Day 3 parasitemia and Plasmodium falciparum Kelch 13 mutations among uncomplicated malaria patients treated with artemether-lumefantrine in Adjumani district, Uganda.

Artemisinin resistance threatens malaria control and elimination efforts globally. Recent studies have reported the emergence of Plasmodium falciparum parasites tolerant to artemisinin agents in sub-Saharan Africa, including Uganda. The current study assessed the day 3 parasite clearance and its correlation with P. falciparum K13 propeller gene (pfkelch13) mutations in P. falciparum parasites isolated from patients with uncomplicated malaria under artemether-lumefantrine (AL) treatment. This study enrolled 100 P. falciparum-positive patients to whom AL was prescribed between 09/September/2022 and 06/November/2022. Blood samples were collected in EDTA tubes before treatment initiation (day 0) and on day 3. Parasitemia was assessed by microscopy from blood smears and quantitative polymerase chain reaction (qPCR) from the DNA extracted. The day 0 parasite K13 gene was sequenced using Sanger sequencing. Sequence data were analysed using MEGA version 11 software. The data were analysed using STATA version 15, and the Mann‒Whitney U test was used to compare PCR parasite clearance on day 3 using the comparative CT value method and pfkelch13 mutations. The prevalence of day 3 parasitaemia was 24% (24/100) by microscopy and 63% (63/100) by qPCR from the AL-treated patients. P. falciparum K13-propeller gene polymorphism was detected in 18.8% (15/80) of the day 0 DNA samples. The K13 mutations found were C469Y, 12.5% (10/80); A675V, 2.5% (2/80); A569S, 1.25%, (1/80), A578S, 1.25%, (1/80) and; F491S, 1.25%, (1/80) a new allele not reported anywhere. The C469Y mutation, compared to the wild-type, was associated with delayed parasite clearance p = 0.0278, Hodges-Lehmann estimation 3.2108 on the log scale, (95%CI 1.7076, 4.4730). There was a high prevalence of day 3 P. falciparum among malaria patients treated using artemether-lumefantrine. We conclude the presence of the K13 mutation associated with artemisinin resistance by P. falciparum in Adjumani district, Uganda, necessitates regular surveillance of the effectiveness and efficacy of artemether-lumefantrine in the country.

Correction: Limitations of rapid diagnostic tests in malaria surveys in areas with varied transmission intensity in Uganda 2017-2019: Implications for selection and use of HRP2 RDTs.

[This corrects the article DOI: 10.1371/journal.pone.0244457.].

Day 3 parasitemia and Plasmodium falciparum Kelch 13 mutations among uncomplicated malaria patients treated with artemether-lumefantrine in Adjumani district, Uganda.

Artemisinin resistance threatens malaria control and elimination efforts globally. Recent studies have reported the emergence of Plasmodium falciparum parasites tolerant to artemisinin agents in sub-Saharan Africa, including Uganda. The current study assessed the day 3 parasite clearance and its correlation with P. falciparum K13 propeller gene (pfkelch13) mutations in P. falciparum parasites isolated from patients with uncomplicated malaria under artemether-lumefantrine (AL) treatment. This study enrolled 100 P. falciparum-positive patients to whom AL was prescribed between 09/September/2022 and 06/November/2022. Blood samples were collected in EDTA tubes before treatment initiation (day 0) and on day 3. Parasitemia was assessed by microscopy from blood smears and quantitative polymerase chain reaction (qPCR) from the DNA extracted. The day 0 parasite K13 gene was sequenced using Sanger sequencing. Sequence data were analysed using MEGA version 11 software. The data were analysed using STATA version 15, and the Mann‒Whitney U test was used to compare PCR parasite clearance on day 3 using the comparative CT value method and pfkelch13 mutations. The prevalence of day 3 parasitaemia was 24% (24/100) by microscopy and 63% (63/100) by qPCR from the AL-treated patients. P. falciparum K13-propeller gene polymorphism was detected in 18.8% (15/80) of the day 0 DNA samples. The K13 mutations found were C469Y, 12.5% (10/80); A675V, 2.5% (2/80); A569S, 1.25%, (1/80), A578S, 1.25%, (1/80) and; F491S, 1.25%, (1/80) a new allele not reported anywhere. The C469Y mutation, compared to the wild-type, was associated with delayed parasite clearance p=0.0278, Hodges-Lehmann estimation 3.2108 on the log scale, (95%CI 1.7076, 4.4730). There was a high prevalence of day 3 P. falciparum among malaria patients treated using artemether-lumefantrine. We conclude that the K13 mutation associated with artemisinin resistance by P. falciparum is present in Adjumani district, Uganda. This necessitates regular surveillance of the effectiveness and efficacy of artemether-lumefantrine in the country.

Access to quality-assured artemisinin-based combination therapy and associated factors among clients of selected private drug outlets in Uganda.

BACKGROUND: Malaria treatment in sub-Saharan Africa is faced with challenges including unreliable supply of efficacious agents, substandard medicines coupled with high price of artemisinin-based combinations. This affects access to effective treatment increasing risk of malaria parasite resistance development and adverse drug events. This study investigated access to quality-assured artemisinin-based combination therapy (QAACT) medicines among clients of selected private drug-outlets in Uganda. METHODS: This was a cross sectional study where exit interviews were conducted among clients of private drug outlets in low and high malaria transmission settings in Uganda. This study adapted the World Health Organization/Health Action International (WHO/HAI) standardized criteria. Data was collected using a validated questionnaire. Data entry screen with checks was created in Epi-data ver 4.2 software and data entered in duplicate. Data was transferred to STATA ver 14.0 and cleaned prior to analysis. The analysis was done at 95% level of significance. RESULTS: A total of 1114 exit interviews were conducted among systematically sampled drug outlet clients. Over half, 54.9% (611/1114) of the participants were males. Majority, 97.2% (1083/1114) purchased an artemisinin-based combination anti-malarial. Most, 55.5% (618/1114) of the participants had a laboratory diagnosis of malaria. Majority, 77.9% (868/1114) of the participants obtained anti-malarial agents without a prescription. Less than a third, 27.7% (309/1114) of the participants obtained a QAACT. Of the participants who obtained QAACT, more than half 56.9% (173/309) reported finding the medicine expensive. The predictors of accessing a QAACT anti-malarial among drug outlet clients include type of drug outlet visited (aPR = 0.74; 95%CI 0.6, 0.91), not obtaining full dose (3-day treatment) of ACT (aPR = 0.49; 95%CI 0.33, 0.73), not finding the ACT expensive (aPR = 1.24; 95%CI 1.03, 1.49), post-primary education (aPR = 1.29; 95%CI 1.07,1.56), business occupation (aPR = 1.24; 95%CI 1.02,1.50) and not having a prescription (aPR = 0.76; 95%CI 0.63, 0.92). CONCLUSION: Less than a third of the private drug outlet clients obtained a QAACT for management of malaria symptoms. Individuals who did not find artemisinin-based combinations to be expensive were more likely to obtain a QAACT anti-malarial. The Ministry of Health needs to conduct regular surveillance to monitor accessibility of QAACT anti-malarial agents under the current private sector copayment mechanism.

Copayment mechanism in selected districts of Uganda: Availability, market share and price of quality assured artemisinin-based combination therapies in private drug outlets.

BACKGROUND: Malaria remains one of the leading causes of morbidity, and mortality in Uganda. A large proportion of malaria symptomatic patients seek healthcare in private sector. However, availability and affordability are major barriers to access to effective treatment. The private sector copayment mechanism in Uganda aims to increase availability and affordability of antimalarial agents. Our study assessed availability, price, and market share of quality assured artemisinin-based combination therapies (QAACTs) in private drug outlets in selected districts during the implementation of copayment mechanism. METHODS: This was a cross-sectional survey of anti-malarial agents in private drug outlets in in selected moderate-to-high (Tororo, and Apac districts) and low (Kabale and Mbarara districts) malaria transmission settings. Following the World Health Organization/Health Action International (WHO/HAI) criteria, an audit of the antimalarial agents was done using a checklist to determine availability, price, and market share of QAACTs. Data were entered in Epi-data and analyzed in STATA ver 14.0 at 95% confidence level. RESULTS: A total of twenty-eight (28) private drug outlets (pharmacies and drug shops) were included in the survey. One in seven (20/144: 95%CI: 9.1, 20.6) of the antimalarial agents in private drug outlets were quality assured artemisinin-based combination therapies (QAACT). Artemether-lumefantrine (AL), 8.9% (11/124) and Artesunate-Amodiaquine (AQ), 7.3% (9/124) were the only QAACTs present in the drug outlets at the time of the survey. The majority, 86.1%% (124/144) of antimalarial agents present in stock in the drug outlets were artemisinin based. The most common, 38.9% (56/144) ACT in the drug outlets was Dihydroartemisinin-Piperaquine (DHP). Most, 69.4% (100/144) of the antimalarial agents were in high malaria transmission settings. The cost of ACT antimalarial agents is high in the country, USD 1.4 (Artemether-Lumefantrine, AL), USD 2.4 (Dihydroartemisinin-Piperaquine, DP), the first line and second-line agents respectively for treatment of uncomplicated malaria in Uganda. There was a statistically significant difference between the dispensing price of 'Green leaf' ACTs (QAACT) and the recommended price (p<0.001). Predictors of availability of QAACT in private drug outlets include pharmacy drug outlet (aPR:0.4; 95%CI: 0.2, 0.9) and dispensing price more than 3000UGX (USD 0.83) (aPR: 0.4, 95%CI: 0.1, 0.51). CONCLUSION: Quality assured artemisinin-based combination therapies (QAACTs) are not common in private drug outlets in selected districts in Uganda. All the drug outlets had at least one ACT antimalarial agent present on the day of the survey. The dispensing price of QAACTs was significantly higher than the recommended markup price. There is need for awareness creation, surveillance, and monitoring of the implementation of Copayment mechanism in the country.

Experiences of healthcare personnel on the efficacy of artemisinin-based combination therapy and malaria diagnosis in hospitals in Uganda.

BACKGROUND: The risk of widespread resistance to artemisinin-based combination therapy (ACT) remains high in Uganda following detection of Plasmodium falciparum parasites with delayed artemisinin clearance genotype and phenotype. Establishment of context specific interventions to mitigate emergence and spread of artemisinin resistance is thus key in the fight against malaria in the country. The aim of this study was to explore the experiences of healthcare personnel on malaria diagnosis and self-reported efficacy of ACT in the management of malaria symptomatic patients in hospitals in low and high malaria transmission settings in Uganda. METHODS: This was a qualitative study in which data was collected from healthcare personnel in hospitals using key informant interviews. The key informant interview guide was developed, pre-tested prior to use and covered the following areas, (i) sociodemographic characteristics, (ii) malaria diagnosis (clinical and parasite based), (iii) quality-assured artemisinin-based combination therapy, (iv) malaria patient follow-up, (v) artemisinin resistance, (vi) anti-malarial self-medication. Data was entered in Atlas.ti ver 9.0 and analysis done following a framework criterion. RESULTS: A total of 22 respondents were interviewed of which 16 (72.7%) were clinicians. Majority, 81.8% (18/22) of the respondents were male. The following themes were developed from the analysis, malaria diagnosis (procedures and challenges), use of malaria laboratory test results, malaria treatment in hospitals, use of quality assured ACT (QAACT) in malaria treatment, and efficacy of ACT in malaria treatment. CONCLUSION: Most healthcare personnel-initiated malaria treatment after a positive laboratory test. Cases of malaria patients who report remaining symptomatic after prior use of ACT exist especially in high malaria transmission settings in Uganda. There is need for regular monitoring of artemisinin resistance emergence and spread in the country.

Pharmacopeial quality of artemether-lumefantrine anti-malarial agents in Uganda.

BACKGROUND: Substandard anti-malarial agents pose a significant challenge to effective malaria control and elimination efforts especially in sub-Saharan Africa. The quality of anti-malarials in most low-and-middle income countries (LMICs) is affected by several factors including inadequate regulation and limited resources. In this study, the pharmacopeial quality of artemether-lumefantrine (AL) in low and high malaria transmission settings in Uganda was assessed. METHODS: This was a cross-sectional study conducted among randomly selected private drug outlets. The AL anti-malarials available in drug outlets were purchased using overt method. The samples were screened for quality using visual inspection, weight uniformity, content assay and dissolution tests. The assay test was done using liquid chromatography-mass spectrometry (LC-MS). The samples were considered substandard if the active pharmaceutical ingredient (API) content was outside 90-110% range of the label claim. Dissolution test was conducted following United States Pharmacopoeia (USP) method. Data was analysed using descriptive statistics and presented as means with standard deviations, frequencies, and proportions. Correlation between medicine quality and independent variables was determined using Fisher's exact test of independence at 95% level of significance. RESULTS: A total of 74 AL anti-malarial samples were purchased from high (49/74; 66.2%) and low (25/74; 33.8%) malaria transmission settings. The most common batch of AL was LONART, 32.4% (24/74), with 33.8% (25/74) being 'Green leaf'. Overall prevalence of substandard quality artemether-lumefantrine was 18.9% (14/74; 95% CI: 11.4-29.7). Substandard quality AL was significantly associated with setting (p = 0.002). A total of 10 samples (13.5%) failed artemether content assay test while, 4 samples (5.4%, 4/74) failed the lumefantrine assay test. One sample from a high malaria transmission setting failed both artemether and lumefantrine assay content test. Of the samples that failed artemether assay test, 90% had low (< 90%) artemether content. All the samples passed visual inspection and dissolution tests. CONCLUSION: Artemether-lumefantrine agents, the recommended first-line treatment for uncomplicated malaria with APIs outside the recommended pharmacopeial content assay limit is common especially in high malaria transmission settings. There is need for continuous surveillance and monitoring of the quality of artemisinin-based anti-malarials across the country by the drug regulatory agency.

East Africa International Center of Excellence for Malaria Research: Summary of Key Research Findings.

The Program for Resistance, Immunology, Surveillance, and Modeling of Malaria (PRISM) has been conducting malaria research in Uganda since 2010 to improve the understanding of the disease and measure the impact of population-level control interventions in the country. Here, we will summarize key research findings from a series of studies addressing routine health facility-based surveillance, comprehensive cohort studies, studies of the molecular epidemiology, and transmission of malaria, evaluation of antimalarial drug efficacy, and resistance across the country, and assessments of insecticide resistance. Among our key findings are the following. First, we found that in historically high transmission areas of Uganda, a combination of universal distribution of long-lasting insecticidal-treated nets (LLINs) and sustained indoor residual spraying (IRS) of insecticides lowered the malaria burden greatly, but marked resurgences occurred if IRS was discontinued. Second, submicroscopic infections are common and key drivers of malaria transmission, especially in school-age children (5-15 years). Third, markers of drug resistance have changed over time, with new concerning emergence of markers predicting resistance to artemisinin antimalarials. Fourth, insecticide resistance monitoring has demonstrated high levels of resistance to pyrethroids, appreciable impact of the synergist piperonyl butoxide to pyrethroid susceptibility, emerging resistance to carbamates, and complete susceptibility of malaria vectors to organophosphates, which could have important implications for vector control interventions. Overall, PRISM has yielded a wealth of information informing researchers and policy-makers on the malaria burden and opportunities for improved malaria control and eventual elimination in Uganda. Continued studies concerning all the types of surveillance discussed above are ongoing.

Prevalence of arps10, fd, pfmdr-2, pfcrt and pfkelch13 gene mutations in Plasmodium falciparum parasite population in Uganda.

In Uganda, Artemether-Lumefantrine and Artesunate are recommended for uncomplicated and severe malaria respectively, but are currently threatened by parasite resistance. Genetic and epigenetic factors play a role in predisposing Plasmodium falciparum parasites to acquiring Pfkelch13 (K13) mutations associated with delayed artemisinin parasite clearance as reported in Southeast Asia. In this study, we report on the prevalence of mutations in the K13, pfmdr-2 (P. falciparum multidrug resistance protein 2), fd (ferredoxin), pfcrt (P. falciparum chloroquine resistance transporter), and arps10 (apicoplast ribosomal protein S10) genes in Plasmodium falciparum parasites prior to (2005) and after (2013) introduction of artemisinin combination therapies for malaria treatment in Uganda. A total of 200 P. falciparum parasite DNA samples were screened. Parasite DNA was extracted using QIAamp DNA mini kit (Qiagen, GmbH, Germany) procedure. The PCR products were sequenced using Sanger dideoxy sequencing method. Of the 200 P. falciparum DNA samples screened, sequencing for mutations in K13, pfmdr-2, fd, pfcrt, arps10 genes was successful in 142, 186, 141, 128 and 74 samples respectively. Overall, we detected six (4.2%, 6/142; 95%CI: 1.4-7.0) K13 single nucleotide polymorphisms (SNPs), of which 3.9% (2/51), 4.4% (4/91) occurred in 2005 and 2013 samples respectively. All four K13 SNPs in 2013 samples were non-synonymous (A578S, E596V, S600C and E643K) while of the two SNPs in 2005 samples, one (Y588N) is non-synonymous and the other (I587I) is synonymous. There was no statistically significant difference in the prevalence of K13 (p = 0.112) SNPs in the samples collected in 2005 and 2013. The overall prevalence of SNPs in pfmdr-2 gene was 39.8% (74/186, 95%CI: 25.1-50.4). Of this, 4.2% (4/95), 76.9% (70/91) occurred in 2005 and 2013 samples respectively. In 2005 samples only one SNP, Y423F (4.2%, 4/95) was found while in 2013, Y423F (38.5%, 35/91) and I492V (38.5%, 35/91) SNPs in the pfmdr-2 gene were found. There was a statistically significant difference in the prevalence of pfmdr-2 SNPs in the samples collected in 2005 and 2013 (p<0.001). The overall prevalence of arps10 mutations was 2.7% (2/72, 95%CI: 0.3-4.2). Two mutations, V127M (4.5%: 1/22) and D128H (4.5%: 1/22) in the arps10 gene were each found in P. falciparum parasite samples collected in 2013. There was no statistically significant difference in the prevalence of arps10 SNPs in the samples collected in 2005 and 2013 (p = 0.238). There were more pfmdr-2 SNPs in P. falciparum parasites collected after introduction of Artemisinin combination therapies in malaria treatment. This is an indicator of the need for continuous surveillance to monitor emergence of molecular markers of artemisinin resistance and its potential drivers in malaria affected regions globally.

Field evaluation of the diagnostic performance of EasyScan GO: a digital malaria microscopy device based on machine-learning.

BACKGROUND: Microscopic examination of Giemsa-stained blood films remains the reference standard for malaria parasite detection and quantification, but is undermined by difficulties in ensuring high-quality manual reading and inter-reader reliability. Automated parasite detection and quantification may address this issue. METHODS: A multi-centre, observational study was conducted during 2018 and 2019 at 11 sites to assess the performance of the EasyScan Go, a microscopy device employing machine-learning-based image analysis. Sensitivity, specificity, accuracy of species detection and parasite density estimation were assessed with expert microscopy as the reference. Intra- and inter-device reliability of the device was also evaluated by comparing results from repeat reads on the same and two different devices. This study has been reported in accordance with the Standards for Reporting Diagnostic accuracy studies (STARD) checklist. RESULTS: In total, 2250 Giemsa-stained blood films were prepared and read independently by expert microscopists and the EasyScan Go device. The diagnostic sensitivity of EasyScan Go was 91.1% (95% CI 88.9-92.7), and specificity 75.6% (95% CI 73.1-78.0). With good quality slides sensitivity was similar (89.1%, 95%CI 86.2-91.5), but specificity increased to 85.1% (95%CI 82.6-87.4). Sensitivity increased with parasitaemia rising from 57% at < 200 parasite/µL, to ≥ 90% at > 200-200,000 parasite/µL. Species were identified accurately in 93% of Plasmodium falciparum samples (kappa = 0.76, 95% CI 0.69-0.83), and in 92% of Plasmodium vivax samples (kappa = 0.73, 95% CI 0.66-0.80). Parasite density estimates by the EasyScan Go were within ± 25% of the microscopic reference counts in 23% of slides. CONCLUSIONS: The performance of the EasyScan Go in parasite detection and species identification accuracy fulfil WHO-TDR Research Malaria Microscopy competence level 2 criteria. In terms of parasite quantification and false positive rate, it meets the level 4 WHO-TDR Research Malaria Microscopy criteria. All performance parameters were significantly affected by slide quality. Further software improvement is required to improve sensitivity at low parasitaemia and parasite density estimations. Trial registration ClinicalTrials.gov number NCT03512678.

Decreased Susceptibility to Dihydrofolate Reductase Inhibitors Associated With Genetic Polymorphisms in Ugandan Plasmodium falciparum Isolates.

BACKGROUND: The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug-susceptibility data for P falciparum field isolates are limited. METHODS: We studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda, from 2016 to 2020, sequenced 383 isolates, and assessed associations between genotypes and drug-susceptibility phenotypes. RESULTS: Median half-maximal inhibitory concentrations (IC50s) were 42 100 nM for pyrimethamine, 1200 nM for cycloguanil, 13000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, 3 PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50s of 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites. CONCLUSIONS: Resistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.

Efficacy and safety of artemether-lumefantrine and dihydroartemisinin-piperaquine for the treatment of uncomplicated Plasmodium falciparum malaria and prevalence of molecular markers associated with artemisinin and partner drug resistance in Uganda.

BACKGROUND: In Uganda, artemether-lumefantrine (AL) is first-line therapy and dihydroartemisinin-piperaquine (DP) second-line therapy for the treatment of uncomplicated malaria. This study evaluated the efficacy and safety of AL and DP in the management of uncomplicated falciparum malaria and measured the prevalence of molecular markers of resistance in three sentinel sites in Uganda from 2018 to 2019. METHODS: This was a randomized, open-label, phase IV clinical trial. Children aged 6 months to 10 years with uncomplicated falciparum malaria were randomly assigned to treatment with AL or DP and followed for 28 and 42 days, respectively. Genotyping was used to distinguish recrudescence from new infection, and a Bayesian algorithm was used to assign each treatment failure a posterior probability of recrudescence. For monitoring resistance, Pfk13 and Pfmdr1 genes were Sanger sequenced and plasmepsin-2 copy number was assessed by qPCR. RESULTS: There were no early treatment failures. The uncorrected 28-day cumulative efficacy of AL ranged from 41.2 to 71.2% and the PCR-corrected cumulative 28-day efficacy of AL ranged from 87.2 to 94.4%. The uncorrected 28-day cumulative efficacy of DP ranged from 95.8 to 97.9% and the PCR-corrected cumulative 28-day efficacy of DP ranged from 98.9 to 100%. The uncorrected 42-day efficacy of DP ranged from 73.5 to 87.4% and the PCR-corrected 42-day efficacy of DP ranged from 92.1 to 97.5%. There were no reported serious adverse events associated with any of the regimens. No resistance-associated mutations in the Pfk13 gene were found in the successfully sequenced samples. In the AL arm, the NFD haplotype (N86Y, Y184F, D1246Y) was the predominant Pfmdr1 haplotype, present in 78 of 127 (61%) and 76 of 110 (69%) of the day 0 and day of failure samples, respectively. All the day 0 samples in the DP arm had one copy of the plasmepsin-2 gene. CONCLUSIONS: DP remains highly effective and safe for the treatment of uncomplicated malaria in Uganda. Recurrent infections with AL were common. In Busia and Arua, the 95% confidence interval for PCR-corrected AL efficacy fell below 90%. Further efficacy monitoring for AL, including pharmacokinetic studies, is recommended. Trial registration The trail was also registered with the ISRCTN registry with study Trial No. PACTR201811640750761.

Genetic diversity and genetic relatedness in Plasmodium falciparum parasite population in individuals with uncomplicated malaria based on microsatellite typing in Eastern and Western regions of Uganda, 2019-2020.

BACKGROUND: Genetic diversity and parasite relatedness are essential parameters for assessing impact of interventions and understanding transmission dynamics of malaria parasites, however data on its status in Plasmodium falciparum populations in Uganda is limited. Microsatellite markers and DNA sequencing were used to determine diversity and molecular characterization of P. falciparum parasite populations in Uganda. METHODS: A total of 147 P. falciparum genomic DNA samples collected from cross-sectional surveys in symptomatic individuals of 2-10 years were characterized by genotyping of seven highly polymorphic neutral microsatellite markers (n = 85) and genetic sequencing of the Histidine Rich Protein 2 (pfhrp2) gene (n = 62). ArcGIS was used to map the geographical distribution of isolates while statistical testing was done using Student's t-test or Wilcoxon's rank-sum test and Fisher's exact test as appropriate at P ≤ 0.05. RESULTS: Overall, 75.5% (95% CI 61.1-85.8) and 24.5% (95% CI14.2-38.9) of parasites examined were of multiclonal (mixed genotype) and single clone infections, respectively. Multiclonal infections occurred more frequently in the Eastern region 73.7% (95% CI 48.8-89.1), P < 0.05. Overall, multiplicity of infection (MOI) was 1.9 (95% CI 1.7-2.1), P = 0.01 that was similar between age groups (1.8 vs 1.9), P = 0.60 and regions (1.9 vs 1.8), P = 0.43 for the < 5 and ≥ 5 years and Eastern and Western regions, respectively. Genomic sequencing of the pfhrp2 exon2 revealed a high level of genetic diversity reflected in 96.8% (60/62) unique sequence types. Repeat type AHHAAAHHATD and HRP2 sequence Type C were more frequent in RDT-/PCR + samples (1.9% vs 1.5%) and (13% vs 8%), P < 0.05 respectively. Genetic relatedness analysis revealed small clusters of gene deleted parasites in Uganda, but no clustering with Eritrean parasites. CONCLUSION: High level of genetic diversity of P. falciparum parasites reflected in the frequency of multiclonal infections, multiplicity of infection and variability of the pfhrp2 gene observed in this study is consistent with the high malaria transmission intensity in these settings. Parasite genetic analysis suggested spontaneous emergence and clonal expansion of pfhrp2 deleted parasites that require close monitoring to inform national malaria diagnosis and case management policies.

Prevalence of Malaria Parasite Infections among U.S.-Bound Congolese Refugees with and without Splenomegaly.

All U.S.-bound refugees from sub-Saharan Africa receive presumptive antimalarial treatment before departing for the United States. Among U.S.-bound Congolese refugees, breakthrough malaria cases and persistent splenomegaly have been reported. In response, an enhanced malaria diagnostic program was instituted. Here, we report the prevalence of plasmodial infection among 803 U.S.-bound Congolese refugees who received enhanced diagnostics. Infections by either rapid diagnostic test (RDT) or PCR were detected in 187 (23%) refugees, with 78 (10%) by RDT only, 35 (4%) by PCR only, and 74 (9%) by both. Infections identified by PCR included 103 monoinfections (87 Plasmodium falciparum, eight Plasmodium ovale, seven Plasmodium vivax, and one Plasmodium malariae) and six mixed infections. Splenomegaly was associated with malaria detectable by RDT (odds ratio: 1.8, 95% CI: 1.0-3.0), but not by PCR. Splenomegaly was not strongly associated with parasitemia, indicating that active malaria parasitemia is not necessary for splenomegaly.

Deletions of pfhrp2 and pfhrp3 genes were uncommon in rapid diagnostic test-negative Plasmodium falciparum isolates from Uganda.

BACKGROUND: Rapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the paralogous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum infections from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on PfHRP2 protein expression, and understanding of the true prevalence of deletions is incomplete. METHODS: Deletions of pfhrp2/pfhrp3 in blood samples were investigated from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. Samples with positive Giemsa-stained thick blood smears, but negative PfHRP2-based RDTs were evaluated by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression of PfHRP2. RESULTS: Of 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum DNA was identified by PCR in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum-positive samples there was a failure to amplify pfhrp2 or pfhrp3: in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum-positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay. CONCLUSION: False negative RDTs were uncommon. Deletions in pfhrp2 and pfhrp3 explained some of these false negatives, but most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

BACKGROUND: In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. METHODS: Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. RESULTS: Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). CONCLUSIONS: We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Limitations of rapid diagnostic tests in malaria surveys in areas with varied transmission intensity in Uganda 2017-2019: Implications for selection and use of HRP2 RDTs.

BACKGROUND: Plasmodium falciparum histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) are exclusively recommended for malaria diagnosis in Uganda; however, their functionality can be affected by parasite-related factors that have not been investigated in field settings. METHODS: Using a cross-sectional design, we analysed 219 RDT-/microscopy+ and 140 RDT+/microscopy+ dried blood spots obtained from symptomatic children aged 2-10 years from 48 districts in Uganda between 2017 and 2019. We aimed to investigate parasite-related factors contributing to false RDT results by molecular characterization of parasite isolates. ArcGIS software was used to map the geographical distribution of parasites. Statistical analysis was performed using chi-square or Fisher's exact tests, with P ≤ 0.05 indicating significance. Odds ratios (ORs) were used to assess associations, while logistic regression was performed to explore possible factors associated with false RDT results. RESULTS: The presence of parasite DNA was confirmed in 92.5% (332/359) of the blood samples. The levels of agreement between the HRP2 RDT and PCR assay results in the (RDT+/microscopy+) and (RDT-/microscopy+) sample subsets were 97.8% (137/140) and 10.9% (24/219), respectively. Factors associated with false-negative RDT results in the (RDT-/microscopy+) samples were parasite density (<1,000/µl), pfhrp2/3 gene deletion and non-P. falciparum species (aOR 2.65, 95% CI: 1.62-4.38, P = 0.001; aOR 4.4, 95% CI 1.72-13.66, P = 0.004; and aOR 18.65, 95% CI: 5.3-38.7, P = 0.001, respectively). Overall, gene deletion and non-P. falciparum species contributed to 12.3% (24/195) and 19.0% (37/195) of false-negative RDT results, respectively. Of the false-negative RDTs results, 80.0% (156/195) were from subjects with low-density infections (< 25 parasites per 200 WBCs or <1,000/µl). CONCLUSION: This is the first evaluation and report of the contributions of pfhrp2/3 gene deletion, non-P. falciparum species, and low-density infections to false-negative RDT results under field conditions in Uganda. In view of these findings, the use of HRP2 RDTs should be reconsidered; possibly, switching to combination RDTs that target alternative antigens, particularly in affected areas, may be beneficial. Future evaluations should consider larger and more representative surveys covering other regions of Uganda.

Associations between Malaria-Preventive Regimens and Plasmodium falciparum Drug Resistance-Mediating Polymorphisms in Ugandan Pregnant Women.

Intermittent preventive treatment in pregnancy (IPTp) with monthly sulfadoxine-pyrimethamine (SP) is recommended for malaria-endemic parts of Africa, but efficacy is compromised by resistance, and, in recent trials, dihydroartemisinin-piperaquine (DP) has shown better antimalarial protective efficacy. We utilized blood samples from a recent trial to evaluate selection by IPTp with DP or SP of Plasmodium falciparum genetic polymorphisms that alter susceptibility to these drugs. The prevalence of known genetic polymorphisms associated with altered drug susceptibility was determined in parasitemic samples, including 375 collected before IPTp drugs were administered, 125 randomly selected from those receiving SP, and 80 from those receiving DP. For women receiving DP, the prevalence of mixed/mutant sequences was greater in samples collected during IPTp than that in samples collected prior to the intervention for PfMDR1 N86Y (20.3% versus 3.9%; P < 0.001), PfMDR1 Y184F (73.0% versus 53.0%; P < 0.001), and PfCRT K76T (46.4% versus 24.0%; P < 0.001). Considering SP, prior to IPTp, the prevalence of all 5 common antifolate mutations was over 92%, and this prevalence increased following exposure to SP, although none of these changes were statistically significant. For two additional mutations associated with high-level SP resistance, the prevalence of PfDHFR 164L (13.7% versus 4.0%; P = 0.004), but not PfDHPS 581G (1.9% versus 3.0%; P = 0.74), was greater in samples collected during IPTp compared to those collected before the intervention. Use of IPTp in Uganda selected for parasites with mutations associated with decreased susceptibility to IPTp regimens. Thus, a potential drawback of IPTp is selection of parasites with decreased drug susceptibility.

Molecular surveillance reveals the presence of pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum parasite populations in Uganda, 2017-2019.

BACKGROUND: Histidine-rich protein-2 (HRP2)-based rapid diagnostic tests (RDTs) are the only RDTs recommended for malaria diagnosis in Uganda. However, the emergence of Plasmodium falciparum histidine rich protein 2 and 3 (pfhrp2 and pfhrp3) gene deletions threatens their usefulness as malaria diagnostic and surveillance tools. The pfhrp2 and pfhrp3 gene deletions surveillance was conducted in P. falciparum parasite populations in Uganda. METHODS: Three-hundred (n = 300) P. falciparum isolates collected from cross-sectional malaria surveys in symptomatic individuals in 48 districts of eastern and western Uganda were analysed for the presence of pfhrp2 and pfhrp3 genes. Presence of parasite DNA was confirmed by PCR amplification of the 18s rRNA gene, msp1 and msp2 single copy genes. Presence or absence of deletions was confirmed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCR. RESULTS: Overall, pfhrp2 and pfhrp3 gene deletions were detected in 29/300 (9.7%, 95% CI 6.6-13.6%) parasite isolates. The pfhrp2 gene was deleted in 10/300 (3.3%, 95% CI 1.6-6.0%) isolates, pfhrp3 in 9/300 (3.0%, 95% CI 1.4-5.6%) while both pfhrp2 and pfhrp3 were deleted in 10/300 (3.3%, 95% CI 1.6-6.0%) parasite isolates. Proportion of pfhrp2/3 deletions was higher in the eastern 14.7% (95% CI 9.7-20.0%) compared to the western region 3.1% (95% CI 0.8-7.7%), p = 0.001. Geographical location was associated with gene deletions aOR 6.25 (2.02-23.55), p = 0.003. CONCLUSIONS: This is the first large-scale survey reporting the presence of pfhrp2/3 gene deletions in P. falciparum isolates in Uganda. Roll out of RDTs for malaria diagnosis should take into consideration the existence of pfhrp2/3 gene deletions particularly in areas where they were detected. Periodic pfhrp2/3 surveys are recommended to inform future decisions for deployment of alternative RDTs.