Characterization of a cohort of Angolan children with sickle cell anemia treated with hydroxyurea

Blood Cells, Molecules and Diseases 105 (2024) 102822 Available online 4 January 2024 1079-9796/© 2024 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Full Length Article Characterization of a cohort of Angolan children with sickle cell anemia treated with hydroxyurea Brígida Santos a , b , d , Catarina Ginete c , Elisˆ angela Gonçalves d , Mariana Delgadinho c , Armandina Miranda e , Paula Faustino e , f , g , Ana Paula Arez d , Miguel Brito a , c , * a Centro de Investigaç˜ ao em Saúde de Angola (CISA), Caxito, Angola b Hospital Pedi´ atrico David Bernardino (HPDB), Luanda, Angola c H&TRC - Health & Technology Research Center, ESTeSL - Escola Superior de Tecnologia da Saúde, Instituto Polit´ ecnico de Lisboa, Lisbon, Portugal d Global Health and Tropical Medicine (GHTM), Associate Laboratory in Translation and Innovation Towards Global Health (LA-REAL), Instituto de Higiene e Medicina Tropical (IHMT), Universidade NOVA de Lisboa (UNL), Lisbon, Portugal e Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), Lisbon, Portugal f Instituto de Saúde Ambiental (ISAMB), Faculdade de Medicina da Universidade de Lisboa, Lisbon, Portugal g Laborat´ orio Associado TERRA, Faculdade de Medicina da Universidade de Lisboa, Lisbon, Portugal ARTICLE INFO Handling Editor Lionel Blanc Keywords: Sickle cell anemia Fetal hemoglobin Hydroxyurea Malaria Angola ABSTRACT Background: Sickle Cell Anemia (SCA) is a monogenic disease, although its severity and response to treatment are very heterogeneous. Objectives: This study aims to characterize a cohort of Angolan children with SCA and evaluate their response to hydroxyurea (HU) treatment and the potential side effects and toxicity. Methods: The study enrolled 215 patients between 3 and 12 years old before and after the administration of HU, at a fix dose of 20 mg/kg/day for 12 months. Results: A total of 157 patients started HU medication and 141 of them completed the 12-month treatment. After initiating HU treatment, the frequency of clinical events decreased (transfusions 53.4 %, hospitalizations 47.1 %). The response to HU medication varied among patients, with some experiencing an increase in fetal hemoglobin (HbF) of <5 %. The mean increase in HbF was 11.9 %, ranging from 1.8 % to 31 %. Responders to HU treatment were 57 %, inadequate responders 38.7 % and non-adherent 4.2 %. No clinical side effects related to HU were reported. Hematological toxicities were transient and reversible. Children naïve to HU and with lower HbF reported higher number of hospitalizations caused by malaria infection. During HU treatment, the frequency of malaria episodes did not appear to be affected by HbF levels. Conclusions: the present study provided a valuable contribution to the understanding of the clinical and laboratory profiles of Angolan children with SCA. These findings support the evidence that the implementation of prophylactic measures and treatment with HU is associated with increased survival in children with SCA. 1. Introduction Sickle cell anemia (SCA) is a genetic disease transmitted through autosomal recessive inheritance. It results from a mutation in position 6 of the β-globin chain, which causes a substitution of glutamic acid for valine (β6Glu → Val) and produces the S-type hemoglobin (HbS). SCA is one of the most common hereditary diseases in the world, with approximately 300,000 births per year. It is estimated that 75 % of these births occur in Sub-Saharan Africa [1]. Although SCA is a monogenic disease, it can present multiple phenotypic expressions and levels of severity among patients, and even throughout each patient's life [2–4]. Despite the scarcity of information on true SCA mortality in Africa, some studies have concluded that this largely neglected disease has an extremely high mortality rate of between 50 and 90 % in children under 5 years of age, mainly caused by infectious diseases and severe anemia [5]. Malaria not only contributes to mortality but also worsens anemia and other SCA manifestations [6–8]. Other causes of death include acute chest syndrome, stroke, and multi-organ failure [9]. Fetal hemoglobin (HbF) is an effective inhibitor of HbS * Corresponding author at: Centro de Investigaç˜ ao em Saúde de Angola (CISA), Caxito, Angola. E-mail address: [email protected] (M. Brito). Contents lists available at ScienceDirect Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd https://doi.org/10.1016/j.bcmd.2023.102822 Received 30 September 2023; Received in revised form 26 December 2023; Accepted 28 December 2023 Blood Cells, Molecules and Diseases 105 (2024) 102822 2 polymerization [10,11]. The concentration of HbF ranges from 1 % to 25 % among patients with SCA [11,12]. Studies have shown that an increase in HbF is associated with a decrease in the number of painful vaso-occlusive events, as well as the number of transfusions and hospitalizations [13,14]. Hydroxyurea (HU) is an HbF-inducing drug that has been approved by both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). The clinical efficacy, tolerance, and safety of HU have been well-documented in adult and pediatric patients with sickle cell disease living in high-income countries in Europe and America [15]. However, in Africa, where the prevalence of SCA is high and coexists with malaria, malnutrition, and deficiencies in health systems, there is little robust experience regarding the treatment with HU. Studies have demonstrated that HU can improve patient survival, reduce the intensity and frequency of pain episodes, decrease emergency room visits, shorten hospitalizations, and decrease the number of required transfusions [16–18]. In a multicentric study conducted in Africa, a reduction in malaria cases was also evident, in addition to the clinical benefits described [19]. HU treatment has also been shown to have benefits on neurological events, as evidenced by the reduction in the velocity of blood flow in the cerebral arteries [18,20–22]. In addition to promoting an increase in HbF, HU is also associated with other laboratory benefits, such as an increase in the concentration of total hemoglobin (Hb), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH), as well as a reduction in the number of neutrophils, platelets, reticulocytes, and other hemolysis parameters [19,23–26]. Mild secondary clinical effects associated with HU have also been reported, including headache, gastrointestinal symptoms (nausea, vomiting and diarrhea), rash, and changes in nail color [27,28]. Laboratory toxicity is mainly hematological with mild to moderate neutropenia and thrombocytopenia, both of which are transient and reversed by discontinuing the medication [27,28]. Biochemical alterations (liver enzymes, urea, and creatinine) are minor, reversible, and reported in insignificant numbers [17,28–31]. Some studies have shown that individual responses to HU treatment are highly variable, with HbF levels ranging from 2 % to >30 %, and approximately 25 % of patients do not respond to HU [17,32–34]. In Angola, 1.5 % of newborns in a total of 36,453 children screened during the neonatal period between 2011 and 2013 were found to have SCA [35]. Unfortunately, HU is not universally available for the treatment of Angolan patients with SCA, even for those with severe clinical courses. Regarding malaria, Angola is an endemic country. Chemoprophylaxis and other measures to control malaria are not commonly implemented. The use of HU in malaria-endemic areas has raised some concerns as it may increase the expression of intercellular adhesion molecule-1 (ICAM-1) in endothelial cells, which could lead to an increase in the cytoadhesion of erythrocytes parasitized with Plasmodium falciparum [33]. However, later studies have shown that HU has a beneficial effect and can decrease the incidence of severe malaria [36]. In this study, our aim was to clinically characterize a cohort of Angolan children with SCA and evaluate their response to treatment with HU. We assessed the clinical and laboratory variations in response to HU treatment, including changes in HbF levels, potential side effects and drug toxicity, and treatment adherence. Moreover, we investigated the occurrence of malarial infections during HU treatment. The objective was to treat SCA patients with HU and evaluate the response to the medication for one year, according to clinical and laboratory variation. 2. Material and methods 2.1. Study population This study enrolled 215 patients between 3 and 12 years old who had a laboratory diagnosis of SCA (confirmed by molecular biology) and were being followed at the Hospital Pedi´ atrico David Bernardino (HPDB) in Luanda and the Hospital Geral do Bengo (HGB) in Caxito. Exclusion criteria included: transfusions within three months prior to enrollment, prior use of HU medication, neoplastic disease, active tuberculosis, HIV infection, and evidence of medullary dysfunction. 2.2. Ethical aspects Participation in the study was voluntary, and the children's parents or legal guardians were requested to sign a broad written informed consent. The study protocol was approved by the Ethics Committees of the Ministry of Health of Angola (CE. N◦040/2018), and by the Escola Superior de Tecnologia da Saúde de Lisboa (CE-ESTeSL-N◦.43-2018). 2.3. Clinical methodology A prospective study was conducted between 2019 and 2022, consisting of two phases - before (pre-HU) and after the administration of HU (post-HU). However, due to the COVID-19 pandemic, there was a partial interruption in 2020 for seven months (April–October), during which only patients who had already started HU medication were followed up clinically. During the pre-HU phase, patient characterization and clinical follow-up were performed every three months for a period of six to nine months to obtain clinical and baseline laboratory data. In the first consultation, an anamnesis was conducted to obtain demographic data, age, details of the first clinical manifestation of the disease, number and causes of hospitalizations and transfusions, complications, and a complete physical examination. In the second phase, patients were prescribed HU at a fix dose of 20 mg/kg/day for 12 months. The weekly dose was calculated to adapt the child's weight to the dosage of available capsules, which were 500 mg. Thus, the number of days of medication was calculated as a function of the number of capsules per week. At each appointment, the medication corresponding to the inter-consultation period was offered. During the first six months of medication, patients were seen monthly, and subsequently, the frequency was reduced to quarterly. In this phase, in addition to the variables described above, possible side effects and toxicity of the medication were evaluated through monthly assessment of hematological parameters and assessment of biochemical parameters every three months. In all follow-up consultations, both in the pre-HU phase and during the medication period, any incidents were recorded, and a physical examination was conducted. Throughout the study period, prophylactic measures were maintained, including the use of folic acid, phenoxymethylpenicillin for children up to 5 years of age, and dietary recommendations. 2.4. Laboratory evaluation In the first consultation, 5 mL of whole blood was obtained from each child to perform the following tests: (a) hematological evaluation (complete blood and reticulocyte count) using a XT-2000i automatic analyzer (Sysmex Corporation's, Kobe, Japan); (b) biochemical evaluation (bilirubin, lactic dehydrogenase, transaminases, urea and creatinine) using Cobas C111 (Roche Diagnostics', Basel, Switzerland) and BA88A (Mindray's, Shenzhen, China) analyzers; (c) quantification of HbF by HPLC (Biorad Variant II Hercules, CA, USA); (d) confirmation of SCA diagnosis by molecular biology; and (e) rapid HIV test. During the pre-HU phase follow-up consultations, which occurred every three months, a complete blood count, reticulocyte count, and the above-mentioned biochemical tests were performed. To identify possible HU toxicity during the post-HU phase, blood and reticulocyte count tests were performed monthly in the first six months of medication. Every three months, biochemical tests including urea, creatinine, alanine aminotransferase, and aspartate aminotransferase were also conducted. Bilirubin and lactic dehydrogenase were quantified to assess therapeutic efficacy. B. Santos et al. Blood Cells, Molecules and Diseases 105 (2024) 102822 3 Quantification of HbF was performed before the start of HU and every three months after treatment initiation. The average values per period were calculated, and the highest HbF value reached was reported. For the evaluation of malarial infection during follow-up consultations, it was proposed to obtain two blood samples from each symptomatic patient (fever, headache, worsening of anemia or jaundice, painful crisis) or asymptomatic individual, coincident with the periods of low and high transmission of malaria. The aim was to identify subpatent infections by Plasmodium spp. However, due to technical reasons, it was not always possible to obtain these samples. A Rapid Diagnostic Test (SD Bioline) was performed, and if positive, the Giemsastained thick smear was observed by optical microscopy. Blood was also stored on filter paper for molecular diagnosis. Parasite DNA was prepared through a saponin/chelex-100 method [37] and subsequently, the detection and identification of Plasmodium species were performed by amplification of the genes that code for the smaller subunit of ribosomal ribonucleic acid (ssrRNA) [38], identification of Plasmodium ovale [39] was done by nested-PCR according to Fuehrer et al. [40]. 2.5. Evaluation variables and operational definitions The baseline concentration of HbF (pre-HU) for each patient was classified as either low or high based on the obtained quartil. The cutoff point of 7.6 % was adopted, according to the 3rd quartil HbF value, with HbF ≤7.6 % considered a low level and HbF >7.6 % considered a high level. To indirectly assess whether patients complied with HU intake, they were asked to show the capsule blisters at each consultation. Medication adherence was assessed taking into account clinical and laboratory parameters. To evaluate the clinical response to the medication, parameters were compared between the two phases of the study (preand post-HU), including the number of pain episodes, transfusions, and hospitalizations. The evaluation of changes in laboratory parameters was based on the variation in the level of HbF and others such as an increase in Hb, MCV, and MCH, and a decrease in the count of leukocytes, neutrophils, platelets, reticulocytes, and bilirubin. Thus, patients were classified as responders if the increase in HbF was equal to or >5 % in relation to the baseline value or an absolute value of 20 %. Inadequate responders were those with an increase in HbF <5 % but with improvements in other laboratory parameters. Non-adherent patients were those who showed no alterations in the HbF level or other laboratory parameters [41]. Clinical manifestations presumably associated with taking HU were defined as HU side effects, such as nausea, vomiting, abdominal pain, asthenia, and headache [42]. Toxicity was defined by laboratory parameters as Hb values <4 g/dL, severe neutropenia (absolute neutrophil count <500/ μ L), mild to moderate neutropenia (absolute neutrophil count 500–1249/ μ L), thrombocytopenia (platelets <80,000 / μ L), and reticulocytes <0.2 %. The values of urea, creatinine, and transaminases were also evaluated, and the toxicity criteria were defined considering the reference values. 2.6. Statistical analysis The data were analyzed using the SPSS program (IBM Corp. Released 2019. IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY: IBM Corp) and were presented using descriptive statistics. The phenotype and laboratorial characteristics were compared using paired sample ttests and qui-square test (p-value <0.05). 3. Results 3.1. Study population The study enrolled a total of 215 children, with 141 followed at the HPDB and 74 at the HGB. The age range of participants was 3 to 12 years old, with a mean age of 6.6 ±2.5 years. Of the participants, 115 (53.5 %) were female and 100 (46.5 %) were male (Table 1). 3.1.1. Clinical characterization at study entry Information regarding personal history was obtained by surveying the patients' parents or caregivers. The age at which patients experienced their SCA first manifestation ranged from 2 to 96 months. Most patients (46.5 %) had their first manifestation between 6 and 11 months of age. Notably, 34 (15.8 %) had their first manifestation before 6 months, and 15 (6.5 %) had it after 36 months. Dactylitis was the most frequent initial manifestation (Table 1). In 193 (89.8 %) patients, laboratory diagnosis was performed following clinical suspicion, and in 22 (10.2 %) by neonatal screening. All patients had confirmed homozygous SS genotype. Twenty-six patients had sequelae of complications from the disease, the most frequent being stroke (5.6 %). A total of 146 (67.9 %) patients had received transfusions, with 121 (56.3 %) having an average frequency of 0.1–0.9 transfusions per year. One hundred and sixty-nine (78.6 %) patients had a history of hospitalization, with severe anemia being the most frequent cause (129, 60 %), followed by painful vasoocclusive crises (91, 42.3 %) (Table 1). Hepatomegaly was observed in 42 children (19.5 %), and the spleen was palpable in 22 (10.2 %) children, of which 13 were older than five years. It may be due to the normal evolution of the spleen in SCA up to 5 years of age, however the cause of splenomegaly after this age has not been investigated. Three children had undergone splenectomy. Despite the presence of hepatomegaly, liver enzyme values were stable, and one patient with splenomegaly had a hemoglobin level below 5 g/dL and another had a platelet count below 80,000/ μ L on one occasion. The mean HbF level was 5.8 %, SD ±4.1, with a minimum of 0.5 %, maximum of 23.8 %. Three children aged three, four, and seven years had baseline HbF values of 23.8 %, 23.6 %, and 22.3 %, respectively. There was no statistical significance between HbF level and sex, although males had slightly lower values. A correlation was made between the mean baseline HbF level (above or below 7.6 %) and demographic and clinical variables, with statistical significance (p <0.05) observed between age classes at study entry, age at the first manifestation of the disease, number of transfusions, and classes of anemia severity (Table 1). The mean baseline total Hb value was 7.3 g/dL, SD ±0.9, with a minimum of 4.2 g/dL and maximum of 10 g/dL. There was no difference in Hb value between age groups. Among the patients, 3 (1.3 %) had mild anemia, 130 (60.4 %) had moderate anemia, and 82 (38.3 %) had severe anemia, according to WHO anemia severity classes. The MCV varied between 56.4 fL and 113.7 fL, with a mean of 77.2 fL and SD ±8.4. The MCH varied between 18.5 pg and 32.1 pg, with a mean of 25.4 pg and SD ±2.9. Overall, 66.9 % of patients had microcytosis, and 53.9 % had hypochromia (Table 1). 3.2. Clinical follow-up 3.2.1. Retention of patients in the study During the study, a total of 73 patients (33.9 %) were lost to followup (Fig. 1). In the pre-HU phase, 58 patients (26.9 %) were lost, with 33 (15.3 %) failing to attend scheduled consultations, 21 (9.7 %) unable to be located to resume consultations after a seven-month interruption of the study due to the COVID-19 pandemic, three deaths (two due to malaria and one due to a road accident), and one child withdrawn due to the father's refusal to continue the study (Fig. 1). A total of 157 patients started HU medication, with 15 (6.9 %) lost during this phase: 13 withdrew from treatment, and two deaths were recorded (one due to malaria at the fifth month of medication, and the other due to severe anemia in an infectious context at the eighth month of medication). Of the patients on medication, 141 completed the 12month treatment (Fig. 1). The daily dose ranged from 17.2 to 22.7 B. Santos et al. Blood Cells, Molecules and Diseases 105 (2024) 102822 4 mg/kg. 3.3. Evaluation of HU medication 3.3.1. Adherence, safety and toxicity The effectiveness of HU medication was evaluated in 141 patients, with 16 cases experiencing two to four weeks of missed medication due to non-attendance at scheduled consultations. By checking the blisters, no capsules were returned. We assumed that patients complied with the prescribed medication dose. No clinical side effects related to HU were reported. Hematological toxicity occurred in 34 patients (24.1 %), including severe neutropenia (the absolute neutrophil count ranged between 230/ μ L and 480/ μ L in three patients), mild to moderate neutropenia (absolute neutrophil count ranged between 700/ μ L and 1140/ μ L) in 23 patients, and thrombocytopenia (platelet count between 33,000/ μ L and 80,000/ μ L) in eight patients. Of the patients who presented hematological toxicity, five had splenomegaly, one of which presented recurrent splenic sequestration (severe anemia, neutropenia and thrombocytopenia). It should be noted that in six patients, without splenomegaly, moderate neutropenia occurred on two times during medication (in the first and second months of medication and then in the sixth and in one case in the twelfth month). No serious bacterial infections or associated hemorrhagic manifestations were recorded. All laboratory toxicities were transient and reversible, and treatment was resumed at the same initial dose following a seven-day interruption of HU. Liver enzymes, urea, and creatinine levels did not show significant variations. 3.4. Clinical events related to HU Comparatively, there was a decrease in the frequency of clinical events from the pre-HU phase to the post-HU phase. Hospitalizations decreased in 53.4 % and transfusions in 47.1 %. During the medication period, four children experienced complications, with three of them having strokes (one of which was a recurrence), and one child had aseptic necrosis of the femoral head. In two children, the HbF variation was >5 %, while in the other two children, it was <5 %. One child had no variation in total Hb, but there was an increase in corpuscular constants (Table 2). Out of the 22 children who had splenomegaly at the beginning of the study, one had a history of recurrent episodes of pain, which subsided after starting HU but continued to experience episodes of splenic sequestration with Hb values <5 g/dL. This patient interrupted HU treatment and underwent splenectomy, subsequently showing clinical and laboratory improvement. 3.5. Effect of HU intake on laboratory parameters 3.5.1. Hematological and biochemical parameters All patients underwent laboratory parameter evaluation while on HU medication. The medication significantly improved HbF level and hematological parameters. Total Hb values and corpuscular constants increased, while values of leukocytes, neutrophils, and platelets decreased. Parameters associated with hemolysis, such as reticulocytes and total bilirubin, showed a significant reduction. LDH also decreased, but not significantly. Liver enzymes, urea, and creatinine, which were used to monitor HU toxicity, showed no significant variation (Table 3). 3.5.2. Fetal hemoglobin During the pre-HU phase, the mean HbF level was 5.94 % with a standard deviation of 3.91 %, while in the post-HU phase, it increased significantly to 12.19 ±6.03 % (with a minimum of 2.10 % and a maximum of 31 %). This increase in HbF was observed in all age groups, with statistical significance (p <0.001) for each group. Specifically, in patients aged ≤5 years, the HbF level increased from 7.2 ±4.5 % to 13.2 Table 1 Demographic and clinical characteristics, correlation with HbF level at study entry. Variables Total (N, %) HbF levels Cutoff point 7.6 % p-Value Low HbF (n,%) High HbF (n, %) Total (n) 215 151 (73.3) 55 (26.7) Age group (years) ≤5 82 (38.1) 49 (32.5) 31 (56.4) 6–9 95 (44.2) 76 (50.3) 16 (29.1) 0.006 ˃9 38 (17.7) 26 (17.2) 8 (14.5) Gender Female 115 (53.5) 77 (50.9) 32 (58.2) 0.361 Male 100 (46.5) 74 (49.1) 23 (41,8) Age 1st manifestation (mos) ˂6 34 (15.8) 28(18.5) 7 (12.7) 6–11 100 (46.5) 78 (51.6) 20 (36.3) 12–23 32 (14.8) 21 (13.9) 9 (16.3) 0.030 24–36 34 (15.8) 18 (11.9) 12 (21.8) ˃36 15 (6.5) 6 (3.9) 7 (12.7) 1st manifestation Dactylitis 149 (69.3) 113(74.8) 33 (60) 0.056 Painful crisis 48 (22.3) 25(16.6) 18 (32.7) 0.009 Severe anemia 16 (7.4) 13 (8.6) 2 (3.6) 0.235 Stroke 1 (0.5) 0 1(1.8) 0.094 Complications None 189 (87.9) 131 (86.8) 52 (94.5) 0.116 Stroke 12 (5.6) 9 (6) 1 (1.8) 0.221 Osteomyelitis 9 (4.8) 8 (5.3) 1 (1.8) 0.450 Hypersplenism 2 (0.9) 1 (0.6) 0 1.000 Femoral head necrosis 1 (0.5) 1 (0.6) 0 1.000 Heart disease 2 (0.9) 1 (0.6) 1 (1.8) 0.464 N◦of hospitalizations/ year 0 37 (18) 22 (14.6) 15 (27.3) 0.1–0.9 140 (68) 105 (69.5) 35 (63.6) 0.080 ˃1 29 (14.1) 24 (15.9) 5 (9.1) Hospitalizations causes Severe anemia 129 (40.7) 99 (41.2) 26 (40.6) 0.017 Painful crisis 91 (28.7) 65 (27) 21 (32.8) 0.531 Non-specific infections 48 (15.1) 36 (15) 11 (17.1) 0.561 Malaria 45 (14.2) 37 (15.4) 6 (9.3) 0.034 Stroke 4 (1.3) 3 (1.2) 0 0.566 N◦of transfusions per year 0 60 (29.1) 33 (21.9) 27 (49.1) 0.1–0.9 121 (58.7) 96 (63.6) 25 (45.5) 0.001 >1 25 (12.1) 22 (14.6) 3 (5.5) Anemia (WHO criteria) a Moderate 56 (26) 26 (17.2) (49) <0.001 Severe 159 (74) 12,5 (82.8) 28(51) MCV (min-max), fL Normocytosis (80–100) 75 (34.9) 48 (31.8) 23 (41.8) 0.447 Microcytosis (<80) 139 (64.7) 102 (67.5) 32 (58.2) Macrocytosis (>100) 1 (0.5) 1 (0.7) 0 (0.0) MCH (min-max), pg Normochromia (26–31.2) 109 (50.9) 71 (47.3) 33 (60.0) 0.073 Hypochromia (18.4–25.9) 105 (49.1) 79 (52.7) 22 (40.0) Comparations performed by chi-square test. Significant values in bold. a Classification of anemia according to WHO criteria (children 5–11 years of age): mild (Hb 11–11.4 g/dL), moderate (8.0–10.9 g/dL), severe (<8 g/dL). B. Santos et al. Blood Cells, Molecules and Diseases 105 (2024) 102822 5 ±6.4 %; in patients aged 6–9 years, it increased from 4.9 ±3.7 % to 10.8 ±5.9 %; and in patients aged >9 years, the HbF level ranged from 4.9 ±3 % to 12 ±5.1 % after HU treatment. Based on the variation in HbF level, hematological parameters, and bilirubin, patients were classified into three groups: responders (57.4 %, 81 patients), inadequate responders (38.3 %, 54 patients), and non-adherent (4.3 %, 6 patients). 3.6. Malaria infection and HU A total of 87 cases of malaria were reported during the study, with 40 cases occurring in the pre-HU phase and 47 cases in the post-HU phase. Out of these cases, 16 (18.4 %) required hospitalization, with 10 cases in the pre-HU phase and six in the post-HU phase. However, some patients sought medical attention outside of their scheduled consultations, going to the nearest health unit or hospital emergency departments, making it impossible to collect a sample to confirm the diagnosis or Plasmodium species identification. Molecular diagnosis was performed on a subgroup of 65 children, with 415 dried blood spots analyzed throughout the study period, corresponding to several samples of the same individuals in different stages of the study (54 samples in the pre-HU phase and 361 in the post-HU phase) (Table 4). Plasmodium infection was detected in 15 samples from 10 patients in both phases of the study, with two patients testing Fig. 1. Patient retention throughout the study. Table 2 Characterization of children who had complications during the period of medication with HU. Children #1 #2 #3 #4 Complications Avascular necrosis Stroke Stroke Stroke Age (years) 6 11 9 8 Gender M M F M Age 1st manifestation (months) 6 20 6 2 Precedent symptoms Severe anemia Osteomyelitis 2 strokes Severe anemia Treatment duration (months) 5 2 12 12 Basal HbF (%) 3.4 1.5 5.9 1 Post-HU HbF (%) 10.2 12.8 10.1 1.8 HbF variation (%) 6.7 11.3 4.2 0.8 Hb pre-/post-HU 7.6/8.5 6.4/7.4 6.3/7.5 7.9/7.7 MCV pre-/post-HU 81.4/90.3 82.5/100.5 84.7/ 93.2 76.7/83.1 MCH pre-/post-HU 26.4/28.8 28.5/34.2 28.7/ 28.4 26.5/28.0 3.7 kb alpha thalassemia genotype αα /− α 3.7 αα / αα αα /− α 3.7 αα /− α 3.7 Table 3 Laboratorial parameters in preand post-HU phases. Laboratory parameters N Pre-HU N Post-HU pValue Mean ±SD Mean ±SD HbF 139 5.94 ±3.91 139 9.9 ±6.03 <0.001 Hemoglobin (g/dL) 141 7.47 ±0.89 141 8.00 ±0.96 <0.001 MCV (fL) 141 76.80 ±7.69 140 85.02 ±8.96 <0.001 MCH (pg) 141 25.39 ±2.91 140 28.88 ±3.47 <0.001 Reticulocytes (%) 141 9.81 ±3.74 141 6.80 ±2.82 <0.001 Leukocytes (×10 3 / μ L) 141 13.38 ±3.82 141 9.91 ±2.36 <0.001 Neutrophils (×10 3 / μ L) 141 5.68 ±1.89 141 4.21 ±1.22 <0.001 Platelets (×10 3 / μ L) 141 446.90 ± 171.85 141 406.19 ± 122.91 <0.001 LDH (U/L) 141 485.42 ± 184.64 141 476.46 ± 115.88 0.570 Total bilirubin (mg/ dL) 140 1.37 ±1.43 141 1.04 ±0.50 0.007 Direct bilirubin (mg/ dL) 141 0.56 ±0.91 141 0.56 ±0.27 0.939 Urea (mg/dL) 134 30.75 ± 18.57 141 33.17 ±9.49 0.139 Creatinine (mg/dL) 141 0.51 ±0.60 141 0.43 ±0.18 0.111 AST (U/L) 141 34.06 ± 16.36 141 35.89 ± 22.76 0.428 ALT (U/L) 141 11.05 ±9.62 141 17.96 ± 28.33 0.006 Comparisons were made by paired sample t-test. B. Santos et al. Blood Cells, Molecules and Diseases 105 (2024) 102822 6 positive on two occasions in the post-HU phase. Four patients who had malaria in the pre-HU phase did not experience any new episodes. The frequency of malaria episodes did not appear to change with the increase in HbF, and multiple episodes were observed in cases with a ≥5 % increase in HbF (Table 4). Three Plasmodium species were identified: P. falciparum, P. malariae, and P. ovale curtisi, with 11 cases occurring in the high transmission season and four in the low transmission season. Of the 15 cases with positive Plasmodium diagnosis, nine patients had some sort of symptom or alteration. Six patients reported pain, and three patients had a decrease in hemoglobin values, but did not require blood transfusions. 4. Discussion SCA affects approximately 1.5 % of the population in Angola [35]. Despite HU demonstrated clinical and laboratory benefits worldwide [13,28], particularly in Africa [18], it is still not available as a comprehensive care option for Angolan patients with SCA. Out of the 215 patients enrolled in this study, 157 (73.02 %) started HU medication, and 141 of them completed the 12-month course. Nonattendance to consultations (25.1 %) resulted in a high number of patients lost, mostly during the pre-HU phase, which may have been influenced by the onset of the COVID-19 pandemic in Angola. The reasons for dropouts were not assessed in this study. In another study, also carried out in an African country, authors identified the frequency and cost of travel as the main reason for loss of follow-up [43]. In our study, we observed that a low baseline HbF level was associated with an earlier age of onset of the disease, a higher number of transfusions, and more severe anemia. Sheehan et al. [25] also reported that a baseline HbF concentration of <10 % was associated with a higher incidence of dactylitis in the first year of life, increased hospitalizations, and transfusions. Additionally, higher levels of HbF (>12 %) were associated with fewer clinical manifestations and a lower risk of chronic organ damage, including stroke, leg ulcers, and priapism [14] In the present study, there was no significant correlation between the baseline HbF concentration and severe complications of the disease. This lack of significant correlation could be attributed to the sample size or other factors that may be involved in the occurrence of major complications, such as, the protective role of alpha thalassemia against stroke, osteomyelitis, spleen, and liver disorders [44,45]; the influence of genetic polymorphisms, in previously identified genes, associated with anemia severity, hemolysis level, hospitalization rate [46] and the incidence of stroke [47]. The medication was well-tolerated, and no clinical side effects presumably associated with HU were reported. The monitoring of medication with laboratory evaluation allowed for the early detection of toxicity markers without clinical implications, which were reversed with a temporary interruption of medication for seven days. The response to HU medication varied among patients, with some experiencing an increase in HbF of <5 %. The mean of HbF increased to 12.19 %, ranging from 2.10 % to 31.00 %. This variability in HbF response is consistent with previous studies, such as Ware et al. [48], who reported a range of 0.1 % to 26.4 % and a mean of 9.6 %. Polymorphisms in genes that regulate HbF and HU metabolism have been suggested as potential factors contributing to the variation in HbF response to HU treatment [49–51]. From a clinical perspective, there were important improvements during the hydroxyurea treatment phase, namely in the reduction of the number transfusions, number of pain crises reported by the patients and in the number of hospitalizations, proving the beneficial effect of HU treatment. These findings support the evidence that the implementation of prophylactic measures and treatment with HU have been associated with increased survival in children with SCA [28,52]. Four patients experienced serious complications during the medication period, including stroke and necrosis of the femoral head. Of the three children who had a stroke, two occurred after 12 months of medication. Upon evaluating laboratory parameters, we found that the increase in HbF was <5 % and hematological parameters varied. Despite HU medication, classic SCA complications were observed in other studies, such as hepatic vaso-occlusive crisis, avascular necrosis of the femoral head, and recurrent splenic sequestration [31]. Although there is no consensus on this matter, some authors suggest that the duration of medication may correlate with the onset of complications, proposing that longer medication duration may lead to lower frequency of SCA complications [16,31,53] . Regarding malaria, most cases were only reported when patients sought care at other health institutions in urgent situations, making it impossible to obtain a blood sample for timely diagnosis. Of the 15 positive cases confirmed by molecular biology, 11 occurred during the peak transmission season, and of the eight positive cases in the post-HU phase, six were registered before six months of medication. In the study by Olupot-Olupot et al. [8], HU medication was associated with a progressive decrease in the incidence of malaria over the course of medication when the maximum tolerated dose was reached. In cases of malaria, the clinical manifestations associated with the SCA phenotype were episodes of pain and a decrease in Hb levels without the need for blood transfusion [54]. In our study, the highest number of hospitalizations was seen in patients with a low baseline HbF level. The mortality rate in this study was 2.3 % (n =5) and was not associated with medication. The deaths occurred in the age group of six to 10 years old and were attributed to malaria (three cases), two of which were in the pre-HU phase. One death was due to anemia in an infectious context in the post-HU phase, and one death was due to an event not related to the disease (a road accident). Patients who died of infectious causes had a low baseline HbF level (HbF ranging from 1.5 % to 5.2 %). It has been previously reported that the level of HbF is inversely proportional to SCA mortality [13]. In Africa the high mortality rate in SCA children is compounded by the prevalence of infectious diseases, especially bacterial and malaria, and this association was also evident in our study. There was a limitation in the study's ability to provide a detailed characterization of clinical episodes due to patients seeking treatment at other hospital units, which made it difficult to Table 4 Malaria Infection as diagnosed by nested PCR and variation of HbF preand post-HU. Patient Pre-HU Post-HU/medication time (months) HbF (%) 2 3 4 5 12 Pre-HU Post-HU 1 P. falciparum Neg Neg Neg Neg P. falciparum 1.1 2.3 2 P. falciparum P. malariae Neg Neg P. malariae Neg 5.9 22.3 3 P. falciparum Neg Neg Neg Neg Neg 10.5 15.5 4 P. malariae Neg Neg Neg Neg Neg 3.5 8.7 5 Neg Neg P. falciparum Neg Neg Neg 7.1 6.6 6 P. falciparum Neg Neg Neg P. falciparum Neg 12.4 12.5 7 P. falciparum Neg Neg Neg Neg Neg 3.9 11 8 P. falciparum Neg Neg Neg Neg Neg 4.9 8 9 Neg Neg Neg Neg P.o. curtisi P.o. curtisi 6.8 11.8 10 Neg Neg Neg P. falciparum Neg Neg 1 1.8 B. Santos et al. Blood Cells, Molecules and Diseases 105 (2024) 102822 7 gather precise information about malaria infection and its influence on the SCA phenotype. A more effective approach for future studies would be to collect blood samples on filter paper during acute episodes, even when patients seek treatment at other institutions. 5. Conclusion The present study provided a valuable contribution to the understanding of the clinical and laboratory profiles of Angolan children with SCA, which can help in the development of guidelines for the use and monitoring of HU in Angola. However, the study also raised important questions about the variability in clinical and laboratory response to HU, as well as the occurrence and recurrence of clinical manifestations in children receiving HU. These questions highlight the need for further studies in Angola to better understand these issues, given the early and severe onset of SCA manifestations in this population. With the anticipated availability of HU in Angola in the short to medium term, it is hoped that more patients will benefit from this medication. Funding This research was funded by Fundaç˜ ao para a Ciˆ encia e Tecnologia Minist´ erio da Ciˆ encia, Tecnologia e Ensino Superior (FCT-MCTES) and Aga Khan Foundation (FCT/MCTES/Aga Khan, project n◦330842553), by FCT/MCTES funding to H&TRC (UIDB/05608/2020, UIDP/05608/ 2020) and to GHTM IHMT NOVA (UID/04413/2020) and LA-REAL – LA/P/0117/2020. CRediT authorship contribution statement Brígida Santos: Writing – original draft, Methodology, Investigation, Formal analysis. Catarina Ginete: Methodology, Investigation, Formal analysis, Data curation. Elisˆ angela Gonçalves: Writing – review & editing, Methodology, Investigation. Mariana Delgadinho: Writing – review & editing, Methodology, Investigation. Armandina Miranda: Writing – review & editing, Methodology, Investigation. Paula Faustino: Writing – review & editing, Methodology, Investigation. Ana Paula Arez: Writing – review & editing, Supervision, Methodology, Investigation. Miguel Brito: Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis. Declaration of competing interest There are no interests to declare. Acknowledgments We would like to express our gratitude to all the children and caregivers who participated in this study by granting permission for sample collection. We also acknowledge the invaluable support of health technicians who made this study possible, specifically Miguel Panzo, Adriano Siatembo, and Felix Lange at CISA. The authors acknowledge the Fundaç˜ ao Calouste Gulbenkian and Cam˜ oes – Instituto da Cooperaç˜ ao e da Língua for logistic support during the project. References [1] F.B. Piel, M.H. Steinberg, D.C. Rees, Sickle cell disease, in: D.L. Longo (Ed.), New England Journal of Medicine vol. 376 (16), Apr 20, 2017, pp. 1561–1573 [Internet]. Available from: http://www.nejm.org/doi/10.1056/NEJMra1510865. [2] A.D. Adekile, S. Al-Sherida, R. Marouf, N. Mustafa, D. Thomas, The sub-phenotypes of sickle cell disease in Kuwait, Hemoglobin 43 (2) (Mar 4, 2019) 83–87. [3] J. Makani, S.F. Ofori-Acquah, O. Nnodu, A. Wonkam, K. Ohene-Frempong, Sickle cell disease: new opportunities and challenges in Africa, Sci. World J. 2013 (2013). [4] N.S. Green, S. Barral, Genetic modifiers of HbF and response to hydroxyurea in sickle cell disease, Pediatr. Blood Cancer 56 (2011) 177–181. [5] G.J. Kato, F.B. Piel, C.D. Reid, M.H. Gaston, K. Ohene-Frempong, L. Krishnamurti, et al., Sickle cell disease, Nat. Rev. Dis. Primers 4 (1) (Mar 15, 2018) 18010 [Internet]. Available from: https://www.nature.com/articles/nrdp201810. [6] S.D. Grosse, I. Odame, H.K. Atrash, D.D. Amendah, F.B. Piel, T.N. Williams, Sickle cell disease in Africa: a neglected cause of early childhood mortality, Am. J. Prev. Med. 41 (2011) (Elsevier Inc.). [7] C.F. McAuley, C. Webb, J. Makani, A. Macharia, S. Uyoga, D.H. Opi, et al., High mortality from Plasmodium falciparum malaria in children living with sickle cell anemia on the coast of Kenya, Blood 116 (10) (Sep 9, 2010) 1663–1668. [8] P. Olupot-Olupot, G. Tomlinson, T.N. Williams, L. Tshilolo, B. Santos, L.R. Smart, et al., Hydroxyurea treatment is associated with lower malaria incidence in children with sickle cell anemia in sub-Saharan Africa, Blood 141 (12) (Mar 23, 2023) 1402–1410 [Internet]. Available from: http://www.ncbi.nlm.nih.gov/pubmed /36375125. [9] G.R. Serjeant, The natural history of sickle cell disease, Cold Spring Harb. Perspect. Med. 3 (10) (2013). [10] M.H. Steinberg, Fetal-like hemoglobin in sickle cell anemia, N. Engl. J. Med. 386 (7) (Feb 17, 2022) 689–691. [11] S.L. Thein, Genetic association studies in-hemoglobinopathies [Internet]. Available from: http://ashpublications.org/hematology/article-pdf/2013/1/354/1250849/ bep00113000354.pdf. [12] M. Delgadinho, C. Ginete, B. Santos, A. Miranda, M. Brito, Genotypic diversity among angolan children with sickle cell anemia, Int. J. Environ. Res. Public Health 18 (10) (May 2, 2021). [13] M.H. Steinberg, F. Barton, O. Castro, C.H. Pegelow, S.K. Ballas, A. Kutlar, et al., Effect of hydroxyurea on mortality and morbidity in adult sickle cell anemia risks and benefits up to 9 years of treatment [Internet]. Available from: https://jamanet work.com/. [14] A. Adekile, The genetic and clinical significance of fetal hemoglobin expression in sickle cell disease, Med. Princ. Pract. 30 (3) (Jun 1, 2021) 201–211 [Internet]. Available from: https://www.karger.com/Article/FullText/511342. [15] R. Thomas, R. Dulman, A. Lewis, B. Notarangelo, E. Yang, Prospective longitudinal follow-up of children with sickle cell disease treated with hydroxyurea since infancy, Pediatr. Blood Cancer 66 (9) (Sep 1, 2019). [16] A. Ferster, P. Tahriri, C. Vermylen, G. Sturbois, F. Corazza, P. Fondu, et al., Five years of experience with hydroxyurea in children and young adults with sickle cell disease, Blood 97 (11) (Jun 1, 2001) 3628–3632 [Internet]. Available from: https ://ashpublications.org/blood/article/97/11/3628/107474/Five-years-of-experie nce-with-hydroxyurea-in. [17] S. Amuel, C. Harache, I.L.T. Errin, I.D.M. Oore, G. Eorge, J.D. Over, et al., Effect of Hydroxyurea on the Frequency of Painful Crises in Sickle Cell Anemia and the Investigators of Them Ulticenter Study of Hydroxyurea in Sickle Cell Anemia * vol. 332, 1995. [18] L. Tshilolo, G. Tomlinson, T.N. Williams, B. Santos, P. Olupot-Olupot, A. Lane, et al., Hydroxyurea for children with sickle cell anemia in Sub-Saharan Africa, N. Engl. J. Med. 380 (2) (Jan 10, 2019) 121–131. [19] T. Kratovil, D. Bulas, M.C. Driscoll, B. Speller-Brown, R. McCarter, C.P. Minniti, Hydroxyurea therapy lowers TCD velocities in children with sickle cell disease, Pediatr. Blood Cancer 47 (7) (Dec 2006) 894–900. [20] I.O. Lagunju, B.J. Brown, A.O. Oyinlade, A. Asinobi, J. Ibeh, A. Esione, et al., Annual stroke incidence in Nigerian children with sickle cell disease and elevated TCD velocities treated with hydroxyurea, Pediatr. Blood Cancer 66 (3) (Mar 1, 2019). [21] S.A. Zimmerman, W.H. Schultz, S. Burgett, N.A. Mortier, R.E. Ware, Hydroxyurea therapy lowers transcranial Doppler flow velocities in children with sickle cell anemia, Available from: www.bloodjournal.org, 2007. [22] M.C. Silva, E.L.T. Shimauti, Efic´ acia e toxicidade da hidroxiur´ eia em crianças com anemia falciforme effectiveness and toxicity of hydroxyurea in children with sickle cell anemia, Rev. Bras. Hematol. Hemoter. 28 (2006). [23] R. Borba, C.S.P. Lima, H.Z.W. Grotto, Reticulocyte parameters and hemoglobin F production in sickle cell disease patients undergoing hydroxyurea therapy, J. Clin. Lab. Anal. 17 (2) (2003) 66–72. [24] R.K. Agrawal, R.K. Patel, V. Shah, L. Nainiwal, B. Trivedi, Hydroxyurea in sickle cell disease: drug review, Indian J. Hematol. Blood Transfus. 30 (2014) 91–96 (Springer India). [25] V.A. Sheehan, Z. Luo, J.M. Flanagan, T.A. Howard, B.W. Thompson, W.C. Wang, et al., Genetic modifiers of sickle cell anemia in the baby hug cohort: influence on laboratory and clinical phenotypes, Am. J. Hematol. 88 (7) (Jul 2013) 571–576. [26] W.C. Wang, L.W. Wynn, Z.R. Rogers, J.P. Scott, P.A. Lane, R.E. Ware, A two-year pilot trial of hydroxyurea in very young children with sickle-cell anemia, J. Pediatr. 139 (6) (Dec 2001) 790–796 [Internet]. Available from: https://linkinghub.else vier.com/retrieve/pii/S002234760191580X. [27] Kinney TR, Helms RW, O'branski EE, Ohene-Frempong K, Wang W, Daeschner C, et al. Safety of Hydroxyurea in Children With Sickle Cell Anemia: Results of the HUG-KIDS Study, a Phase I/II Trial. [28] Lopes de Castro, C. Lobo, J.F.C. Pinto, E.M. Nascimento, P.G. Moura, G.P. Cardoso, J.S. Hankins, The effect of hydroxcarbamide therapy on survival of children with sickle cell disease, Br. J. Haematol. 161 (6) (Jun 2013) 852–860. [29] E. Voskaridou, D. Christoulas, A. Bilalis, E. Plata, K. Varvagiannis, G. Stamatopoulos, et al., The effect of prolonged administration of hydroxyurea on morbidity and mortality in adult patients with sickle cell syndromes: results of a 17-year, single-center trial (LaSHS), Blood 115 (12) (Mar 25, 2010) 2354–2363. [30] J.J. Strouse, S. Lanzkron, M.C. Beach, C. Haywood, H. Park, C. Witkop, et al., Hydroxyurea for sickle cell disease: a systematic review for efficacy and toxicity in children, Pediatrics 122 (6) (Dec 2008) 1332–1342. B. Santos et al. Blood Cells, Molecules and Diseases 105 (2024) 102822 8 [31] M. Maier-Redelsperger, M. de Montalembert, A. Flahault, M.G. Neonato, R. Ducrocq, M.P. Masson, et al., Fetal hemoglobin and F-cell responses to long-term hydroxyurea treatment in young sickle cell patients, Blood 91 (12) (Jun 15, 1998) 4472–4479 [Internet]. Available from: https://ashpublications.org/blood/article /91/12/4472/260859/Fetal-Hemoglobin-and-FCell-Responses-to-LongTerm. [32] S.A. Zimmerman, W.H. Schultz, J.S. Davis, C.V. Pickens, N.A. Mortier, T. A. Howard, et al., Sustained long-term hematologic efficacy of hydroxyurea at maximum tolerated dose in children with sickle cell disease, Blood 103 (6) (Mar 15, 2004) 2039–2045. [33] E.C. Aneni, D.H. Hamer, C.J. Gill, Systematic review of current and emerging strategies for reducing morbidity from malaria in sickle cell disease, Tropical Med. Int. Health 18 (2013) 313–327. [34] J.N. Anyanwu, O. Williams, C.L. Sautter, P. Kasirye, H. Hume, R.O. Opoka, et al., Novel use of hydroxyurea in an African region with malaria: protocol for a randomized controlled clinical trial, JMIR Res. Protoc. 5 (2) (Jun 23, 2016) e110. [35] P.T. Mcgann, M.G. Ferris, U. Ramamurthy, B. Santos, V. de Oliveira, L. Bernardino, et al., A prospective newborn screening and treatment program for sickle cell anemia in Luanda, Angola, Am. J. Hematol. 88 (12) (Dec 2013) 984–989. [36] R.O. Opoka, C.M. Ndugwa, T.S. Latham, A. Lane, H.A. Hume, P. Kasirye, et al., Novel use of hydroxyurea in an African Region with malaria (NOHARM): a trial for children with sickle cell anemia, Available from: www.clinicaltrials.gov, 2017. [37] C.V. Plowe, A. Djimde, M. Bouare, O. Doumbo, T.E. Wellems, Pyrimethamine and proguanil resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase: polymerase chain reaction methods for surveillance in Africa, Am. J. Trop. Med. Hyg. 52 (6) (1995) 565–568. [38] G. Snounou, S. Viriyakosola, X. Ping Zhua, W. Jarraa, L. Pinheirob, V.E. do Rosariob, et al., High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction, Mol. Biochem. Parasitol. 61 (2) (1993) 315–332. [39] C.J. Sutherland, N. Tanomsing, D. Nolder, M. Oguike, C. Jennison, S. Pukrittayakamee, et al., Two nonrecombining sympatric forms of the human malaria parasite plasmodium ovale occur globally, J. Infect. Dis. 201 (10) (May 15, 2010) 1544–1550. [40] H.P. Fuehrer, M.T. Stadler, K. Buczolich, I. Bloeschl, H. Noedl, Two techniques for simultaneous identification of Plasmodium ovale curtisi and Plasmodium ovale wallikeri by use of the small-subunit rRNA gene, J. Clin. Microbiol. 50 (12) (Dec 2012) 4100–4102. [41] A.R. Chand, H. Xu, L.G. Wells, B. Clair, C. Neunert, A.E. Spellman, et al., Are there true non-responders to hydroxyurea in sickle cell disease? A multiparameter analysis, Blood 124 (21) (Dec 6, 2014) 4073. [42] P.T. McGann, R.E. Ware, Hydroxyurea therapy for sickle cell anemia, in: Expert Opinion on Drug Safety vol. 14, Taylor and Francis Ltd, 2015, pp. 1749–1758. [43] R.I. Chianumba, A.O.D. Ofakunrin, J. Morrice, O. Olanrewaju, O. Oniyangi, A. Kuliya-Gwarzo, et al., Outcome of hydroxyurea use in SCD and evaluation of patients’ perception and experience in Nigeria, Front. Genet. 13 (Mar 24, 2022). [44] O.O. Ojewunmi, T.A. Adeyemo, A.I. Oyetunji, Y. Benn, M.G. Ekpo, B.A. Iwalokun, Association of alpha-thalassemia and Glucose-6-Phosphate Dehydrogenase deficiency with transcranial Doppler ultrasonography in Nigerian children with sickle cell anemia, J. Clin. Lab. Anal. 35 (6) (Jun 1, 2021). [45] B. Santos, M. Delgadinho, J. Ferreira, I. Germano, A. Miranda, A.P. Arez, et al., CoInheritance of alpha-thalassemia and sickle cell disease in a cohort of Angolan pediatric patients, Mol. Biol. Rep. 47 (7) (Jul 1, 2020) 5397–5402. [46] I. Germano, B. Santos, M. Delgadinho, C. Ginete, P. Lopes, A.P. Arez, et al., Genetic modulation of anemia severity, hemolysis level, and hospitalization rate in Angolan children with Sickle Cell Anemia, Mol. Biol. Rep. 49 (11) (Nov 1, 2022) 10347–10356. [47] J.M. Flanagan, D.M. Frohlich, T.A. Howard, W.H. Schultz, C. Driscoll, R. Nagasubramanian, et al., Genetic predictors for stroke in children with sickle cell anemia, Blood 117 (24) (Jun 16, 2011) 6681–6684. [48] R.E. Ware, B. Eggleston, R. Redding-Lallinger, W.C. Wang, K. Smith-Whitley, C. Daeschner, et al., Predictors of fetal hemoglobin response in children with sickle cell anemia receiving hydroxyurea therapy [Internet]. Available from: http://ash publications.org/blood/article-pdf/99/1/10/1679428/h80102000010.pdf, 2002. [49] M.M. Aleluia, R.P. Santiago, C.C. da Guarda, T.C.C. Fonseca, F.I. Neves, R. S. Quinto, et al., Genetic modulation of fetal hemoglobin in hydroxyurea-treated sickle cell anemia, Am. J. Hematol. 92 (2017) E70–E72 (Wiley-Liss Inc.). [50] R.R. Sales, B.L. Nogueira, J.A.G. Tosatti, K.B. Gomes, M.R. Luizon, Do genetic polymorphisms affect fetal hemoglobin (HbF) levels in patients with sickle cell Anemia treated with hydroxyurea? A systematic review and pathway analysis, Front. Pharmacol. 12 (2022) (Frontiers Media S.A.). [51] C. Ginete, M. Delgadinho, B. Santos, V. Pinto, C. Silva, A. Miranda, et al., Are genetic modifiers the answer to different responses to hydroxyurea treatment?-a pharmacogenetic study in sickle cell anemia Angolan children, Int. J. Mol. Sci. 24 (10) (May 1, 2023). [52] G.A.O. Arduini, L.P. Rodrigues, A.B. Trov´ o de Marqui, Mortality by sickle cell disease in Brazil, Rev. Bras. Hematol. Hemoter. 39 (2017) 52–56 (Elsevier Editora Ltda). [53] R.E. Ware, S.A. Zimmerman, W.H. Schultz, Hydroxyurea as an alternative to blood transfusions for the prevention of recurrent stroke in children with sickle cell disease [Internet]. Available from: http://ashpublications.org/blood/article-pdf/9 4/9/3022/1659039/3022.pdf. [54] M. Muszlak, S. Pissard, C. Badens, A. Chamouine, O. Maillard, I. Thuret, Genetic modifiers of sickle cell disease: a genotype-phenotype relationship study in a cohort of 82 children on Mayotte Island, Hemoglobin 39 (3) (Jun 1, 2015) 156–161. B. Santos et al.