Prevalence of G6PD deficiency and distribution of its genetic variants among malaria-suspected patients visiting Metehara health centre, Eastern Ethiopia.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) is cytosolic enzyme, which has a vital role for the integrity and functioning of red blood cells. Lower activity of this enzyme leads to the occurrence of acute haemolytic anaemia after exposure to oxidative stressors like primaquine. Primaquine is an important drug for the radical cure of Plasmodium vivax and blocking transmission of Plasmodium falciparum, and thereby enhancing malaria elimination. However, there is a need to identify G6PD deficient individuals and administer the drug with caution due to its haemolytic side effects. The main objective of this study is to determine the prevalence of G6PD deficiency among malaria-suspected individuals. METHODS: A facility-based cross-sectional study was conducted from September 2020 to September 2021 in Metehara Health Centre, Eastern Ethiopia. A structured questionnaire was used to collect the socio-demographic and clinical information of the study participants. Capillary and venous blood samples were collected based on standard procedures for onsite screening, dried blood spot preparation, and malaria microscopy. The G6PD enzyme activity was measured by careSTART™ G6PD biosensor analyzer. Data was entered and analysed by SPSS. RESULTS: A total of 498 study participants were included in the study, of which 62% (309) were males. The overall prevalence of G6PD deficiency based on the biosensor screening was 3.6% (18/498), of which 2.9% and 4.8% were males and females, respectively. Eleven of the G6PD deficient samples had mutations confirmed by G6PD gene sequencing analysis. Mutations were detected in G267 + 119C/T, A376T, and ChrX:154535443. A significant association was found in sex and history of previous malaria infection with G6PD deficiency. CONCLUSIONS: The study showed that the G6PD deficient phenotype exists in Metehara even if the prevalence is not very high. G267 + 119C/T mutation is the predominant G6PD variant in this area. Therefore, malaria patient treatment using primaquine should be monitored closely for any adverse effects.

Prevalence and Epidemiological Characteristics of Asymptomatic Malaria Based on Ultrasensitive Diagnostics: A Cross-sectional Study.

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

Plasmodium falciparum resistant to artemisinin and diagnostics have emerged in Ethiopia.

Diagnosis and treatment of Plasmodium falciparum infections are required for effective malaria control and are pre-requisites for malaria elimination efforts; hence we need to monitor emergence, evolution and spread of drug- and diagnostics-resistant parasites. We deep sequenced key drug-resistance mutations and 1,832 SNPs in the parasite genomes of 609 malaria cases collected during a diagnostic-resistance surveillance study in Ethiopia. We found that 8.0% (95% CI 7.0-9.0) of malaria cases were caused by P. falciparum carrying the candidate artemisinin partial-resistance kelch13 (K13) 622I mutation, which was less common in diagnostic-resistant parasites mediated by histidine-rich proteins 2 and 3 (pfhrp2/3) deletions than in wild-type parasites (P = 0.03). Identity-by-descent analyses showed that K13 622I parasites were significantly more related to each other than to wild type (P < 0.001), consistent with recent expansion and spread of this mutation. Pfhrp2/3-deleted parasites were also highly related, with evidence of clonal transmissions at the district level. Of concern, 8.2% of K13 622I parasites also carried the pfhrp2/3 deletions. Close monitoring of the spread of combined drug- and diagnostic-resistant parasites is needed.

Plasmodium falciparum is evolving to escape malaria rapid diagnostic tests in Ethiopia.

In Africa, most rapid diagnostic tests (RDTs) for falciparum malaria recognize histidine-rich protein 2 antigen. Plasmodium falciparum parasites lacking histidine-rich protein 2 (pfhrp2) and 3 (pfhrp3) genes escape detection by these RDTs, but it is not known whether these deletions confer sufficient selective advantage to drive rapid population expansion. By studying blood samples from a cohort of 12,572 participants enroled in a prospective, cross-sectional survey along Ethiopia's borders with Eritrea, Sudan and South Sudan using RDTs, PCR, an ultrasensitive bead-based immunoassay for antigen detection and next-generation sequencing, we estimate that histidine-rich protein 2-based RDTs would miss 9.7% (95% confidence interval 8.5-11.1) of P. falciparum malaria cases owing to pfhrp2 deletion. We applied a molecular inversion probe-targeted deep sequencing approach to identify distinct subtelomeric deletion patterns and well-established pfhrp3 deletions and to uncover recent expansion of a singular pfhrp2 deletion in all regions sampled. We propose a model in which pfhrp3 deletions have arisen independently multiple times, followed by strong positive selection for pfhrp2 deletion owing to RDT-based test-and-treatment. Existing diagnostic strategies need to be urgently reconsidered in Ethiopia, and improved surveillance for pfhrp2 deletion is needed throughout the Horn of Africa.

The Galabat-Metema cross-border onchocerciasis focus: The first coordinated interruption of onchocerciasis transmission in Africa.

BACKGROUND: Onchocerciasis transmission across international borders is not uncommon, yet a coordinated cross border stops mass drug administration (MDA) decision has not been documented. METHODS/PRINCIPLE FINDINGS: The Galabat-Metema focus involves neighboring districts on the border between Sudan and Ethiopia. Mass drug administration (MDA) was provided once and subsequently twice per year in this focus, with twice-per-year beginning in Ethiopia's Metema subfocus in 2016 and in the Sudan's Galabat subfocus in 2008. Ov16 ELISA-based serosurveys were conducted in 6072 children under 10 years of age in the Metema subfocus in 2014, and 3931 in the Galabat in 2015. Between 2014 and 2016, a total of 27,583 vector Simulium damnosum flies from Metema and 9,148 flies from Galabat were tested by pool screen PCR for Onchocerca volvulus O-150 DNA. Only 8 children were Ov16 seropositive (all in the Metema subfocus); all were negative by skin snip PCR. The upper limit of the 95% confidence interval (UCL) for Ov16 seropositive was <0.1% for the overall focus and 0.14 positive fly heads per 2000 (UCL = 0.39/2000). However, an entomological 'hotspot' was detected on the Wudi Gemzu river in Metema district. The hotspot was confirmed when 4 more positive fly pools were found on repeat testing in 2017 (1.04 L3/2000 flies (UCL = 2.26/2000). Information exchange between the two countries led to stopping MDA in a coordinated fashion in 2018, with the exception of the hotspot at Wudi Gemzu, where MDA with ivermectin was increased to every three months to hasten interruption of transmission. CONCLUSION: Coordinated stop MDA decisions were made by Sudan and Ethiopia based on data satisfying the World Health Organization's criteria for interruption of onchocerciasis transmission. Definitions of entomological 'hotspots' and buffer zones around the focus are proposed.

Evaluation of an OV-16 IgG4 Enzyme-Linked Immunosorbent Assay in Humans and Its Application to Determine the Dynamics of Antibody Responses in a Non-Human Primate Model of Onchocerca volvulus Infection.

Onchocerciasis is a neglected parasitic disease targeted for elimination. Current World Health Organization guidelines for elimination include monitoring antibody responses to the recombinant Onchocerca volvulus antigen OV-16 in children to demonstrate the absence of transmission. We report the performance characteristics of a modified OV-16 enzyme-linked immunosorbent assay (ELISA) and describe anti-OV-16 responses in serum samples from laboratory-inoculated nonhuman primates (NHPs) in relation to microfilariae (mf) in skin snip biopsies. This OV-16 IgG4 ELISA had sensitivity and specificity of 88.2% and 99.7%, respectively, as determined by receiver operator characteristic analysis using a serum panel of 110 positive and 287 negative samples from people infected with other filariae or other parasitic infections. Anti-OV-16 responses in inoculated NHP (N = 9) were evaluated at quarterly intervals for IgM and the four IgG subclasses. Enzyme-linked immunosorbent assay results showed a well-defined IgG4 reactivity pattern and moderate IgG1 antibody responses. Meanwhile, the reactivity by IgG2, IgG3, or IgM did not show a clear pattern. Temporal evolution of IgG4 reactivity was evaluated through monthly testing, showing that NHPs developed anti-OV-16 IgG4 on average at 15 months postinoculation (range: 10-18 months). The average time to detectable mf was also 15 months (range: 11-25). The OV-16 ELISA used in this study was robust and allowed the detection of IgG4 responses, which were observed only among animals with detectable mf (N = 5), four of which showed declines in antibody responses once mf cleared. These findings also confirmed that the most informative antibody subclass responses to OV-16 are IgG4.

Characterizing Reactivity to Onchocerca volvulus Antigens in Multiplex Bead Assays.

Multiplex bead assays (MBAs) may provide a powerful integrated tool for monitoring, evaluation, and post-elimination surveillance of onchocerciasis and co-endemic diseases; however, the specificity and sensitivity of Onchocerca volvulus antigens have not been characterized within this context. An MBA was developed to evaluate three antigens (OV-16, OV-17, and OV-33) for onchocerciasis. Receiver operating characteristics (ROC) analyses were used to characterize antigen performance using a panel of 610 specimens: 109 O. volvulus-positive specimens, 426 non-onchocerciasis controls with filarial and other confirmed parasitic infection, and 75 sera from patients with no other parasitic infection. The IgG and IgG4 assays for OV-16 demonstrated sensitivities of 95.4% and 96.3%, and specificities of 99.4% and 99.8%, respectively. The OV-17 IgG and IgG4 assays had sensitivities of 86.2% and 76.1% and specificities of 79.2% and 82.8%. For OV-33, the IgG and IgG4 assays had sensitivities of 90.8% and 96.3%, and specificities of 96.8% and 98.6%. The OV-16 IgG4-based MBA had the best assay characteristics, followed by OV-33 IgG4. The OV-16 IgG4 assay would be useful for monitoring and evaluation using the MBA platform. Further evaluations are needed to review the potential use of OV-33 as a confirmatory test in the context of program evaluations.