Primaquine radical cure in patients with Plasmodium falciparum malaria in areas co-endemic for P falciparum and Plasmodium vivax (PRIMA): a multicentre, open-label, superiority randomised controlled trial.

BACKGROUND: In areas co-endemic for Plasmodium vivax and Plasmodium falciparum there is an increased risk of P vivax parasitaemia following P falciparum malaria. Radical cure is currently only recommended for patients presenting with P vivax malaria. Expanding the indication for radical cure to patients presenting with P falciparum malaria could reduce their risk of subsequent P vivax parasitaemia. METHODS: We did a multicentre, open-label, superiority randomised controlled trial in five health clinics in Bangladesh, Indonesia, and Ethiopia. In Bangladesh and Indonesia, patients were excluded if they were younger than 1 year, whereas in Ethiopia patients were excluded if they were younger than 18 years. Patients with uncomplicated P falciparum monoinfection who had fever or a history of fever in the 48 h preceding clinic visit were eligible for enrolment and were required to have a glucose-6-dehydrogenase (G6PD) activity of 70% or greater. Patients received blood schizontocidal treatment (artemether-lumefantrine in Ethiopia and Bangladesh and dihydroartemisinin-piperaquine in Indonesia) and were randomly assigned (1:1) to receive either high-dose short-course oral primaquine (intervention arm; total dose 7 mg/kg over 7 days) or standard care (standard care arm; single dose oral primaquine of 0·25 mg/kg). Random assignment was done by an independent statistician in blocks of eight by use of sealed envelopes. All randomly assigned and eligible patients were included in the primary and safety analyses. The per-protocol analysis excluded those who did not complete treatment or had substantial protocol violations. The primary endpoint was the incidence risk of P vivax parasitaemia on day 63. This trial is registered at ClinicalTrials.gov, NCT03916003. FINDINGS: Between Aug 18, 2019, and March 14, 2022, a total of 500 patients were enrolled and randomly assigned, and 495 eligible patients were included in the intention-to-treat analysis (246 intervention and 249 control). The incidence risk of P vivax parasitaemia at day 63 was 11·0% (95% CI 7·5-15·9) in the standard care arm compared with 2·5% (1·0-5·9) in the intervention arm (hazard ratio 0·20, 95% CI 0·08-0·51; p=0·0009). The effect size differed with blood schizontocidal treatment and site. Routine symptom reporting on day 2 and day 7 were similar between groups. In the first 42 days, there were a total of four primaquine-related adverse events reported in the standard care arm and 26 in the intervention arm; 132 (92%) of all 143 adverse events were mild. There were two serious adverse events in the intervention arm, which were considered unrelated to the study drug. None of the patients developed severe anaemia (defined as haemoglobin <5 g/dL). INTERPRETATION: In patients with a G6PD activity of 70% or greater, high-dose short-course primaquine was safe and relatively well tolerated and reduced the risk of subsequent P vivax parasitaemia within 63 days by five fold. Universal radical cure therefore potentially offers substantial clinical, public health, and operational benefits, but these benefits will vary with endemic setting. FUNDING: Australian Academy of Science Regional Collaborations Program, Bill & Melinda Gates Foundation, and National Health and Medical Research Council.

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

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

Weekly primaquine for radical cure of patients with Plasmodium vivax malaria and glucose-6-phosphate dehydrogenase deficiency.

BACKGROUND: The World Health Organization recommends that primaquine should be given once weekly for 8-weeks to patients with Plasmodium vivax malaria and glucose-6-phosphate dehydrogenase (G6PD) deficiency, but data on its antirelapse efficacy and safety are limited. METHODS: Within the context of a multicentre, randomised clinical trial of two primaquine regimens in P. vivax malaria, patients with G6PD deficiency were excluded and enrolled into a separate 12-month observational study. They were treated with a weekly dose of 0.75 mg/kg primaquine for 8 weeks (PQ8W) plus dihydroartemisinin piperaquine (Indonesia) or chloroquine (Afghanistan, Ethiopia, Vietnam). G6PD status was diagnosed using the fluorescent spot test and confirmed by genotyping for locally prevalent G6PD variants. The risk of P. vivax recurrence following PQ8W and the consequent haematological recovery were characterized in all patients and in patients with genotypically confirmed G6PD variants, and compared with the patients enrolled in the main randomised control trial. RESULTS: Between July 2014 and November 2017, 42 male and 8 female patients were enrolled in Afghanistan (6), Ethiopia (5), Indonesia (19), and Vietnam (20). G6PD deficiency was confirmed by genotyping in 31 patients: Viangchan (14), Mediterranean (4), 357A-G (3), Canton (2), Kaiping (2), and one each for A-, Chatham, Gaohe, Ludhiana, Orissa, and Vanua Lava. Two patients had recurrent P. vivax parasitaemia (days 68 and 207). The overall 12-month cumulative risk of recurrent P. vivax malaria was 5.1% (95% CI: 1.3-18.9) and the incidence rate of recurrence was 46.8 per 1000 person-years (95% CI: 11.7-187.1). The risk of P. vivax recurrence was lower in G6PD deficient patients treated with PQ8W compared to G6PD normal patients in all treatment arms of the randomised controlled trial. Two of the 26 confirmed hemizygous males had a significant fall in haemoglobin (>5g/dl) after the first dose but were able to complete their 8 week regimen. CONCLUSIONS: PQ8W was highly effective in preventing P. vivax recurrences. Whilst PQ8W was well tolerated in most patients across a range of different G6PD variants, significant falls in haemoglobin may occur after the first dose and require clinical monitoring. TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov (NCT01814683).

El Niño and other climatic drivers of epidemic malaria in Ethiopia: new tools for national health adaptation plans.

BACKGROUND: Ethiopia has a history of climate related malaria epidemics. An improved understanding of malaria-climate interactions is needed to inform malaria control and national adaptation plans. METHODS: Malaria-climate associations in Ethiopia were assessed using (a) monthly climate data (1981-2016) from the Ethiopian National Meteorological Agency (NMA), (b) sea surface temperatures (SSTs) from the eastern Pacific, Indian Ocean and Tropical Atlantic and (c) historical malaria epidemic information obtained from the literature. Data analysed spanned 1950-2016. Individual analyses were undertaken over relevant time periods. The impact of the El Niño Southern Oscillation (ENSO) on seasonal and spatial patterns of rainfall and minimum temperature (Tmin) and maximum temperature (Tmax) was explored using NMA online Maprooms. The relationship of historic malaria epidemics (local or widespread) and concurrent ENSO phases (El Niño, Neutral, La Niña) and climate conditions (including drought) was explored in various ways. The relationships between SSTs (ENSO, Indian Ocean Dipole and Tropical Atlantic), rainfall, Tmin, Tmax and malaria epidemics in Amhara region were also explored. RESULTS: El Niño events are strongly related to higher Tmax across the country, drought in north-west Ethiopia during the July-August-September (JAS) rainy season and unusually heavy rain in the semi-arid south-east during the October-November-December (OND) season. La Niña conditions approximate the reverse. At the national level malaria epidemics mostly occur following the JAS rainy season and widespread epidemics are commonly associated with El Niño events when Tmax is high, and drought is common. In the Amhara region, malaria epidemics were not associated with ENSO, but with warm Tropical Atlantic SSTs and higher rainfall. CONCLUSION: Malaria-climate relationships in Ethiopia are complex, unravelling them requires good climate and malaria data (as well as data on potential confounders) and an understanding of the regional and local climate system. The development of climate informed early warning systems must, therefore, target a specific region and season when predictability is high and where the climate drivers of malaria are sufficiently well understood. An El Niño event is likely in the coming years. Warming temperatures, political instability in some regions, and declining investments from international donors, implies an increasing risk of climate-related malaria epidemics.

Durability of long-lasting insecticidal nets (LLINs) in Ethiopia.

BACKGROUND: The functional survival time of long-lasting insecticidal nets (LLINs), which varies across different field contexts, is critical for the successful prevention of malaria transmission. However, there is limited data on LLIN durability in field settings in Ethiopia. METHODS: A three-year longitudinal study was conducted to monitor attrition, physical integrity, and bio-efficacy and residual chemical concentration of LLINs in four regions in Ethiopia. World Health Organization (WHO) guidelines were used to determine sample size, measure physical integrity, and calculate attrition rates, and functional survival time. Yearly bio-efficacy testing was done on randomly selected LLINs. An excel tool developed by vector works project was used to calculate the median functional survival time of the LLINs. Predictors of functional survival were identified by fitting binary and multivariate cox proportional hazards model. RESULTS: A total of 3,396 LLINs were included in the analysis. A total of 3,396 LLINs were included in the analysis. By the end of 36 months, the proportion of LLINs functionally surviving was 12.9% [95% confidence interval (CI) 10.5, 15.6], the rates of attrition due to physical damage and repurposing were 48.8% [95% confidence interval (CI) 45.0, 52.6] and 13.8% [95% confidence interval (CI) 11.6, 14.6], respectively. The estimated median functional survival time was 19 months (95%CI 17, 21). Factors associated with shorter functional survival time include being in a low malaria transmission setting [Adjusted Hazards Ratio (AHR) (95%CI) 1.77 (1.22, 2.55)], rural locations [AHR (95%CI) 1.83 (1.17, 2.84)], and in a room where cooking occurs [AHR (95%CI) 1.28 (1.05, 1.55)]. Bioassay tests revealed that 95.3% (95%CI 86.4, 98.5) of the LLINs met the WHO criteria of bio-efficacy after 24 months of distribution. CONCLUSION: The LLIN survival time was shorter than the expected three years due to high attrition rates and rapid loss of physical integrity. National malaria programmes may consider, procuring more durable LLINs, educating communities on how to prevent damage of LLINs, and revising the current three-year LLIN distribution schedule to ensure sufficient protection is provided by LLINs against malaria transmission. While this paper contributes to the understanding of determinants impacting functional survival, further research is needed to understand factors for the rapid attrition rates and loss of physical integrity of LLINs in field settings.

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

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

Epidemiological, Entomological, and Climatological Investigation of the 2019 Dengue Fever Outbreak in Gewane District, Afar Region, North-East Ethiopia.

Dengue Fever (DF) is an important arthropod-borne viral infection that has repeatedly occurred as outbreaks in eastern and northeastern Ethiopia since 2013. A cross-sectional epidemiological outbreak investigation was carried out from September to November 2019 on febrile patients (confirmed malaria negative) who presented with suspected and confirmed DF at both public and private health facilities in Gewane District, Afar Region, northeastern Ethiopia. Entomological investigation of containers found in randomly selected houses belonging to DF-positive patients was undertaken to survey for the presence of Aedes larvae/pupae. A total of 1185 DF cases were recorded from six health facilities during the 3-month study period. The mean age of DF cases was 27.2 years, and 42.7% of cases were female. The most affected age group was 15−49 years old (78.98%). The total case proportions differed significantly across age groups when compared to the population distribution; there were approximately 15% and 5% higher case proportions among those aged 15−49 years and 49+ years, respectively. A total of 162 artificial containers were inspected from 62 houses, with 49.4% found positive for Aedes aegypti larva/pupae. Aedes mosquitoes were most commonly observed breeding in plastic tanks, tires, and plastic or metal buckets/bowls. World Health Organization entomological indices classified the study site as high risk for dengue virus outbreaks (House Index = 45.2%, Container Index = 49.4%, and Breteau Index = 129). Time series climate data, specifically rainfall, were found to be significantly predictive of AR (p = 0.035). Study findings highlight the importance of vector control to prevent future DF outbreaks in the region. The scarcity of drinking water and microclimatic conditions may have also contributed to the occurrence of this outbreak.

Reducing the risk of Plasmodium vivax after falciparum infections in co-endemic areas-a randomized controlled trial (PRIMA).

BACKGROUND: Plasmodium vivax forms dormant liver stages that can reactivate weeks or months following an acute infection. Recurrent infections are often associated with a febrile illness and can cause a cumulative risk of severe anaemia, direct and indirect mortality, and onward transmission of the parasite. There is an increased risk of P. vivax parasitaemia following falciparum malaria suggesting a rationale for universal use of radically curative treatment in patients with P. falciparum malaria even in the absence of detectable P. vivax parasitaemia in areas that are co-endemic for both species. METHODS: This is a multicentre, health care facility-based, randomized, controlled, open-label trial in Bangladesh, Indonesia and Ethiopia. Patients with uncomplicated falciparum malaria, G6PD activity of ≥70% of the adjusted male median (AMM) and haemoglobin levels ≥8g/dl are recruited into the study and randomized to either receive standard schizonticidal treatment plus 7-day high dose primaquine (total dose 7mg/kg) or standard care in a 1:1 ratio. Patients are followed up weekly until day 63. The primary endpoint is the incidence risk of any P. vivax parasitemia on day 63. Secondary endpoints include incidence risk on day 63 of symptomatic P. vivax malaria and the risk of any P. falciparum parasitaemia. Secondary safety outcomes include the proportion of adverse events and serious adverse events, the incidence risk of severe anaemia (Hb<5g/dl and <7g/dl) and/or the risk for blood transfusion, the incidence risk of ≥ 25% fall in haemoglobin with and without haemoglobinuria, and the incidence risk of ≥ 25% fall in haemoglobin to under 7g/dl with and without haemoglobinuria. DISCUSSION: This study evaluates the potential benefit of a universal radical cure for both P. vivax and P. falciparum in different endemic locations. If found safe and effective universal radical cure could represent a cost-effective approach to clear otherwise unrecognised P. vivax infections and hence accelerate P. vivax elimination. TRIAL REGISTRATION: NCT03916003 . Registered on 12 April 2019.

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

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

The effect of long-lasting insecticidal nets (LLINs) physical integrity on utilization.

BACKGROUND: In Ethiopia, despite improvements in coverage and access, utilization of long-lasting insecticidal nets (LLINs) remains a challenge. Different household-level factors have been identified as associated with LLIN use. However, the contribution of LLIN physical integrity to their utilization is not well investigated and documented. This study aimed to assess the association between the physical integrity of LLINs and their use. METHODS: This study employed a nested case-control design using secondary data from the Ethiopian LLIN durability monitoring study conducted from May 2015 to June 2018. LLINs not used the night before the survey were identified as cases, while those used the previous night were categorized as controls. The physical integrity of LLINs was classified as no holes, good, acceptable, and torn using the proportionate hole index (pHI). A Generalized Estimating Equation (GEE) model was used to assess and quantify the association between LLIN physical integrity and use. The model specifications included binomial probabilistic distribution, logit link, exchangeable correlation matrix structure, and robust standard errors. The factors included in the model were selected first by fitting binary regression, and then by including all factors that showed statistical significance at P-value less than 0.25 and conceptually relevant variables into the multivariate regression model. RESULTS: A total of 5277 observations fulfilled the inclusion criteria. Out of these 1767 observations were cases while the remaining 3510 were controls. LLINs that were in torn physical condition had higher odds (AOR [95% CI] = 1.76 [1.41, 2.19]) of not being used compared to LLINs with no holes. Other factors that showed significant association included the age of the LLIN, sleeping place type, washing status of LLINs, perceptions towards net care and repair, LLIN to people ratio, economic status, and study site. CONCLUSION AND RECOMMENDATION: LLINs that have some level of physical damage have a relatively higher likelihood of not being used. Community members need to be educated about proper care and prevention of LLIN damage to delay the development of holes as long as possible and use available LLINs regularly.

Baseline malaria prevalence at the targeted pre-elimination districts in Ethiopia.

BACKGROUND: Encouraged by the previous success in malaria control and prevention strategies, the Ethiopian ministry of health launched malaria elimination with a stepwise approach by primarily targeting the low-transmission Districts and their adjacent areas/zones in order to shrink the country's malaria map progressively. Hence, this community survey was conducted to establish baseline malaria information at the preliminary phase of elimination at targeted settings. METHODS: A community-based cross-sectional survey was conducted at 20 malaria-elimination targeted Districts selected from five Regional states and one city administration in Ethiopia. The GPS-enabled smartphones programmed with Open Data Kit were used to enumerate 9326 study households and collect data from 29,993 residents. CareStart™ Malaria PAN (pLDH) Rapid Diagnostic Tests (RDTs) were used for blood testing at the field level. Armpit digital thermometers were used to measure axillary temperature. RESULT: Overall malaria prevalence by RDTs was 1.17% (339/28973). The prevalence at District levels ranged from 0.0 to 4.7%. The proportion of symptomatic cases (axillary temperature > 37.5oc) in the survey was 9.2% (2760/29993). Among the 2510 symptomatic individuals tested with RDTs, only 3.35% (84/2510) were malaria positive. The 75.2% (255/339) of all malaria positives were asymptomatic. Of the total asymptomatic malaria cases, 10.2% (26/255) were under-five children and 89.8% (229/255) were above 5 years of age. CONCLUSION: The study shows a decrease in malaria prevalence compared to the reports of previous malaria indicator surveys in the country. The finding can be used as a baseline for measuring the achievement of ongoing malaria elimination efforts. Particularly, the high prevalence of asymptomatic individuals (0.88%) in these transmission settings indicates there may be sustaining hidden transmission. Therefore, active case detection with more sensitive diagnostic techniques is suggested to know more real magnitude of residual malaria in the elimination-targeted areas.

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

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

Establishment of COVID-19 testing laboratory in resource-limited settings: challenges and prospects reported from Ethiopia.

The Coronavirus pandemic is recording unprecedented deaths worldwide. The temporal distribution and burden of the disease varies from setting to setting based on economic status, demography and geographic location. A rapid increase in the number of COVID-19 cases is being reported in Africa as of June 2020. Ethiopia reported the first COVID-19 case on 13 March 2020. Limited molecular laboratory capacity in resource constrained settings is a challenge in the diagnosis of the ever-increasing cases and the overall management of the disease. In this article, the Ethiopian Public Health Institute (EPHI) shares the experience, challenges and prospects in the rapid establishment of one of its COVID-19 testing laboratories from available resources. The first steps in establishing the COVID-19 molecular testing laboratory were i) identifying a suitable space ii) renovating it and iii) mobilizing materials including consumables, mainly from the Malaria and Neglected Tropical Diseases (NTDs) research team at the EPHI. A chain of experimental design was set up with distinct laboratories to standardize the extraction of samples, preparation of the master mix and detection. At the commencement of sample reception and testing, laboratory contamination was among the primary challenges faced. The source of the contamination was identified in the master mix room and resolved. In summary, the established COVID-19 testing lab has tested more than 40,000 samples (August 2020) and is the preferred setting for research and training. The lessons learned may benefit the further establishment of emergency testing laboratories for COVID-19 and/or other epidemic/pandemic diseases in resource-limited settings.

National mapping of soil-transmitted helminth and schistosome infections in Ethiopia.

BACKGROUND: An accurate understanding of the geographical distributions of both soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, and the hookworms Necator americanus and Ancylostoma duodenale) and schistosomes (SCH; Schistosoma mansoni and S. haematobium) is pivotal to be able to effectively design and implement mass drug administration (MDA) programmes. The objective of this study was to provide up-to-date data on the distribution of both STH and SCH in Ethiopia to inform the design of the national control program and to be able to efficiently achieve the 75% MDA coverage target set by the WHO. METHODS: Between 2013 and 2015, we assessed the distributions of STH and SCH infections in a nationwide survey covering 153,238 school-aged children (aged 5-15 years), from 625 woredas (districts), representing all nine Regional States and two City Administrations of Ethiopia. Nationwide disease maps were developed at the woreda level to enable recommendations on the design of the national MDA programme. RESULTS: The prevalence of any STH infection across the study population was 21.7%, with A. lumbricoides (12.8%) being the most prevalent STH, followed by hookworms (7.6%) and T. trichiura (5.9%). The prevalence for any SCH was 4.0% in areas where both SCH species were evaluated. Schistosoma mansoni was the most prevalent SCH (3.5 vs 0.3%). STHs were more prevalent in southwest Ethiopia, whereas SCH was found mostly in the west and northeast of the country. The prevalence of moderate-to-heavy intensity infections was 2.0% for STHs and 1.6% for SCH. For STH, a total of 251 woredas were classified as moderately (n = 178) or highly endemic (n = 73), and therefore qualify for an annual and biannual MDA program, respectively. For SCH, 67 woredas were classified as endemic and 8 as highly endemic, and hence they require every two years and annual MDA programme, respectively. CONCLUSIONS: The results confirm that Ethiopia is endemic for both STHs and SCH, posing a significant public health problem. Following the WHO recommendations on mass drug administration, 18 and 14 million school-aged children are in need of MDA for STHs and SCH, respectively, based on the number of SACs that live on the eligible geographical areas.

Malaria infection clustered into small residential areas in lowlands of southern Ethiopia.

Malaria is a complex disease and its distribution is not random in endemic areas, and hence areas with low malaria transmission require fine spatial sampling and careful follow-up to identify the hot spots for effective resource utilization to control malaria. The present study is aimed to assess malaria infection in both humans and mosquitoes in a small residential lowland area of southern Ethiopia from July to December 2016. A repeated cross-sectional household survey was conducted in Kolla-Shara Kebele (village) to describe the distribution of malaria and infectious mosquitoes. For the parasitological surveys, a total of 90 households were randomly selected from five sub-villages in equal proportion. About a quarter of the total households included for the surveys were randomly selected for entomological surveys. A P-value of <0.05 was used as a cut-off point for statistical significance. More than a third (35.1%, 46 of 131) febrile cases were microscopically confirmed malaria positive. Above half (58.7%, 27 of 46) of those positive cases were due to P. falciparum and the rest (41.3%, 19 of 46) were due to P. vivax. This study identified two of the five sub-villages as independent clusters with higher risk of malaria infection. Four times higher relative risk (RR) of malaria infection was documented in Abullo sub-village compared to the others (RR = 3.87; P = 0.002). Most of the falciparum malaria cases were aggregated in these sub-villages. About six infectious bites of An. arabiensis per person was recorded during the survey. The infectious bite per person was 17.0 in Abullo and 10.6 in Erze clusters where higher human infections were detected. It is clearly indicated that a smaller portion of the population carry higher malaria cases and infectious bites. Malaria interventions targeting such areas could be effective in the context of malaria elimination strategy in Ethiopia, which consider district as a planning and implementing unit. Future research would preferably be designed to perform long duration of follow-up to identify the appropriate period for interventions and more participants with more heterogeneous villages and districts.

Case Report: Primaquine Failure for Radical Cure of Plasmodium vivax Malaria in Gambella, Ethiopia.

Failures of primaquine for the treatment of relapsed Plasmodium vivax malaria is a serious challenge to malaria elimination in Ethiopia, where P. vivax accounts for up to 40% of malaria infections. We report here occurrence of a total of 15 episodes of primaquine treatment failure for radical cure in three historical P. vivax malaria patients from Gambella, Ethiopia, during 8-16 months of follow-up in 1985-1987. The total primaquine doses received were 17.5 mg/kg, 25.8 mg/kg, and 35.8 mg/kg, respectively. These total doses are much higher than in previous reports of patients with treatment failure in Ethiopia and East Africa. The possibility of new infection was excluded for these cases as the treatment and follow-up were carried out in Addis Ababa, a malaria-free city. Recrudescences were unlikely, considering the short duration pattern of the recurrences. The cytochrome P450 2D6 (CYP2D6) status for these patients is unknown, but polymorphisms have been described in Ethiopia and may have contributed to primaquine treatment failures. It is suggested that further studies be carried out in Ethiopia to determine the prevalence and distribution of primaquine treatment failures in different ethnic groups, considering the impact of CYP2D6 polymorphisms and the potential value of increasing the primaquine dose to avoid relapse.

The effect of ivermectin® on fertility, fecundity and mortality of Anopheles arabiensis fed on treated men in Ethiopia.

BACKGROUND: Insecticide resistance is a growing threat to malaria vector control. Ivermectin, either administered to humans or animals, may represent an alternate strategy to reduce resistant mosquito populations. The aim of this study was to assess the residual or delayed effect of administering a single oral dose of ivermectin to humans on the survival, fecundity and fertility of Anopheles arabiensis in Ethiopia. METHODS: Six male volunteers aged 25-40 years (weight range 64-72 kg) were recruited; four of them received a recommended single oral dose of 12 mg ivermectin and the other two individuals were untreated controls. A fully susceptible insectary colony of An. arabiensis was fed on treated and control participants at 1, 4, 7, 10 and 13 days post ivermectin-administration. Daily mosquito mortality was recorded for 5 days. An. arabiensis fecundity and fertility were measured from day 7 post treatment, by dissection to examine the number of eggs per mosquito, and by observing larval hatching rates, respectively. RESULTS: Ivermectin treatment induced significantly higher An. arabiensis mortality on days 1 and 4, compared to untreated controls (p = 0.02 and p < 0.001, respectively). However, this effect had declined by day 7, with no significant difference in mortality between treated and control groups (p = 0.06). The mean survival time of mosquitoes fed on day 1 was 2.1 days, while those fed on day 4 survived 4.0 days. Mosquitoes fed on the treatment group at day 7 and 10 produced significantly lower numbers of eggs compared to the untreated controls (p < 0.001 and p = 0.04, respectively). An. arabiensis fed on day 7 on treated men also had lower larval hatching rates than mosquitoes fed on days 10 and 13 (p = 0.003 and p = 0.001, respectively). CONCLUSION: A single oral dose of ivermectin given to humans can induce mortality and reduce survivorship of An. arabiensis for 7 days after treatment. Ivermectin also had a delayed effect on fecundity of An. arabiensis that took bloodmeals from treated individuals on day 7 and 10. Additional studies are warranted using wild, insecticide-resistant mosquito populations, to confirm findings and a phase III evaluation among community members in Ethiopia is needed to determine the impact of ivermectin on malaria transmission.

Pooled Deep Sequencing of Drug Resistance Loci from Plasmodium falciparum Parasites across Ethiopia.

Although Ethiopia has an overall lower prevalence of Plasmodium falciparum among countries in Africa, the emergence of drug resistance could seriously hinder elimination efforts. Using samples collected from five therapeutic efficacy studies conducted in 2007-11, we evaluated the prevalence of putative drug resistance mutations in the pfcrt, pfmdr1, and kelch13 genes at the time of those studies, as well as the ama1 gene for genetic relatedness using a pooled amplicon deep sequencing approach. Among all sites, the kelch13 gene showed no mutations, whereas the pfcrt CVIET genotype was fixed in all populations. By contrast, the mdr1 gene demonstrated frequencies of resistant genotypes ranging from 10 to 100% at amino acid position 86 and from 0% to 57.8% at amino acid position 1246. Although we observed a low degree of haplotype sharing between sites, we did observe considerable haplotype sharing within sites over time. This suggests that P. falciparum populations in Ethiopia are isolated and able to persist through time.

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.