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Background: Artemether–lumefantrine (AL) has been the primary anti-malarial drug used to treat uncomplicated Plasmodium falciparum malaria in Ethiopia since 2004. However, there have been recent reports of AL resistance mutations in different African countries, including Ethiopia. This is concerning and requires periodic monitoring of anti-malarial drug resistance. Therefore, the current study aimed to evaluate the therapeutic efficacy of AL in treating uncomplicated P. falciparum malaria in the Arba Minch Zuria District, Gamo Zone, Southwest Ethiopia. Methods: A single-arm prospective study with a 28-day follow-up period was conducted from July to October 2022. Capillary blood samples were collected for RDT and microscopic examination. The study enrolled monoinfected P. falciparum patients aged ≥ 18 years at Ganta Sira Health Post. Sociodemographic and clinical data were recorded, and a dried blood spot (DBS) was prepared for each participant. Nested polymerase chain reaction (nPCR) genotyping of the msp-1 and msp-2 genes was only performed for recurrent cases to distinguish between recurrence and reinfection. Data entry and analysis were performed using the WHO Excel spreadsheet and SPSS version 26. Results: A total of 89 patients were enrolled, and 67 adequately completed the 28-day follow-up period. AL showed a 100% clearance rate for fever on day 2 and asexual parasites on day 3. Gametocytes were detected in 13.5% (12/89) of the participants. The gametocyte clearance rate was 58.3% (7/12) until day 7 and 100% (12/12) until day 14. Five participants developed recurrent malaria, three of whom experienced relapse and two of whom experienced reinfection. Based on the Kaplan–Meier survival analysis, the PCR-uncorrected and PCR-corrected cumulative incidence of success were 93.7% (95% CI 85.5–97.3) and 96.2% (95% CI 85.5–98.7), respectively. Conclusion: AL was efficacious in treating uncomplicated P. falciparum malaria in the study area. However, the detection of recurrent patients highlights the need for continuous efficacy studies in this area. [ABSTRACT FROM AUTHOR]

Background: Rapid diagnostic tests (RDTs) provide quick, easy, and convenient early diagnosis of malaria ensuring better case management particularly in resource-constrained settings. Nevertheless, the efficiency of HRP2-based RDT can be compromised by Plasmodium falciparum histidine-rich protein 2/3 gene deletion and genetic diversity. This study explored the genetic diversity of PfHRP2/3 in uncomplicated malaria cases from Ethiopia. Methods: A cross-sectional study was conducted from June 2022 to March 2023 at Metehara, Zenzelema and Kolla Shele health centres, Ethiopia. Finger-prick blood samples were collected for RDT testing and microscopic examination. For molecular analysis, parasite genomic DNA was extracted from venous blood. Plasmodium falciparum was confirmed using VarATS real time PCR. Additionally, PfHRP2/3 was amplified, and DNA amplicons were sequenced using Oxford Nanopore technology. Results: PfHRP2/3 sequences revealed small variations in the frequency and number of amino acid repeat types per isolate across the three health centres. Twelve and eight types of amino acid repeats were identified for PfHRP2 and PfHRP3, respectively, which had been previously characterized. Repeat type 1, 4 and 7 were present in both PfHRP2 and PfHRP3 amino acid sequences. Type 2 and 7 repeats were commonly dispersed in PfHRP2, while repeat types 16 and 17 were found only in PfHRP3. A novel 17 V repeat type variant, which has never been reported in Ethiopia, was identified in six PfHRP3 amino acid sequences. The majority of the isolates, as determined by the Baker's logistic regression model, belonged to group C, of which 86% of them were sensitive to PfHRP2-based RDT. Likewise, PfHRP2-based RDT detected 100% of the isolates in group A (product of type 2 × type 7 repeats ≥ 100) and 85.7% in group B (product of types 2 × type 7 repeats 50–99) at a parasitaemia level > 250 parasite/μl. Conclusion: This study highlights the significant diversity observed in PfHRP2 and PfHRP3 among clinical isolates of Plasmodium falciparum in Ethiopia. This emphasizes the necessity for monitoring of PfHRP2- based RDT efficacy and their repeat type distribution using a large sample size and isolates from various ecological settings. [ABSTRACT FROM AUTHOR]

Background: There is a paucity of data regarding resting habits and comparison of the physiological states of Anopheles mosquito populations from indoor and outdoor collection venues in Ethiopia. Therefore, this study aims to investigate resting behavior and age structure of the malaria vectors in Wonji Sugar Estate. Methods: Longitudinal entomological study was conducted at Wonji Sugar Estate from July 2018 to June 2020. Pyrethrum spray catches (PSC), Pit Shelter (PIT), and Oral Aspirators were used to collect adult Anopheles species from various resting sites. Mosquitoes were identified to the species using morphological keys. Finally, the resting behavior of Anopheles mosquitoes were determined using standard protocol. Results: A total of 1406 mosquitoes were collected from indoor and outdoor resting venues, with the outdoor resting population being significantly higher (F = 8.296, P =.04). The number of half-gravid/gravid Anopheles species collected from outdoors was higher than that from indoor resting venues (F = 35.612, P =.00). Eight two percent of the indoor sampled Anopheles species left the indoor venue before completing their gonotrophic cycle. Anopheles pharoensis and An. coustani cx. exhibited exophily rate of 83.4% and 100%, respectively. The overall parity rate of Anopheles gambiae s.l. was 45.7%. The highest parity rate, daily survival rate, and longevity of Anopheles gambiae s.l. was 66.7%, 0.874, and 7.399 days, respectively. Anopheles gambiae s.l. reached the infective stages for Plasmodium falciparum and P. vivax in February 2019. Conlusion: Anopheles mosquitoes, including the major malaria vector Anopheles gambiae s.l., showed predominantly exophilic habits, underscoring the need for outdoor vector control strategies in addition to indoor-based interventions. The increase in the nulliparous population indicates a higher risk of vector-human contact. Meanwhile, the extended lifespan of Anopheles gambiae s.l. observed during the dry season could increase the risk of infectious bites. [ABSTRACT FROM AUTHOR]

Background: The protective effectiveness of vector control in malaria relies on how the implemented tools overlap with mosquito species-specific compositions and bionomic traits. In Ethiopia, targeted entomological data enabling strategic decision-making are lacking around high-risk migrant worker camps in the lowlands and resident communities in the highlands—resulting in suboptimal malaria control strategies for both populations. This study investigates spatial and temporal mosquito behavior, generating baseline evidence that will improve malaria control for both migrant workers in the lowlands and their home communities in the highlands. Methods: Hourly Centers for Disease Control and Prevention (CDC) light trap collections were performed indoors and outdoors during the peak (October to December 2022) and minor (March to May 2023) malaria transmission seasons. These seasons coincide with the post-long rain and post-short rain seasons, respectively. Eight resident households were sampled from each of four villages in the highlands and eight households/farm structures on and near farms in four villages in the lowlands. The sampling occurred between 18:00 and 06:00. Spatiotemporal vector behaviors and hourly indoor and outdoor mosquito capture rates, used as a proxy for human biting rates, were calculated for overall catches and for individual species. Adult mosquitoes were identified using morphological keys, and a subset of samples were confirmed to species by sequencing ribosomal DNA internal transcribed spacer region 2 (ITS2) and/or mitochondrial DNA cytochrome c oxidase subunit 1 (Cox1). Results: In the highlands, 4697 Anopheles mosquitoes belonging to 13 morphologically identified species were collected. The predominant species of Anopheles identified in the highlands was An. gambiae sensu lato (s.l.) (n = 1970, 41.9%), followed by An. demeilloni (n = 1133, 24.1%) and An. cinereus (n = 520, 11.0%). In the lowland villages, 3220 mosquitoes belonging to 18 morphological species were collected. Anopheles gambiae s.l. (n = 1190, 36.9%), An. pretoriensis (n = 899, 27.9%), and An. demeilloni (n = 564, 17.5%) were the predominant species. A total of 20 species were identified molecularly, of which three could not be identified to species through comparison with published sequences. In highland villages, the indoor Anopheles mosquito capture rate was much greater than the outdoor rate. This trend reversed in the lowlands, where the rate of outdoor captures was greater than the indoor rate. In both highlands and lowlands, Anopheles mosquitoes showed early biting activities in the evening, which peaked between 18:00 and 21:00, for both indoor and outdoor locations. Conclusions: The high diversity of Anopheles vectors and their variable behaviors result in a dynamic and resilient transmission system impacting both exposure to infectious bites and intervention effectiveness. This creates gaps in protection allowing malaria transmission to persist. To achieve optimal control, one-size-fits-all strategies must be abandoned, and interventions should be tailored to the diverse spatiotemporal behaviors of different mosquito populations. [ABSTRACT FROM AUTHOR]

Asymptomatic malaria can impact existing malaria control and elimination efforts around the world, particularly in Africa, where the majority of malaria cases and death occurs. This is a cross-sectional study aimed to determine the prevalence and predictors of asymptomatic malaria among migrant farmworkers from June to July 2020 in the Upper Awash Agro-industry, East Shewa zone, Oromia Regional State, Ethiopia. A total of 254 migrant farmworkers without signs and symptoms of malaria were enrolled. Data on socio-demographic characteristics and malaria prevention practices were obtained through a structured questionnaire. Venous blood samples were collected and diagnosed using microscopy, rapid diagnostic tests, and polymerase chain reaction (PCR). Data were coded, entered, and analyzed using SPSS version-21 statistical software. Multivariable logistic regression was used to assess associated factors. A p < 0.05 was considered statistically significant. The overall prevalence of asymptomatic malaria among farmworkers in this study was 5.1% [95% CI 1.6, 6.7]. The proportions of Plasmodium falciparum was 90.0% (9/10) while it was 10.0% (1/10) for Plasmodium vivax. Out of the microscopy and/or RDT-confirmed malaria cases, (n = 9; 100%) were confirmed to be P. falciparum by nested PCR, while (n = 3/122; 2.46%) were found to be P. falciparum among 50% negative cases with the microscopy and/or RDT. The gametocyte stage was detected in 40% of microscopically positive cases out of which 44.4% belongs to P. falciparum. Home area/origin of migrant laborers [AOR = 6.08, (95% CI 1.08, 34.66)], family history of malaria [AOR = 8.15, (95% CI 1.43, 46.44)], and outdoor sleeping [AOR = 10.14, (95% CI 1.15, 89.14)] were significantly associated with asymptomatic malaria. In conclusion, asymptomatic malaria was detected among farmworkers in the study area and it was significantly associated with outdoor sleeping, home area, and family history of malaria. Prevention tools and control strategies, particularly focusing on migrant farmworkers, should be considered to support the ongoing malaria control and elimination effort in Ethiopia. [ABSTRACT FROM AUTHOR]

Background Rapid diagnostic tests (RDTs) play a signifcant role in expanding case management in peripheral healthcare systems. Histidine-rich protein-2 (HRP2) antigen detection RDTs are predominantly used to diagnose Plasmodium falciparum infection. However, the evolution and spread of P. falciparum parasite strains with deleted hrp2/3 genes, causing false-negative results, have been reported. This study assessed the diagnostic performance of HRP2- detecting RDTs for P. falciparum cases and the prevalence of pfhrp2/3 deletions among symptomatic patients seeking malaria diagnosis at selected health facilities in southern Ethiopia. Methods A multi-health facilities-based cross-sectional study was conducted on self-presenting febrile patients seeking treatment in southern Ethiopia from July to September 2022. A purposive sampling strategy was used to enroll patients with microscopically confrmed P. falciparum infections. A capillary blood sample was obtained to prepare a blood flm for microscopy and a RDT using the SD Bioline™ Malaria Pf/Pv Test. Dried blood spot samples were collected for further molecular analysis. DNA was extracted using gene aid kits and amplifcation was performed using nested PCR assay. Exon 2 of hrp2 and hrp3, which are the main protein-coding regions, was used to confrm its deletion. The diagnostic performance of RDT was evaluated using PCR as the gold standard test for P. falciparum infections. Results Of 279 P. falciparum PCR-confrmed samples, 249 (89.2%) had successful msp-2 amplifcation, which was then genotyped for hrp2/3 gene deletions. The study revealed that pfhrp2/3 deletions were common in all health centres, and it was estimated that 144 patients (57.8%) across all health facilities had pfhrp2/3 deletions, leading to falsenegative PfHRP2 RDT results. Deletions spanning exon 2 of hrp2, exon 2 of hrp3, and double deletions (hrp2/3) accounted for 68 (27.3%), 76 (30.5%), and 33 (13.2%) of cases, respectively. The study fndings revealed the prevalence of P. falciparum parasites lacking a single pfhrp2-/3-gene and that both genes varied across the study sites. This study also showed that the sensitivity of the SD Bioline PfHRP2-RDT test was 76.5% when PCR was used as the reference test. Conclusion This study confrmed the existence of widespread pfhrp2/3- gene deletions, and their magnitude exceeded the WHO-recommended threshold (>5%). False-negative RDT results resulting from deletions in Pfhrp2/3- afect a country’s attempts at malaria control and elimination. Therefore, the adoption of non-HRP2-based RDTs as an alternative measure is required to avoid the consequences associated with the continued use of HRP-2-based RDTs, in the study area in particular and in Ethiopia in general. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Despite Ethiopia's concerted efforts to eliminate malaria by 2030, the disease continues to pose a significant public health and socioeconomic challenge in the country. The year 2021 witnessed 2.78 million malaria cases and 8041 associated deaths, emphasizing the persistent threat. Monitoring the prevalence trend of malaria is crucial for devising effective control and elimination strategies. This study aims to assess the trend of malaria prevalence at the Metehara Health Centre in the East Shoa Zone, Ethiopia. Methods: A retrospective study, spanning from February to September 2023, utilized malaria registration laboratory logbooks at Metehara Health Centre to evaluate the prevalence of malaria from 2017/18 to 2022/23. Malaria and related data were collected using a pre-designed data collection sheet. Descriptive statistics were employed for data summarization, presented through graphs and tables. Results: Out of 59,250 examined blood films, 17.4% confirmed the presence of Plasmodium infections. Among the confirmed cases, 74.3%, 23.8%, and 1.84% were attributed to Plasmodium falciparum, Plasmodium vivax, and mixed infections, respectively. The trend of malaria exhibited a steady decline from 2017/18 to 2021/22, reaching 9.8% prevalence. However, an abrupt increase to 26.5% was observed in 2022/23. Males accounted for a higher proportion (66%) of cases compared to females (34%). The age group 15–24 years experienced the highest malaria incidence at 42%. Notably, malaria cases peaked during autumn (September to November) at 43% and reached the lowest percentage during spring (March to May) at 13%. Conclusion: Malaria persists as a significant health challenge in and around Metehara, central Ethiopia, predominantly driven by Plasmodium falciparum. The five-year declining trend was interrupted by a notable upsurge in 2022/23, indicating a resurgence of malaria in the study area. It is imperative to adopt a reverse strategy to sustain the progress achieved by the national malaria control plan. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Schoolchildren with asymptomatic malaria infections often go undiagnosed and untreated, serving as reservoirs for infection that hamper malaria control and elimination efforts. In this context, little is known about the magnitude of asymptomatic malaria infections in apparently healthy schoolchildren in Ethiopia. This study was aimed at determining the prevalence of asymptomatic malaria infection and its associated factors in apparently healthy schoolchildren in Ethiopia. Methods: From September 2021 to January 2022, a school-based cross-sectional study was conducted on 994 apparently healthy schoolchildren (aged 6–15 years) selected from 21 primary schools in the Gomma district, of Jimma zone, southwestern Oromia, Ethiopia. A multi-stage sampling technique was used to select schools and participants. After allocating the total sample proportionally to each school and then to each grade, participants were selected using the lottery method from a list of student records (rosters). Finger-pricked blood samples were collected for microscopy blood film preparation and malaria rapid diagnostic test (RDT) (SD Bioline Malaria Ag Pf/Pv). Moreover, dry blood spots (DBSs) were prepared onto filter papers for quantitative real time polymerase chain reaction (qPCR) analysis. Results: As determined by RDT and microscopy, the prevalence of asymptomatic malaria was 2.20% and 1.51%, respectively. Using qPCR, the overall prevalence was 5.03% (50/994). Of this, Plasmodium falciparum, Plasmodium vivax and mixed infections accounted for 90%, 6% and 4%, respectively. Submicroscopic asymptomatic malaria infection was also accounted for 70% (35/50) of the overall prevalence. Household head age, nighttime outdoor activities of household heads, family history of malaria, absence of insecticide-treated nets (ITN), and presence of stagnant water around the houses are all significantly associated with asymptomatic malaria infections among schoolchildren. Conclusions: This study found that both RDT and microscopy underestimated the prevalence of asymptomatic malaria in schoolchildren. However, qPCR was able to detect even low levels of parasitaemia and revealed a higher prevalence of asymptomatic submicroscopic malaria infections. The findings imply that schoolchildren with asymptomatic malaria infection are potential hotspot for malaria reservoir that fuels ongoing transmission. Therefore, it is imperative to include schoolchildren and schools in malaria intervention package and equally important is the adoption of more advanced and sensitive diagnostic tools, which would be crucial for successful malaria control and elimination efforts. Targeted interventions for asymptomatic malaria-infected schoolchildren can provide invaluable support to the National Malaria Control Programme in controlling and eventually eliminating the disease. [ABSTRACT FROM AUTHOR]

Background Urbanization generally improves health outcomes of residents and is one of the potential factors that might contribute to reducing malaria transmission. However, the expansion of Anopheles stephensi, an urban malaria vector, poses a threat for malaria control and elimination efforts in Africa. In this paper, malaria trends in urban settings in Ethiopia from 2014 to 2019 are reported with a focus on towns and cities where An. stephensi surveys were conducted. Methods A retrospective study was conducted to determine malaria trends in urban districts using passive surveillance data collected at health facilities from 2014 to 2019. Data from 25 towns surveyed for An. stephensi were used in malaria trend analysis. Robust linear models were used to identify outliers and impute missing and anomalous data. The seasonal Mann-Kendal test was used to test for monotonic increasing or decreasing trends. Results A total of 9,468,970 malaria cases were reported between 2014 and 2019 through the Public Health Emergency Management (PHEM) system. Of these, 1.45 million (15.3%) cases were reported from urban settings. The incidence of malaria declined by 62% between 2014 and 2018. In 2019, the incidence increased to 15 per 1000 population from 11 to 1000 in 2018. Both confirmed (microscopy or RDT) Plasmodium falciparum (67%) and Plasmodium vivax (28%) were reported with a higher proportion of P. vivax infections in urban areas. In 2019, An. stephensi was detected in 17 towns where more than 19,804 malaria cases were reported, with most of the cases (56%) being P. falciparum. Trend analysis revealed that malaria cases increased in five towns in Afar and Somali administrative regions, decreased in nine towns, and had no obvious trend in the remaining three towns. Conclusion The contribution of malaria in urban settings is not negligible in Ethiopia. With the rapid expansion of An. stephensi in the country, the receptivity is likely to be higher for malaria. Although the evidence presented in this study does not demonstrate a direct linkage between An. stephensi detection and an increase in urban malaria throughout the country, An. stephensi might contribute to an increase in malaria unless control measures are implemented as soon as possible. Targeted surveillance and effective response are needed to assess the contribution of this vector to malaria transmission and curb potential outbreaks. [ABSTRACT FROM AUTHOR]

Background: Malaria, transmitted by the bite of infective female Anopheles mosquitoes, remains a global public health problem. The presence of invasive Anopheles stephensi, capable of transmitting Plasmodium vivax and Plasmodium falciparum, was first reported in Ethiopia in 2016. The ecology of this mosquito species differs from that of Anopheles arabiensis, the primary malaria vector in Ethiopia. This study aimed to evaluate the efficacy of selected insecticides, which are used in indoor residual spraying (IRS) and selected long-lasting insecticidal nets (LLINs) for malaria vector control against adult An. stephensi. Methods: Anopheles stephensi mosquitoes were collected as larvae and pupae from Awash Subah Kilo Town and Haro Adi village, Ethiopia. Adult female An. stephensi, reared from larvae and pupae collected from the field, aged 3–5 days were exposed to impregnated papers of IRS insecticides (propoxur 0.1%, bendiocarb 0.1%, pirimiphos-methyl 0.25%), and insecticides used in LLINs (alpha-cypermethrin 0.05%, deltamethrin 0.05% and permethrin 0.75%), using diagnostic doses and WHO test tubes in a bio-secure insectary at Aklilu Lemma Institute of Pathobiology, Addis Ababa University. For each test and control tube, batches of 25 female An. stephensi were used to test each insecticide used in IRS. Additionally, cone bioassay tests were conducted to expose An. stephensi from the reared population to four brands of LLINs, MAGNet™ (alpha-cypermethrin), PermaNet® 2.0 (deltamethrin), DuraNet© (alpha-cypermethrin) and SafeNet® (alpha-cypermethrin). A batch of ten sugar-fed female mosquitoes aged 2–5 days was exposed to samples taken from five positions/sides of a net. The data from all replicates were pooled and descriptive statistics were used to describe features of the data. Results: All An. stephensi collected from Awash Subah Kilo Town and Haro Adi village (around Metehara) were resistant to all tested insecticides used in both IRS and LLINs. Of the tested LLINs, only MAGNet™ (alpha-cypermethrin active ingredient) caused 100% knockdown and mortality to An. stephensi at 60 min and 24 h post exposure, while all other net brands caused mortality below the WHO cut-off points (< 90%). All these nets, except SafeNet®, were collected during LLIN distribution for community members through the National Malaria Programme, in December 2020. Conclusions: Anopheles stephensi is resistant to all tested insecticides used in IRS and in the tested LLIN brands did not cause mosquito mortality as expected, except MAGNet. This suggests that control of this invasive vector using existing adult malaria vector control methods will likely be inadequate and that alternative strategies may be necessary. [ABSTRACT FROM AUTHOR]

Background: The interaction between the Plasmodium vivax Duffy-binding protein and the corresponding Duffy Antigen Receptor for Chemokines (DARC) is primarily responsible for the invasion of reticulocytes by P. vivax. The Duffy-negative host phenotype, highly prevalent in sub-Saharan Africa, is caused by a single point mutation in the GATA-1 transcription factor binding site of the DARC gene promoter. The aim of this study was to assess the Duffy status of patients with P. vivax infection from different study sites in Ethiopia. Methods: A cross-sectional study was conducted from February 2021 to September 2022 at five varying eco-epidemiological malaria endemic sites in Ethiopia. Outpatients who were diagnosed with P. vivax infection (pure and mixed P. vivax/P. falciparum) by microscopy and Rapid Diagnostic Test (RDT) were subjected to PCR genotyping at the DARC promoter. The associations between P. vivax infection, host genotypes and other factors were evaluated. Result: In total, 361 patients with P. vivax infection were included in the study. Patients with pure P. vivax infections accounted for 89.8% (324/361), while the remaining 10.2% (37/361) had mixed P. vivax/P. falciparum infections. About 95.6% (345/361) of the participants were Duffy-positives (21.2% homozygous and 78.8%, heterozygous) and 4.4% (16/361) were Duffy-negatives. The mean asexual parasite density in homozygous and heterozygous Duffy-positives was 12,165 p/μl (IQR25-75: 1,640–24,234 p/μl) and11,655 p/μl (IQR25-75: 1,676–14,065 p/μl), respectively, significantly higher than that in Duffy-negatives (1,227p/μl; IQR25-75: 539–1,732p/μl). Conclusion: This study confirms that Duffy-negativity does not provide complete protection against P. vivax infection. The development of P. vivax-specific elimination strategies, including alternative antimalarial vaccines should be facilitated by a better understanding of the epidemiological landscape of vivax malaria in Africa. More importantly, low parasitemia associated with P. vivax infections in Duffy-negative patients may represent hidden reservoirs of transmission in Ethiopia. Author summary: Plasmodium vivax generally receives less attention than P. falciparum and was neglected in sub-Saharan Africa. However, the characteristics of P. vivax infection in Duffy-negative individuals, and the distribution of Duffy blood group in different eco-epidemiological zones and ethnic groups of Ethiopia are not well documented. Here, we determined the Duffy genotypes of P. vivax infected patients across broad regions of Ethiopia. It is clear that Duffy negative individuals in Ethiopia are not fully protected against P. vivax infection, and that these infections in Duffy negatives are often associated with low parasitemia. Our findings lend support to the notion that P. vivax may have developed a Duffy-independent erythrocyte invasion pathway and/or evolution in host immune responses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

One of the key obstacles to malaria elimination is largely attributed to Plasmodium vivax's ability to form resilient hypnozoites in the host liver that cause relapsing infections. As a result, interruption of P. vivax transmission is difficult. P. vivax transmission occurs in Duffy-positive individuals and have been mainly thought to be absent in Africa. However, increasing studies using molecular tools detected P. vivax among Duffy-negative individuals in various African countries. Studies on the African P. vivax has been severely limited because most of malaria control program focus mainly on falciparum malaria. In addition, there is a scarcity of laboratory infrastructures to overcome the biological obstacles posed by P. vivax. Herein, we established field transmission of Ethiopian P. vivax for routine sporozoite supply followed by liver stage infection in Mali. Furthermore, we evaluated local P. vivax hypnozoites and schizonts susceptibilities to reference antimalarial drugs. The study enabled the assessment of local African P. vivax hypnozoite production dynamics. Our data displayed the ability of the African P. vivax to produce hypnozoite forms ex-vivo at different rates per field isolate. We report that while tafenoquine (1µM) potently inhibited both hypnozoites and schizont forms; atovaquone (0.25µM) and the phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691 (0.5µM) showed no activity against hypnozoites forms. Unlike hypnozoites forms, P. vivax schizont stages were fully susceptible to both atovaquone (0.25µM) and the (PI4K)-specific inhibitor KDU691 (0.5µM). Together, the data revealed the importance of the local platform for further biological investigation and implementation of drug discovery program on the African P. vivax clinical isolates. [ABSTRACT FROM AUTHOR]

Background: Pfcrt gene has been associated with chloroquine resistance and the pfmdr1 gene can alter malaria parasite susceptibility to lumefantrine, mefloquine, and chloroquine. In the absence of chloroquine (CQ) and extensive use of artemether–lumefantrine (AL) from 2004 to 2020 to treat uncomplicated falciparum malaria, pfcrt haplotype, and pfmdr1 single nucleotide polymorphisms (SNPs) were determined in two sites of West Ethiopia with a gradient of malaria transmission. Methods: 230 microscopically confirmed P. falciparum isolates were collected from Assosa (high transmission area) and Gida Ayana (low transmission area) sites, of which 225 of them tested positive by PCR. High-Resolution Melting Assay (HRM) was used to determine the prevalence of pfcrt haplotypes and pfmdr1 SNPs. Furthermore, the pfmdr1 gene copy number (CNV) was determined using real-time PCR. A P-value of less or equal to 0.05 was considered significant. Results: Of the 225 samples, 95.5%, 94.4%, 86.7%, 91.1%, and 94.2% were successfully genotyped with HRM for pfcrt haplotype, pfmdr1-86, pfmdr1-184, pfmdr1-1042 and pfmdr1-1246, respectively. The mutant pfcrt haplotypes were detected among 33.5% (52/155) and 80% (48/60) of isolates collected from the Assosa and Gida Ayana sites, respectively. Plasmodium falciparum with chloroquine-resistant haplotypes was more prevalent in the Gida Ayana area compared with the Assosa area (COR = 8.4, P = 0.00). Pfmdr1-N86Y wild type and 184F mutations were found in 79.8% (166/208) and 73.4% (146/199) samples, respectively. No single mutation was observed at the pfmdr1-1042 locus; however, 89.6% (190/212) of parasites in West Ethiopia carry the wild-type D1246Y variants. Eight pfmdr1 haplotypes at codons N86Y–Y184F–D1246Y were identified with the dominant NFD 61% (122/200). There was no difference in the distribution of pfmdr1 SNPs, haplotypes, and CNV between the two study sites (P > 0.05). Conclusion: Plasmodium falciparum with the pfcrt wild-type haplotype was prevalent in high malaria transmission site than in low transmission area. The NFD haplotype was the predominant haplotype of the N86Y–Y184F–D1246Y. A continuous investigation is needed to closely monitor the changes in the pfmdr1 SNPs, which are associated with the selection of parasite populations by ACT. [ABSTRACT FROM AUTHOR]

Background: Measuring risk of malaria transmission is complex, especially in case of Plasmodium vivax. This may be overcome using membrane feeding assays in the field where P. vivax is endemic. However, mosquito-feeding assays are affected by a number of human, parasite and mosquito factors. Here, this study identified the contributions of Duffy blood group status of P. vivax-infected patients as a risk of parasite transmission to mosquitoes. Methods: A membrane feeding assay was conducted on a total of 44 conveniently recruited P. vivax infected patients in Adama city and its surroundings in East Shewa Zone, Oromia region, Ethiopia from October, 2019 to January, 2021. The assay was performed in Adama City administration. Mosquito infection rates were determined by midgut dissections at seven to 8 days post-infection. Duffy genotyping was defined for each of the 44 P. vivax infected patients. Results: The infection rate of Anopheles mosquitoes was 32.6% (296/907) with 77.3% proportion of infectious participants (34/44). Infectiousness of participants to Anopheles mosquitoes appeared to be higher among individuals with homozygous Duffy positive blood group (TCT/TCT) than heterozygous (TCT/CCT), but the difference was not statistically significant. The mean oocyst density was significantly higher among mosquitoes fed on blood of participants with FY*B/FY*BES than other genotypes (P = 0.001). Conclusion: Duffy antigen polymorphisms appears to contribute to transmissibility difference of P. vivax gametocytes to Anopheles mosquitoes, but further studies are required. [ABSTRACT FROM AUTHOR]

Background: One of the major roadblocks to the falciparum malaria elimination programme is the presence of a portion of the population, such as school children, with asymptomatic malaria infection. Targeting such reservoirs of infections is critical to interrupting transmission and enhancing elimination efforts. The NxTek™ Eliminate Malaria Pf test is a highly sensitive rapid diagnostic test (hsRDT) for the detection of HRP-2. However, knowledge gaps exist in Ethiopia on the diagnostic performance of hsRDT for the detection of Plasmodium falciparum in school children with asymptomatic malaria. Methods: A school-based cross-sectional study was conducted from September 2021 to January 2022 on 994 healthy school children (aged 6–15 years). Finger-pricked whole blood samples were collected for microscopy, hsRDT, conventional RDT (cRDT or SD Bioline Malaria Ag Pf/P.v), and QuantStudio™ 3 Real—Time PCR system (qPCR). The hsRDT was compared to cRDT and microscopy. qPCR and microscopy were used as reference methods. Results: The prevalence of Plasmodium falciparum was 1.51%, 2.2%. 2.2% and 4.52%, by microscopy, hsRDT, cRDT and qPCR, respectively. Using qPCR as reference, the sensitivity of hsRDT was higher (48.89%) than the microscopy (33.3%), and showed 100% specificity and a positive predictive value (PPV). Microscopy showed similar specificity and PPV as hsRDT. Using microscopy as a reference, the diagnostic perforrmances of both hsRDT and cRDT were similar. Both RDTs demonstrated identical diagnostic performances in both comparison methods. Conclusions: hsRDT has the same diagnostic performance as cRDT but improved diagnostic characteristics than microscopy for detection of P. falciparum in school children with asymptomatic malaria. It can be a useful tool for the national malaria elimination plan of Ethiopia. [ABSTRACT FROM AUTHOR]

Background: Malaria, transmitted by the bite of infective female Anopheles mosquitoes, remains a global public health problem. The presence of an invasive Anopheles stephensi, capable of transmitting Plasmodium vivax and Plasmodium falciparum parasites was first reported in Ethiopia in 2016. The ecology of An. stephensi is different from that of Anopheles arabiensis, the primary Ethiopian malaria vector, and this suggests that alternative control strategies may be necessary. Larviciding may be an effective alternative strategy, but there is limited information on the susceptibility of Ethiopian An. stephensi to common larvicides. This study aimed to evaluate the efficacy of temephos and Bacillus thuringiensis var. israelensis (Bti) larvicides against larvae of invasive An. stephensi. Methods: The diagnostic doses of two larvicides, temephos (0.25 ml/l) and Bti (0.05 mg/l) were tested in the laboratory against the immature stages (late third to early fourth stages larvae) of An. stephensi collected from the field and reared in a bio-secure insectary. Larvae were collected from two sites (Haro Adi and Awash Subuh Kilo). For each site, three hundred larvae were tested against each insecticide (as well as an untreated control), in batches of 25. The data from all replicates were pooled and descriptive statistics prepared. Results: The mortality of larvae exposed to temephos was 100% for both sites. Mortality to Bti was 99.7% at Awash and 100% at Haro Adi site. Conclusions: Larvae of An. stephensi are susceptible to temephos and Bti larvicides suggesting that larviciding with these insecticides through vector control programmes may be effective against An. stephensi in these localities. [ABSTRACT FROM AUTHOR]

Background. The genetic variation of Plasmodium falciparum has been studied to assess local malaria transmission genetic profile using evidence-based intervention measures. However, there are no known previous reports of P. falciparum polymorphism in Badewacho and Boset districts, Southern Ethiopia. The purpose of this study was to determine the genetic diversity of the merozoite surface protein-1 and -2 (msp-1 and msp-2) allelic families in P. falciparum isolates from an asymptomatic populations. Methods. This study was conducted from finger-prick blood samples spotted on 3 mm Whatman filter paper collected during a community-based cross-sectional study. Nested polymerase chain reaction amplification was used to type the allelic variants of msp-1 and msp-2. Results. From 669 asymptomatic study participants, a total of 50 samples positive for P. falciparum were included for molecular analysis. Of 50 positive samples, 43 P. falciparum isolates were successfully amplified for the msp-1 and msp-2 allelic families. A total of twelve different allele sizes (75–250 bp) were identified within the three allelic families of msp-1, whereas ten different allele sizes (250–500 bp) were detected within the two allelic families of msp-2. MAD20 had a higher allelic proportion, 65% among allelic families of msp-1, whereas the 3D7 allelic family 90.7% was higher in msp-2. A slightly higher frequency of polyclonal infection 53.5% was found in msp-2 allelic family, whereas a low proportion polyclonal infection 46.5% was found in msp-1 allelic family. The overall mean multiplicity of infection (MOI) for msp-1 and msp-2 was identical (MOI = 1.56). Correspondingly, the expected heterozygosity (He) value for msp-1 (He = 0.23) and msp-2 (He = 0.22) was almost similar. Conclusions. The findings of this study revealed low genetic diversity of the msp-1 and msp-2 allelic families in P. falciparum isolates. However, continued monitoring status of the local genetic diversity profile in the P. falciparum population is required to support current malaria control and elimination strategies. [ABSTRACT FROM AUTHOR]

Background: Genetic diversity of malaria parasites can inform the intensity of transmission and poses a major threat to malaria control and elimination interventions. Characterization of the genetic diversity would provide essential information about the ongoing control efforts. This study aimed to explore allelic polymorphism of merozoite surface protein 1 (msp1) and merozoite surface protein 2 (msp2) to determine the genetic diversity and multiplicity of Plasmodium falciparum infections circulating in high and low transmission sites in western Ethiopia. Methods: Parasite genomic DNA was extracted from a total of 225 dried blood spots collected from confirmed uncomplicated P. falciparum malaria-infected patients in western Ethiopia. Of these, 72.4% (163/225) and 27.6% (62/225) of the samples were collected in high and low transmission areas, respectively. Polymorphic msp1 and msp2 genes were used to explore the genetic diversity and multiplicity of falciparum malaria infections. Genotyping of msp1 was successful in 86.5% (141/163) and 88.7% (55/62) samples collected from high and low transmission areas, respectively. Genotyping of msp2 was carried out among 85.3% (139/163) and 96.8% (60/62) of the samples collected in high and low transmission sites, respectively. Plasmodium falciparum msp1 and msp2 genes were amplified by nested PCR and the PCR products were analysed by QIAxcel ScreenGel Software. A P-value of less or equal to 0.05 was considered significant. Results: High prevalence of falciparum malaria was identified in children less than 15 years as compared with those ≥ 15 years old (AOR = 2.438, P = 0.005). The three allelic families of msp1 (K1, MAD20, and RO33) and the two allelic families of msp2 (FC27 and 3D7), were observed in samples collected in high and low transmission areas. However, MAD 20 and FC 27 alleles were the predominant allelic families in both settings. Plasmodium falciparum isolates circulating in western Ethiopia had low genetic diversity and mean MOI. No difference in mean MOI between high transmission sites (mean MOI 1.104) compared with low transmission area (mean MOI 1.08) (p > 0.05). The expected heterozygosity of msp1 was slightly higher in isolates collected from high transmission sites (He = 0.17) than in those isolates from low transmission (He = 0.12). However, the heterozygosity of msp2 was not different in both settings (Pfmsp2: 0.04 in high transmission; pfmsp2: 0.03 in low transmission). Conclusion: Plasmodium falciparum from clinical malaria cases in western Ethiopia has low genetic diversity and multiplicity of infection irrespective of the intensity of transmission at the site of sampling. These may be signaling the effectiveness of malaria control strategies in Ethiopia; although further studies are required to determine how specific intervention strategies and other parameters that drive the pattern. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum resistance to series of anti-malarial drugs is a major challenge in efforts to control and/or eliminate malaria globally. In 1998, following the widespread of chloroquine (CQ) resistant P. falciparum, Ethiopia switched from CQ to sulfadoxine–pyrimethamine (SP) and subsequently in 2004 from SP to artemether–lumefantrine (AL) for the treatment of uncomplicated falciparum malaria. Data on the prevalence of CQ resistance markers after more than two decades of its removal is important to map the selection pressure behind the targets codons of interest. The present study was conducted to determine the prevalence of mutations in Pfcrt K76T and Pfmdr1 N86Y codons among malaria-infected patients from Adama, Olenchiti and Metehara sites of East Shewa zone, Oromia Regional State, Ethiopia. Methods: Finger-prick whole blood samples were collected on 3MM Whatman ® filter papers from a total of 121 microscopically confirmed P. falciparum infected patients. Extraction of parasite DNA was done by Chelex-100 method from dried blood spot (DBS). Genomic DNA template was used to amplify Pfcrt K76T and Pfmdr1 N86Y codons by nested PCR. Nested PCR products were subjected to Artherobacter protophormiae-I (APoI) restriction enzyme digestion to determine mutations at codons 76 and 86 of Pfcrt and Pfmdr1 genes, respectively. Results: Of 83 P. falciparum isolates successfully genotyped for Pfcrt K76T, 91.6% carried the mutant genotypes (76T). The prevalence of Pfcrt 76T was 95.7%, 92.5% and 84.5% in Adama, Metehara and Olenchiti, respectively. The prevalence of Pfcrt 76T mutations in three of the study sites showed no statistical significance difference (χ2 = 1.895; P = 0.388). On the other hand, of the 80 P. falciparum samples successfully amplified for Pfmdr1, all carried the wild-type genotypes (Pfmdr1 N86). Conclusion: Although CQ officially has been ceased for the treatment of falciparum malaria for more than two decades in Ethiopia, greater proportions of P. falciparum clinical isolates circulating in the study areas carry the mutant 76T genotypes indicating the presence of indirect CQ pressure in the country. However, the return of Pfmdr1 N86 wild-type allele may be favoured by the use of AL for the treatment of uncomplicated falciparum malaria. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The increase in detections of Plasmodium vivax infection in Duffy-negative individuals in Africa has challenged the dogma establishing the unique P. vivax Duffy Binding Protein-Duffy antigen receptor for chemokines (PvDBP-DARC) pathway used by P. vivax merozoites to invade reticulocytes. Information on the impact of Duffy antigen polymorphisms on the epidemiology of P. vivax malaria remains elusive. The objective of this study was to determine the distribution of asexual parasitaemia of P. vivax according to the Duffy antigen polymorphisms in Ethiopia. Methods: DNA was extracted from dried blood spots (DBS) collected from prospectively recruited 138 P. vivax-infected patients from health centres. The identification and estimation of P. vivax asexual parasitaemia were performed by microscopic examination and quantitative real-time polymerase chain reaction (PCR). Duffy genotyping was conducted by DNA sequencing in a total of 138 P.vivax infected samples. Results: The proportion of Duffy-negatives (FY*BES/FY*BES) in P. vivax infected patients was 2.9% (4/138). Duffy genotype FY*B/FY*BES (48.6%) was the most common, followed by FY*A/FY*BES genotype (25.4%). In one patient, the FY*02 W.01/FY*02 N.01 genotype conferring a weak expression of the Fyb antigen was observed. All P.vivax infected Duffy-negative patients showed low asexual parasitaemia (≤ 110 parasites/µL). The median P. vivax parasitaemia in Duffy-negative patients (53 parasites/µL) was significantly lower than those found in homozygous and heterozygous individuals (P < 0.0001). Conclusion: Plasmodium vivax in Duffy-negative patients shows invariably low asexual parasitaemia. This finding suggests that the pathway used by P. vivax to invade Duffy-negative reticulocytes is much less efficient than that used in Duffy-positives. Moreover, the low asexual parasitaemia observed in Duffy-negative individuals could constitute an 'undetected silent reservoir', thus likely delaying the elimination of vivax malaria in Ethiopia. [ABSTRACT FROM AUTHOR]

Background. Despite significant progress achieved globally in reducing malaria burden, still it is one of the major public health and economic problems in Ethiopia. Investigation of the local genetic polymorphism of P. falciparum, the most virulent and predominant malaria parasite primarily targeted in malaria control and elimination program, is paramount to assess intensity of parasite transmission. Analysis of the block 3 region of the msp-2 gene of P. falciparum provides strong molecular evidence to evaluate the real picture of malaria epidemiology to fine-tune the ongoing control and elimination programs in the region. Thus, this study was aimed at examining the status of such polymorphic gene and its implications in Adama and its surroundings. Methods. 148 isolates from patients with uncomplicated falciparum malaria were collected in the study from September 2019 to August 2020. Tween® 20 and the Chelex method were employed for parasite DNA extraction. msp-2 allelic families were genotyped by using nested polymerase chain reaction targeting its 3D7 and FC27 allelic variants followed by gel electrophoresis for fragment analysis. Results. Seventeen different polymorphic forms of msp-2 allelic fragments were detected in the study area. Moreover, 47 (31.8%) and 41(27.7%) were detected for 3D7 and FC27 allelic families, respectively. Furthermore, the multiclonal allele type accounted for 60 (40.5%). The mean MOI was 1.4, and the heterogeneity index (He) is 0.49 indicating nearly intermediate malaria transmission in the study area. Conclusions. The study revealed nearly intermediate genetic diversity and mean MOI of P. falciparum in the study area, demanding further scale up of the ongoing control and elimination efforts. [ABSTRACT FROM AUTHOR]

Introduction: Malaria continues to strike hardest against the health and economic development in Ethiopia. The peak of malaria incidence follows the main rainfall season in each year; however, its transmission tends to be highly heterogeneous within or between years and from area to area. Thus, this study was aimed to determine the trend prevalence of malaria in Mojo town, central Ethiopia. Methods: A retrospective study was conducted in Mojo town, East Shoa zone, Ethiopia from February to March 2021. Malaria cases and related data documented between 2016 and 2020 were carefully reviewed from laboratory registration logbooks. The collected data were analyzed using descriptive statistics. Results: A total of 19,106 blood films were examined from malaria-suspected patients. The overall microscopically confirmed prevalence of malaria was 4.2% (793/19,106). Plasmodium vivax was the predominant species accounting for 76.2% (604/793) of positive samples. Malaria cases declined from 259 in 2016 to 77 in 2020. The proportion of malaria was higher among males (64.8%, 514/793) than females (35.2%, 279/793) (P < 0.0001). Higher malaria cases were observed from the age group 15– 24 years followed by the age group of 25– 34 (P < 0.0001). The number of malaria cases was at a high level from September to November and lowest from December to February. Conclusion: Despite a declining trend of malaria prevalence, it remains a public health burden in the area. P. vivax, the predominant species, should get attention during prevention and control strategies for the successful progress of the malaria elimination program. [ABSTRACT FROM AUTHOR]

The unique biological features of Plasmodium vivax not only make it difficult to control but also to eliminate. For the transmission of the malaria parasite from infected human to the vector, gametocytes play a major role. The transmission potential of a malarial infection is inferred based on microscopic detection of gametocytes and molecular screening of genes in the female gametocytes. Microscopy-based detection methods could grossly underestimate the reservoirs of infection as gametocytes may occur as submicroscopic or as micro- or macro-gametocytes. The identification of genes that are highly expressed and polymorphic in male and female gametocytes is critical for monitoring changes not only in their relative proportions but also the composition of gametocyte clones contributing to transmission over time. Recent transcriptomic study revealed two distinct clusters of highly correlated genes expressed in the P. vivax gametocytes, indicating that the male and female terminal gametocytogeneses are independently regulated. However, the detective power of these genes is unclear. In this study, we compared genetic variations of 15 and 11 genes expressed, respectively, in the female and male gametocytes among P. vivax isolates from Southeast Asia, Africa, and South America. Further, we constructed phylogenetic trees to determine the resolution power and clustering patterns of gametocyte clones. As expected, Pvs 25 (PVP01_0616100) and Pvs 16 (PVP01_0305600) expressed in the female gametocytes were highly conserved in all geographical isolates. In contrast, genes including 6-cysteine protein Pvs230 (PVP01_0415800) and upregulated in late gametocytes ULG8 (PVP01_1452800) expressed in the female gametocytes, as well as two CPW-WPC family proteins (PVP01_1215900 and PVP01_1320100) expressed in the male gametocytes indicated considerably high nucleotide and haplotype diversity among isolates. Parasite samples expressed in male and female gametocyte genes were observed in separate phylogenetic clusters and likely represented distinct gametocyte clones. Compared to Pvs 25, Pvs230 (PVP01_0415800) and a CPW-WPC family protein (PVP01_0904300) showed higher expression in a subset of Ethiopian P. vivax samples. Thus, Pvs230 , ULG8 , and CPW-WPC family proteins including PVP01_0904300, PVP01_1215900, and PVP01_1320100 could potentially be used as novel biomarkers for detecting both sexes of P. vivax gametocytes in low-density infections and estimating transmission reservoirs. [ABSTRACT FROM AUTHOR]

Recent genome-wide association studies (GWASs) of severe malaria have identified several association variants. However, much about the underlying biological functions are yet to be discovered. Here, we systematically predicted plausible candidate genes and pathways from functional analysis of severe malaria resistance GWAS summary statistics (N = 17,000) meta-analysed across 11 populations in malaria endemic regions. We applied positional mapping, expression quantitative trait locus (eQTL), chromatin interaction mapping, and gene-based association analyses to identify candidate severe malaria resistance genes. We further applied rare variant analysis to raw GWAS datasets (N = 11,000) of three malaria endemic populations including Kenya, Malawi, and Gambia and performed various population genetic structures of the identified genes in the three populations and global populations. We performed network and pathway analyses to investigate their shared biological functions. Our functional mapping analysis identified 57 genes located in the known malaria genomic loci, while our gene-based GWAS analysis identified additional 125 genes across the genome. The identified genes were significantly enriched in malaria pathogenic pathways including multiple overlapping pathways in erythrocyte-related functions, blood coagulations, ion channels, adhesion molecules, membrane signalling elements, and neuronal systems. Our population genetic analysis revealed that the minor allele frequencies (MAF) of the single nucleotide polymorphisms (SNPs) residing in the identified genes are generally higher in the three malaria endemic populations compared to global populations. Overall, our results suggest that severe malaria resistance trait is attributed to multiple genes, highlighting the possibility of harnessing new malaria therapeutics that can simultaneously target multiple malaria protective host molecular pathways. [ABSTRACT FROM AUTHOR]

Background: Rapid diagnostic tests (RDT) are commonly used for the diagnosis of malaria caused by Plasmodium falciparum. However, false negative results of RDT caused by genetic variation of P. falciparum histidine-rich protein 2 and 3 genes (pfhrp2/3) threaten existing malaria case management and control efforts. The main objective of this study was to investigate the genetic variations of the pfhrp2/3 genes. Methods: A cross-sectional study was conducted from malaria symptomatic individuals in 2018 in Assosa zone, Ethiopia. Finger-prick blood samples were collected for RDT and microscopic examination of thick and thin blood films. Dried blood spots (DBS) were used for genomic parasite DNA extraction and molecular detection. Amplification of parasite DNA was made by quantitative PCR. DNA amplicons of pfhrp2/3 were purified and sequenced. Results: The PfHRP2 amino acid repeat type isolates were less conserved compared to the PfHRP3 repeat type. Eleven and eight previously characterized PfHRP2 and PfHRP3 amino acid repeat types were identified, respectively. Type 1, 4 and 7 repeats were shared by PfHRP2 and PfHRP3 proteins. Type 2 repeats were found only in PfHRP2, while types 16 and 17 were found only in PfHRP3 with a high frequency in all isolates. 18 novel repeat types were found in PfHRP2 and 13 novel repeat types were found in PfHRP3 in single or multiple copies per isolate. The positivity rate for PfHRP2 RDT was high, 82.9% in PfHRP2 and 84.3% in PfHRP3 sequence isolates at parasitaemia levels > 250 parasites/µl. Using the Baker model, 100% of the isolates in group A (If product of types 2 × type 7 repeats ≥ 100) and 73.7% of the isolates in group B (If product of types 2 × type 7 repeats 50–99) were predicted to be detected by PfHRP2 RDT at parasitaemia level > 250 parasite/μl. Conclusion: The findings of this study indicate the presence of different PfHRP2 and PfHRP3 amino acid repeat including novel repeats in P. falciparum from Ethiopia. These results indicate that there is a need to closely monitor the performance of PfHRP2 RDT associated with the genetic variation of the pfhrp2 and pfhrp3 gene in P. falciparum isolates at the country-wide level. [ABSTRACT FROM AUTHOR]

Background: One of the major challenges in developing an effective vaccine against asexual stages of Plasmodium falciparum is genetic polymorphism within parasite population. Understanding the genetic polymorphism like block 2 region of merozoite surface protein-1 (msp-1) gene of P. falciparum enlighten mechanisms underlining disease pathology, identification of the parasite clone profile from the isolates, transmission intensity and potential deficiencies of the ongoing malaria control and elimination efforts in the locality. Detailed understanding of local genetic polymorphism is an input to pave the way for better management, control and elimination of malaria. The aim of this study was to detect the most frequent allelic variant of the msp-1 gene of P. falciparum clinical isolates from selected health facilities in Adama town and its surroundings, Oromia, Ethiopia. Methods: One hundred thirty-nine clinical isolates were successfully amplified for msp-1 gene using specific primers. Nested PCR amplification was conducted targeting K1, MAD20, and R033 alleles followed by gel electrophoresis for fragment analysis. Based on the detection of a PCR fragment, infections were classified as monoclonal or multiple infections. Results: 19 different size polymorphism of msp-1 gene were identified in the study, with 67(48%) MAD20, 18 (13%) K-1 and 18 (13%) RO33 allelic family. Whereas, the multiple infections were 21(15%), 8 (5.8%), 4(2.9%), 3(2.2%) for MAD20 + K-1, MAD20 + RO33, K-1 + RO33, and MAD20 + K-1, RO33, respectively. The overall Multiplicity of infection (MOI) was 1.3 and the expected heterozygosity (He) was 0.39 indicating slightly low falciparum malaria transmission. Conclusion: The status of msp-1 allele size polymorphism, MOI and He observed in the study revealed the presence of slightly low genetic diversity of P. falciparum clinical isolates. However, highly frequent MAD20 allelic variant was detected from clinical isolates in the study area. Moreover, the driving force that led to high predominance of MAD20 allelic variant revealed in such malaria declining region demands further research. [ABSTRACT FROM AUTHOR]

Background: Red blood cell invasion by the Plasmodium vivax merozoite requires interaction between the Duffy antigen receptor for chemokines (DARC) and the P. vivax Duffy-binding protein II (PvDBPII). Given that the disruption of this interaction prevents P. vivax blood-stage infection, a PvDBP-based vaccine development has been well recognized. However, the polymorphic nature of PvDBPII prevents a strain transcending immune response and complicates attempts to design a vaccine. Methods: Twenty-three P. vivax clinical isolates collected from three areas of Ethiopia were sequenced at the pvdbpII locus. A total of 392 global pvdbpII sequences from seven P. vivax endemic countries were also retrieved from the NCBI archive for comparative analysis of genetic diversity, departure from neutrality, linkage disequilibrium, genetic differentiation, PvDBP polymorphisms, recombination and population structure of the parasite population. To establish a haplotype relationship a network was constructed using the median joining algorithm. Results: A total of 110 variable sites were found, of which 44 were parsimony informative. For Ethiopian isolates there were 12 variable sites of which 10 were parsimony informative. These parsimony informative variants resulted in 10 nonsynonymous mutations. The overall haplotype diversity for global isolates was 0.9596; however, the haplotype diversity was 0.874 for Ethiopia. Fst values for genetic revealed Ethiopian isolates were closest to Indian isolates as well as to Sri Lankan and Sudanese isolates but further away from Mexican, Papua New Guinean and South Korean isolates. There was a total of 136 haplotypes from the 415 global isolates included for this study. Haplotype prevalence ranged from 36.76% to 0.7%, from this 74.2% were represented by single parasite isolates. None of the Ethiopian isolates grouped with the Sal I reference haplotype. From the total observed nonsynonymous mutations 13 mapped to experimentally verified epitope sequences. Including 10 non-synonymous mutations from Ethiopia. However, all the polymorphic regions in Ethiopian isolates were located away from DARC, responsible for junction formation. Conclusion: The results of this study are concurrent with the multivalent vaccine approach to design an effective treatment. However, the presence of novel haplotypes in Ethiopian isolates that were not shared by other global sequences warrant further investigation. [ABSTRACT FROM AUTHOR]

Background: Rapid diagnostic tests (RDTs) targeting histidine rich protein 2(HRP2) are widely used for diagnosis of Plasmodium falciparum infections. Besides PfHRP2, the PfHRP3 antigen contributes to the detection of P. falciparum infections in PfHRP2 RDTs. However, the performance HRP2-based RDT is affected by pfhrp2/3 gene deletions resulting in false-negative test results. The objective of this study was to determine the presence and prevalence of pfhrp2/3 gene deletions including the respective flanking regions among symptomatic patients in Assosa zone, Northwest Ethiopia. Methods: A health-facility based cross-sectional study was conducted in febrile patients seeking a malaria diagnosis in 2018. Blood samples were collected by finger-prick for microscopic examination of blood smears, malaria RDT, and molecular analysis using dried blood spots (DBS) prepared on Whatman filter paper. A total of 218 P. falciparum positive samples confirmed by quantitative PCR were included for molecular assay of pfhrp2/3 target gene. Results: Of 218 P. falciparum positive samples, exon 2 deletions were observed in 17.9% of pfhrp2 gene and in 9.2% of pfhrp3 gene. A high proportion of deletions in short segments of pfhrp2 exon1-2 (50%) was also detected while the deletions of the pfhrp3 exon1-2 gene were 4.1%. The deletions were extended to the downstream and upstream of the flanking regions in pfhrp2/3 gene (above 30%). Of eighty-six PfHRP2 RDT negative samples, thirty-six lacked pfhrp2 exon 2. Five PfHRP2 RDT negative samples had double deletions in pfhrp2 exon 2 and pfhrp3 exon2. Of these double deletions, only two of the samples with a parasite density above 2000 parasite/µl were positive by the microscopy. Three samples with intact pfhrp3 exon2 in the pfhrp2 exon2 deleted parasite isolates were found to be positive by PfHRP2 RDT and microscopy with a parasite density above 10,000/µl. Conclusion: This study confirms the presence of deletions of pfhrp2/3 gene including the flanking regions. Pfhrp2/3 gene deletions results in false-negative results undoubtedly affect the current malaria control and elimination effort in the country. However, further countrywide investigations are required to determine the magnitude of pfhrp2/3 gene deletions and its consequences on routine malaria diagnosis. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum parasite populations in Ethiopia have been experiencing local selective pressures from drugs and immunity, leading to evolutionary adaptation. However, there was a paucity of data on genomic characterization and evolutionary adaptations of P. falciparum isolates from the central area of Ethiopia. Methods: Whole-genome analysis of 25 P. falciparum isolates from central Ethiopia, specifically from West Arsi, were studied to determine their genetic diversity, population structures, and signatures of selection in known drug resistance alleles against global isolates from Cambodia, Thailand, DR Congo, and Malawi. Results: A total of 18,517 high-quality single-nucleotide polymorphisms (SNPs) were identified in Ethiopian P. falciparum isolates. About 84% of the Ethiopian P. falciparum isolates had a FWS value > 0.95 showing a dominant single genotype infection in most isolates at the time of collection with little potential for out-crossing as expected in areas with low transmission intensity. Within-host diversity of Ethiopian infections was significantly different from East African (p < 0.001), but not Southeast Asian infections (P > 0.05). A significant population structure has been observed by PCA and population differentiation between Ethiopian parasites and East African (Fst ~ 10%) and Southeast Asian populations (Fst ~ 18%), suggesting limited gene flow and the independent evolution of the Ethiopian parasite population. Moreover, a total of 125 genes under balancing selection was found that include ama1, trap, eba175, and lsa3, previously identified as targets of human host immunity. Recent directional selection analysis using integrated standardized haplotype score (IHS) did not detect any selection signatures in the Pfcrt, Pfdhfr, Pfdhps, Pfmdr1, and PfK13 genes. However, known drug resistance-conferring mutations analysis showed that at least one SNP marker was fixed in these genes, but not in Pfdhps and PfK13. Conclusion: Plasmodium falciparum populations in the central region of Ethiopia was structurally diverged from both Southeast Asian and other East African populations. Malaria infections in Ethiopia had low within-host diversity, and parasites carry fixed chloroquine resistance markers despite the withdrawal of this drug for the treatment of P. falciparum. [ABSTRACT FROM AUTHOR]

Background: Rapid diagnostic tests (RDTs) targeting histidine rich protein 2(HRP2) are widely used for diagnosis of Plasmodium falciparum infections. Besides PfHRP2, the PfHRP3 antigen contributes to the detection of P. falciparum infections in PfHRP2 RDTs. However, the performance HRP2-based RDT is affected by pfhrp2/3 gene deletions resulting in false-negative test results. The objective of this study was to determine the presence and prevalence of pfhrp2/3 gene deletions including the respective flanking regions among symptomatic patients in Assosa zone, Northwest Ethiopia. Methods: A health-facility based cross-sectional study was conducted in febrile patients seeking a malaria diagnosis in 2018. Blood samples were collected by finger-prick for microscopic examination of blood smears, malaria RDT, and molecular analysis using dried blood spots (DBS) prepared on Whatman filter paper. A total of 218 P. falciparum positive samples confirmed by quantitative PCR were included for molecular assay of pfhrp2/3 target gene. Results: Of 218 P. falciparum positive samples, exon 2 deletions were observed in 17.9% of pfhrp2 gene and in 9.2% of pfhrp3 gene. A high proportion of deletions in short segments of pfhrp2 exon1-2 (50%) was also detected while the deletions of the pfhrp3 exon1-2 gene were 4.1%. The deletions were extended to the downstream and upstream of the flanking regions in pfhrp2/3 gene (above 30%). Of eighty-six PfHRP2 RDT negative samples, thirty-six lacked pfhrp2 exon 2. Five PfHRP2 RDT negative samples had double deletions in pfhrp2 exon 2 and pfhrp3 exon2. Of these double deletions, only two of the samples with a parasite density above 2000 parasite/µl were positive by the microscopy. Three samples with intact pfhrp3 exon2 in the pfhrp2 exon2 deleted parasite isolates were found to be positive by PfHRP2 RDT and microscopy with a parasite density above 10,000/µl. Conclusion: This study confirms the presence of deletions of pfhrp2/3 gene including the flanking regions. Pfhrp2/3 gene deletions results in false-negative results undoubtedly affect the current malaria control and elimination effort in the country. However, further countrywide investigations are required to determine the magnitude of pfhrp2/3 gene deletions and its consequences on routine malaria diagnosis. [ABSTRACT FROM AUTHOR]

Deletions in Plasmodium falciparum histidine rich protein 2(pfhrp2) gene threaten the usefulness of the most widely used HRP2-based malaria rapid diagnostic tests (mRDTs) that cross react with its structural homologue, PfHRP3. Parasites with deleted pfhrp2/3 genes remain undetected and untreated due to 'false-negative' RDT results. As Ethiopia recently launched malaria elimination by 2030 in certain selected areas, the availability of RDTs and the scale of their use have rapidly increased in recent years. Thus, it is important to explore the presence and prevalence of deletion in the target genes, pfhrp2 and pfhrp3. From a total of 189 febrile patients visited Adama Malaria Diagnostic centre, sixty-four microscopically-and polymerase chain reaction (PCR)-confirmed P. falciparum clinical isolates were used to determine the frequency of pfhrp2/3 gene deletions. Established PCR assays were applied to DNA extracted from blood spotted onto filter papers to amplify across pfhrp2/3 exons and flanking regions. However, analysis of deletions in pfhrp2, pfhrp3 and flanking genomic regions was successful for 50 of the samples. The pfhrp2 gene deletion was fixed in the population with all 50(100%) isolates presenting a deletion variant. This deletion extended downstream towards the Pf3D7 0831900 (MAL7PI.230) gene in 11/50 (22%) cases. In contrast, only 2/50 (4%) of samples had deletions for the Pf3D7 0831700 (MALPI.228) gene, upstream of pfhrp2. Similarly, the pfhrp3 gene was deleted in all isolates (100%), while 40% of the isolates had an extension of the deletion to the downstream flanking region that codes for Pf3D7 13272400 (MAL13PI.485).The pfhrp3 deletion also extended upstream to Pf3D7 081372100 (MAL13PI.475) region in 49/50 (95%) of the isolates, exhibiting complete absence of the locus. Although all samples showed deletions of pfhrp2 exon regions, amplification of an intron region was successful in five cases. Two different repeat motifs in the intron regions were observed in the samples tested. Pfhrp2/3 gene deletions are fixed in Ethiopia and this will likely reduce the effectiveness of PfHRP2-based mRDTs. It will be important to determine the sensitivity PfHRP 2/3-based RDTs in these populations and conduct a countrywide survey to determine the extent of these deletions and its effect on routine RDT-based malaria diagnosis. [ABSTRACT FROM AUTHOR]

Introduction: Historically, the global community has focused on the control of symptomatic malaria. However, interest in asymptomatic malaria has been growing, particularly in the context of malaria elimination.Areas covered: We undertook a comprehensive PubMed literature review on asymptomatic malaria as it relates to detection and elimination with emphasis between 2014 and 2019. Diagnostic tools with a low limit of detection (LOD) have allowed us to develop a more detailed understanding of asymptomatic malaria and its impact. These highly sensitive diagnostics have demonstrated that the prevalence of asymptomatic malaria is greater than previously thought. In addition, it is now possible to detect the malaria reservoir in the community, something that was previously not feasible. Asymptomatic malaria has previously not been treated, but research has begun to examine whether treating individuals with asymptomatic malaria may lead to health benefits. Finally, we have begun to understand the importance of asymptomatic malaria in ongoing transmission.Expert opinion: Therefore, with malaria elimination back on the agenda, asymptomatic malaria can no longer be ignored, especially in light of new ultra-sensitive diagnostic tools. [ABSTRACT FROM AUTHOR]

Background As the global public-health objectives for malaria evolve from malaria control towards malaria elimination, there is increasing interest in the significance of asymptomatic infections and the optimal diagnostic test to identify them. Method We conducted a cross-sectional study of asymptomatic individuals (N = 562) to determine the epidemiological characteristics associated with asymptomatic malaria. Participants were tested by rapid diagnostic tests (CareStart, Standard Diagnostics [SD] Bioline, and Alere ultrasensitive RDT [uRDT]), loop-mediated isothermal amplification (LAMP), and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to determine malaria positivity. Hemoglobin values were recorded, and anemia was defined as a binary variable, according to World Health Organization guidelines. Results Compared to reference qRT-PCR, LAMP had the highest sensitivity (92.6%, 95% confidence interval [CI] 86.4–96.5), followed by uRDT Alere Malaria (33.9%, 95% CI 25.5–43.1), CareStart Malaria (14.1%, 95% CI 8.4–21.5), microscopy (5.0%, 95% CI 1.8–10.5), and SD Bioline (5.0%, 95% CI 1.8–10.5). For Plasmodium falciparum specimens only, the sensitivity for uRDT Alere Malaria was 50.0% (95% CI 38.8–61.3) and SD Bioline was 7.3% (95% CI 2.7–15.3). Based on multivariate regression analysis with qRT-PCR as the gold standard, for every 3.2% increase in the prevalence of asymptomatic malaria, hemoglobin decreased by 1 gram per deciliter (prevalence ratio 0.968, 95% CI 0.940–0.997; P =.032). Deletions (4.8%) in hrp2 were noted. Conclusions While uRDT Alere Malaria has superior sensitivity to rapid diagnostic tests and microscopy in detecting asymptomatic malaria, LAMP is superior still. Ultrasensitive diagnostics provide the accurate prevalence estimates of asymptomatic malaria required for elimination. [ABSTRACT FROM AUTHOR]

Background: Malaria is more often considered a problem of the rural poor and the disease has been overlooked in urban settings for centuries due to the assumption that economic development in urban areas results in better life conditions, such as improved housing, drainage system and environmental changes that makes urban areas not conducive for breeding of the malaria vector. But, for many African countries, including Ethiopia, in most urban areas, although there are rapid developments, they are characterized by poor housing, lack of sanitation and drainage of surface water that would provide favourable conditions for vector breeding. Limited studies have been conducted as far as urban malaria is concerned in Ethiopia. The purpose of this study was to assess the status of falciparum and vivax malaria transmission in Adama City, Eastern Shoa Zone, Oromia, Ethiopia. Understanding the local epidemiology of malaria will help policy makers and other stakeholders to design and implement tailored cost effective and efficient intervention strategies targeting urban malaria. Methods: The study was designed to analyse 5-year trends of malaria burden by two co-endemic species in Ethiopia (Plasmodium falciparum and Plasmodium vivax) and its annual and seasonal transmission pattern in the city, by using retrospective data on malaria burden by species, malaria related inpatient department (IPD) and outpatient department (OPD) consultations from 2013/14 to 2017/18. Results: OPD retrospective data analysis indicated that adolescents and adults (≥ 15 years of age) were most affected by P. vivax 43.5% (2986/6862) and P. falciparum 31.7% (2179/6862). Plasmodium vivax was found to be a predominant species in causing malaria burden in the city exhibiting less seasonal occurrence, and the relative burden of P. vivax is gradually increasing from year to year over P. falciparum. Conclusion: Malaria is endemic to the city showing a public health problem. The productive group of the community, adolescents and adults, were most affected exacerbating poverty. Plasmodium vivax was found to be the highest malaria burden in the city and the observed epidemiological shift from P. falciparum to P. vivax calls for additional tailored intervention strategies to reduce the associated burden. [ABSTRACT FROM AUTHOR]

Background: Knowledge of the distribution of ABO-Rh(D) blood groups in a locality is vital for safe blood services. However, the distribution of these blood systems among Ethiopians in general is little explored. This study was, therefore, designed to determine the ABO-Rh(D) blood group distribution among patients attending Gambella hospital, southwestern Ethiopia. Methods: A cross-sectional study was conducted between November and December 2013 (N = 449). The patients were grouped into two broad categories. Those who originally moved from different parts of Ethiopia and currently residing in Gambella are named 'highlanders' (n = 211). The other group consisted of natives (Nilotics) to the locality (n = 238). ABO-Rh(D) blood groups were typed by agglutination, open-slide test method, using commercial antisera (Biotech laboratories Ltd, Ipswich, Suffolk, UK). Results: Overall, majority of the participants (41.20%) had blood type 'O' followed by types 'A' (34.96%), 'B' (20.48%) and 'AB' (3.34%). However, blood type 'A' was the most frequent (44.07%) blood group among the 'highlanders' and 50.42% of Nilotic natives had type 'O'. The proportion of participants devoid of the Rh factor was 19.37%. Conclusions: While the ABO blood group distribution is similar to previous reports, the Rh(D) frequency is much higher than what was reported so far for Ethiopia and continental Africa. [ABSTRACT FROM AUTHOR]

Background: Malaria is ranked as the leading communicable disease in Ethiopia, where Plasmodium falciparum and Plasmodium vivax are co-endemic. The incidence of P. vivax is usually considered to be less seasonal than P. falciparum. Clinical cases of symptomatic P. falciparum exhibit notable seasonal variation, driven by rainfall-dependent variation in the abundance of Anopheles mosquitoes. A similar peak of clinical cases of P. vivax is usually observed during the rainy season. However, the ability of P. vivax to relapse causing new blood-stage infections weeks to months after an infectious mosquito bite can lead to substantial differences in seasonal patterns of clinical cases. These cannot be detected with currently available diagnostic tools and are not cleared upon treatment with routinely administered anti-malarial drugs. Methods: A health- facility based cross-sectional study was conducted in Adama malaria diagnostic centre from May 2015 to April 2016. Finger-prick blood samples were collected for thin and thick blood film preparation from participants seeking treatment for suspected cases of febrile malaria. Informed consent was obtained from each study participant or their guardians. Seasonal patterns in malaria cases were analysed using statistical models, identifying the peaks in cases, and the seasonally varying proportion of P. vivax cases attributable to relapses. Results: The proportion of patients with malaria detectable by light microscopy was 36.1% (1141/3161) of which P. vivax, P. falciparum, and mixed infections accounted for 71.4, 25.8 and 2.8%, respectively. Of the febrile patients diagnosed, 2134 (67.5%) were males and 1919 (60.7%) were urban residents. The model identified a primary peak in P. falciparum and P. vivax cases from August to October, as well as a secondary peak of P. vivax cases from February to April attributable to cases arising from relapses. During the secondary peak of P. vivax cases approximately 77% (95% CrI 68, 84%) of cases are estimated to be attributable to relapses. During the primary peak from August to October, approximately 40% (95% CrI 29, 57%) of cases are estimated to be attributable to relapses. Discussion: It is not possible to diagnose whether a P. vivax case has been caused by blood-stage infection from a mosquito bite or a relapse. However, differences in seasonal patterns of P. falciparum and P. vivax cases can be used to estimate the population-level proportion of P. vivax cases attributable to relapses. These observations have important implications for the epidemiological assessment of vivax malaria, and initiating therapy that is effective against both blood stages and relapses. [ABSTRACT FROM AUTHOR]

Background: The presence of asymptomatic infections has serious implications for malaria elimination campaigns. Since asymptomatic carriers do not seek treatment for their infection and may become gametocyte carriers, they undoubtedly contribute to the persistence of malaria transmission in a population. The presence of asymptomatic parasitemias was noted in areas with seasonal malaria transmission. In Ethiopia there is a paucity of data regarding the prevalence of asymptomatic malaria carriage. This study was undertaken to assess the presence and prevalence of asymptomatic Plasmodium falciparum and Plasmodium vivax infections in south-central Oromia, Ethiopia. Methods: A total of 1094 apparently healthy individuals ≥ 2 years of age in south-central Oromia, Ethiopia, an area with seasonal and unstable malaria transmission, were screened for the presence of asymptomatic plasmodial infections. Finger-prick blood samples were taken from each participant for blood film preparation for microscopy and the rapid diagnostic test (RDT). Blood samples were also spotted on Whatman 3MM filter paper for parasite DNA extraction. Results: The prevalence of asymptomatic Plasmodium carriage (P. falciparum, P. vivax and mixed species) was 5.0 % (55/1,094) as determined by microscopy, while the prevalence as determined using RDT was 8.2 % (90/1,094). PCR was done on 47 of 55 microscopy-confirmed and on 79 of 90 RDT-confirmed samples. PCR detected parasite DNA in 89.4 % (42/47) of the microscopy-positive samples and in 77.2 % (61/79) of the RDT-positive samples. No significant difference was observed in the prevalence of asymptomatic P. falciparum or P. vivax infections in the study area (P > 0.1). However, the prevalence of asymptomatic parasitaemia was significantly associated with gender (OR = 0.47, P = 0.015; being higher in males than females) and age (X² = 25, P < 0.001; being higher in younger than in older individuals). Age and parasite densities had an inverse relationship. Conclusions: This study confirms the presence of asymptomatic P. falciparum and P. vivax infections in south-central Oromia, an area with low, seasonal and unstable malaria transmission in Ethiopia. Of 55 microscopically confirmed asymptomatic infections, P. falciparum monoinfection accounted for 45.5 % and of 90 RDT positive asymptomatic infections, 66.7 % were P. falciparum. Although not statistically significant, P. falciparum accounted for a relatively large number of the asymptomatic infections as determined by both tests. The prevalence of asymptomatic parasitaemia was highest in the younger age group. HRP-2-based RDTs specific for P. falciparum showed high false positivity rate compared to Plasmodium lactate dehydrogenase (pLDH) specific to P. vivax. Although microscopy and RDT detected substantial numbers of asymptomatic infections in apparently healthy inhabitants, the use of a highly sensitive molecular diagnostics offers a more accurate assessment of the magnitude of asymptomatic infections. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum resistance to anti-malarials is a major drawback in effective malaria control and elimination globally. Artemisinin-combination therapy (ACT) is currently the key first-line treatment for uncomplicated falciparum malaria. Plasmodium falciparum genetic signatures at pfmdr-1, pfcrt, and pfubp-1 loci are known to modulate in vivo and in vitro parasite response to ACT. The objective of this study was to assess the distribution of these resistance gene markers in isolates collected from different malaria transmission intensity in Ethiopia and Tanzania. Methods: Plasmodium falciparum clinical isolates were collected from different regions of Ethiopia and Tanzania. Genetic polymorphisms in the genes pfcrt, pfmdr-1 and pfubp-1 were analysed by PCR and sequencing. Frequencies of the different alleles in the three genes were compared within and between regions, and between the two countries. Results: The majority of the isolates from Ethiopia were mutant for the pfcrt 76 and wild-type for pfmdr-1 86. In contrast, the majority of the Tanzanian samples were wild-type for both pfcrt and pfmdr-1 loci. Analysis of a variable linker region in pfmdr-1 showed substantial variation in isolates from Tanzania as compared to Ethiopian isolates that had minimal variation. Direct sequencing of the pfubp-1 region showed that 92.8% (26/28) of the Ethiopian isolates had identical genome sequence with the wild type reference P. falciparum strain 3D7. Of 42 isolates from Tanzania, only 13 (30.9%) had identical genome sequences with 3D7. In the Tanzanian samples, 10 variant haplotypes were identified. Conclusion: The majority of Ethiopian isolates carried the main marker for chloroquine (CQ) resistance, while the majority of the samples from Tanzania carried markers for CQ susceptibility. Polymorphic genes showed substantially more variation in Tanzanian isolates. The low variability in the polymorphic region of pfmdr-1 in Ethiopia may be a consequence of low transmission intensity as compared to high transmission intensity and large variations in Tanzania. [ABSTRACT FROM AUTHOR]

Background: The presence of asymptomatic infections has serious implications for malaria elimination campaigns. Since asymptomatic carriers do not seek treatment for their infection and may become gametocyte carriers, they undoubtedly contribute to the persistence of malaria transmission in a population. The presence of asymptomatic parasitemias was noted in areas with seasonal malaria transmission. In Ethiopia there is a paucity of data regarding the prevalence of asymptomatic malaria carriage. This study was undertaken to assess the presence and prevalence of asymptomatic Plasmodium falciparum and Plasmodium vivax infections in south-central Oromia, Ethiopia. Methods: A total of 1094 apparently healthy individuals ≥ 2 years of age in south-central Oromia, Ethiopia, an area with seasonal and unstable malaria transmission, were screened for the presence of asymptomatic plasmodial infections. Finger-prick blood samples were taken from each participant for blood film preparation for microscopy and the rapid diagnostic test (RDT). Blood samples were also spotted on Whatman 3MM filter paper for parasite DNA extraction. Results: The prevalence of asymptomatic Plasmodium carriage (P. falciparum, P. vivax and mixed species) was 5.0 % (55/1,094) as determined by microscopy, while the prevalence as determined using RDT was 8.2 % (90/1,094). PCR was done on 47 of 55 microscopy-confirmed and on 79 of 90 RDT-confirmed samples. PCR detected parasite DNA in 89.4 % (42/47) of the microscopy-positive samples and in 77.2 % (61/79) of the RDT-positive samples. No significant difference was observed in the prevalence of asymptomatic P. falciparum or P. vivax infections in the study area (P > 0.1). However, the prevalence of asymptomatic parasitaemia was significantly associated with gender (OR = 0.47, P = 0.015; being higher in males than females) and age (X2 = 25, P < 0.001; being higher in younger than in older individuals). Age and parasite densities had an inverse relationship. This study confirms the presence of asymptomatic P. falciparum and P. vivax infections in south-central Oromia, an area with low, seasonal and unstable malaria transmission in Ethiopia. Of 55 microscopically confirmed asymptomatic infections, P. falciparum monoinfection accounted for 45.5 % and of 90 RDT positive asymptomatic infections, 66.7 % were P. falciparum. Although not statistically significant, P. falciparum accounted for a relatively large number of the asymptomatic infections as determined by both tests. The prevalence of asymptomatic parasitaemia was highest in the younger age group. HRP-2-based RDTs specific for P. falciparum showed high false positivity rate compared to Plasmodium lactate dehydrogenase (pLDH) specific to P. vivax. Although microscopy and RDT detected substantial numbers of asymptomatic infections in apparently healthy inhabitants, the use of a highly sensitive molecular diagnostics offers a more accurate assessment of the magnitude of asymptomatic infections. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum resistance to anti-malarials is a major drawback in effective malaria control and elimination globally. Artemisinin-combination therapy (ACT) is currently the key first-line treatment for uncomplicated falciparum malaria. Plasmodium falciparum genetic signatures at pfmdr-1, pfcrt, and pfubp-1 loci are known to modulate in vivo and in vitro parasite response to ACT. The objective of this study was to assess the distribution of these resistance gene markers in isolates collected from different malaria transmission intensity in Ethiopia and Tanzania. Methods: Plasmodium falciparum clinical isolates were collected from different regions of Ethiopia and Tanzania. Genetic polymorphisms in the genes pfcrt, pfmdr-1 and pfubp-1 were analysed by PCR and sequencing. Frequencies of the different alleles in the three genes were compared within and between regions, and between the two countries. Results: The majority of the isolates from Ethiopia were mutant for the pfcrt 76 and wild-type for pfmdr-1 86. In contrast, the majority of the Tanzanian samples were wild-type for both pfcrt and pfmdr-1 loci. Analysis of a variable linker region in pfmdr-1 showed substantial variation in isolates from Tanzania as compared to Ethiopian isolates that had minimal variation. Direct sequencing of the pfubp-1 region showed that 92.8% (26/28) of the Ethiopian isolates had identical genome sequence with the wild type reference P. falciparum strain 3D7. Of 42 isolates from Tanzania, only 13 (30.9%) had identical genome sequences with 3D7. In the Tanzanian samples, 10 variant haplotypes were identified. Conclusion: The majority of Ethiopian isolates carried the main marker for chloroquine (CQ) resistance, while the majority of the samples from Tanzania carried markers for CQ susceptibility. Polymorphic genes showed substantially more variation in Tanzanian isolates. The low variability in the polymorphic region of pfmdr-1 in Ethiopia may be a consequence of low transmission intensity as compared to high transmission intensity and large variations in Tanzania. [ABSTRACT FROM AUTHOR]

Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13- propeller mutations in sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Background: Evidence for decreasing chloroquine (CQ) efficacy against Plasmodium vivax has been reported from many endemic countries in the world. In Ethiopia, P. vivax accounts for 40% of all malaria cases and CQ is the first-line drug for vivax malaria. Mutations in multidrug resistance 1 (pvmdr-1) and K10 insertion in the pvcrt-o genes have been identified as possible molecular markers of CQ-resistance (CQR) in P. vivax. Despite reports of CQ treatment failures, no data are currently available on the prevalence of molecular markers of P. vivax resistance in Ethiopia. The objective of this study was to determine the prevalence of mutations in the pvmdr-1 and K10 insertion in the pvcrt-o genes. Methods: A total of 36 P. vivax clinical isolates were collected from West Arsi district in Ethiopia. Sequencing was used to analyse polymorphisms of the pvcrt-o and pvmdr-1 genes. Results: Sequencing results of the pvmdr-1 fragment showed the presence of two non-synonymous mutations at positions 976 and 1076. The Y → F change at codon 976 (TAC → TTC) was observed in 21 (75%) of 28 the isolates while the F → L change (at codon 1076), which was due to a single mutation (TTT → CTT), was observed in 100% of the isolates. Of 33 samples successfully amplified for the pvcrt-o, the majority of the isolates (93.9%) were wild type, without K10 insertion. Conclusions: High prevalence of mutations in candidate genes conferring CQR in P. vivax was identified. The fact that CQ is still the first-line treatment for vivax malaria, the significance of mutations in the pvcrt-o and pvmdr-1 genes and the clinical response of the patients' to CQ treatment and whether thus an association exists between point mutations of the candidate genes and CQR requires further research in Ethiopia. [ABSTRACT FROM AUTHOR]

Background Glucose-6-phosphate dehydrogenase deficiency (G6PDd) is widespread across malaria endemic regions. G6PD-deficient individuals are at risk of haemolysis when exposed, among other agents, to primaquine and tafenoquine, which are capable of blocking malaria transmission by killing Plasmodium falciparum gametocytes and preventing Plasmodium vivax relapses by targeting hypnozoites. It is evident that no measures are currently in place to ensure safe delivery of these drugs within the context of G6PDd risk. Thus, determining G6PDd prevalence in malarious areas would contribute towards avoiding possible complications in malaria elimination using the drugs. This study, therefore, was aimed at determining G6PDd prevalence in Gambella hospital, southwest Ethiopia, using CareStart™ G6PDd fluorescence spot test. Methods Venous blood samples were collected from febrile patients (n = 449) attending Gambella hospital in November-December 2013. Malaria was diagnosed using blood films and G6PDd was screened using CareStart™ G6PDd fluorescence spot test. Haematological parameters were also measured. The association of G6PD phenotype with sex, ethnic group and malaria smear positivity was tested. Results Malaria prevalence was 59.2% (96.6% of the cases being P. falciparum). Totally 33 participants (7.3%) were G6PD-deficient with no significant difference between the sexes. The chance of being G6PD-deficient was significantly higher for the native ethnic groups (Anuak and Nuer) compared to the 'highlanders'/settlers (odds ratio (OD) = 3.9, 95% confidence interval (CI) 0.481-31.418 for Anuak vs 'highlanders'; OD = 4.9, 95% CI 0.635- 38.00 for Nuer vs 'highlanders'). G6PDd prevalence among the Nuer (14.3%) was significantly higher than that for the Anuak (5.1%). Conclusions G6PDd prevalence in the area is substantial with 30 (90.9%) of the 33 deficient individuals having malaria suggesting the non-protective role of the disorder at least from clinical malaria. The indigenous Nilotic people tend to have a higher chance of being G6PD-deficient as 32 (96.9%) of the total 33cases occurred among them. [ABSTRACT FROM AUTHOR]

Background: As a result of extensive chloroquine resistance (CQR) in Plasmodium falciparum in late 1990s, Ethiopia replaced CQ with sulphadoxine-pyrimethamine (SP) as first-line drug, which in turn was replaced by artemisinin combination therapy in 2004. Plasmodium falciparum resistance to CQ is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene. Understanding diversity in the P. falciparum genome is crucial since it has the potential to influence important phenotypes of the parasite such as drug resistance. Limited data is available regarding the type of pfcrt mutant allelic type, the effect of CQ withdrawal and diversity of the parasite population in south-central Oromia, Ethiopia. Methods: Finger-pricked blood spotted on Whatman 3MM filter papers were collected from falciparum malaria patients. Parasite DNA was extracted from individual blood spots on the filter papers. The presence of K76T mutations was determined using nested PCR for all isolates. Complete sequencing of mutations in pfcrt 72-76 was done for a set of randomly selected resistant isolates. Four microsatellite (MS) markers were analysed to determine the heterozygosity. Results: Although CQ was withdrawn for more than a decade, 100% of the parasites still carried the pfcrt K76T mutation. All isolates were mutant at the K76T polymorphism. Based on combinations of MS markers, seven different Ethiopian CQR variants (E1-E7) were identified. Heterozygosity (He) for MS flanking the pfcrt chloroquine resistance allele ranged from 0.00 (mscrt -29, -29.268 kb) to 0.21 (mscrt -2, -2.814 kb). He ranged from 0.00 (msint 3, 0 kb) to 0.19 (msint 2, 0 kb) for MS within the pfcrt gene. Both intronic and MS flanking the pfcrt gene showed low levels of diversity. Conclusion: pfcrt CQR allele seems to be fixed in the study area. Of the different haplotypes associated with CQR, only the CVIET genotype was identified. No reversal to the wild-type has occurred in Ethiopia unlike in many Africa countries where CQR parasites declined after cessation of CQ use. Decreased diversity in CQR isolates surrounding pfcrt suggests CQ selection and homogenization among CQR parasite population. While mutation in msint 3 and mscrt -29 of the mutant pfcrt allele is being fixed, it seems that mutations in msint 2 and mscrt -2 are still evolving and may indicate the start of re-diversification of the population from a fixed 76 T population. [ABSTRACT FROM AUTHOR]