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.

Ecology and Infection Status of Sand Flies in Rural and Urban Cutaneous Leishmaniasis Endemic Areas in Northwest Ethiopia.

Cutaneous leishmaniasis (CL) caused by Leishmania aethiopica is transmitted by Phlebotomus longipes in northern Ethiopia. No studies have been conducted to investigate the transmission dynamics of CL, despite its high endemicity in both rural and urban settings. Evidence on the ecology and behavior of the vector from this area are required to develop integrated disease control strategies. Sand flies were collected in the dry and wet seasons in 2021 in CL-endemic rural Gindmeteaye and urban Addis-Alem in northwest Ethiopia. Trapping was performed with sticky and Centers for Disease Control and Prevention (CDC) light traps in three habitats, including inside patients' houses, peridomestic areasand in caves/rocky areas. Sand flies were morphologically identified to species level. Female Phlebotomus species were categorized according to blood feeding status and tested by spliced-leader (SL-) ribonucleic acid (RNA) polymerase chain reaction (PCR) to screen for Leishmania infection. Of 1161 sand flies, the majority (77%) were P. longipes, six (0.5%) were P. orientalis and the remaining were Sergentomyia. The abundance of the 430 female P. longipes was significantly linked to seasonality (p < 0.001), with the majority in the dry season occurring in the outdoor rocky (37%) and peridomestic (34%) sites, while, in the wet season, most (62%) were captured indoors. This seasonality was more pronounced in rural Gindmeteaye, where housing construction is poor. The number of blood-fed and gravid P. longipes was significantly higher in the wet (31%; 22%), compared to the dry season (13%; 8%), and their proportion was highest indoors. Eighteen (4%) female P. longipes were Leishmania positive, with highest infection prevalence in caves (7% compared to 3% indoors, p = 0.022), and in the dry season (6%, p < 0.001). Phlebotomus orientalis specimens were all captured in May in rural Gindmeteaye, five indoors and one in a peridomestic site. Further research should be conducted to investigate the absolute contribution of humans and indoor transmission to the transmission cycle of CL. Inhabitants of endemic villages should be made aware that evening outdoor activities near caves may increase their exposure to infectious sand flies. Whether P. orientalis can breed and become infected at high altitudes should be further studied.

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

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

Mosquito odour-baited mass trapping reduced malaria transmission intensity: a result from a controlled before-and-after intervention study.

BACKGROUND: Conventional vector control strategies have significantly reduced the malaria burden. The sustainability of these methods is currently challenged. Odour-based traps are emerging technologies that can complement the existing tools. Implementation of odour-based traps for mass trapping is limited due to the restricted range of vectors caught with available carbon dioxide-dependent lures, and the lack of comprehensive field studies. The objective of this study was to assess the impact of odour-mediated mass trapping targeting outdoor vectors, using a synthetic cattle urine lure that attracts a wide range of vector species in a variety of physiological states, on malaria prevalence and entomological parameters to determine malaria transmission intensities. METHODS: A controlled before-and-after study was conducted in two rural communities in southern Ethiopia. Baseline monthly entomological and seasonal cross-sectional malaria prevalence surveys were conducted in both communities for a year. Then, mass trapping of mosquitoes was conducted in one of the villages, while the monthly entomological surveillance and seasonal malaria prevalence surveys continued in both villages. Generalised linear mixed models were constructed and tested to determine which factors were significantly affected by the intervention. RESULTS: Mass trapping contributed to the reduction of the population of the principal malaria vector, Anopheles arabiensis, and the associated entomological indicators, the human bite rate (HBR) and the entomological inoculation rate (EIR), in the intervention village compared to the control village. The intervention village had an average HBR by An. arabiensis of 3.0 (95% CI 1.4-4.6) during the peak malaria transmission season, compared to 10.5 (95% CI - 0.5-21.5; P < 0.0001) in the control village. The intervention village (mean 0.02, 95% CI - 0.05-0.4.8) had a daily EIR eight times lower than the control village (mean 0.17, 95% CI), which likely contributed to the reduced malaria prevalence in the intervention community following its introduction by ca. 60% (95% CI 55-63). CONCLUSIONS: The combined use of odour-based mass trapping and conventional control strategies coincided with a reduction of human-vector contact and malaria prevalence, providing support for odour-baited technologies as a viable option for next-generation vector control tools. Further cluster-randomised control studies are recommended in different eco-epidemiological settings with varying malaria transmission intensities.

The potential of addressing asymptomatic malaria in the context of malaria elimination in Ethiopia: Scoping review.

Objectives: Currently, evidence synthesis targeting asymptomatic malaria infections in Ethiopia are scarce. This review intended to collect and organize information on asymptomatic malaria. Methods: A Joanna Briggs Institute, scoping review protocol was used. Searches for peer-reviewed articles published between 01 January 2010 and 10 August 2022, were done through a variety of databases, and gray literatures. Results: 17 articles were included out of 7672 articles identified. There was no any longitudinal study to trace forward these asymptomatic malaria cases. The reviewed studies did not address how asymptomatic malaria could be treated. Moreover, living in index houses, their neighbours and family sizes were the main predictors and more associated with onward transmission of malaria. Asymptomatic malaria (ASM) infection might persist in all seasons except June-August, for which data is lacking. Conclusions: Therefore, as implication of research and policy, it would be necessary to focus on index families and their neighbours in prevention of ASM, conducting longitudinal studies to ascertain when and how many asymptomatic malaria cases without fever during diagnosis would develop clinical malaria. As well, establishing a more sensitive diagnostic technique of malaria surveillance. It is also necessary to provide information regarding the feasibility of treating asymptomatic malaria cases in Ethiopia.

Plasmodium infections and associated risk factors among parturients in Jawi district, northwest Ethiopia: a cross-sectional study.

BACKGROUND: Pregnant women have an increased risk of Plasmodium infections and disease. Malaria in pregnancy is a major public health problem in endemic areas. Assessment of the burden and risk factors of malaria in pregnancy across different malaria transmission settings is required to guide control strategies and for malaria elimination. Thus, the current study is generating such evidence from parturient women in northwest Ethiopia. METHODS: A cross-sectional study was conducted among 526 pregnant women admitted to the delivery rooms of selected health facilities in Jawi district, northwest Ethiopia, between November 2021 and July 2022. Data on the socio-demographic, clinical, obstetric, and malaria prevention practices of pregnant women were collected using interviewer-administered questionnaires and from women's treatment cards. Malaria was diagnosed by light microscopy, rapid diagnostic test, and multiplex real-time polymerase chain reaction. Risk factors for malaria were evaluated using bivariable and multivariable logistic regression models. A P-value of < 0.05 was considered statistically significant. RESULTS: Among the examined parturient women, 14.3% (95% CI 11.4-17.5%) had Plasmodium infections. The prevalence of peripheral, placental, and congenital malaria was 12.2% (95% CI 9.5-15.3%), 10.9% (95% CI 8.2-14.1%), and 3.7% (95% CI 2.3-6.1%), respectively. About 90.6% of peripheral and 92% of placental Plasmodium infections were asymptomatic. Plasmodium infection at parturiency was independently predicted by maternal illiteracy (AOR = 2.03, 95% CI 1.11-3.74), primigravidity (AOR = 1.88, 95% CI 1.01-3.49), lack of antenatal care follow-up (AOR = 2.28, 95% CI 1.04-5.03), and history of symptomatic malaria during pregnancy (AOR = 4.2, 95% CI 2.32-7.59). Moreover, the blood group O phenotype was significantly associated with placental malaria among the primiparae. CONCLUSIONS: Overall, asymptomatic Plasmodium infections were prevalent among parturients in northwest Ethiopia. Maternal illiteracy, primigravidity, lack of antenatal care follow-up, and history of symptomatic malaria during pregnancy were the risk factors for malaria during parturiency. Thus, promotion of a healthy pregnancy through ANC follow-up, strengthening malaria prevention and control practices, and screening of malaria in asymptomatic pregnant women are suggested to reduce its burden in pregnancy.

Potential hidden Plasmodium vivax malaria reservoirs from low parasitemia Duffy-negative Ethiopians: Molecular evidence.

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.

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

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

Malaria positivity rate trend analysis at water resources development project of Wonji Sugar Estate Oromia, Ethiopia.

Evidence on the trends of the proportion of malaria infections detected by routine passive case detection at health facilities is important for public health decision making especially in areas moving towards elimination. The objective was to assess nine years of trends on clinical malaria infections detected at health facility and its associated climate factors, in the water resource development set up of Wonji sugar estate, Oromia, Ethiopia. Retrospective data were collected from malaria-suspected patient recording logbook at Wonji sugar factory's primary hospital. Monthly average meteorological data were obtained from the estate meteorological station. Data were collected from April through June 2018 and January 2022. The data were analyzed using Stata version 16.0 software for Chi-square and regression analysis. Over the last nine years, 34,388 cases were legible for analysis with complete data. Of these, 11.75% (4039/34388) were positive for clinical malaria. Plasmodium vivax test positivity was the highest proportion (8.2%, n = 2820) followed by Plasmodium falciparum (3.48%, n = 1197) and mixed infections (P. falciparum and P. vivax, 0.06%, n = 21). The odds of being positive for malaria was highest in males (AOR = 1.46; 95%CI = 1.36-1.52; P < 0.001) compared to females and in older individuals of above 15 years old (AOR = 4.55, 95%CI = 4.01-5.17, P < 0.001) followed by school-aged children (5-15 years old) (AOR = 2.16; 95%CI = 1.88-2.49, P < 0.001). There was no significant variation in the proportion of malaria-positive cases in the dry and wet seasons (P = 0.059). Malaria test positivity rates were associated with average monthly rainfall (AdjIRR = 1.00; 95%CI = 1.00-1.001, P < 0.001) while negatively associated with average monthly minim temperature (adjIRR = 0.94; 95%CI = 0.94-0.95; P < 0.001) and average monthly relative humidity (adjIRR = 0.99, 95%CI = 0.99-1.00, P = 0.023). There was year-round malaria transmission, adults especially males and school children frequently tested malaria positive. Hence, alternative vector management tools like larval source management have to be deployed besides ITNs and IRS in such water development areas to achieve the malaria elimination goal.

Resistance of Anopheles stephensi to selected insecticides used for indoor residual spraying and long-lasting insecticidal nets in Ethiopia.

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.

Cutaneous leishmaniasis in Kutaber District, Ethiopia: Prevalence, sand fly fauna and community knowledge, attitude and practices.

Background: Cutaneous leishmaniasis (CL) affects 25% of the population living in the highlands of Ethiopia. CL intervention has not decreased the number of leishmaniasis patients. A cross-sectional study was conducted to determine CL prevalence, community's knowledge, attitude and practices (KAP), and the sand fly fauna in Kutaber district, northeast Ethiopia. Methods: A retrospective, community-based cross-sectional study was conducted in Boru Meda Hospital from December 2014-March 2021 to study CL prevalence of Kutaber district. A Pre-tested, well-structured questionnaire was used to collect data on the participants' socio-demographic characteristics, KAP towards CL and knowledge about sand fly vectors. Chi-square test and logistic regression analysis were used in the study, and data were analyzed using SPSS version 23 (p < 0.05). Results: A total of 10,002 (14.02%), of which 71,325 samples were confirmed as positive for CL. The infection rate of CL in females (7.1%) was a little bit higher than males (7.0%). More cases were recorded among 15-29 age category. The study also revealed that 77.1% of the respondents had poor knowledge about CL treatment, prevention, clinical presentation and disease transmission. Farmers tended to have poorer knowledge about sand flies than non-workers and students (32.7 vs. 35 and 44.1%; P = 0.049). Housewives had poorer knowledge about sand flies than farmers and workers (22.2 vs. 32.7 and 33.3%; P = 0.023). Phlebotomus longipes comprised the highest composition (80%) of the sand fly species identified in Kutaber district. Conclusions: The data showed that the community had poor knowledge about CL, vector, and transmission mode. CL preventive measures were prevalent, implying the need to raise CL awareness. Phlebotomus longipes was identified as the most dominant sand fly species which accounted for CL. The findings can be used in developing an effective control strategy to reduce CL transmission in the study area and elsewhere in Ethiopia.

Sero-prevalence of visceral leishmaniasis and its associated factors among asymptomatic individuals visiting Denan health center, southeastern Ethiopia.

BACKGROUND: In the Somali region of Ethiopia, visceral leishmaniasis (VL) is a public health concern. However, VL epidemiology and sand fly vectors have not been well studied in various areas of the regional state, including Denan district. Therefore, this study was conducted to determine the sero-prevalence, associated factors, and distribution of sand fly vectors of VL in Denan district, south-eastern Ethiopia. METHODS: A facility-based cross-sectional study was conducted from April to September 2021 among VL patients with classic signs and symptoms visiting Denan Health Center in south-eastern Ethiopia. Using a convenience sampling method, 187 blood samples were collected from individuals who visited Denan Health Center during the study period. Blood samples were subjected to Direct Agglutination Test for the detection of antibodies to VL. A pre-tested structured questionnaire was also used to gather information on risk factors and other characteristics of knowledge and attitude assessment. Sand flies were also collected from indoor, peri-domestic, mixed forest, and termite mounds using light and sticky traps to determine the fauna and abundance. RESULTS: The overall sero-prevalence rate was 9.63% (18/187). The sero-prevalence was significantly associated with outdoor sleeping (OR = 2.82), the presence of damp floors (OR = 7.76), and sleeping outdoor near animals (OR = 3.22). Around 53.48% of the study participants had previously heard about VL. Study participants practiced different VL control methods, including bed nets (42%), insecticide spraying (32%), smoking plant parts (14%), and environmental cleaning (8%). In total, 823 sand fly specimens, comprising 12 species in two genera (Phlebotomus and Sergentomyia), were trapped and identified. The most abundant species was Sergentomyia clydei (50.18%), followed by Phlebotomus orientalis (11.42%). Also, a higher proportion of P. orientalis was found in termite mounds (65.43%), followed by mixed forest (37.8%) and peri-domestic (20.83%) habitats. CONCLUSION: The study demonstrated a 9.63% sero-positivity of VL and a remarkable gap in knowledge, attitude, and practices towards VL. P. orientalis was also detected, which could be a probable vector in this area. Thus, public education should be prioritized to improve the community's awareness of VL and its public health impact. In addition, detailed epidemiological and entomological studies are recommended.

Clinical isolates of uncomplicated falciparum malaria from high and low malaria transmission areas show distinct pfcrt and pfmdr1 polymorphisms in western Ethiopia.

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.

Laboratory-based efficacy evaluation of Bacillus thuringiensis var. israelensis and temephos larvicides against larvae of Anopheles stephensi in ethiopia.

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.

Low genetic diversity of Plasmodium falciparum merozoite surface protein 1 and 2 and multiplicity of infections in western Ethiopia following effective malaria interventions.

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.

Insecticidal effects of some selected plant extracts against Anopheles stephensi (Culicidae: Diptera).

BACKGROUND: The use of synthetic insecticides against mosquitoes may lead to resistance development and potential health hazards in humans and the environment. Consequently, a paradigm needs to shift towards the alternative use of botanical insecticides that could strengthen an insecticide resistance management programme. This study aimed to assess the insecticidal effects aqueous, hexane, and methanol crude leaf extracts of Calpurnia aurea, Momordica foetida, and Zehneria scabra on an insectary colony of Anopheles stephensi larvae and adults. METHODS: Fresh leaves of C. aurea, M. foetida and Z. scabra were collected and dried, then separately ground to powder. Powdered leaves of test plants were extracted using sonication with aqueous, hexane, and methanol solvents. The extracts were concentrated, and a stock solution was prepared. For comparison, Temephos (Abate®) and control solutions (a mixture of water and emulsifier) were used as the positive and negative controls, respectively. Different test concentrations for the larvae and the adults were prepared and tested according to WHO (2005) and CDC (2010) guidelines to determine lethal concentration (LC) values. Mortality was observed after 24 h exposure. The statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software (Kruskal-Wallis test) and R software (a generalized linear model was used to determine LC50 and LC90 values of the extracts). RESULTS: The lowest LC50 values were observed in aqueous extracts of M. foetida followed by Z. scabra extract and C. aurea leaves at 34.61, 35.85, and 38.69 ppm, respectively, against the larvae. Larval mortality was not observed from the hexane extracts and negative control, while the standard larvicide (temephos) achieved 100% mortality. Further, the adulticidal efficacy was greatest for aqueous extract of Z. scabra with LC50 = 176.20 ppm followed by aqueous extract of C. aurea (LC50 = 297.75 ppm). CONCLUSION: The results suggest that the leaf extracts of the three test plants have the potential of being used for the control of vector An. stephensi larvae and adult instead of synthetic mosquitocides. Further studies need to be conducted to identify the active ingredients and their mode of action.

Persistence of Residual Submicroscopic P. falciparum Parasitemia following Treatment of Artemether-Lumefantrine in Ethio-Sudan Border, Western Ethiopia.

The emergence of artemisinin-resistant parasites in Africa has had a devastating impact, causing most malaria cases and related deaths reported on the continent. In Ethiopia, artemether-lumefantrine (AL) is the first-line drug for the treatment of uncomplicated falciparum malaria. This study is one of the earliest evaluations of artemether-lumefantrine (AL) efficacy in western Ethiopia, 17 years after the introduction of this drug in the study area. This study aimed at assessing PCR- corrected clinical and parasitological responses at 28 days following AL treatment. Sixty uncomplicated falciparum malaria patients were enrolled, treated with standard doses of AL, and monitored for 28 days with clinical and parasitological assessments from September 15 to December 15, 2020. Microscopy was used for patient recruitment and molecular diagnosis of P. falciparum was performed by Var gene acidic terminal sequence (varATS) real-time PCR on dried blood spots collected from each patient from day 0 and on follow-up days 1, 2, 3, 7, 14, 21, and 28. MspI and msp2 genotyping was done to confirm occurrence of recrudescence. Data entry and analysis were done by using the WHO-designed Excel spreadsheet and SPSS version 20 for Windows. A P value of less or equal to 0.05 was considered significant. From a total of 60 patients enrolled in this efficacy study, 10 were lost to follow-up; the results were analyzed for 50 patients. All the patients were fever-free on day 3. The asexual parasite positivity rate on day 3 was zero. However; 60% of the patients were PCR positive on day 3. PCR positivity on day 3 was more common among patients <15 years old as compared with those ≥15 years old (AOR = 6.44, P = 0.027). Only two patients met the case definition of treatment failure. These patients were classified as a late clinical failure as they showed symptoms of malaria and asexual stages of the parasite detected by microscopy on day 14 of their follow-ups. Hence, the Kaplan-Meier analysis of PCR- corrected adequate clinical and parasitological response (ACPR) rate of AL among study participants was 96% (95% CI: 84.9-99). In seven patients, the residual submicroscopic parasitemia persists from day 0 to day 28 of the follow-up. In addition, 16% (8/50) of patients were PCR- and then turned PCR+ after day 7 of the follow-up. AL remains efficacious for the treatment of uncomplicated falciparum malaria in the study area. However, the persistence of PCR-detected residual submicroscopic parasitemia following AL might compromise this treatment and need careful monitoring.

Intestinal helminthiasis survey with emphasis on schistosomiasis in Koga irrigation scheme environs, northwest Ethiopia.

BACKGROUND: Distribution of schistosomiasis is more focal due to spatial heterogeneities in intermediate host snail dynamics and water contact behavior of humans. This makes the search for new transmission foci of schistosomiasis and its connection with malacologically receptive water bodies essential for effective control of its transmission. This study was intended to assess the prevalence of intestinal helminth infections among schoolchildren and Schistosoma mansoni transmission in Koga irrigation scheme surroundings, northwest Ethiopia. MATERIALS AND METHODS: Cross-sectional parasitological and malacological surveys were conducted in three schools and nearby water bodies, respectively around Koga irrigation scheme. Stool specimens were collected from 421 randomly selected schoolchildren and microscopically examined using Kato-Katz and formol-ether concentration methods. Malacological surveys were carried out and the identified Biomphalaria pfeifferi snails were screened for schistosome infection. Swiss albino mice were exposed to schistosome cercariae shed by Biomphalaria pfeifferi for definite identification of Schistosoma species. RESULTS: Among the examined schoolchildren, 22.6% (95% CI: 18.7%-26.9%) were positive for at least one intestinal helminths species. Ascaris lumbricoides was the most frequent intestinal helminth detected among forty (9.5%) children. Schistosoma mansoni was detected among 4.8% (95% CI: 2.9%-7.2%) of children and its prevalence was significantly higher among male children (p = 0.038) and those attending in Mengesha Jemberie Primary School (p = 0.044). Biomphalaria pfeifferi snails were identified in water bodies in close proximity to Mengesha Jemberie and Wotete Abay Primay schools. Schistosoma mansoni adult worms were harvested after exposure of mice to cercariae shed from Biomphalaria pfeifferi snails collected from water bodies nearby Mengesha Jemberie Primary School. CONCLUSIONS: Schistosoma mansoni infection of schoolchildren, findings of schistosome infected snails and establishment of mice infection confirm that transmission is taking place in the study areas. Hence, snail control and other measures such as provision of sanitary facilities and health education are recommended.