Indigenous emergence and spread of kelch13 C469Y artemisinin-resistant Plasmodium falciparum in Uganda.

Artemisinin-based combination therapies (ACTs) were introduced as the standard of care for uncomplicated malaria in Africa almost two decades ago. Recent studies in East Africa have reported a gradual increase in kelch13 (k13) mutant parasites associated with reduced artesunate efficacy. As part of the Community Access to Rectal Artesunate for Malaria project, we collected blood samples from 697 children with signs of severe malaria in northern Uganda between 2018 and 2020, before and after the introduction of rectal artesunate (RAS) in 2019. K13 polymorphisms were assessed, and parasite editing and phenotyping were performed to assess the impact of mutations on parasite resistance. Whole-genome sequencing was performed, and haplotype networks were constructed to determine the geographic origin of k13 mutations. Of the 697 children, 540 were positive for Plasmodium falciparum malaria by PCR and were treated with either RAS or injectable artesunate monotherapy followed in most cases by ACT. The most common k13 mutation was C469Y (6.7%), which was detected more frequently in samples collected after RAS introduction. Genome editing confirmed reduced in vitro susceptibility to artemisinin in C469Y-harboring parasites compared to wild-type controls (P < 0.001). The haplotypic network showed that flanking regions of the C469Y mutation shared the same African genetic background, suggesting a single and indigenous origin of the mutation. Our data provide evidence of selection for the artemisinin-resistant C469Y mutation. The realistic threat of multiresistant parasites emerging in Africa should encourage careful monitoring of the efficacy of artemisinin derivatives and strict adherence to ACT treatment regimens.

Evidence of Artemisinin-Resistant Malaria in Africa.

BACKGROUND: In the six Southeast Asian countries that make up the Greater Mekong Subregion, Plasmodium falciparum has developed resistance to derivatives of artemisinin, the main component of first-line treatments for malaria. Clinical resistance to artemisinin monotherapy in other global regions, including Africa, would be problematic. METHODS: In this longitudinal study conducted in Northern Uganda, we treated patients who had P. falciparum infection with intravenous artesunate (a water-soluble artemisinin derivative) and estimated the parasite clearance half-life. We evaluated ex vivo susceptibility of the parasite using a ring-stage survival assay and genotyped resistance-related genes. RESULTS: From 2017 through 2019, a total of 14 of 240 patients who received intravenous artesunate had evidence of in vivo artemisinin resistance (parasite clearance half-life, >5 hours). Of these 14 patients, 13 were infected with P. falciparum parasites with mutations in the A675V or C469Y allele in the kelch13 gene. Such mutations were associated with prolonged parasite clearance half-lives (geometric mean, 3.95 hours for A675V and 3.30 hours for C469Y, vs. 1.78 hours for wild-type allele; P<0.001 and P = 0.05, respectively). The ring-stage survival assay showed a higher frequency of parasite survival among organisms with the A675V allele than among those with the wild-type allele. The prevalence of parasites with kelch13 mutations increased significantly, from 3.9% in 2015 to 19.8% in 2019, due primarily to the increased frequency of the A675V and C469Y alleles (P<0.001 and P = 0.004, respectively). Single-nucleotide polymorphisms flanking the A675V mutation in Uganda were substantially different from those in Southeast Asia. CONCLUSIONS: The independent emergence and local spread of clinically artemisinin-resistant P. falciparum has been identified in Africa. The two kelch13 mutations may be markers for detection of these resistant parasites. (Funded by the Japan Society for the Promotion of Science and others.).

The Impact of Sequestration on Artemisinin-Induced Parasite Clearance in Plasmodium falciparum Malaria in Africa.

BACKGROUND: Artemisinin-resistant Plasmodium falciparum is spreading in Southeast Asia and Africa. In vivo susceptibility to artemisinin is studied by looking at the rate of decline of peripheral parasitemia (parasite clearance half-life). However, parasites that are adhered/sequestered to the endothelium and undetectable in the peripheral blood are not considered in the estimation of parasite clearance. Here, we evaluated the influence of sequestration on in vivo artemisinin efficacy in Uganda, where artemisinin resistance is spreading. METHODS: We analyzed 133 patients with P. falciparum malaria included in an in vivo study on artemisinin efficacy in northern Uganda in 2018 and 2019. The parasite clearance half-life was estimated from peripheral parasitemia after artemisinin monotherapy. P. falciparum histidine-rich protein 2 (PfHRP2) was measured in pretreatment plasma. The number of sequestered parasites was estimated from PfHRP2 concentration and peripheral parasitemia. RESULTS: The estimated number of sequestered parasites per plasma volume ranged from 0 to 2 564 000/µL. Inflammation, thrombocytopenia, and dyslipidemia were significantly associated with sequestration independent of peripheral parasitemia. The median parasite clearance half-lives were 1.65 hours in patients infected with Pfkelch13 wild-type parasites (n = 104) and 3.95 hours in those with A675V artemisinin-resistant mutant (n = 18). In the multivariable model for the wild-type population, 1 000 000/µL of sequestered parasites were estimated to delay parasite clearance by 16.8% (95% confidence interval, 5.1%-28.5%), although it was not clear in the A675V population. CONCLUSIONS: In patients with P. falciparum malaria without artemisinin-resistant mutations, intensive sequestration delays parasite clearance after treatment, which may contribute to reduced artemisinin efficacy.

Circulation of an Artemisinin-Resistant Malaria Lineage in a Traveler Returning from East Africa to France.

A returned traveler to Uganda presented with a Plasmodium falciparum kelch13 A675V mutant infection that exhibited delayed clearance under artesunate therapy. Parasites were genetically related to recently reported Ugandan artemisinin-resistant A675V parasites. Adequate malaria prevention measures and clinical and genotypic surveillance are important tools to avoid and track artemisinin resistance.

Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda.

BACKGROUND: Usage of chloroquine was discontinued from the treatment of Plasmodium falciparum infection in almost all endemic regions because of global spread of resistant parasites. Since the first report in Malawi, numerous epidemiological studies have demonstrated that the discontinuance led to re-emergence of chloroquine-susceptible P. falciparum, suggesting a possible role in future malaria control. However, most studies were cross-sectional, with few studies looking at the persistence of chloroquine recovery in long term. This study fills the gap by providing, for a period of at least 6 years, proof of persistent re-emergence/stable recovery of susceptible parasite populations using both molecular and phenotypic methods. METHODS: Ex vivo drug-susceptibility assays to chloroquine (n = 319) and lumefantrine (n = 335) were performed from 2013 to 2018 in Gulu, Northern Uganda, where chloroquine had been removed from the official malaria treatment regimen since 2006. Genotyping of pfcrt and pfmdr1 was also performed. RESULTS: Chloroquine resistance (≥ 100 nM) was observed in only 3 (1.3%) samples. Average IC50 values for chloroquine were persistently low throughout the study period (17.4-24.9 nM). Parasites harbouring pfcrt K76 alleles showed significantly lower IC50s to chloroquine than the parasites harbouring K76T alleles (21.4 nM vs. 43.1 nM, p-value = 3.9 × 10-8). Prevalence of K76 alleles gradually increased from 71% in 2013 to 100% in 2018. CONCLUSION: This study found evidence of stable persistence of chloroquine susceptibility with the fixation of pfcrt K76 in Northern Uganda after discontinuation of chloroquine in the region. Accumulation of similar evidence in other endemic areas in Uganda could open channels for possible future re-use of chloroquine as an option for malaria treatment or prevention.

Artemisinin-Resistant Plasmodium falciparum with High Survival Rates, Uganda, 2014-2016.

Because ≈90% of malaria cases occur in Africa, emergence of artemisinin-resistant Plasmodium falciparum in Africa poses a serious public health threat. To assess emergence of artemisinin-resistant parasites in Uganda during 2014-2016, we used the recently developed ex vivo ring-stage survival assay, which estimates ring-stage-specific P. falciparum susceptibility to artemisinin. We conducted 4 cross-sectional surveys to assess artemisinin sensitivity in Gulu, Uganda. Among 194 isolates, survival rates (ratio of viable drug-exposed parasites to drug-nonexposed controls) were high (>10%) for 4 isolates. Similar rates have been closely associated with delayed parasite clearance after drug treatment and are considered to be a proxy for the artemisinin-resistant phenotype. Of these, the PfKelch13 mutation was observed in only 1 isolate, A675V. Population genetics analysis suggested that these possibly artemisinin-resistant isolates originated in Africa. Large-scale surveillance of possibly artemisinin-resistant parasites in Africa would provide useful information about treatment outcomes and help regional malaria control.

A longer-chain acylated derivative of Dictyostelium differentiation-inducing factor-1 enhances the antimalarial activity against Plasmodium parasites.

The spread of malarial parasites resistant to first-line treatments such as artemisinin combination therapies is a global health concern. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one) originally found in the cellular slime mould Dictyostelium discoideum. We previously showed that some derivatives of DIF-1, particularly DIF-1(+2) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) octan-1-one), exert potent antimalarial activities. In this study, we synthesised DIF-1(+3) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) nonan-1-one). We then evaluated the effects of DIF-1(+3) in vitro on Plasmodium falciparum and in vivo over 7 days (50-100 mg/kg/day) in a mouse model of Plasmodium berghei. DIF-1(+3) exhibited a half-maximal inhibitory concentration of approximately 20-30 % of DIF-1(+2) in three laboratory strains with a selectivity index > 263, including in strains resistant to chloroquine and artemisinin. Parasite growth and multiplication were almost completely suppressed by treatment with 100 mg/kg DIF-1(+3). The survival time of infected mice was significantly increased (P = 0.006) with no apparent adverse effects. In summary, addition of an acyl group to DIF-1(+2) to prepare DIF-1(+3) substantially enhanced antimalarial activity, even in drug-resistant malaria, indicating the potential of applying DIF-1(+3) for malaria treatment.

Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in Northern Uganda.

In Uganda, artemether-lumefantrine was introduced as an artemisinin-based combination therapy (ACT) for malaria in 2006. We have previously reported a moderate decrease in ex vivo efficacy of lumefantrine in Northern Uganda, where we also detected ex vivo artemisinin-resistant Plasmodium falciparum. Therefore, it is necessary to search for candidate partner alternatives for ACT. Here, we investigated ex vivo susceptibility to four ACT partner drugs as well as quinine and chloroquine, in 321 cases between 2013 and 2018. Drug-resistant mutations in pfcrt and pfmdr1 were also determined. Ex vivo susceptibility to amodiaquine, quinine, and chloroquine was well preserved, whereas resistance to mefloquine was found in 45.8%. There were few cases of multi-drug resistance. Reduced sensitivity to mefloquine and lumefantrine was significantly associated with the pfcrt K76 wild-type allele, in contrast to the association between chloroquine resistance and the K76T allele. Pfmdr1 duplication was not detected in any of the cases. Amodiaquine, a widely used partner drug for ACT in African countries, may be the first promising alternative in case lumefantrine resistance emerges. Therapeutic use of mefloquine may not be recommended in this area. This study also emphasizes the need for sustained monitoring of antimalarial susceptibility in Northern Uganda to develop proper treatment strategies.

Application of a cell microarray chip system for accurate, highly sensitive, and rapid diagnosis for malaria in Uganda.

Accurate, sensitive, rapid, and easy operative diagnosis is necessary to prevent the spread of malaria. A cell microarray chip system including a push column for the recovery of erythrocytes and a fluorescence detector was employed for malaria diagnosis in Uganda. The chip with 20,944 microchambers (105 µm width and 50 µm depth) was made of polystyrene. For the analysis, 6 µl of whole blood was employed, and leukocytes were practically removed by filtration through SiO2-nano-fibers in a column. Regular formation of an erythrocyte monolayer in each microchamber was observed following dispersion of an erythrocyte suspension in a nuclear staining dye, SYTO 21, onto the chip surface and washing. About 500,000 erythrocytes were analyzed in a total of 4675 microchambers, and malaria parasite-infected erythrocytes could be detected in 5 min by using the fluorescence detector. The percentage of infected erythrocytes in each of 41 patients was determined. Accurate and quantitative detection of the parasites could be performed. A good correlation between examinations via optical microscopy and by our chip system was demonstrated over the parasitemia range of 0.0039-2.3438% by linear regression analysis (R(2) = 0.9945). Thus, we showed the potential of this chip system for the diagnosis of malaria.

Evidence of long term benefit of morbidity reduction due to praziquantel treatment against schistosoma mansoni in kigungu fishing village in entebbe, Uganda.

Praziquantel (PZQ) is efficacious against Schistosoma mansoni. This was prospective cohort study. This study was carried out at Kigungu fishing village, Entebbe, Uganda. The goal of the study was to establish cost effective regiment for mass drug administration (MDA) of Praziquentel in the morbidity reduction of S.mansoni infection. In January 2004, nine hundred and forty five (945) participants were registered in this study. Our analysis was based on examining microscopically three slides prepared from each of 945 stool specimens delivered by each of the participant using modified Kato/Katz method. These included male and female, children and adults living in Kigungu fishing village in Entebbe Uganda. In total 901, cohorts were re-examined for infections clearance six months later in July 2004 and 18 months later in June 2005, 625 cohorts were again re-evaluated for S.mansoni infections after the baseline study. At baseline, (448) of 945 (47.5%) cohorts were S. mansoni positive. All these participants were treatment with a single oral dose of praziquantel at 40mg/kg. At the same time, 495 (52.5%) were S. mansoni negative. Of the 625 (66.3%) cohorts who came back for final review, 80 (12.8%) were still positive for S. mansoni while 210 (33.6%) remained negative after the base line treatment with praziquantel. On the other hand 103 (16.3%) of cohorts who were initially negative at the base line became S.mansoni positive after 18 months and 213(34.1%) remained negative for S.mansoni. The force of re-infection after six months was significant {(P=0.0001), (OR 0.47) CI at 95% (0.31-0.71)}. Nevertheless the force of reinfection was not significant after 18 months {(P=0.766), (OR 0.95) CI at 95% (0.68-1.34)}.The geometric mean eggs excretion of the 80 cohorts who were S.mansoni positive at 18 months was 151.967.This did not reach the geometric mean egg excreted by the same cohorts at baseline which was 285.05. The egg excretion was reduced by 46.8%. Similarly there was marked decrease in clinical symptoms amongst the cohorts. Our study suggests evidence of long-term benefit of praziquantel in Kigungu and that a yearly administration of praziquantel to the community could be a regiment for mass drug administration (MAD) for this community to control schistosomiasis morbidity.

Substitution of malachite green with nigrosin-eosin yellow stain in the Kato-Katz method: microscopical appearance of the helminth eggs.

BACKGROUND: The Kato-Katz thick smear technique is the standard technique recommended by the World Health Organisation for the quantitative diagnosis of Schistosoma mansoni and other intestinal helminth infections. The major problem of the technique is that a few hours after the preparation of slides hookworm eggs over clear and disappear due glycerin. OBJECTIVE: To illustrate clear visibility of different helminth eggs microscopically in Odongo-Aginya method, substitution of malachite green with 7.5% nigrosin in 10% formalin and 5% eosin in 10% formalin. METHOD: Measured, strained stool specimen was stained with mixture of nigrosin/eosin and covered with cellophane cover slips. The prepared slide was examined immediately microscopically. RESULT: Slides prepared with Odongo-Aginya method can be examined immediately or later without compromising the visibility of parasite eggs and larvae. Hookworm eggs remain visible for a long time. CONCLUSION: The present publication shows microscopic appearance of the helminth eggs using the Odongo-Aginya modification.

High prevalence and morbidity of Schistosoma mansoni along the Albert Nile in Uganda.

An epidemiological cross sectional study of Schistosoma mansoni was conducted in two hyper endemic fishing villages of Rhino Camp and Obongi both in West Nile district in northern Uganda in 1991 and 1992. People with various water contacts were registered. A small group of civil servants and clergies with less water contact in the river Nile were studied for control of infection and morbidity. An overall prevalence of 81.5% of the 1367 people studied in both fishing villages of Rhino Camp and Obongi were excreting from 100 to > or = 500 Schistosoma mansoni eggs per gram (epg). 253 18.5% did not have Schistosoma mansoni eggs in their faeces. The influence of socioeconomic factors on infections in the study population was high among poorer illiterates who have frequent water contacts activities with River Nile. The sonomorphological abnormalities of periportal thickening (PT) due to Schistosoma mansoni were performed using ultrasound. 664 patients were found to have various stages of (PT stages 0, I, II and III). A total of 703 (51.4%) patients did not have any periportal thickening (PT 0) in their livers despite the fact that 450 (32.9%) of them had Schistosoma. mansoni eggs in their faeces. The gravities of schistosomiasis in the two villages were similar showing greater morbidity in the younger adults.