Emergence of Plasmodium falciparum strains with artemisinin partial resistance in East Africa and the Horn of Africa: is there a need to panic?

The emergence and spread of artemisinin partial resistance in East and Horn of Africa is alarming. However, artemisinin-based combination therapy (ACT) generally remains efficacious for the treatment of falciparum malaria. The emergence of partial artemisinin resistance does not currently meet the criteria to initiate change on treatment guidelines nor affect ACT routine procurement and distribution. It is high time for scientists and transitional researchers to be more critical and vigilant on further changes so that national programmes will be able to make informed decisions as well as remain alert and prepared for any change that may be required in the future.

Genetic differentiation of Plasmodium vivax duffy binding protein in Ethiopia and comparison with other geographical isolates.

BACKGROUND: Plasmodium vivax Duffy binding protein (PvDBP) is a merozoite surface protein located in the micronemes of P. vivax. The invasion of human reticulocytes by P. vivax merozoites depends on the parasite DBP binding domain engaging Duffy Antigen Receptor for Chemokine (DARC) on these red blood cells (RBCs). PvDBPII shows high genetic diversity which is a major challenge to its use in the development of a vaccine against vivax malaria. METHODS: A cross-sectional study was conducted from February 2021 to September 2022 in five study sites across Ethiopia. A total of 58 blood samples confirmed positive for P. vivax by polymerase chain reaction (PCR) were included in the study to determine PvDBPII genetic diversity. PvDBPII were amplified using primers designed from reference sequence of P. vivax Sal I strain. Assembling of sequences was done using Geneious Prime version 2023.2.1. Alignment and phylogenetic tree constructions using MEGA version 10.1.1. Nucleotide diversity and haplotype diversity were analysed using DnaSP version 6.12.03, and haplotype network was generated with PopART version 1.7. RESULTS: The mean age of the participants was 25 years, 5 (8.6%) participants were Duffy negatives. From the 58 PvDBPII sequences, seven haplotypes based on nucleotide differences at 8 positions were identified. Nucleotide diversity and haplotype diversity were 0.00267 ± 0.00023 and 0.731 ± 0.036, respectively. Among the five study sites, the highest numbers of haplotypes were identified in Arbaminch with six different haplotypes while only two haplotypes were identified in Gambella. The phylogenetic tree based on PvDBPII revealed that parasites of different study sites shared similar genetic clusters with few exceptions. Globally, a total of 39 haplotypes were identified from 223 PvDBPII sequences representing different geographical isolates obtained from NCBI archive. The nucleotide and haplotype diversity were 0.00373 and 0.845 ± 0.015, respectively. The haplotype prevalence ranged from 0.45% to 27.3%. Two haplotypes were shared among isolates from all geographical areas of the globe. CONCLUSIONS: PvDBPII of the Ethiopian P. vivax isolates showed low nucleotide but high haplotype diversity, this pattern of genetic variability suggests that the population may have undergone a recent expansion. Among the Ethiopian P. vivax isolates, almost half of the sequences were identical to the Sal-I reference sequence. However, there were unique haplotypes observed in the Ethiopian isolates, which does not share with isolates from other geographical areas. There were two haplotypes that were common among populations across the globe. Categorizing population haplotype frequency can help to determine common haplotypes for designing an effective blood-stage vaccine which will have a significant role for the control and elimination of P. vivax.

Therapeutic efficacy and safety of artemether-lumefantrine for uncomplicated Plasmodium falciparum malaria treatment in Metehara, Central-east Ethiopia.

BACKGROUND: Malaria remains a major global health problem although there was a remarkable achievement between 2000 and 2015. Malaria drug resistance, along with several other factors, presents a significant challenge to malaria control and elimination efforts. Numerous countries in sub-Saharan Africa have documented the presence of confirmed or potential markers of partial resistance against artemisinin, the drug of choice for the treatment of uncomplicated Plasmodium falciparum malaria. The World Health Organization (WHO) recommends regular surveillance of artemisinin therapeutic efficacy to inform policy decisions. METHODS: This study aimed to evaluate the therapeutic efficacy of artemether-lumefantrine (AL), which is the first-line treatment for uncomplicated P. falciparum malaria in Ethiopia since 2004. Using a single-arm prospective evaluation design, the study assessed the clinical and parasitological responses of patients with uncomplicated P. falciparum malaria in Metehara Health Centre, central-east Ethiopia. Out of 2332 malaria suspects (1187 males, 1145 females) screened, 80 (50 males, 30 females) were enrolled, followed up for 28 days, and 73 (44 males, 29 females) completed the follow up. The study was conducted and data was analysed by employing the per-protocol and Kaplan-Meier analyses following the WHO Malaria Therapeutic Efficacy Evaluation Guidelines 2009. RESULTS: The results indicated rapid parasite clearance and resolution of clinical symptoms, with all patients achieving complete recovery from asexual parasitaemia and fever by day (D) 3. The prevalence of gametocytes decreased from 6.3% on D0 to 2.5% on D2, D3, D7, and ultimately achieving complete clearance afterward. CONCLUSION: The overall cure rate for AL treatment was 100%, demonstrating its high efficacy in effectively eliminating malaria parasites in patients. No serious adverse events related to AL treatment were reported during the study, suggesting its safety and tolerability among the participants. These findings confirm that AL remains a highly efficacious treatment for uncomplicated P. falciparum malaria in the study site after 20 years of its introduction in Ethiopia.

Detection of P. malariae using a new rapid isothermal amplification lateral flow assay.

BACKGROUND: While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. METHODS: Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo. RESULTS: Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min. CONCLUSION: Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

Chloroquine has shown high therapeutic efficacy against uncomplicated Plasmodium vivax malaria in southern Ethiopia: seven decades after its introduction.

BACKGROUND: Plasmodium vivax malaria is a leading cause of morbidity in Ethiopia. The first-line treatment for P. vivax is chloroquine (CQ) and primaquine (PQ), but there have been local reports of CQ resistance. A clinical study was conducted to determine the efficacy of CQ for the treatment of P. vivax malaria in southern Ethiopia. METHODS: In 2021, patients with P. vivax mono-infection and uncomplicated malaria were enrolled and treated with 25 mg/kg CQ for 3 consecutive days. Patients were followed for 28 days according to WHO guidelines. The data were analysed using per-protocol (PP) and Kaplan‒Meier (K‒M) analyses to estimate the risk of recurrent P. vivax parasitaemia on day 28. RESULTS: A total of 88 patients were enrolled, 78 (88.6%) of whom completed the 28 days of follow-up. Overall, 76 (97.4%) patients had adequate clinical and parasitological responses, and two patients had late parasitological failures. The initial therapeutic response was rapid, with 100% clearance of asexual parasitaemia within 48 h. CONCLUSION: Despite previous reports of declining chloroquine efficacy against P. vivax, CQ retains high therapeutic efficacy in southern Ethiopia, supporting the current national treatment guidelines. Ongoing clinical monitoring of CQ efficacy supported by advanced molecular methods is warranted to inform national surveillance and ensure optimal treatment guidelines.

Significant number of Plasmodium vivax mono-infections by PCR misidentified as mixed infections (P. vivax/P. falciparum) by microscopy and rapid diagnostic tests: malaria diagnostic challenges in Ethiopia.

BACKGROUND: Plasmodium vivax malaria is now recognized as a cause of severe morbidity and mortality, resulting in a substantial negative effect on health especially in endemic countries. Accurate and prompt diagnosis and treatment of P. vivax malaria is vital for the control and elimination of the disease. METHODS: A cross-sectional study was conducted from February 2021 to September 2022 at five malaria endemic sites in Ethiopia including Aribaminch, Shewarobit, Metehara, Gambella, and Dubti. A total of 365 samples that were diagnosed positive for P. vivax (mono and mixed infection) using RDT, site level microscopists and expert microscopists were selected for PCR. Statistical analyses were performed to calculate the proportions, agreement (k), frequencies, and ranges among different diagnostic methods. Fisher's exact tests and correlation test were used to detect associations and relationship between different variables. RESULTS: Of the 365 samples, 324 (88.8%), 37(10.1%), 2 (0.5%), and 2 (0.5%) were P. vivax (mono), P. vivax/Plasmodium falciparum (mixed), P. falciparum (mono) and negative by PCR, respectively. The overall agreement of rapid diagnostic test (RDT), site level microscopy and expert microscopists result with PCR was 90.41% (k: 0.49), 90.96% (k: 0.53), and 80.27% (k: 0.24). The overall prevalence of sexual (gametocyte) stage P. vivax in the study population was 215/361 (59.6%). The majority of these 215 samples (180; 83.7%) had below 1000 parasites/µl, with only four samples (1.9%) had ≥ 5000 parasites/µl. The gametocyte density was found to be weakly positive but statically significant with asexual parasitaemia (r = 0.31; p < 0.001). CONCLUSION: Both microscopy and RDT showed moderate agreement with PCR in the detection and identification of P. vivax (mono) and P. vivax/P. falciparum (mixed) infections. Therefore, to achieve malaria elimination goals, strengthening routine malaria diagnostic methods by implementing diagnostic tools with a good performance in detecting and accurately identifying malaria species in clinical settings is recommended.

Amplicon deep sequencing of five highly polymorphic markers of Plasmodium falciparum reveals high parasite genetic diversity and moderate population structure in Ethiopia.

BACKGROUND: Plasmodium falciparum genetic diversity can add information on transmission intensity and can be used to track control and elimination interventions. METHODS: Dried blood spots (DBS) were collected from patients who were recruited for a P. falciparum malaria therapeutic efficacy trial in three malaria endemic sites in Ethiopia from October to December 2015, and November to December 2019. qPCR-confirmed infections were subject to amplicon sequencing of polymorphic markers ama1-D3, csp, cpp, cpmp, msp7. Genetic diversity, the proportion of multiclonal infections, multiplicity of infection, and population structure were analysed. RESULTS: Among 198 samples selected for sequencing, data was obtained for 181 samples. Mean MOI was 1.38 (95% CI 1.24-1.53) and 17% (31/181) of infections were polyclonal. Mean He across all markers was 0.730. Population structure was moderate; populations from Metema and Metehara 2015 were very similar to each other, but distinct from Wondogent 2015 and Metehara 2019. CONCLUSION: The high level of parasite genetic diversity and moderate population structure in this study suggests frequent gene flow of parasites among sites. The results obtained can be used as a baseline for additional parasite genetic diversity and structure studies, aiding in the formulation of appropriate control strategies in Ethiopia.

Safety and therapeutic efficacy of artemether-lumefantrine in the treatment of uncomplicated Plasmodium falciparum malaria at Shecha health centre, Arba Minch, Ethiopia.

BACKGROUND: In 2004, Ethiopia adopted artemether-lumefantrine (AL, Coartem®) as first-line treatment for the management of uncomplicated Plasmodium falciparum malaria. Continuous monitoring of AL therapeutic efficacy is crucial in Ethiopia, as per the World Health Organization (WHO) recommendation. This study aimed to assess the therapeutic efficacy of AL in the treatment of uncomplicated P. falciparum infection. METHODS: A 28 day onearm, prospective evaluation of the clinical and parasitological response to AL was conducted at Shecha Health Centre, Arba Minch town, Southern Ethiopia. Patients were treated with six-dose regimen of AL over three days and monitored for 28 days with clinical and laboratory assessments. Participant recruitment and outcome classification was done in accordance with the 2009 WHO methods for surveillance of anti-malarial drug efficacy guidelines. RESULTS: A total of 88 study participants were enrolled and 69 of them completed the study with adequate clinical and parasitological response. Two late parasitological failures were observed, of which one was classified as a recrudescence by polymerase chain reaction (PCR). The PCRcorrected cure rate was 98.6% (95% CI 92.3-100). AL demonstrated a rapid parasite and fever clearance with no parasitaemia on day 2 and febrile cases on day 3. Gametocyte clearance was complete by day three. No serious adverse events were reported during the 28 days follow-up. CONCLUSION: The study demonstrated high therapeutic efficacy and good safety profile of AL. This suggests the continuation of AL as the first-line drug for the treatment of uncomplicated P. falciparum malaria in Ethiopia. Periodic therapeutic efficacy studies and monitoring of markers of resistance are recommended for early detection of resistant parasites.

Therapeutic efficacy of pyronaridine-artesunate (Pyramax®) against uncomplicated Plasmodium falciparum infection at Hamusit Health Centre, Northwest Ethiopia.

BACKGROUND: Early case detection and prompt treatment are important malaria control and elimination strategies. However, the emergence and rapid spread of drug-resistant strains present a major challenge. This study reports the first therapeutic efficacy profile of pyronaridine-artesunate against uncomplicated Plasmodium falciparum in Northwest Ethiopia. METHODS: This single-arm prospective study with 42-day follow-up period was conducted from March to May 2021 at Hamusit Health Centre using the World Health Organization (WHO) therapeutic efficacy study protocol. A total of 90 adults ages 18 and older with uncomplicated falciparum malaria consented and were enrolled in the study. A standard single-dose regimen of pyronaridine-artesunate was administered daily for 3 days, and clinical and parasitological outcomes were assessed over 42 days of follow-up. Thick and thin blood films were prepared from capillary blood and examined using light microscopy. Haemoglobin was measured and dried blood spots were collected on day 0 and on the day of failure. RESULTS: Out of 90 patients, 86/90 (95.6%) completed the 42-day follow-up study period. The overall PCR-corrected cure rate (adequate clinical and parasitological response) was very high at 86/87 (98.9%) (95% CI: 92.2-99.8%) with no serious adverse events. The parasite clearance rate was high with fast resolution of clinical symptoms; 86/90 (95.6%) and 100% of the study participants cleared parasitaemia and fever on day 3, respectively. CONCLUSION: Pyronaridine-artesunate was highly efficacious and safe against uncomplicated P. falciparum in this study population.

The heterogeneity of symptom reporting across study sites: a secondary analysis of a randomised placebo-controlled multicentre antimalarial trial.

INTRODUCTION: Symptoms reported following the administration of investigational drugs play an important role in decisions for registration and treatment guidelines. However, symptoms are subjective, and interview methods to quantify them are difficult to standardise. We explored differences in symptom reporting across study sites of a multicentre antimalarial trial, with the aim of informing trial design and the interpretation of safety and tolerability data. METHODS: Data were derived from the IMPROV trial, a randomised, placebo-controlled double blinded trial of high dose primaquine to prevent Plasmodium vivax recurrence conducted in eight study sites in Afghanistan, Ethiopia, Indonesia and Vietnam. At each follow up visit a 13-point symptom questionnaire was completed. The number and percentage of patients with clinically relevant symptoms following the administration of primaquine or placebo, were reported by study site including vomiting, diarrhoea, anorexia, nausea, abdominal pain and dizziness. Multivariable logistic regression was used to estimate the confounder-adjusted site-specific proportion of each symptom. RESULTS: A total of 2,336 patients were included. The greatest variation between sites in the proportion of patients reporting symptoms was for anorexia between day 0 and day 13: 97.3% (361/371) of patients in Arba Minch, Ethiopia, reported the symptom compared with 4.7% (5/106) of patients in Krong Pa, Vietnam. Differences attenuated slightly after adjusting for treatment arm, age, sex, day 0 parasite density and fever; with the adjusted proportion for anorexia ranging from 4.8% to 97.0%. Differences between sites were greater for symptoms graded as mild or moderate compared to those rated as severe. Differences in symptom reporting were greater between study sites than between treatment arms within the same study site. CONCLUSION: Despite standardised training, there was large variation in symptom reporting across trial sites. The reporting of severe symptoms was less skewed compared to mild and moderate symptoms, which are likely to be more subjective. Trialists should clearly distinguish between safety and tolerability outcomes. Differences between trial arms were much less variable across sites, suggesting that the relative difference in reported symptoms between intervention and control group is more relevant than absolute numbers and should be reported when possible. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01814683; March 20th, 2013.

Spatial Distribution of Plasmodium falciparum and Plasmodium vivax in Northern Ethiopia by Microscopic, Rapid Diagnostic Test, Laboratory Antibody, and Antigen Data.

BACKGROUND: Determining malaria transmission within regions of low, heterogenous prevalence is difficult. A variety of malaria tests exist and range from identification of diagnostic infection to testing for prior exposure. This study describes the concordance of multiple malaria tests using data from a 2015 household survey conducted in Ethiopia. METHODS: Blood samples (n=2279) from 3 regions in northern Ethiopia were assessed for Plasmodium falciparum and Plasmodium vivax by means of microscopy, rapid diagnostic test, multiplex antigen assay, and multiplex assay for immunoglobulin G (IgG) antibodies. Geospatial analysis was conducted with spatial scan statistics and kernel density estimation to identify malaria hot spots by different test results. RESULTS: The prevalence of malaria infection was low (1.4% by rapid diagnostic test, 1.0% by microscopy, and 1.8% by laboratory antigen assay). For P. falciparum, overlapping spatial clusters for all tests and an additional 5 unique IgG clusters were identified. For P. vivax, clusters identified with bead antigen assay, microscopy, and IgG partially overlapped. CONCLUSIONS: Assessing the spatial distribution of malaria exposure using multiple metrics can improve the understanding of malaria transmission dynamics in a region. The relative abundance of antibody clusters indicates that in areas of low transmission, IgG antibodies are a more useful marker to assess malaria exposure.

Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions from Persons with Symptomatic Malaria Infection in Ethiopia, Kenya, Madagascar, and Rwanda.

Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests detect Plasmodium falciparum malaria and are used throughout sub-Saharan Africa. However, deletions in the pfhrp2 and related pfhrp3 (pfhrp2/3) genes threaten use of these tests. Therapeutic efficacy studies (TESs) enroll persons with symptomatic P. falciparum infection. We screened TES samples collected during 2016-2018 in Ethiopia, Kenya, Rwanda, and Madagascar for HRP2/3, pan-Plasmodium lactate dehydrogenase, and pan-Plasmodium aldolase antigen levels and selected samples with low levels of HRP2/3 for pfhrp2/3 genotyping. We observed deletion of pfhrp3 in samples from all countries except Kenya. Single-gene deletions in pfhrp2 were observed in 1.4% (95% CI 0.2%-4.8%) of Ethiopia samples and in 0.6% (95% CI 0.2%-1.6%) of Madagascar samples, and dual pfhrp2/3 deletions were noted in 2.0% (95% CI 0.4%-5.9%) of Ethiopia samples. Although this study was not powered for precise prevalence estimates, evaluating TES samples revealed a low prevalence of pfhrp2/3 deletions in most sites.

Temporal dynamics of Plasmodium falciparum population in Metehara, east-central Ethiopia.

BACKGROUND: Plasmodium falciparum is the most serious, genetically most complex and fastest-evolving malaria parasite. Information on genetic diversity of this parasite would guide policy decision and malaria elimination endeavors. This study explored the temporal dynamics of P. falciparum population in two time points in Metehara, east-central Ethiopia. METHODS: The participants were quantitative real-time polymerase chain reaction-confirmed patients who were recruited for uncomplicated falciparum malaria therapeutic efficacy test in 2015 and 2019. Dry blood spot samples were analysed by the nested PCR to genotype P. falciparum merozoite surface protein (msp1, msp2) and glutamate-rich protein (glurp) genes. RESULTS: While msp1, msp2 and glurp genotypes were successfully detected in 26(89.7%), 24(82.8%) and 14(48.3%) of 2015 samples (n = 29); the respective figures for 2019 (n = 41) were 31(68.3%), 39(95.1%), 25(61.0%). In 2015, the frequencies of K1, MAD20 and RO33 allelic families of msp1, and FC27 and IC/3D7 of msp2 were 19(73.1%), 8(30.6%), 14(53.8%), 21(87.5%), 12(50.5%); and in 2019 it was 15(48.4%), 19(61.3%), 15(48.4%), 30(76.9%), 27(69.2%) respectively. MAD20 has shown dominance over both K1 and RO33 in 2019 compared to the proportion in 2015. Similarly, although FC27 remained dominant, there was shifting trend in the frequency of IC/3D7 from 50.5% in 2015 to 69.2% in 2019. The multiplicity of infection (MOI) and expected heterozygosity index (He) in 2015 and 2019 were respectively [1.43 ± 0.84] and [1.15 ± 0.91], 0.3 and 0.03 for msp1. However, there was no significant association between MOI and age or parasitaemia in both time points. CONCLUSION: The lower genetic diversity in P. falciparum population in the two time points and overall declining trend as demonstrated by the lower MOI and He may suggest better progress in malaria control in Metehara. But, the driving force and selective advantage of switching to MAD20 dominance over the other two msp1 allelic families, and the dynamics within msp2 alleles needs further investigation.

Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia.

BACKGROUND: Malaria incidence has declined in Ethiopia in the past 10 years. Current malaria diagnostic tests, including light microscopy and rapid antigen-detecting diagnostic tests (RDTs) cannot reliably detect low-density infections. Studies have shown that nucleic acid amplification tests are highly sensitive and specific in detecting malaria infection. This study took place with the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria using patient samples collected from health facilities located at malaria elimination targeted low transmission settings in Ethiopia. METHODS: A health facility-based, cross-sectional survey was conducted in selected malaria sentinel sites. Malaria-suspected febrile outpatients referred to laboratory for malaria testing between December 2019 and March 2020 was enrolled into this study. Sociodemographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs. Additionally, five circles of dry blood spot (DBS) samples on Whatman filter paper and thick and thin smear were prepared for molecular testing and microscopic examination, respectively. Multiplex real time PCR assay was performed at Ethiopian Public Health Institute (EPHI) malaria laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the diagnosis of malaria was compared and evaluated against each other. RESULTS: Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as Plasmodium falciparum infection, 16 as Plasmodium vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as P. falciparum, 18 as P. vivax, and RDT detected 43 as P. falciparum, 17 as P. vivax, and one mixed infection. Using light microscopy as reference test, the sensitivity and specificity of multiplex real time PCR were 100% (95% CI (93-100)) and 83.2% (95% CI (77.6-87.9)), respectively. Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58% (95% CI 46.9-68.4) and 67% (95% CI 56.2-76.7); and 100% (95% CI 98-100) and 98.9% (95% CI 96-99.9), respectively. Substantial level of agreement was reported between microscopy and multiplex real time PCR results with kappa value of 0.65. CONCLUSIONS: Multiplex real-time PCR had an advanced performance in parasite detection and species identification on febrile patients' samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination programme, particularly for community based epidemiological samples. Although microscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities.

Investigation of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions and performance of a rapid diagnostic test for identifying asymptomatic malaria infection in northern Ethiopia, 2015.

BACKGROUND: Rapid diagnostic tests (RDTs) are widely used for malaria diagnosis of both symptomatic and asymptomatic infections. Although RDTs are a reliable and practical diagnostic tool, the sensitivity of histidine-rich protein 2 (HRP2)-based RDTs can be reduced if pfhrp2 or pfhrp3 (pfhrp2/3) gene deletions exist in the Plasmodium falciparum parasite population. This study evaluated dried blood spot (DBS) samples collected from a national household survey to investigate the presence of pfhrp2/3 deletions and the performance of the RDT used in the cross-sectional survey in a low transmission setting. METHODS: The 2015 Ethiopia Malaria Indicator Survey tested household members by RDT and collected DBS samples. DBS (n = 2648) from three regions in northern Ethiopia were tested by multiplex bead-based antigen detection assay after completion of the survey. The multiplex assay detected pan-Plasmodium lactate dehydrogenase (LDH), pAldolase, and HRP2 antigens in samples. Samples suspected for pfhrp2/3 gene deletions (pLDH and/or pAldolase positive but low or absent HRP2) were further investigated by molecular assays for gene deletions. Antigen results were also compared to each individual's RDT results. Dose-response logistic regression models were fit to estimate RDT level of detection (LOD) antigen concentrations at which 50, 75, 90, and 95% of the RDTs returned a positive result during this survey. RESULTS: Out of 2,648 samples assayed, 29 were positive for pLDH or pAldolase antigens but low or absent for HRP2 signal, and 15 of these samples (51.7%) were successfully genotyped for pfhrp2/3. Of these 15 P. falciparum infections, eight showed single deletions in pfhrp3, one showed a single pfhrp2 deletion, and six were pfhrp2/3 double-deletions. Six pfhrp2 deletions were observed in Tigray and one in Amhara. Twenty-five were positive for HRP2 by the survey RDT while the more sensitive bead assay detected 30 HRP2-positive samples. A lower concentration of HRP2 antigen generated a positive test result by RDT compared to pLDH (95% LOD: 16.9 ng/mL vs. 319.2 ng/mL, respectively). CONCLUSIONS: There is evidence of dual pfhrp2/3 gene deletions in the Tigray and Amhara regions of Ethiopia in 2015. As the prevalence of malaria was very low (< 2%), it is difficult to make strong conclusions on RDT performance, but these results challenge the utility of biomarkers in household surveys in very low transmission settings.

Therapeutic efficacy of dihydroartemisinin-piperaquine for the treatment of uncomplicated Plasmodium vivax malaria in Seacha area, Arbaminch Zuria District, South West Ethiopia.

BACKGROUND: Declining efficacy of chloroquine against Plasmodium vivax malaria has been documented in Ethiopia. Thus, there is a need to assess the efficacy of alternative schizontocidal anti-malarials such as dihydroartemisinin-piperaquine (DHA-PPQ) in P. vivax malaria-infected patients. This study was conducted to evaluate the therapeutic efficacy of DHA-PPQ drug in South West Ethiopia. METHODS: This is a single-arm, prospective therapeutic efficacy study in patients with uncomplicated P. vivax malaria. The study was conducted from May 2021 to August 2021, based on the standard World Health Organization study protocol for surveillance of anti-malarial therapeutic efficacy. The study endpoint was adequate clinical and parasitological response on day 42. RESULTS: A total of 86 patients with uncomplicated vivax malaria were enrolled. Of these, 79 patients completed the scheduled follow up; all showing adequate clinical and parasitological responses to day 42, with a successful cure rate of 100% (95% CI 96-100). Parasitaemias were cleared rapidly (86% by day 1 and 100% by day 3), as were clinical symptoms (100% by day 1). Gametocyte carriage decreased from 44% on Day 0 to 1% on day 1 and 0% on Day 2. Mean haemoglobin concentrations increased between day 0 (mean 12.2 g/dL) and day 42 (mean 13.3 g/dL). Treatment was well tolerated and no severe adverse events were observed. CONCLUSION: In summary, treatment with DHA-PPQ demonstrated excellent efficacy for uncomplicated P. vivax, with no recurrences to day 42, and no safety concerns. This treatment, which is also effective against P. falciparum, appears to be an ideal alternative for P. vivax as part of the malaria elimination programme.

Efficacy and safety of pyronaridine-artesunate (Pyramax®) for the treatment of uncomplicated Plasmodium vivax malaria in Northwest Ethiopia.

BACKGROUND: Declining efficacy of chloroquine for the treatment Plasmodium vivax malaria has been reported in different endemic settings in Ethiopia. This highlights the need to assess alternative options for P. vivax treatment with artemisinin-based combination therapy, such as pyronaridine-artesunate. This treatment regimen has shown high efficacy for uncomplicated malaria in both Africa and Asia. However, limited data are available from Ethiopia. This study was conducted to assess the efficacy and safety of pyronaridine-artesunate for the treatment of uncomplicated P. vivax malaria in Northwest Ethiopia. METHODS: A single arm prospective efficacy study was conducted in the Hamusite area, Northwest Ethiopia. Fifty-one febrile adult patients with uncomplicated P. vivax malaria were enrolled between March and July 2021. Patients were treated with pyronaridine-artesunate once daily for three days. Clinical and parasitological parameters were monitored over a 42-day follow-up period using the standard World Health Organization protocol for therapeutic efficacy studies. RESULTS: A total of 4372 febrile patients were screened with 51 patients enrolled and 49 completing the 42-day follow-up period. The PCR-uncorrected adequate clinical and parasitological response (ACPR) was 95.9% (47/49; 95% CI 84.9-99.0) on day 42. Two patients had recurrences [4.0% (2/49); 95% CI 0.7-12.1] on days 35 and 42. The parasite clearance rate was rapid with fast resolution of clinical symptoms; 100% of participants had cleared parasitaemia on day 1 and fever on day 2. All 16 (31.4%) patients with gametocyte carriage on day 0 had cleared by day 1. There were no serious adverse events. CONCLUSION: In this small study, pyronaridine-artesunate was efficacious and well-tolerated for the treatment of uncomplicated P. vivax malaria. In adults in the study setting, it would be a suitable alternative option for case management.

Therapeutic efficacies of artemether-lumefantrine and dihydroartemisinin-piperaquine for the treatment of uncomplicated Plasmodium falciparum and chloroquine and dihydroartemisinin-piperaquine for uncomplicated Plasmodium vivax infection in Ethiopia.

BACKGROUND: Routine monitoring of anti-malarial drugs is recommended for early detection of drug resistance and to inform national malaria treatment guidelines. In Ethiopia, the national treatment guidelines employ a species-specific approach. Artemether-lumefantrine (AL) and chloroquine (CQ) are the first-line schizonticidal treatments for Plasmodium falciparum and Plasmodium vivax, respectively. The National Malaria Control and Elimination Programme in Ethiopia is considering dihydroartemisinin-piperaquine (DHA/PPQ) as an alternative regimen for P. falciparum and P. vivax. METHODS: The study assessed the clinical and parasitological efficacy of AL, CQ, and DHA/PPQ in four arms. Patients over 6 months and less than 18 years of age with uncomplicated malaria mono-infection were recruited and allocated to AL against P. falciparum and CQ against P. vivax. Patients 18 years or older with uncomplicated malaria mono-infection were recruited and randomized to AL or dihydroartemisinin-piperaquine (DHA/PPQ) against P. falciparum and CQ or DHA/PPQ for P. vivax. Patients were followed up for 28 (for CQ and AL) or 42 days (for DHA/PPQ) according to the WHO recommendations. Polymerase chain reaction (PCR)-corrected and uncorrected estimates were analysed by Kaplan Meier survival analysis and per protocol methods. RESULTS: A total of 379 patients were enroled in four arms (n = 106, AL-P. falciparum; n = 75, DHA/PPQ- P. falciparum; n = 142, CQ-P. vivax; n = 56, DHA/PPQ-P. vivax). High PCR-corrected adequate clinical and parasitological response (ACPR) rates were observed at the primary end points of 28 days for AL and CQ and 42 days for DHA/PPQ. ACPR rates were 100% in AL-Pf (95% CI: 96-100), 98% in CQ-P. vivax (95% CI: 95-100) at 28 days, and 100% in the DHA/PPQ arms for both P. falciparum and P. vivax at 42 days. For secondary endpoints, by day three 99% of AL-P. falciparum patients (n = 101) cleared parasites and 100% were afebrile. For all other arms, 100% of patients cleared parasites and were afebrile by day three. No serious adverse events were reported. CONCLUSION: This study demonstrated high therapeutic efficacy for the anti-malarial drugs currently used by the malaria control programme in Ethiopia and provides information on the efficacy of DHA/PPQ for the treatment of P. falciparum and P. vivax as an alternative option.

Genetic polymorphisms of Plasmodium falciparum isolates from Melka-Werer, North East Ethiopia based on the merozoite surface protein-2 (msp-2) gene as a molecular marker.

BACKGROUND: The characterization of parasite populations circulating in malaria endemic areas is necessary to evaluate the success of ongoing interventions and malaria control strategies. This study was designed to investigate the genetic diversity of Plasmodium falciparum isolates from the semi-arid area in North East Ethiopia, using the highly polymorphic merozoite surface protein-2 (msp2) gene as a molecular marker. METHODS: Dried blood spot isolates were collected from patients with P. falciparum infection between September 2014 and January 2015 from Melka-Werer, North East Ethiopia. Parasite DNA was extracted and genotyped using allele-specific nested polymerase chain reactions for msp2. RESULTS: 52 isolates were collected with msp2 identified in 41 (78.8%) isolates. Allele typing of the msp2 gene detected the 3D7/IC allelic family in 54% and FC27 allelic family in 46%. A total of 14 different msp2 genotypes were detected including 6 belonging to the 3D7/IC family and 8 to the FC27 family. Forty percent of isolates had multiple genotypes and the overall mean multiplicity of infections (MOI) was 1.2 (95%CI 0.96-1.42). The heterozygosity index was 0.50 for the msp2 locus. There was no difference in MOI between age groups. A negative correlation between parasite density and multiplicity of infection was found (p = 0.02). CONCLUSION: Plasmodium falciparum isolates from the semi-arid area of North East Ethiopia are mainly monoclonal with low MOI and limited genetic diversity in the study population.

Mentorship on malaria microscopy diagnostic service in Ethiopia: baseline competency of microscopists and performance of health facilities.

BACKGROUND: In Ethiopia, malaria cases are declining as a result of proven interventions, and in 2017 the country launched a malaria elimination strategy in targeted settings. Accurate malaria diagnosis and prompt treatment are the key components of the strategy to prevent morbidity and stop the continuation of transmission. However, the quality of microscopic diagnosis in general is deteriorating as malaria burden declines. This study was carried out to evaluate the competency of microscopists and the performance of health facilities on malaria microscopic diagnosis. METHODS: A cross-sectional study was conducted from 1 August to 30 September, 2019 in 9 regional states and one city administration. A standard checklist was used for on-site evaluation, archived patient slides were re-checked and proficiency of microscopists was tested using a WHO-certified set of slides from the national slide bank at the Ethiopian Public Health Institute (EPHI). The strength of agreement, sensitivity, specificity, and positive and negative predictive values were calculated. RESULTS: In this study, 102 health facilities (84 health centres and 18 hospitals) were included, from which 202 laboratory professionals participated. In slide re-checking, moderate agreement (agreement (A): 76.0%; Kappa (K): 0.41) was observed between experts and microscopists on malaria detection in all health facilities. The sensitivity and specificity of routine slide reading and the re-checking results were 78.1 and 80.7%, respectively. Likewise, positive predictive value of 65.1% and negative predictive value of 88.8% were scored in the routine diagnosis. By panel testing, a substantial overall agreement (A: 91.8%; K: 0.79) was observed between microscopists and experts in detecting malaria parasites. The sensitivity and specificity in the detection of malaria parasites was 92.7 and 89.1%, respectively. In identifying species, a slight agreement (A: 57%; K: 0.18) was observed between microscopists and experts. CONCLUSION: The study found significant false positive and false negative results in routine microscopy on slide re-checking of Plasmodium parasites. Moreover, reduced grade in parasite species identification was reported on the panel tests. Implementing comprehensive malaria microscopy mentorship, in-service training and supportive supervision are key strategies to improve the overall performance of health facilities in malaria microscopy.