A digital microscope for the diagnosis of Plasmodium falciparum and Plasmodium vivax, including P. falciparum with hrp2/hrp3 deletion.

Sensitive and accurate malaria diagnosis is required for case management to accelerate control efforts. Diagnosis is particularly challenging where multiple Plasmodium species are endemic, and where P. falciparum hrp2/3 deletions are frequent. The Noul miLab is a fully automated portable digital microscope that prepares a blood film from a droplet of blood, followed by staining and detection of parasites by an algorithm. Infected red blood cells are displayed on the screen of the instrument. Time-to-result is approximately 20 minutes, with less than two minutes hands-on time. We evaluated the miLab among 659 suspected malaria patients in Gondar, Ethiopia, where P. falciparum and P. vivax are endemic, and the frequency of hrp2/3 deletions is high, and 991 patients in Ghana, where P. falciparum transmission is intense. Across both countries combined, the sensitivity of the miLab for P. falciparum was 94.3% at densities >200 parasites/µL by qPCR, and 83% at densities >20 parasites/µL. The miLab was more sensitive than local microscopy, and comparable to RDT. In Ethiopia, the miLab diagnosed 51/52 (98.1%) of P. falciparum infections with hrp2 deletion at densities >20 parasites/µL. Specificity of the miLab was 94.0%. For P. vivax diagnosis in Ethiopia, the sensitivity of the miLab was 97.0% at densities >200 parasites/µL (RDT: 76.8%, microscopy: 67.0%), 93.9% at densities >20 parasites/µL, and specificity was 97.6%. In Ethiopia, where P. falciparum and P. vivax were frequent, the miLab assigned the wrong species to 15/195 mono-infections at densities >20 parasites/µL by qPCR, and identified only 5/18 mixed-species infections correctly. In conclusion, the miLab was more sensitive than microscopy and thus is a valuable addition to the toolkit for malaria diagnosis, particularly for areas with high frequencies of hrp2/3 deletions.

Plasmodium falciparum transmission in the highlands of Ethiopia is driven by closely related and clonal parasites.

Malaria cases are frequently recorded in the Ethiopian highlands even at altitudes above 2000 m. The epidemiology of malaria in the Ethiopian highlands, and, in particular, the role of importation by human migration from the highly endemic lowlands is not well understood. We sequenced 187 Plasmodium falciparum samples from two sites in the Ethiopian highlands, Gondar (n = 159) and Ziway (n = 28), using a multiplexed droplet digital PCR (ddPCR)-based amplicon sequencing method targeting 35 microhaplotypes and drug resistance loci. Here, we characterize the parasite population structure and genetic relatedness. We identify moderate parasite diversity (mean HE : 0.54) and low infection complexity (74.9% monoclonal). A significant percentage of infections share microhaplotypes, even across transmission seasons and sites, indicating persistent local transmission. We identify multiple clusters of clonal or near-clonal infections, highlighting high genetic relatedness. Only 6.3% of individuals diagnosed with P. falciparum reported recent travel. Yet, in clonal or near-clonal clusters, infections of travellers were frequently observed first in time, suggesting that parasites may have been imported and then transmitted locally. 31.1% of infections are pfhrp2-deleted and 84.4% pfhrp3-deleted, and 28.7% have pfhrp2/3 double deletions. Parasites with pfhrp2/3 deletions and wild-type parasites are genetically distinct. Mutations associated with resistance to sulphadoxine-pyrimethamine or suggested to reduce sensitivity to lumefantrine are observed at near-fixation. In conclusion, genomic data corroborate local transmission and the importance of intensified control in the Ethiopian highlands.

Highland Malaria Transmission Dynamics in Space and Time Before Pre-elimination Era, Northwest Ethiopia.

BACKGROUND: Currently, the district-level malaria transmission stratification has indicated the Northern, Northwestern, Southern, and rift valley lowland and surrounding highland districts are almost entirely classified as high or moderate malaria transmission zones. Conducting malaria surveillance to track, test, and treat all malaria cases cannot be implemented in Ethiopia in the current situation. OBJECTIVE: To show malaria transmission dynamics in different health facilities located from 1800 to 2772 m altitudes during 2018-2021 in Northwest Ethiopia. METHODS: A total of 3.5 years (2018-2021) retrospective confirmed and treated malaria cases in 43 kebeles health posts and clinics in Gondar Zuria district were used for analysis. RESULT: The total malaria count was 5893 for 2019 compared to 31, 550 for 2020 and 33, 248 for 2021. Mean monthly malaria incidence/1000 people in 2019 was 2.39 ± 5.4 and increased to 10.64 ± 16.99 in 2020 and 11.19 ± 16.59 in 2021. Annual malaria incidence increased from 24 cases/1000 people in 2019 to 139.08 cases/1000 people in 2021 and is alarming danger in malaria elimination program in the district or the country as a whole. Poisson and Negative binomial regressions models indicated 5.78- and 5.26-fold malaria cases increase, respectively, in 2021 compared to 2019. The sudden increase in malaria incidences (counts) in 2020 and 2021 coincided with the interruption of residual insecticide application in Gondar Zuria district during the transition period towards the malaria pre-elimination stage implicating the role of malaria control tools in suppressing transmission. Study on climate variability also indicated that the rainfall variability in different months might have also favored high malaria transmission in 2020 and 2021 compared to 2019. Thus, in addition to re-starting the use of malaria control tools, giving attention to climate anomalies (variability) that favors malaria transmission, for prompt interventional actions, is required. The malaria elimination program in Ethiopia might have not reached a pre-elimination stage as malaria cases per 1000 people have not decreased below five in the majority of Ethiopian districts. Tracing, confirming, and treating individual cases to stop further transmission is, almost, impossible. In a situation like this, the Ethiopian malaria elimination program should work intensively towards understanding malaria epidemiology at the district level to re-design a localized malaria control strategy. The renewed malaria control program should also consider altitudes above 2000 m.

Intestinal Parasitosis and its Association with CD4+ T Cell Count and Viral Load among People Living with HIV in Parasite Endemic Settings of Northwest Ethiopia.

PURPOSE: To study intestinal parasitosis and its association with viral load and CD4+ T cell count in HIV-infected individuals at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia. METHODS: A cross-sectional study was conducted from March to June 2019. Three hundred and sixteen study participants were selected using systematic random sampling technique. Sociodemographic and clinical data were collected using structured questionnaire. Stool samples were collected and examined using direct saline, formol ether concentration technique and modified acid fast staining. CD4+ T cell counts and viral load were determined by fluorescence-activated cell sorting (BD FACS) and COBAS Ampliprep/COBAS TaqMan HI2CAP assay, respectively. Data were entered into Epi Data 3.1 and transferred to SPSS version 20 software for analysis. Bivariable and multivariable analyses were performed using a binary logistic regression model. P values of less than 0.05 were considered statistically significant. RESULTS: The overall prevalence of intestinal parasitosis was 24.7% (78/316). The most commonly detected parasite was Cryptosporidium species with 5.4% (17/316), followed by Ascaris lumbricoides with 5.1% (16/316). There was a significant association with low CD4+ T cell count (AOR: 3.207; 95% CI: 1.237, 8.317), high viral load (AOR: 2.933; 95% CI: 1.326, 6.489), individuals aged 31-40 years (AOR: 0.305; 95% CI: 0.124, 0.751) and individuals aged 41-50 years (AOR: 0.261; 95% CI: 0.101, 0.671). CONCLUSION: In this study, prevalence of intestinal parasitic infections was high and was associated with low CD4+ T cell count and high viral load. Therefore, screening of HIV patients, especially those with low CD4+ T-cell count and high viral load, particularly for opportunistic intestinal parasitic infections would be of utmost importance in the efforts to prevent and control opportunistic infections in HIV patients.

Synergetic antimicrobial effects of mixtures of ethiopian honeys and ginger powder extracts on standard and resistant clinical bacteria isolates.

Purpose. To evaluate antimicrobial effects of mixtures of Ethiopian honeys and ginger rhizome powder extracts on Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Staphylococcus aureus (MRSA), Escherichia coli (R), and Klebsiella pneumoniae (R). Methods. Agar diffusion and broth assays were performed to determine susceptibility of these standard and resistant clinical bacteria isolates using honey-ginger powder extract mixtures. Results. Honey-ginger powder extract mixtures produced the highest mean inhibition (25.62 mm ± 2.55) compared to the use of honeys (21.63 mm ± 3.30) or ginger extracts (19.23 mm ± 3.42) individually. The ranges of inhibitions produced by honey-ginger extract mixtures on susceptible test organisms (26-30 mm) and resistant strains (range: 19-27 mm) were higher compared to 7-22 mm and 0-14 mm by standard antibiotic discs. Minimum inhibitory concentrations (MIC) of mixture of honeys-ginger extracts were 6.25% (0.625 v/mL) on the susceptible bacteria compared to 75% for resistant clinical isolates. Minimum bactericidal concentration (MBC) of honey-ginger extracts was 12.5% (0.125 g/mL) for all the test organisms. Conclusion. The result of this study showed that honey-ginger powder extract mixtures have the potential to serve as cheap source of antibacterial agents especially for the drug resistant bacteria strains.

Antibacterial effects of Apis mellifera and stingless bees honeys on susceptible and resistant strains of Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae in Gondar, Northwest Ethiopia.

BACKGROUND: Honey is a natural substance produced by honeybees and has nutritional and therapeutic uses. In Ethiopia, honeys are used traditionally to treat wounds, respiratory infections and diarrhoea. Recent increase of drug resistant bacteria against the existing antibiotics forced investigators to search for alternative natural remedies and evaluate their potential use on scientific bases. Thus, the aim of this study was to evaluate the antibacterial effects of different types of honeys in Ethiopia which are used traditionally to treat different types of respiratory and gastrointestinal infections. METHODS: Mueller Hinton agar (70191) diffusion and nutrient broth culture medium assays were performed to determine susceptibility of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and resistant clinical isolates (Methicillin resistant Staphylococcus aureus(MRSA), Escherichia coli(R) and Klebsiella pneumoniae (R), using honeys of Apis mellifera and stingless bees in northern and north western Ethiopia. RESULTS: Honey of the stingless bees produced the highest mean inhibition (22.27 ± 3.79 mm) compared to white honey (21.0 ± 2.7 mm) and yellow honey (18.0 ± 2.3 mm) at 50% (v/v) concentration on all the standard and resistant strains. Stingless bees honey was found to have Minimum Inhibitory Concentration (MIC) of 6.25% (6.25 mg/ml) for 80% of the test organisms compared to 40% for white and yellow Apis mellifera honeys. All the honeys were found to have minimum bactericidal concentration (MBC) of 12.5% (12.5 mg/ml) against all the test organisms. Staphylococcus aureus (ATCC 25923) was susceptible to amoxicillin, methicillin, kanamycine, tetracycline, and vancomycine standard antibiotic discs used for susceptibility tests. Similarly, Escherichia coli (ATCC 25922) was found susceptible for kanamycine, tetracycline and vancomycine. Escherichia coli (ATCC 25922) has not been tested for amoxicillin ampicillin and methicillin. The susceptibility tests performed against Staphylococcus aureus (MRSA), Escherichia coli (R) and Klebsiella pneumoniae (R) using three of methicillin, erythromycin, ampicillin, Penicillin and amoxicillin discs were resistant. But, these drug resistant strains were susceptible to antibacterial agents found in the honeys and inhibited from 16 mm to 20.33 mm. CONCLUSIONS: Honeys in Ethiopia can be used as therapeutic agents for drug resistant bacteria after pharmaceutical standardization and clinical trials.