Escalating Plasmodium falciparum K13 marker prevalence indicative of artemisinin resistance in southern Rwanda.

In 2023, we updated data collected since 2010 on Plasmodium falciparum K13 and MDR1 drug resistance markers in Huye district, southern Rwanda. Artemisinin resistance-associated PfK13 markers occurred in 17.5% of 212 malaria patients (561H, 9.0%; 675V, 5.7%; and 469F, 2.8%), nearly double the frequency from 2019. PfMDR1 N86, linked with lumefantrine tolerance, was close to fixation at 98%. In southern Rwanda, markers signaling resistance to artemisinin and lumefantrine are increasing, albeit at a relatively slow rate.

ESBL-producing Enterobacteriaceae in a rural Rwandan community: Carriage among community members, livestock, farm products and environment.

OBJECTIVES: Extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) are spreading globally. However, respective data from African communities including livestock and environmental specimens are rare. In a rural community of southern Rwanda, we assessed intestinal carriage of ESBL-PE among residents and livestock as well as presence in household specimens and examined associated factors. METHODS: Samples of humans and livestock (both rectal swabs), soil, water, vegetables and animal products were collected within 312 community households in Sovu, Southern Rwanda. Specimens were screened for ESBL-PE on chromogenic agar, and susceptibility to common antibiotics was determined by disc diffusion assays. Socio-demographic information was collected with questionnaires focusing on the socio-economic background, alimentation, living conditions, hygiene measures and medical history of the participants. RESULTS: Data and specimens from 312 randomly selected households including 617 human beings, 620 livestock and of approximately each 300 kitchen vegetables, animal products, soil and drinking water were analysed. Overall, 14.8% of 2508 collected samples were positive for ESBL-PE; figures were highest for humans (37.9%) and livestock (15.6%), lower for vegetables (3.8%) and animal products (3.3%), and lowest for soil (1.6%) and water (0.6%). Most detected ESBL-PE were Escherichia coli (93.5%) in addition to Klebsiella pneumoniae (6.5%). Cross-resistance to ampicillin-sulbactam, ciprofloxacin and co-trimoxazole was common. Logistic regression identified increasing age, another ESBL-PE positive household member, prolonged time for fetching water, current diarrhoea and the ability to pay school fees as independent predictors of intestinal ESBL-PE carriage among community members. CONCLUSIONS: ESBL-PE carriage is common in a rural Rwandan farming community. Carriage in livestock is not associated with human carriage. Associated factors suggest few addressable risk factors. The data indicate that in southern Rwanda, ESBL-PE are no longer primarily hospital-based but circulate in the community.

Plasmodium vivax Malaria in Duffy-Positive Patients in Rwanda.

Plasmodium vivax is the second-most common malaria pathogen globally, but is considered very rare in the predominantly Duffy-negative sub-Saharan African population. In 259 malaria patients from highland southern Rwanda, we assessed Plasmodium species and Duffy blood group status by polymerase chain reaction (PCR). Plasmodium falciparum, P. vivax, Plasmodium malariae, and Plasmodium ovale were seen in 90.7%, 8.1%, 11.6%, and 5.0%, respectively. Plasmodium vivax occurred more frequently as a monoinfection than in combination with P. falciparum. All P. vivax-infected individuals showed heterozygous Duffy positivity, whereas this was the case for only 3.1% of patients with P. falciparum monoinfection and malaria-negative control subjects (P < 0.01). Based on PCR diagnosis, P. vivax is not rare in southern Rwanda. All episodes of P. vivax were observed in heterozygous Duffy-positive patients, whereas elsewhere in Africa, P. vivax is also reported in Duffy-negative individuals. Refined mapping of Plasmodium species is required to establish control and elimination strategies including all malaria species.

In Vitro Confirmation of Artemisinin Resistance in Plasmodium falciparum from Patient Isolates, Southern Rwanda, 2019.

Artemisinin resistance in Plasmodium falciparum is conferred by mutations in the kelch 13 (K13) gene. In Rwanda, K13 mutations have increased over the past decade, including mutations associated with delayed parasite clearance. We document artemisinin resistance in P. falciparum patient isolates from Rwanda carrying K13 R561H, A675V, and C469F mutations.

Changing Pattern of Plasmodium falciparum pfmdr1 Gene Polymorphisms in Southern Rwanda.

Plasmodium falciparum multidrug resistance-1 gene (pfmdr1) polymorphisms associate with altered antimalarial susceptibility. Between 2010 and 2018/2019, we observed that the prevalence of the wild-type allele N86 and the wild-type combination NYD increased 10-fold (4% versus 40%) and more than 2-fold (18% versus 44%), respectively. Haplotypes other than NYD or NFD declined by up to >90%. Our molecular data suggest the pfmdr1 pattern shifted toward one associated with artemether-lumefantrine resistance.

Increase in Kelch 13 Polymorphisms in Plasmodium falciparum, Southern Rwanda.

Artemisinin resistance in Plasmodium falciparum is associated with nonsynonymous mutations in the Kelch 13 (K13) propeller domain. We found that 12.1% (8/66) of clinical P. falciparum isolates from Huye district, Rwanda, exhibited K13 mutations, including R561H, a validated resistance marker. K13 mutations appear to be increasing in this region.

Co-infections with Plasmodium, Ascaris and Giardia among Rwandan schoolchildren.

OBJECTIVES: Co-infections with Plasmodium, Ascaris and Giardia are common in sub-Saharan Africa but epidemiological and clinical data are rare. We examined factors associated with co-infections and their clinical manifestation among Rwandan schoolchildren. METHODS: Schoolchildren aged 6-10 years attending 12 schools in Huye district, Rwanda, were recruited preceding routine deworming. Data on socioeconomic status (SES) and children's histories were obtained, and children were clinically and anthropometrically examined. Blood and stool samples were collected, and infections with Plasmodium, Ascaris and Giardia were determined by microscopy and PCR assays. RESULTS: Among 878 schoolchildren, Plasmodium, Ascaris and Giardia were present in 22%, 35% and 36%, respectively. Co-infections with two or more parasites were found in 24%; only one-third of the children did not harbour any of the parasites examined. Factors associated with parasite (co-)infections largely overlapped and reflected low SES, in addition to a few specific risk factors. Clinically, most children were asymptomatic but anaemia (38%), underweight (17%), and reported signs and symptoms in the preceding 2 weeks (46%) were common. Many of the reported and assessed signs and symptoms were associated with Plasmodium infection, and co-infection with Ascaris and/or Giardia did basically not modify the clinical picture. One exception was malnutrition, which was pronounced in Ascaris-Giardia co-infection vs. individual mono-infections. CONCLUSIONS: Parasitic co-infections are common in Rwandan schoolchildren, and are associated with a rather silent clinical manifestation that nevertheless may affect school performance and long-term development. School-based health interventions should target such co-infections in an integrated manner.

OBJECTIFS: Les coinfections par Plasmodium, Ascaris et Giardia sont courantes en Afrique subsaharienne, mais les données épidémiologiques et cliniques sont rares. Nous avons examiné les facteurs associés aux coinfections et leurs manifestations cliniques chez les écoliers rwandais. MÉTHODES: Des écoliers âgés de 6 à 10 ans fréquentant 12 écoles du district de Huye au Rwanda ont été recrutés avant le déparasitage de routine. Les données sur le statut socioéconomique (SSE) et les antécédents des enfants ont été obtenues et les enfants ont été examinés cliniquement et anthropométriquement. Des échantillons de sang et de selles ont été recueillis et les infections à Plasmodium, Ascaris et Giardia ont été déterminées par microscopie et par PCR. RÉSULTATS: sur 878 écoliers, Plasmodium, Ascaris et Giardia étaient présents chez 22%, 35% et 36%, respectivement. Des coinfections avec deux parasites ou plus ont été trouvées chez 24%; seul un tiers des enfants n'hébergeait aucun des parasites examinés. Les facteurs associés aux (co)infections parasitaires se chevauchaient largement et reflétaient un faible statut SSE, en plus de quelques facteurs de risque spécifiques. Sur le plan clinique, la plupart des enfants étaient asymptomatiques mais l'anémie (38%), l'insuffisance pondérale (17%) et les signes et symptômes rapportés au cours des deux semaines précédentes (46%) étaient fréquents. De nombreux signes et symptômes rapportés et évalués étaient associés à l'infection au Plasmodium et la coinfection par Ascaris et/ou Giardia n'a fondamentalement pas modifié le tableau clinique. Une exception était la malnutrition, qui était prononcée dans la coinfection Ascaris-Giardia par rapport aux mono-infections individuelles. CONCLUSIONS: Les coinfections parasitaires sont courantes chez les écoliers rwandais et sont associées à une manifestation clinique plutôt silencieuse qui peut néanmoins affecter les performances scolaires et le développement à long terme. Les interventions de santé en milieu scolaire devraient cibler ces coinfections de manière intégrée.

Comment on "The optimal timing of post-treatment sampling for the assessment of anthelminthic drug efficacy against Ascaris infections in humans".

A recent publication by Levecke et al. (Int. J. Parasitol, 2018, 8, 67-69) provides important insights into the kinetics of worm expulsion from humans following treatment with albendazole. This is an important aspect of determining the optimal time-point for post treatment sampling to examine anthelmintic drug efficacy. The authors conclude that for the determination of drug efficacy against Ascaris, samples should be taken not before day 14 and recommend a period between days 14 and 21. Using this recommendation, they conclude that previous data (Krücken et al., 2017; Int. J. Parasitol, 7, 262-271) showing a reduction of egg shedding by 75.4% in schoolchildren in Rwanda and our conclusions from these data should be interpreted with caution. In reply to this, we would like to indicate that the very low efficacy of 0% in one school and 52-56% in three other schools, while the drug was fully efficient in other schools, cannot simply be explained by the time point of sampling. Moreover, there was no correlation between the sampling day and albendazole efficacy. We would also like to indicate that we very carefully interpreted our data and, for example, nowhere claimed that we found anthelmintic resistance. Rather, we stated that our data indicated that benzimidazole resistance may be suspected in the study population. We strongly agree that the data presented by Levecke et al. suggests that recommendations for efficacy testing of anthelmintic drugs should be revised.

Reduced efficacy of albendazole against Ascaris lumbricoides in Rwandan schoolchildren.

Control of human soil-transmitted helminths (STHs) relies on preventive chemotherapy of schoolchildren applying the benzimidazoles (BZ) albendazole or mebendazole. Anthelmintic resistance (AR) is a common problem in nematodes of veterinary importance but for human STHs, information on drug efficacy is limited and routine monitoring is rarely implemented. Herein, the efficacy of single dose albendazole (400 mg) was evaluated in 12 schools in the Huye district of Rwanda where Ascaris is the predominant STH. Ascaris eggs were detected by wet mount microscopy and the Mini-FLOTAC method to assess cure rate (CR) and faecal egg count reduction (FECR). Blood and faecal samples were analysed for co-infections with Plasmodium sp. and Giardia duodenalis, respectively. Ascaris positive samples collected before and after treatment were analysed for putatively BZ-resistance associated ß-tubulin gene single nucleotide polymorphisms. The overall CR was 69.9% by Mini-FLOTAC and 88.6% by wet mount microscopy. The FECR was 75.4% and the 95% calculated confidence intervals were 50.4-87.8% using sample variance, 55.4-88.8% by bootstrapping, and 75.0-75.7% applying a Markov Chain Monte Carlo Bayesian approach. FECR varied widely between 0 and 96.8% for individual schools. No putative BZ-resistance associated polymorphisms were found in the four Ascaris ß-tubulin isotype genes examined. Since FECRs <95% indicate reduced efficacy, these findings raise the suspicion of BZ resistance. In the absence of respective molecular evidence, heritable AR in the local Ascaris populations cannot be formally proven. However, since FECRs <95% indicate reduced efficacy, BZ resistance may be suspected which would be alarming and calls for further analyses and routine monitoring in preventive chemotherapy programs.

Asymptomatic only at first sight: malaria infection among schoolchildren in highland Rwanda.

BACKGROUND: Plasmodium infection and malaria in school children are increasingly recognized as a relevant public health problem, but data on actual prevalence and health consequences are insufficient. The present study from highland southern Rwanda aimed at estimating infection prevalence among children attending school, at identifying associated factors and at assessing the clinical consequences of these infections. METHODS: In a survey including 12 schools in the Huye district of Rwanda, 1089 children aged 6-10 years were clinically and anthropometrically examined, malaria parasites were diagnosed by microscopy and PCR, haemoglobin concentrations were measured, and socio-economic and behavioural parameters as well as medical histories were obtained. RESULTS: Upon examination, the vast majority of children was asymptomatic (fever 2.7%). Plasmodium infection was detected in 22.4% (Plasmodium falciparum, 18.8%); 41% of these were submicroscopic. Independent predictors of infection included low altitude, higher age, preceding antimalarial treatment, and absence of electricity or a bicycle in the household. Plasmodium infection was associated with anaemia (mean haemoglobin difference of -1.2 g/dL; 95% CI, -0.8 to -1.5 g/dL), fever, underweight, clinically assessed malnutrition and histories of fever, tiredness, weakness, poor appetite, abdominal pain, and vomiting. With the exception of underweight, these conditions were also increased at submicroscopic infection. CONCLUSION: Malaria infection is frequent among children attending school in southern highland Rwanda. Although seemingly asymptomatic in the vast majority of cases, infection is associated with a number of non-specific symptoms in the children´s histories, in addition to the impact on anaemia. This argues for improved malaria surveillance and control activities among school children.