms of action remain incompletely defined and the HbF response is highly variable. To identify pathways of hydroxycarbamide activity, we performed microarray expression analyses of early reticulocyte RNA obtained from children with SCA enrolled in the HydroxyUrea Study of Long-term Effects (NCT00305175) and examined the effects of hydroxycarbamide exposure in vivo. Hydroxycarbamide affected a large number of erythroid genes, with significant decreases in the expression of genes involved in translation, ribosome assembly and chromosome organization, presumably reflecting the daily cytotoxic pulses of hydroxycarbamide. Hydroxycarbamide also affected expression of numerous genes associated with HbF including BCL11A, a key regulator of baseline HbF levels. Together, these data indicate that hydroxycarbamide treatment for SCA leads to substantial changes in erythroid gene expression, including BCL11A and other potential signalling pathways associated with HbF induction.

A novel laboratory technique demonstrating the influences of RHD zygosity and the RhCcEe phenotype on erythrocyte D antigen expression.

D antigen is the most immunogenic and clinically relevant antigen within the complex Rh blood group system. Variability of D antigen expression was first described decades ago but has rarely been investigated quantitatively, particularly in the context of RHD zygosity along with RhCcEe serological phenotype. With IRB approval, 107 deidentified blood samples were analyzed. Rh phenotypes were determined serologically by saline technique using monoclonal antibodies against D, C, c, E, and e antigens. RHD zygosity was determined using both PCR-restriction fragment length polymorphisms and quantitative real-time PCR techniques. A novel and robust method was developed for quantitation of erythrocyte D antigen sites using calibrated microspheres and flow cytometry, allowing correlation of D antigen density with RHD zygosity and expression of Rh CcEe antigens. Subjects homozygous for RHD expressed nearly twice the number of D antigen sites compared with RHD hemizygotes (33,560 ± 8,222 for DD versus 17,720 ± 4,471 for Dd, P < 0.0001). Expression of c or E antigens was associated with significantly increased erythrocyte D antigen expression, whereas presence of C or e antigens reduced expression. These data and this novel quantitation method will be important for future studies investigating the clinical relevance of D antigen variability.

Genotoxicity associated with hydroxyurea exposure in infants with sickle cell anemia: results from the BABY-HUG Phase III Clinical Trial.

BACKGROUND: The laboratory and clinical benefits of hydroxyurea therapy for children with sickle cell anemia (SCA) are well recognized, but treatment in young patients is limited in part by concerns about long-term genotoxicity, and specifically possible carcinogenicity. PROCEDURE: The Pediatric Hydroxyurea Phase III Clinical Trial (BABY HUG) was a multicenter double-blinded placebo-controlled randomized clinical trial (NCT00006400) testing whether hydroxyurea could prevent chronic organ damage in very young patients with SCA. An important secondary objective was the measurement of acquired genotoxicity using three laboratory assays: chromosomal karyotype, illegitimate VDJ recombination events, and micronucleated reticulocyte formation. RESULTS: Our data indicate that hydroxyurea treatment was not associated with any significant increases in genotoxicity compared to placebo treatment. CONCLUSIONS: These data provide additional support to the safety profile of hydroxyurea for young patients with SCA, and suggest that genotoxicity in this patient population is low.

Chromosome damage and repair in children with sickle cell anaemia and long-term hydroxycarbamide exposure.

Hydroxycarbamide (hydroxyurea) provides laboratory and clinical benefits for adults and children with sickle cell anaemia (SCA). Given its mechanism of action and prior reports of genotoxicity, concern exists regarding long-term toxicities and possible carcinogenicity. We performed cross-sectional analyses of chromosome stability using peripheral blood mononuclear cells (PBMC) from 51 children with SCA and 3-12 years of hydroxycarbamide exposure (mean age 13·2 ± 4·1 years), compared to 28 children before treatment (9·4 ± 4·7 years). Chromosome damage was less for children receiving hydroxycarbamide than untreated patients (0·8 ± 1·2 vs. 1·9 ± 1·5 breaks per 100 cells, P = 0·004). There were no differences in repairing chromosome breaks after in vitro radiation; PBMC from children taking hydroxycarbamide had equivalent 2 Gy-induced chromosome breaks compared to untreated patients (30·8 ± 16·1 vs. 31·7 ± 8·9 per 100 cells, P = not significant). Radiation plus hydroxycarbamide resulted in similar numbers of unrepaired breaks in cells from children on hydroxycarbamide compared to untreated patients (95·8 ± 44·2 vs. 76·1 ± 23·1 per 100 cells, P = 0·08), but no differences were noted with longer exposure (97·9 ± 42·8 breaks per 100 cells for 3-6 years of hydroxycarbamide exposure vs. 91·2 ± 48·4 for 9-12 years of exposure). These observations provide important safety data regarding long-term risks of hydroxycarbamide exposure for children with SCA, and suggest low in vivo mutagenicity and carcinogenicity.

Operational analysis of the national sickle cell screening programme in the Republic of Uganda.

BACKGROUND: Sickle cell anaemia is a common global life-threatening haematological disorder. Most affected births occur in sub-Saharan Africa where children usually go undiagnosed and die early in life. Uganda's national sickle cell screening programme was developed in response to a 2014 sickle cell surveillance study that documented a high disease prevalence. OBJECTIVE: This study describes the temporal and financial aspects of Uganda's 2014-2019 sickle cell screening programme. METHODS: National sickle cell screening data from Uganda's Central Public Health Laboratories were used to calculate turn-around times (TATs) from sample collection to delivery, testing, and result reporting for blood samples collected from February 2014 to March 2019. The parameters affecting specific TATs were assessed. The exact programme expenditures were analysed to determine cost per test and per positive sickle cell disease case detected. RESULTS: A total of 278 651 samples were analysed. The median TAT from sample collection to laboratory receipt was 8 days (interquartile range [IQR]: 6-12), receipt to testing was 3 days (IQR: 1-7), and testing to result reporting was 6 days (IQR: 3-12). Altogether, the sample continuum averaged 16 days (IQR: 11-24). Lower level healthcare facilities were associated with longer sample delivery TATs. Calendar months (January and December) and larger sample volumes impacted testing and result reporting TATs. The cost per test was $4.46 (United States dollars [USD]) and $483.74 USD per positive case detected. CONCLUSION: Uganda's sickle cell screening programme is efficient and cost-effective. Universal newborn screening is the best strategy for detecting sickle cell anaemia in Uganda.

Hydroxyurea Pharmacokinetics in Pediatric Patients After Total Pancreatectomy With Islet Autotransplantation.

Total pancreatectomy with islet autotransplantation is a complex surgical approach for acute recurrent or chronic pancreatitis that frequently triggers extreme thrombocytosis (platelets ≥ 1000 × 109 /L). Thrombocytosis can be prothrombotic, so cytoreductive hydroxyurea is often initiated after this surgery; however, optimal dosing strategy and efficacy are unknown. This prospective pilot study characterized the pharmacokinetics of hydroxyurea after this procedure in children. It also compared them with previously published pediatric parameters in sickle cell anemia (SCA), the disease in which pediatric hydroxyurea pharmacokinetics have primarily been studied. Plasma hydroxyurea levels were quantified in 14 participants aged 4-19 years using high-performance liquid chromatography. Blood collections were scheduled 20 minutes, 1 hour, and 4 hours after the first dose, on pharmacokinetic day 1 (PK1), and again 2-3 months later if still on hydroxyurea (PK2). Six participants had PK1 and PK2 data at all 3 postdose timed collections, 5 only had PK1 samples, and 3 only had PK2 samples. Total pancreatectomy with islet autotransplantation participants had reduced and delayed absorption compared with sickle cell anemia participant data from the Hydroxyurea Study of Long-Term Effects, regardless of timing or dosing methodology. Total pancreatectomy with islet autotransplantation participants had different pharmacokinetic profiles at PK1 versus PK2, with lower dose-normalized exposures than previously reported in sickle cell anemia. These results suggest variability exists in hydroxyurea absorption and bioavailability in total pancreatectomy with islet autotransplantation patients, suspected to be primarily because of Roux-en-Y reconstruction, and suggest that more pharmacokinetic data are needed for scenarios when hydroxyurea is prescribed to children without sickle cell anemia.

Assessment of genotoxicity associated with hydroxyurea therapy in children with sickle cell anemia.

Hydroxyurea induces fetal hemoglobin, improves laboratory parameters, and ameliorates clinical complications of sickle cell anemia (SCA), but its long-term efficacy and safety in this patient population remain incompletely defined. Although generally considered non-DNA reactive, an important safety concern is that hydroxyurea may indirectly cause genotoxic damage. To better address this safety issue of hydroxyurea in patients with SCA, we measured the production of micronuclei (MN) in red blood cells (RBCs) as a marker of genotoxicity. Blood samples were collected from children with SCA enrolled in the Hydroxyurea Study of Long-term Effects (ClinicalTrials.gov NCT00305175). Flow cytometry quantified circulating MN-containing erythrocyte sub-populations before and during hydroxyurea exposure. The frequency of micronucleated reticulocytes (MN-CD71(+)) and micronucleated mature erythrocytes (MN-RBC) was then tested for associations with laboratory and clinical data. In cross-sectional analysis of 293 blood samples from 105 children with SCA and a median of 2 years of hydroxyurea therapy, exposure to hydroxyurea was associated with significantly increased frequencies of MN-CD71(+) and MN-RBC compared to baseline. The increases were evident by 3 months of therapy, and did not escalate further with up to 12 years of continuous drug exposure. In prospective longitudinal analysis, substantial inter-individual variation in the effect of hydroxyurea on %MN-CD71(+) was observed that was associated with the expected laboratory effects of hydroxyurea. In conclusion, clinically relevant exposure to hydroxyurea is associated with increased MN production consistent with erythroblast genotoxicity but with substantial inter-patient variability. Associations between increased %MN-CD71(+) and laboratory benefits suggest that hydroxyurea effects on MN production may be related to individual patient sensitivity to hydroxyurea within the bone marrow.