Abstract The Consortium on Newborn Screening in Africa (CONSA), launched by the American Society of Hematology in 2020, is designed to initiate and expand sustainable newborn screening (NBS) programs for sickle cell disease (SCD) across sub-Saharan Africa. This multiyear pilot program includes 11 clinical sites in 7 countries, namely Ghana, Kenya, Liberia, Nigeria, Tanzania, Uganda, and Zambia. After extensive training of laboratory and clinical personnel, dried blood spots were collected from newborns and tested by isoelectric focusing at central laboratories within each country. Positive samples were confirmed, and the affected infants were invited into clinical care for penicillin prophylaxis, malaria prevention, routine immunizations, and family education. As of November 2025, almost 175 000 samples have been collected and assessed. The overall prevalence of SCD was 1.46%, with the highest prevalence in Mwanza, Tanzania (2.00%). The majority of positive screening results were homozygous HbSS (81.5%), along with compound heterozygous HbSC (11.0%) and HbSβ+ thalassemia (7.5%). Hemoglobin S trait was common throughout the countries with an average of 16.17%, whereas hemoglobin C trait had an incidence of 1.59% and was found primarily in Ghana and Nigeria. Additional hemoglobin variants were also detected in several countries. Confirmatory samples have been documented in about one-third of infants with a positive screening result, with 87.8% of those confirmed to have SCD. Fewer than half of the affected infants have documented clinical follow-up at CONSA sites for various logistical and financial reasons. CONSA has made great strides in promoting NBS for SCD in sub-Saharan Africa, but gaps in the confirmatory testing and enrollment into clinical care persist.

Introduction: In 2018, the American Society of Hematology (ASH) established the Consortium on Newborn Screening in Africa (CONSA) for sickle cell disease (SCD), a program designed to implement standardized hemoglobinopathy newborn screening (NBS) coupled with early clinical management for children with SCD across sub-Saharan Africa. After a rigorous application and evaluation process, seven countries with large clinical catchment areas in sub-Saharan Africa were included into CONSA. We now present the initial cumulative diagnostic screening results, which represent the feasibility, training, and proof-of-principle stages of the consortium. Methods: ASH members and staff work directly with national coordinators and local laboratory scientists to implement NBS using standardized isoelectric focusing (IEF) techniques. Blood samples are collected by heel prick in newborns and young infants at local hospitals and clinics; dried blood spots (DBS) are prepared and then transported to a central laboratory for testing. The laboratory techniques for conducting and scoring the IEF gels follow a Standard Operating Procedure (SOP) developed by CONSA and include known controls for hemoglobin A, F, S, and C. All DBS samples that are scored as SCD (hemoglobin FS or FSC) are initially repeated to verify the abnormal result; a subsequent new sample is then collected at the first clinical visit to confirm the diagnosis of SCD. Results: To date, ten screening centers have been established in seven countries across sub-Saharan Africa: Ghana, Kenya, Liberia, Nigeria, Tanzania, Uganda, and Zambia. As of April 2024, a total of 73,903 unique DBS samples have been tested and scored with satisfactory results, which have been curated and uploaded to the ASH CONSA database. Sickle cell trait has been identified in 11,281 (15.43%) of DBS tested, ranging from a low value of 5.7% in Monrovia, Liberia to a high value of 23.7% in Kaduna, Nigeria. Sickle cell anemia has been identified in 1055 (1.44%) of DBS tested, ranging from a low value of 0.4% in Monrovia, Liberia to 2.8% in Jinja and Lira, Uganda. Hemoglobin variants have been recorded in 412 (0.56%) DBS samples with the vast majority of these found in Uganda and Kenya. Of the SCD cases identified, a total of 392 have been confirmed with an additional blood sample and the mothers and caregivers have been counselled. Efforts are ongoing to locate and enroll these infants into clinical management. Upcoming steps to increase the efficiency of SCD diagnosis include using point-of-care devices for disease confirmation, after a positive IEF result from the initial DBS sample. Conclusion: The ASH-initiated CONSA program has demonstrated, and will continue to demonstrate, that NBS is feasible to conduct in high-burden low-resource areas within multiple countries across sub-Saharan Africa. The process of rigorous and repeated laboratory and technical training, coupled with a high-quality standardized SOP, has documented the high burden of sickle cell trait and disease across this region. Ongoing efforts to link children diagnosed with SCD with early clinical care will continue. Communication of these results with local, national, and donor partners will help raise awareness and should increase efforts toward universal NBS screening.

More than 75% of the global burden of sickle cell disease (SCD) occurs in sub-Saharan Africa, where scarce diagnostic and health care services result in unacceptably high under-five mortality rates. To address this challenge the American Society of Hematology (ASH) initiated the Consortium on Newborn Screening in Africa (CONSA) comprising seven countries: Ghana, Liberia, Nigeria, Kenya, Tanzania, Uganda and Zambia. CONSA seeks to demonstrate the benefits of hemoglobinopathy newborn screening and early clinical interventions for babies diagnosed with SCD. Dried blood spots (DBS), collected by heel prick of newborns or young infants at local hospitals and clinics are transported to a central laboratory at each site for testing. Ten sites in these seven countries initiated screening by December 2021. An accurate diagnosis of SCD is critical for CONSA's success, and here we highlight the strategy and outcome of six key measures implemented to build local capacity and infrastructure. 1) Public private partnerships. ASH engaged with a commercial partner, which facilitated the subsidized procurement of the same isoelectric focusing (IEF) equipment and reagents for all ten laboratory sites. In other partnerships an App was developed to capture data electronically, and to upload gel results onto an ASH datahub. 2) Standard procedures. ASH members of the CONSA Laboratory and Diagnostics subcommittee prepared SOPs covering all aspects of the diagnostic process to ensure uniform experimental procedures and documentation of the data. In addition, a central laboratory facility was designated at each site to perform IEF on all DBS collected from the surrounding clinical facilities. 3) Staff empowerment. The national coordinator of each country recruited laboratory staff who underwent rigorous and repeated training and mentorship both in-person and virtually to run and interpret IEF gels of newborns according to the CONSA SOPs. Several training sessions were conducted and in September 2023 a hands-on refresher training workshop was hosted in Ghana, where two laboratory staff from each site attended. In addition to honing their technical skills and building confidence, this allowed the staff to meet and network with their peers. 4) Quality control. Several lanes of a standard commercial control are included on each gel to facilitate accurate identification of hemoglobin A, F, S and C. Each gel is interpreted independently by two staff members to ensure accuracy of the results and to minimize transcription errors. ASH subcommittee members performed spot checks on gels and provided written feedback followed by a virtual meeting with each site to discuss errors and to institute appropriate corrective action. Each sample that is identified as SCD is verified by repeat testing with a new punch from the original DBS. The caregiver of the affected baby is called to bring the baby to the clinic where a new DBS sample is collected for a confirmatory test. Babies with confirmed SCD are enrolled in a preventive clinical care program. 5) Site visits. ASH subcommittee members visited each site to understand the contextual realities. These visits were highly informative since they provided insight into the local conditions and challenges faced by each site. Gaps could be identified, and solutions formulated by contributions from all team members. They also built rapport and fostered cooperation between the ASH subcommittee members and the laboratory staff / national coordinators. 6) ASH staff support. Since the inception of CONSA, ASH staff have provided invaluable logistic and administrative support to ensure efficient functioning of the consortium. In conclusion, CONSA has demonstrated the feasibility of setting up sickle cell newborn screening centers of excellence in resource-constrained sub-Saharan African countries with a high burden of SCD. Skills development, mentorship and quality control measures have enabled local staff to effectively screen newborns using IEF and to accurately identify infants with SCD or sickle cell trait. Babies with SCD are enrolled in a clinical care program from a very early age, which should improve their survival and quality of life. This consortium offers hope for alleviating the burden of this devastating disease and may pave the way to sustainable universal newborn screening in low- and middle-income countries, early clinical intervention and reduced morbidity and mortality.

Background The recent worldwide increase in malaria cases highlights the need for renewed efforts to eliminate malaria. The World Health Organization advocates that malaria surveillance becomes a core intervention. Current methods to estimate the malaria burden rely on clinical malaria case reports and surveys of asymptomatic parasite infection mainly from children < 5 years. In this study the hypothesis was that screening blood donors for malaria parasites would provide real-time information on the asymptomatic reservoir of parasites in the adult population and mirror other surveillance data. Methods This study was conducted in Malawi, a high malaria burden country, at the Malawi Blood Transfusion Service, which collects blood units at donation sites countrywide. A secondary analysis was conducted on data obtained from a prior Sysmex XN-31 analyser malaria diagnostic evaluation study utilizing residual donor blood samples. XN-31 malaria results, donor age, sex, geographical location, and collection date, were analysed using standard statistical methods. Results The malaria parasite prevalence in blood donors was 11.6% (614/5281 samples) increasing seasonally from December (8.6%) to April (18.3%). The median age was 21 years and 45.9% of donors were from urban areas, which showed a lower prevalence compared to non-urban regions. The Central administrative region had the highest and the Northern region the lowest malaria parasite prevalence. The donors were predominantly male (80.2%), 13.1% of whom had malaria parasites, which was significantly higher (p < 0.0001) than for female donors (7.4%). Multivariable logistic regression analysis showed that age, location, and collection month were significant predictors of malaria positivity in males, whereas in females only location was significant. There was no gender difference in parasite density nor gametocyte carriage. Conclusions This study demonstrates the powerful utility of screening blood donors for malaria parasites using the XN-31, which not only improves the safety of blood transfusion, but provides valuable complementary surveillance data for malaria control, especially targeting males, who are generally excluded from periodic household surveys. Blood donations are sourced countrywide, year-round, and thus provide dynamic, real-time information on the malaria burden. Furthermore, the XN-31 identifies the asymptomatic human reservoir of infectious gametocytes, which must be targeted to eliminate malaria.

Sickle cell disease (SCD) is a common condition within sub-Saharan Africa and associated with high under-5 mortality (U5M). The American Society of Hematology instituted the Consortium on Newborn Screening in Africa (CONSA) for SCD, a 7-country network of sites to implement standardized newborn hemoglobinopathy screening and early intervention for children with SCD in sub-Saharan Africa. CONSA's overall hypothesis is that early infant SCD screening and entry into standardized, continuous care will reduce U5M compared with historical estimates in the region. Primary trial objectives are to determine the population-based birth incidence of SCD and effectiveness of early standardized care for preventing early mortality consortium-wide at each country's site(s). Secondary objectives are to establish universal screening and early interventions for SCD within clinical networks of CONSA partners and assess trial implementation. Outcomes will be evaluated from data collected using a shared patient registry. Standardized trial procedures will be implemented among designated birth populations in 7 African countries whose programs met eligibility criteria. Treatment protocol includes administering antibacterial and antimalarial prophylaxis and standard childhood vaccinations against infections commonly affecting children with SCD. Infants with a positive screen and confirmation of SCD within the catchment areas defined by each consortium partner will be enrolled in the clinical intervention protocol and followed regularly until age of 5 years. Effectiveness of these early interventions, along with culturally appropriate family education and counseling, will be evaluated by comparing U5M in the enrolled cohort to estimated preprogram data. Here, we describe the methodology planned for this trial.

A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (<50 nM) together with strong gametocyte sterilizing properties that translated to transmission-blocking activity in the standard membrane feeding assay. Mechanistic studies through resistance selection with one of the analogues followed by whole-genome sequencing implicated the P. falciparum cyclic amine resistance locus in the mode of resistance.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium . Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H + -ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

To the Editor: Co-existence of red blood cell (RBC) membrane disorders and hemoglobinopathies are rare and may present a diagnostic challenge due to overlap of clinical findings. We describe a novel mechanism for the phenotype of hereditary pyropoikilocytosis (HPP), associated with dominant β-thalassemia due to a novel β-globin gene (HBB) mutation, exacerbated by co-inheritance of two α-spectrin gene (SPTA1) mutations in trans. Beta-thalassemia is generally an autosomal recessive disorder caused by HBB mutations, which affect the production of the β-globin subunit of hemoglobin. Frameshift or nonsense mutations in HBB exons 1 and 2 are common, resulting in abnormal termination codons, which trigger nonsense-mediated mRNA decay (NMD) that eliminates the defective mRNA.1 Individuals who are heterozygous for mutations in HBB generally have β-thalassemia minor or a trait with microcytosis and mild or no anemia. However, there are rare HBB mutations that lead to a dominantly inherited form of β-thalassemia causing moderate to severe symptomatic anemia. Splenomegaly is often present and hemolysis may be prominent. More than 40 of these mutations have been described, ranging from heterogeneous missense mutations to frameshift mutations, caused by small insertions and deletions, resulting in truncated or elongated β-globin variants.1 Most of these mutations are located in the distal part of HBB exon 2 or 3 and tend to escape NMD. This leads to an accumulation of defective peptides, which are hyper-unstable, non-functional and toxic when combined with normal α/β globin dimers. These variants may exhibit heterogeneous clinical phenotypes within families.2 Hereditary pyropoikilocytosis is a rare severe hemolytic disorder characterized by unique poikilocytic and microspherocytic erythrocytes.3 The HPP patients are typically homozygous or compound heterozygotes for mutations in SPTA1, which markedly reduce the “horizontal interactions” of spectrin αβ heterodimers to form tetramers, the main structural unit of the RBC membrane skeleton, that regulates the shape and deformability of the cell.4 These abnormalities severely weaken the skeleton resulting in poikilocyte formation during circulatory shear stress. Hereditary pyropoikilocytosis patients also exhibit a decreased amount of spectrin, which interferes with the “vertical interactions” between the skeleton and the lipid bilayer and leads to loss of membrane and microspherocytes. This combination of qualitative and quantitative defects of spectrin is a hallmark of HPP.4 Here we describe a 59-year-old Dominican male who presented with life-long history of severe microcytic hypochromic anemia and jaundice, with his hemoglobin ranging from 5–7 g/dL requiring occasional RBC transfusions, without iron deficiency. The clinical and laboratory findings are shown in Figure 1(A). His peripheral blood smear (Figure 1(B)) revealed typical HPP RBC morphology. After initial evaluation, the proband became progressively severely anemic. Ten months later, he underwent bone marrow biopsy that was compatible with myelodysplastic syndrome, confirmed by cytogenetic abnormalities. He was then lost to follow up. We performed several investigations as outlined in supplementary data. Sequencing of the HBB gene revealed a novel pathogenic frameshift β-thalassemia mutation in exon 2, HBB282delT, coding for β-globin Cys93fs. No large deletions or duplications were detected by multiplex ligation-dependent probe amplification (MLPA) analysis. Computational algorithms predicted that intron 2 splicing is not affected, and the mutant HBB transcript was confirmed by Sanger sequencing using cDNA obtained from the patient's peripheral blood granulocyte RNA. As we could not obtain sufficient reticulocyte RNA, we performed in vitro expansion of erythroid progenitors in 3-week liquid erythroid cultures. Initially, most of the peripheral blood mononuclear cells were lymphoid cells. At day 7, most lymphoid cells were dead, and the remaining cells were erythroid progenitors. The cells expressing CD71 at day 7 were counted showing that the difference in cell number between samples was minimal. Progenitor cells were harvested at day 14 and we detected a mutant HBB transcript missing the T in codon 93 of exon 2. Equal amounts of the mutant and wild type HBB transcripts were present, indicating that NMD was not induced. The mutation is predicted to result in an elongated protein of 156 amino acids, referred to as Hb Santo Domingo, containing ten additional residues at the C-terminal end, and altering all the amino acids downstream of the frameshift until a new stop codon terminates translation (Figure S1). The mutant sequence is enriched in hydrophobic amino acids, including nine tryptophan residues, which have bulky side chains that influence the structural aspects and stability of the peptide. Secondary structure predictions revealed that the α helices normally present in the C-terminal end of β-globin are disrupted and replaced by random coils and extended strands. Four acidic amino acids (two aspartic and two glutamic acid residues) that are important for electrostatic interactions with α-globin are lacking, and ten other amino acids that are critical for α/β dimer formation have been lost, implying that the elongated mutant β-globin is unable to participate in the assembly of a stable globin tetramer. The new hydrophobic C-terminal may render the mutant peptide less susceptible to effective proteolysis, resulting in an accumulation of defective peptides. The excess single β-globin chains form insoluble aggregates that precipitate, together with redundant α-globin, as intracellular inclusions,5 evidenced by the presence of numerous Heinz bodies in the patient. The isopropanol test was positive for unstable hemoglobin. However, no mutant hemoglobin could be detected by cation exchange HPLC, reverse phase HPLC or capillary electrophoresis, most likely due to the hyper-instability of the aberrant β-globin molecule and Heinz body formation, which prevents in vitro solubility and laboratory detection. These inclusions likely contribute to accelerated RBC destruction. The in vitro analysis of erythroid progenitors revealed a marked decrease in both proliferation (Figure 1(C)) and differentiation (Figure 1(D)) as evaluated by temporal changes of glycophorin and transferrin receptor positivity and intensity in the proband. The hemoglobinization of maturing erythroid progenitors was not discernible at day 14 while the control had readily discernible “red” erythroid progenitors. These data suggest that Hb Santo Domingo compromises erythropoiesis and that the mutant protein is toxic to erythroid progenitors. The HPP morphology of the proband prompted further investigations. Of note, EMA analysis showed a mild decrease in fluorescence of 13% indicative of an RBC membrane protein defect. By exome and targeted Sanger sequencing, two mutations were detected in SPTA1. High-throughput sequencing revealed no pathogenic mutations in genes coding for the other major RBC membrane proteins and enzymes, as well as other genes relevant to hemolytic anemia. Theproband was heterozygous for spectrin Jendouba (SPTA1D791E[c.2373C > A]),6 in the SPTA1 gene in exon 17. This mutation resides in the α8 repeat within the αII domain of α-spectrin, which is far away from the dimer self-association site at the N-terminal in the αI domain, and thus has minimal effect on spectrin tetramer formation. Morphologically, it is associated with infrequent elliptocytes.6 The SPTA1 hypomorphic αLELY polymorphism7 was present in trans to spectrin Jendouba. This αLELY is a low expression allele composed of three variants (c.5572C > G, c.6531-12C > T, c.6549-12G > A) in SPTA1 and has a frequency of 20%–31% in different populations.7 On its own αLELY does not cause disease, neither in heterozygotes nor homozygotes. The αLELY allele causes exon skipping in 50% of the SPTA1 transcripts, which results in only 50% of the normal amount of α-spectrin being produced, but since α-spectrin is synthesized in excess of β-spectrin, there is sufficient α-spectrin to interact with β-spectrin to form heterodimers and tetramers. Inheritance of αLELY in trans with other pathogenic SPTA1 mutations associated with defects in the spectrin self-association site, can lead to moderate to severe hemolysis. However, co-inheritance in trans with spectrin Jendouba only resulted in a marginal increase in spectrin dimers6 and would thus not give rise to the HPP morphology in our patient. A more plausible explanation for the RBC membrane budding and microspherocytosis is that the hyper-unstable β-globin thalassemia mutation promotes the formation of Heinz bodies, which bind to the RBC membrane8 and destabilize the underlying abnormal spectrin skeleton. It has been shown that there is a reduction in the quantity of spectrin in patients with unstable hemoglobin and Heinz bodies, due to oxidative damage to the membrane.9 A decreased amount of spectrin would thus explain the microspherocytes on the peripheral smear, as well as the relatively mild reduction in the binding of EMA to exposed residues of the macromolecular band 3 complex of the membrane. We propose that the combination of defects in β-globin and α-spectrin enhances the clinical severity and exacerbates the thalassemic RBC morphological abnormalities leading to an HPP phenotype. The inheritance of Hb Santo Domingo and the SPTA1 alleles was traced in family studies (Figure 1(A) and Figure S2), which were limited by the paucity of material obtained and inability to perform follow up studies. The mother had sickle cell trait and passed the HbS gene onto her three children, but only two children inherited Hb Santo Domingo. Their peripheral blood morphology was complicated by numerous sickle cells, but their RBCs did not show the typical HPP red cell morphology evident in the father. Child one showed delayed erythroid maturation compared to child two (Figure 1(D)), although the reason for this intriguing finding is not clear. Both children inherited Hb Santo Domingo and HbS, but they have different SPTA1 alleles, which, however, are either asymptomatic or have a minimal effect on the mature RBC membrane skeleton. The consequences during erythropoiesis are not known. This study illustrates the diagnostic challenge in a case of co-inheritance of defects in the RBC membrane and hemoglobin. A few such rare cases have been described10, 11 where the presence of hereditary spherocytosis or elliptocytosis enhances the severity of β-thalassemia, and also adversely influences the RBC morphology. In this we describe not only a novel HBB mutation leading to autosomal dominant β-thalassemia, but also provide a novel etiology of the HPP phenotype.10, 11 [Correction added on March 17, 2021, after first online publication: references #10 and #11 are added after original publication.] We propose that this is due to a complex interplay between mutant β-globin peptides and an abnormal RBC membrane skeleton, which reduces the spectrin content of the membrane and exacerbates the effect of the α-spectrin elliptocytosis mutation and low expression of the SPTA1 allele. S.K. and J.S. designed and performed most experiments, analyzed data and wrote the manuscript; S.R. designed and performed some experiments, and analyzed data. J.L. cared for the patient, obtained the sample and provided clinical data; A.K. performed some hemoglobinopathy experiments and analyzed data and edited and approved manuscript; A.A. assessed and evaluated clinical data, analyzed pathology, found novel beta globin mutation, and wrote the manuscript; T.L.C. performed computer modeling, analyzed data, conceptually analyzed RBC cytoskeleton data, and wrote the manuscript; J.T.P. conceived and designed the study, and interpreted all experimental and clinical data and wrote the manuscript. All authors read and approved the final version of the manuscript. This research was supported by VA Merit Review Award (J.T.P) for funding and the National Institutes of Health under Ruth L. Kirschstein National Research Service Award 2T32HL007576-31 from the National Heart, Lung, and Blood Institute (JS). The authors declare no conflict of interest. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants involved in the study. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1 Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

BACKGROUND:Early and accurate diagnosis of malaria is a critical aspect of efforts to control the disease, and several diagnostic tools are available. Microscopic assessment of a peripheral blood smear enables direct visualization of parasites in infected red blood cells and is the clinical diagnostic gold standard. However, it is subjective and requires a high level of skill. Numerous indirect detection methods are in use, but are not ideal since surrogate markers of infection are measured. This study describes the first clinical performance evaluation of the automated Sysmex XN-30 analyser, which utilizes fluorescence flow cytometry to directly detect and quantitate parasite-infected red blood cells.RESULTS:Residual EDTA blood samples from suspected malaria cases referred for routine diagnosis were analysed on the XN-30. Parasitaemia was reported as a percentage, as well as absolute numbers of infected red blood cells, and scattergrams provided a visual image of the parasitized red blood cell clusters. The results reported by the XN-30 correlated with microscopy and the analyser demonstrated 100% sensitivity and specificity. Measurements were reproducible and storage of samples at room temperature did not affect the parameters. Several Plasmodium species were detected, including Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale. The XN-30 also identified the transmissible gametocytes as a separate cluster on the scattergrams. Abnormal red blood cell indices (low haemoglobin and raised reticulocyte counts), haemoglobinopathies and thrombocytopenia did not interfere with the detection of parasites. The XN-30 also generated a concurrent full blood count for each sample.CONCLUSIONS:The novel technology of the Sysmex XN-30 provides a robust, rapid, automated and accurate platform for diagnosing malaria in a clinical setting. The objective enumeration of red blood cells infected with Plasmodium species makes it suitable for global use and allows monitoring of the parasite load once therapy has been initiated, thereby providing an early marker of drug resistance. The automated generation of a full blood count for each sample provides an opportunity for detecting unsuspected cases. Asymptomatic carriers can also be identified, which will be useful in blood transfusion centres, and will enable treatment of these individuals to prevent the spread of the disease.

A drug repositioning approach was leveraged to derivatize astemizole (AST), an antihistamine drug whose antimalarial activity was previously identified in a high-throughput screen. The multistage activity potential against the Plasmodium parasite's life cycle of the subsequent analogues was examined by evaluating against the parasite asexual blood, liver, and sexual gametocytic stages. In addition, the previously reported contribution of heme detoxification to the compound's mode of action was interrogated. Ten of the 17 derivatives showed half-maximal inhibitory concentrations (IC50s) of <0.1 μM against the chloroquine (CQ)-sensitive Plasmodium falciparum NF54 ( PfNF54) strain while maintaining submicromolar potency against the multidrug-resistant strain, PfK1, with most showing low likelihood of cross-resistance with CQ. Selected analogues ( PfNF54-IC50 < 0.1 μM) were tested for cytotoxicity on Chinese hamster ovarian (CHO) cells and found to be highly selective (selectivity index > 100). Screening of AST and its analogues against gametocytes revealed their moderate activity (IC50: 1-5 μM) against late stage P. falciparum gametocytes, while the evaluation of activity against P. berghei liver stages identified one compound (3) with 3-fold greater activity than the parent AST compound. Mechanistic studies showed a strong correlation between in vitro inhibition of β-hematin formation by the AST derivatives and their antiplasmodium IC50s. Analyses of intracellular inhibition of hemozoin formation within the parasite further yielded signatures attributable to a possible perturbation of the heme detoxification machinery.

Structure−activity relationship studies involving N-aryl-3-trifluoromethyl pyrido[1,2-a]benzimidazoles (PBI) identified several compounds possessing potent in vitro activities against the asexual blood, liver, and gametocyte stages of the Plasmodium parasite with no cross-resistance to chloroquine. Frontrunner lead compounds with good in vitro absorption, distribution, metabolism, and excretion (ADME) profiles were subjected to in vivo proof-of-concept studies in NMRI mice harboring the rodent P. berghei infection. This led to the identification of compounds 10 and 49, effecting 98% and 99.93% reduction in parasitemia with mean survival days of 12 and 14, respectively, at an oral dose of 4 × 50 mg/kg. In vivo pharmacokinetics studies on 10 revealed slow absorption, low volume of distribution, and low clearance profiles. Furthermore, this series displayed a low propensity to inhibit the human ether-a-go-go-related gene (hERG) potassium ion channel whose inhibition is associated with cardiotoxicity.

Christel Brunschwig,a Nina Lawrence,a Dale Taylor,a Efrem Abay,a Mathew Njoroge,a Gregory S. Basarab,a Claire Le Manach,b Tanya Paquet,b Diego Gonzàlez Cabrera,b Aloysius T. Nchinda,b Carmen de Kock,a Lubbe Wiesner,c Paolo Denti,c David Waterson,d Benjamin Blasco,d Didier Leroy,d Michael J. Witty,d Cristina Donini,d James Duffy,d Sergio Wittlin,e,f Karen L. White,g Susan A. Charman,g Maria Belén Jiménez-Díaz,h Iñigo Angulo-Barturen,h Esperanza Herreros,h Francisco Javier Gamo,h Rosemary Rochford,i Dalu Mancama,j Theresa L. Coetzer,k Mariëtte E. van der Watt,l Janette Reader,l Lyn-Marie Birkholtz,l Kennan C. Marsh,m Suresh M. Solapure,n† John E. Burke,z Jacob A. McPhail,z Manu Vanaerschot,o David A. Fidock,o,p Paul V. Fish,q Peter Siegl,r Dennis A. Smith,s Grennady Wirjanata,t Rintis Noviyanti,u Ric N. Price,t,v Jutta Marfurt,t Kigbafori D. Silue,w Leslie J. Street,b Kelly Chibaleb,x,y

The 2-aminopyridine MMV048 was the first drug candidate inhibiting Plasmodium phosphatidylinositol 4-kinase (PI4K), a novel drug target for malaria, to enter clinical development. In an effort to identify the next generation of PI4K inhibitors, the series was optimized to improve properties such as solubility and antiplasmodial potency across the parasite life cycle, leading to the 2-aminopyrazine UCT943.

Objectives:Novel chemical tools to eliminate malaria should ideally target both the asexual parasites and transmissible gametocytes. Several imidazopyridazines (IMPs) and 2-aminopyridines (2-APs) have been described as potent antimalarial candidates targeting lipid kinases. However, these have not been extensively explored for stage-specific inhibition of gametocytes in Plasmodium falciparum parasites. Here we provide an in-depth evaluation of the gametocytocidal activity of compounds from these chemotypes and identify novel starting points for dual-acting antimalarials. Methods:We evaluated compounds against P. falciparum gametocytes using several assay platforms for cross-validation and stringently identified hits that were further profiled for stage specificity, speed of action and ex vivo efficacy. Physicochemical feature extraction and chemogenomic fingerprinting were applied to explore the kinase inhibition susceptibility profile. Results:We identified 34 compounds with submicromolar activity against late stage gametocytes, validated across several assay platforms. Of these, 12 were potent at <100 nM (8 were IMPs and 4 were 2-APs) and were also active against early stage gametocytes and asexual parasites, with >1000-fold selectivity towards the parasite over mammalian cells. Front-runner compounds targeted mature gametocytes within 48 h and blocked transmission to mosquitoes. The resultant chemogenomic fingerprint of parasites treated with the lead compounds revealed the importance of targeting kinases in asexual parasites and gametocytes. Conclusions:This study encompasses an in-depth evaluation of the kinase inhibitor space for gametocytocidal activity. Potent lead compounds have enticing dual activities and highlight the importance of targeting the kinase superfamily in malaria elimination strategies.

In neonates presenting with a non-immune haemolytic anaemia, a high index of suspicion is raised for hereditary red cell membrane disorders. The distinction between red cell membrane disorders, however, is often difficult in neonates in the absence of a complete family history. We describe a case of a 26-day-old female twin who presented with jaundice and severe haemolysis, which required multiple red cell transfusions. Laboratory investigations revealed a non-immune haemolysis. Red cell membrane extraction and sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis, including spectrin analysis, revealed the presence of mutant spectrin αI/74. A diagnosis of hereditary elliptocytosis with transient infantile poikilocytosis was favoured. On follow-up at 4 months, a decline in haemolysis was observed.

Structure-activity relationship studies involving N-aryl-3-trifluoromethyl pyrido[1,2- a]benzimidazoles (PBI) identified several compounds possessing potent in vitro activities against the asexual blood, liver, and gametocyte stages of the Plasmodium parasite with no cross-resistance to chloroquine. Frontrunner lead compounds with good in vitro absorption, distribution, metabolism, and excretion (ADME) profiles were subjected to in vivo proof-of-concept studies in NMRI mice harboring the rodent P. berghei infection. This led to the identification of compounds 10 and 49, effecting 98% and 99.93% reduction in parasitemia with mean survival days of 12 and 14, respectively, at an oral dose of 4 × 50 mg/kg. In vivo pharmacokinetics studies on 10 revealed slow absorption, low volume of distribution, and low clearance profiles. Furthermore, this series displayed a low propensity to inhibit the human ether-a-go-go-related gene (hERG) potassium ion channel whose inhibition is associated with cardiotoxicity.

Human migration has reached an unprecedented level. Progressive globalization has integrated people and facilitated the introduction of deleterious genes into populations in which they were originally absent, thus impacting negatively on public health. A great challenge encountered by clinicians in the era of "migration hematology" lies in the hemoglobinopathies, a group of conditions representing the commonest, life-threatening, monogenic disorders globally. Of the ±400 000 newborn children affected annually, ~65% have sickle cell disease (SCD), a disorder that has spread far beyond its origins because of slave trade and contemporary population movements. More than 70% of all global SCD cases are due to autosomal recessive homozygous inheritance of a βS-mutation (missense Glu6Val) in the HBB gene, creating sickle hemoglobin (HbS), a structural variant of adult hemoglobin (HbA) and causing the most severe form of SCD, sickle cell anemia (SCA). Other forms result from inheritance of HbS in combination with different mutations, most commonly a second structural β-globin variant, βC (S/C) or one of many that lead to decreased β-globin synthesis (S/β-thalassemia). Clinical consequences are due to polymerization of deoxygenated HbS and its deleterious effects on erythrocytes. It is a debilitating syndrome characterized by chronic hemolysis and vaso-occlusive crises, manifesting as acute painful episodes, organ infarction and eventual multi-organ damage. Early diagnosis of SCD is vital so that appropriate management can be initiated timeously. Current diagnostic tests include hemoglobin electrophoresis (HE), high performance liquid chromatography (HPLC), isoelectric focusing (IEF) and DNA analysis. These specialized expensive modalities have been successfully incorporated into antenatal/neonatal screening programmes of some high-income countries such as in the UK. However, implementation of similar initiatives in various other regions has been difficult due to practical, political and economic challenges. The present study outlines a simple algorithm for use in the pediatric population to identify children at risk for SCD. Residual EDTA-anticoagulated capillary blood samples were analyzed in a tertiary hospital laboratory using an automated benchtop hematology instrument (Sysmex XN-450) that provides a CBC, differential white cell count and reticulocyte percentage (RET%). Patients with known SCD (85% SCA), confirmed by HE and HPLC, presenting for routine out-patient clinic review, were included (n=107; 1 month to 18 years old; 54% male). Samples were processed within 12 hours of collection and those who had received blood transfusions in the preceding 12 weeks were excluded. The mean ± SD was determined for each CBC parameter and RET%. Of these, 5 parameters were used to establish an algorithm with reference limits specific for each age category of patients with SCD (Table 1). When compared to the population-based normal reference ranges, these limits were statistically significantly different (p<0.05). The algorithm was validated against 107 random blood results previously obtained on routine follow up of pediatric patients with SCD. If a single outlying parameter was allowed, the algorithm demonstrated a sensitivity of 95.3% for SCD. The 4.7% of outlying cases (n=5) exhibited severe microcytosis and had a coexisting thalassemia component. The sensitivity with a complete match, i.e. 5/5 parameters, was 86.9%. Other CBC parameters were not informative. To combat the burden of SCD early and accurate diagnosis is critical. The algorithm described here is a cost-effective and sensitive filtering tool that can quickly be used to identify children at risk for SCD at any age and exclude those who do not require definitive testing, thereby limiting unnecessary expenditure. It will be beneficial in resource-constrained low-income countries; regions where SCD is not historically present and clinical suspicion is low; or in scenarios where it is difficult to obtain a medical history due to a language barrier and/or health-related stigma. Since using the algorithm only requires a CBC and RET%, it is suitable for application at any healthcare level. Assessment may be conveniently conducted in line with a child's immunization schedule prior to symptomatic presentation, and if combined with the Mentzer index and a family origin questionnaire, the sensitivity can be enhanced. No relevant conflicts of interest to declare.

Malaria remains a global health threat with 91 countries still endemic for the disease. Despite a recent substantial decline in morbidity and mortality, the magnitude of disease burden is still enormous as indicated by the World Health Organisation (WHO) report of an estimated 212 million new cases and approximately 429 000 deaths in 2015. Sub-Saharan Africa bears the brunt of the disease with 92% of all deaths. Plasmodium falciparum is the most lethal of the 5 parasite species that infect humans, and causes 99% of all deaths. In patients infected with P. falciparum , parasitemia can dramatically escalate every 48 hours or less and can be rapidly fatal if left untreated. Accurate, timely diagnosis is thus a critical first step in the management of malaria and the WHO recommends that all suspected cases must have diagnostic confirmation either by microscopy or rapid diagnostic testing (RDT), prior to initiation of artemisinin combination therapy. Microscopic assessment of peripheral blood is the current gold standard but is subjective and requires a high level of skill, whereas RDTs indirectly detect parasite antigen and can potentially give false positive/negative results. Other methods include PCR and the detection of monocytes with ingested hemozoin, which is a surrogate marker for malaria parasites. This study describes novel technology in the form of an automated Sysmex XN-30 analyzer, which utilizes a blue laser and flow cytometry to detect and count malaria-infected red blood cells. To evaluate the performance of the XN-30, 189 residual EDTA blood samples from suspected malaria cases referred for routine diagnosis by microscopy and RDT, were analyzed within 24 hours of collection. A total of 127 P. falciparum positive samples and 62 negative samples were processed over a 6-month period. Discrepancies between the analyzer and microscopy/RDT were resolved by PCR using dried blood spots prepared at the time of sample reception. The analyzer showed excellent specificity (98.5%) and sensitivity (98.4%). Measurements were reproducible with a precision of 1.24% and showed no carryover between samples. The percentage parasitemia that was accurately detected ranged from 0.003 to 27%. Positive samples were stored at room temperature and 4°C up to 7 days and daily measurements indicated that the parasite count was stable. To exclude interference by non-malaria factors, separate malaria negative samples (20 per index) with low hemoglobin, high reticulocyte count, thrombocytopenia and hemoglobinopathies (thalassemia, sickle cell anemia) were analyzed, but these had no effect on the malaria gates. Other Plasmodium species were also detected, including P. vivax, P. ovale and P. malariae. The direct detection and quantitation of Plasmodium parasites with the XN-30 holds great promise and has many advantages over microscopy/RDT/PCR. It is automated, easy to use and provides rapid, robust and objective diagnosis of malaria, which will ensure prompt treatment and thus undoubtedly improve the quality of healthcare and alleviate the disease burden in endemic countries. Since the XN-30 provides a CBC with each analysis, it may also serve as a valuable diagnostic aid in detecting unsuspected cases with fever of unknown origin. This scenario is becoming more common as international travel to endemic countries increases and tourists often present with symptoms when returning to their home country, where clinicians may not have a high index of suspicion for malaria, but would request a CBC as part of a diagnostic workup. The decline in parasitemia can be monitored by the XN-30 and if the clinical response to therapy is delayed, this may serve as an early warning sign of drug resistance. This will yield valuable data on the spread of artemisinin resistance, which is alarmingly prevalent in the Greater Mekong sub-region. The analyzer can differentiate gametocytes making it useful for detection of asymptomatic carriers who can be treated to prevent the transmission of parasites to the mosquito vector and thus protect communities. This is a vital step in disrupting the lifecycle of the parasite and is in line with global initiatives to eliminate malaria. It can also process finger prick samples making it suitable for infants and population studies, as well as monitoring efficacy of vaccines or new drugs in clinical trials. The XN-30 is currently for research use only but further development and clinical studies are in progress. Disclosures Pillay: Sysmex Corporation: Research Funding. Coetzer: Sysmex Corporation: Research Funding.

Background:Huntington's Disease-like 2 (HDL2) is classified as a neuroacanthocytosis; however, this remains unverified. We aim to determine if acanthocytes are present in HDL2 and whether acanthocytes can differentiate HDL2 from Huntington's disease (HD).Methods:We prospectively compared 13 HD and 12 HDL2 cases against 21 unaffected controls in Johannesburg. Blood smears were prepared using international standards and reviewed by at least two blinded reviewers. An acanthocytosis rate of greater than 1.2% in the dry smear or greater than 3.7% in the wet smear was designated a priori as the threshold for clinical significance based on previously established standards. Flow cytometry was performed on all but four of the cases. Red cell membrane protein analysis was performed on all participants.Results:There were 12 HDL2, 13 HD, and 21 controls enrolled. None of the HD or HDL2 participants had defined acanthocytosis or other morphological abnormalities. None of the HD or HDL2 cases had evidence of an abnormal band 3.Discussion:Acanthocytosis was not identified in either HDL2 or HD in our patient population. Our results, based on the first prospective study of acanthocytes in HDL2 or HD, suggest that screening for acanthocytes will not help establish the diagnosis of HD or HDL2, nor differentiate between the two disorders and raises the question if HDL2 should be placed within the neuroacanthocytosis syndromes.

Further structure-activity relationship (SAR) studies on the recently identified pyrido[1,2-a]benzimidazole (PBI) antimalarials have led to the identification of potent, metabolically stable compounds with improved in vivo oral efficacy in the P. berghei mouse model and additional activity against parasite liver and gametocyte stages, making them potential candidates for preclinical development. Inhibition of hemozoin formation possibly contributes to the mechanism of action.