Prevalence and Characteristics of Plasmodium vivax Gametocytes in Duffy-Positive and Duffy-Negative Populations across Ethiopia.

Plasmodium parasites replicate asexually in human hosts. The proportion of infections that carries gametocytes is a proxy for human-to-mosquito transmissibility. It is unclear which proportion of Plasmodium vivax infections in Duffy-negative populations carries gametocytes. We determined the prevalence and characteristics of P. vivax gametocytes in Duffy-positive and -negative populations across broad regions of Ethiopia. Finger-prick blood samples were collected for microscopic and molecular screening of Plasmodium parasites and Duffy status of individuals. Molecular screening of Plasmodium species and Duffy blood group genotyping was done using SYBR green and the Taqman quantitative polymerase chain reaction method. Of the 447 febrile patients who were shown to be P. vivax smear positive, 414 (92.6%) were confirmed by molecular screening as P. vivax and 16 (3.9%) of them were from Duffy-negative individuals. Of these, 5 of 16 (31.3%) Duffy-negative P. vivax-infected samples were detected with gametocytes. Of the 398 Duffy-positive P. vivax-infected samples, 150 (37.7%) were detected with gametocytes, slightly greater than that in Duffy-negative samples. This study highlights the presence of P. vivax gametocytes in Duffy-negative infections, suggestive of human-to-mosquito transmissibility. Although P. vivax infections in Duffy-negative individuals were commonly associated with low parasitemia, some of these infections were shown to have relatively high parasitemia and may represent a prominent erythrocyte invasion capability of P. vivax, and hidden reservoirs that can contribute to transmission. A better understanding of P. vivax transmission biology and gametocyte function particularly in Duffy-negative populations would aid future treatment and management of P. vivax malaria in Africa.

Widespread pfhrp2/3 deletions and HRP2-based false-negative results in southern Ethiopia.

BACKGROUND: Rapid diagnostic tests (RDTs) play a significant 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 confirmed P. falciparum infections. A capillary blood sample was obtained to prepare a blood film 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 amplification was performed using nested PCR assay. Exon 2 of hrp2 and hrp3, which are the main protein-coding regions, was used to confirm 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-confirmed samples, 249 (89.2%) had successful msp-2 amplification, 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 false-negative 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 findings 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 confirmed 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- affect 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.

Cytotaxonomic characterization and estimation of migration patterns of onchocerciasis vectors (Simulium damnosum sensu lato) in northwestern Ethiopia based on RADSeq data.

BACKGROUND: While much progress has been made in the control and elimination of onchocerciasis across Africa, the extent to which vector migration might confound progress towards elimination or result in re-establishment of endemism in areas where transmission has been eliminated remains unclear. In Northern Ethiopia, Metema and Metekel-two foci located near the Sudan border-exhibit continuing transmission. While progress towards elimination has been faster in Metema, there remains a problematic hotspot of transmission. Whether migration from Metekel contributes to this is currently unknown. METHODOLOGY/PRINCIPLE FINDINGS: To assess the role of vector migration from Metekel into Metema, we present a population genomics study of 151 adult female vectors using 47,638 RADseq markers and mtDNA CoI sequencing. From additional cytotaxonomy data we identified a new cytoform in Metema, closely related to S. damnosum s.str, here called the Gondar form. RADseq data strongly indicate the existence of two distinctly differentiated clusters within S. damnosum s.l.: one genotypic cluster found only in Metema, and the second found predominantly in Metekel. Because blackflies from both clusters were found in sympatry (in all four collection sites in Metema), but hybrid genotypes were not detected, there may be reproductive barriers preventing interbreeding. The dominant genotype in Metema was not found in Metekel while the dominant genotype in Metekel was found in Metema, indicating that (at the time of sampling) migration is primarily unidirectional, with flies moving from Metekel to Metema. There was strong differentiation between clusters but little genetic differentiation within clusters, suggesting migration and gene flow of flies within the same genetic cluster are sufficient to prevent genetic divergence between sites. CONCLUSIONS/SIGNIFICANCE: Our results confirm that Metekel and Metema represent different transmission foci, but also indicate a northward movement of vectors between foci that may have epidemiological importance, although its significance requires further study.

Field performance of Plasmodium falciparum lactate dehydrogenase rapid diagnostic tests during a large histidine-rich protein 2 deletion survey in Ethiopia.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) have expanded diagnostic service to remote endemic communities in Ethiopia, where 70% of malaria services per annum are reliant on them. However, diagnostic strategies are threatened by Plasmodium falciparum parasites with deletions of the histidine-rich protein 2 and/or 3 (pfhrp2/3) genes. Studies have reported pfhrp2/3 gene deletion prevalence in Ethiopia that exceeds the WHO recommended threshold to switch to non-HRP2 targeted RDTs for detection of P. falciparum. Therefore, RDTs that target alternative antigens, such as P. falciparum lactate dehydrogenase (PfLDH) are increasingly in programmatic use. METHODS: Malaria suspected patients visiting health facilities of Amhara, Tigray, Gambella, and Oromia regions of Ethiopia were screened by community health workers using Carestart Pf/Pv (HRP2/Pv-LDH) and SD-Bioline Pf (HRP2 for Pf/LDH for Pf) RDTs. Dried blood spot (DBS) samples were collected from selected patients for molecular and serological analysis. The clinical data and RDT results were recorded on standard forms, entered into EpiInfo, and analysed using STATA. The Pf-LDH detecting RDT results were compared with real-time PCR and bead-based immunoassay to determine their diagnostic performance. RESULTS: The 13,172 (56% male and 44% female, median age of 19 years ranging from 1 to 99 year) study participants were enrolled and tested with PfHRP2 and PfLDH detection RDTs; 20.6% (95% CI: 19.6 to 21.6) were P. falciparum RDT positive. A subset of samples (n = 820) were previously tested using P. falciparum lactate dehydrogenase (pfldh) quantitative real-time PCR, and 456 of these further characterized using bead-based immunoassay. The proportion of samples positive for P. falciparum by the PfHRP2 Carestart and SD-Bioline RDTs were 66% (539/820) and 59% (481/820), respectively; 68% (561/820) were positive for the PfLDH band on the SD-Bioline RDT. The sensitivity and specificity of the PfLDH RDT band were 69% and 38%, respectively, versus pfldh qPCR; and 72% and 36%, respectively, versus PfLDH detection by immunoassay. Among samples with results for RDT, qPCR, and immunoassay, higher proportions of P. falciparum were recorded by pfldh qPCR (90%, 411/456) and PfLDH immunoassay (88%, 363/413) compared to the PfLDH band on the SD-Bioline RDT (74.6%, 340/456). CONCLUSION AND RECOMMENDATION: Both PfHRP2 RDTs detected fewer P. falciparum cases than PfLDH, and fewer cases than qPCR or immunoassay. The poor sensitivity and specificity of the PfLDH RDT compared to qPCR and to immunoassay in this study raises concern. Continuous operator training and RDTs quality assurance programme to ensure quality diagnostic services are recommended.

Sero-identification of the aetiologies of human malaria exposure (Plasmodium spp.) in the Limu Kossa District of Jimma Zone, South western Ethiopia.

BACKGROUND: Malaria remains a very important public health problem in Ethiopia. Currently, only Plasmodium falciparum and Plasmodium vivax are considered in the malaria diagnostic and treatment policies. However, the existence and prevalence of Plasmodium ovale spp. and Plasmodium malariae in Ethiopia have not been extensively investigated. The objective of this study was to use a multiplex IgG antibody detection assay to evaluate evidence for exposure to any of these four human malaria parasites among asymptomatic individuals. METHODS: Dried blood spots (DBS) were collected from 180 healthy study participants during a 2016 onchocerciasis survey in the Jimma Zone, southwest Ethiopia. IgG antibody reactivity was detected using a multiplex bead assay for seven Plasmodium antigens: P. falciparum circumsporozoite protein (CSP), P. falciparum apical membrane antigen-1 (AMA1), P. falciparum liver stage antigen-1 (LSA1), and homologs of the merozoite surface protein-1 (MSP1)-19kD antigens that are specific for P. falciparum, P. vivax, P. ovale spp. and P. malariae. RESULTS: One hundred six participants (59%) were IgG seropositive for at least one of the Plasmodium antigens tested. The most frequent responses were against P. falciparum AMA1 (59, 33%) and P. vivax (55, 28%). However, IgG antibodies against P. ovale spp. and P. malariae were detected in 19 (11%) and 13 (7%) of the participants, respectively, providing serological evidence that P. malariae and P. ovale spp., which are rarely reported, may also be endemic in Jimma. CONCLUSION: The findings highlight the informative value of multiplex serology and the need to confirm whether P. malariae and P. ovale spp. are aetiologies of malaria in Ethiopia, which is critical for proper diagnosis and treatment.

Review of Ethiopian Onchocerciasis Elimination Programme.

Onchocerciasis is a severe parasitic infection which causes disabling skin and subcutaneous tissue changes. The disease is endemic in many African countries including Ethiopia. In 2013, Ethiopia launched Onchocerciasis elimination program with the goal of attaining interruption of onchocerciasis transmission nationwide by 2020. The country has successfully scaled up interventions and achieved 100% geographic coverage in all known endemic districts. The main strategy for interrupting the disease is mass drug administration (MDA) delivered two times per year. The treatment coverage for the last five years has been maintained at more than 80%. Despite many years of ivermectin MDA the transmission of onchocerciasis in many districts remained unabated. To achieve the 2020 goal, sustained high geographic and therapeutic coverage is required which is validated by coverage surveys. The programme should aim to improve the knowledge and attitude of the community towards the programme in order to improve drug compliance. The partnership between the relevant stakeholders should be strengthened to facilitate open discussions regarding the programme implementation and any challenges that may arise in the control and elimination of the disease. It is also important to consider intensified vector control.

Epidemiological Mapping of Human Onchocerciasis in Transmission Suspected Districts of Bale, Borena, and West Arsi Zones of Eastern Ethiopia.

Onchocerciasis is mainly found in western part of Ethiopia and there is no evidence of transmission in the east ward. However, some zones (Bale, Borena, and West Arsi) are suspected for transmission given the area has fast flowing rivers and is covered with vegetation. Therefore, this study was conducted to map onchocerciasis transmission in those zones. About 19 villages were selected based on proximity to the rivers, representation of districts, zones, and vegetation covers, whereas the study participants, all village residents of age > 5 years with good health condition, were skin sniped and examined using microscopy. In this study a total of 2560 study participants were surveyed of which 1332 were female (52%) and 122 were male (48%). The age group of 21-30 years was highest (34.4%) and that of age > 51 years was the lowest (3.1%) study participants. The survey result revealed that none of the study participants regardless of age, sex, and location demonstrated skin snip Onchocerca microfilariae. The prevalence of microfilariae and community microfilarial load (CMFL) were 0% and 0 mf/s, respectively. The finding implied that there is no onchocerciasis in the area and, therefore, there is no need for interventions. Black fly distribution, cytotaxonomic study, and intraborder cross transmission monitoring are recommended.