Genetic polymorphism of merozoite surface protein 2 and prevalence of K76T pfcrt mutation in Plasmodium falciparum field isolates from Congolese children with asymptomatic infections

<p>Abstract</p> <p>Background</p> <p>In order to prepare the field site for future interventions, the prevalence of asymptomatic <it>Plasmodium falciparum </it>infection was evaluated in a cohort of children living in Brazzaville. <it>Plasmodium falciparum </it>merozoite surface protein 2 gene (<it>msp</it>2) was used to characterize the genetic diversity and the multiplicity of infection. The prevalence of mutant <it>P. falciparum </it>chloroquine resistance transporter (<it>pfcrt</it>) allele in isolates was also determined.</p> <p>Methods</p> <p>Between April and June 2010, 313 children below 10 years of age enrolled in the cohort for malaria surveillance were screened for <it>P. falciparum </it>infection using microscopy and polymerase chain reaction (PCR). The children were selected on the basis of being asymptomatic. <it>Plasmodium falciparum msp2 </it>gene was genotyped by allele-specific nested PCR and the <it>pfcrt </it>K76T mutation was detected using nested PCR followed by restriction endonuclease digestion.</p> <p>Results</p> <p>The prevalence of asymptomatic <it>P. falciparum </it>infections was 8.6% and 16% by microscopy and by PCR respectively. Allele typing of the <it>msp2 </it>gene detected 55% and 45% of 3D7 and FC27 allelic families respectively. The overall multiplicity of infections (MOI) was 1.3. A positive correlation between parasite density and multiplicity of infection was found. The prevalence of the mutant <it>pfcrt </it>allele (T76) in the isolates was 92%.</p> <p>Conclusion</p> <p>This is the first molecular characterization of <it>P. falciparum </it>field isolates in Congolese children, four years after changing the malaria treatment policy from chloroquine (CQ) to artemisinin-based combination therapy (ACT). The low prevalence of asymptomatic infections and MOI is discussed in the light of similar studies conducted in Central Africa.</p

IN VITRO EVALUATION OF ANTIPLASMODIAL ACTIVITY OF EXTRACTS OF ACANTHOSPERMUM HISPIDUM DC (ASTERACEAE) AND FICUS THONNINGII BLUME (MORACEAE), TWO PLANTS USED IN TRADITIONAL MEDICINE IN THE REPUBLIC OF CONGO.

The aim of this study was to evaluate extracts from two medicinal plants, Acanthospermum hispidum and Ficus thonningii, used in traditional medicine in Congo Brazzaville, for in vitro antiplasmodial activities against two laboratory strains of Plasmodium falciparum: the chloroquine sensitive 3D7 and the chloroquine resistant Dd2. ELISA HRP2 assay was used to evaluate the in vitro inhibitory activity of the extracts alone or in combination with chloroquine. Cytotoxicity was assessed on human HeLa cell line and reflected by the selectivity index. Methanolic extract of Acanthospermum hispidum exhibited a strong and a moderate inhibitory activity on the growth of Dd2 and 3D7 at 2.8 µg/ml and 9.2 µg/ml concentrations respectively with a selectivity index >10. The combination of the most active extract (methanolic extract of Acanthospermum hispidum) with chloroquine showed a synergistic interaction on both strains. The good selectivity index of Acanthospermum hispidum on HeLa cells reflects the safety of this plant. Extracts from Ficus thonningii did not show any promising antiplasmodial activity on both 3D7 and Dd2. Except the methanolic extract which exhibited a slight antiplasmodial activity with inhibitory concentration and selectivity index corresponding to 9.61 µg/ml and 11.16 respectively. Methanolic extract of Acanthospermum hispidum exhibited moderate to high inhibitory activity on 3D7 and Dd2 laboratory strains and a synergistic antimalarial effect when combined with chloroquine. Ficus thonningii seems to have no antimalarial activity. Phytochemical analysis, in vivo investigations using animal models and later clinical trials in collaboration with traditional practitioners are necessary to clarify the potential antimalarial activity of both plants

Elevated Plasmodium sporozoite infection and multiple insecticide resistance in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon

Background: Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance, Plasmodium infection rate, insecticide resistance profiles and underlying resistance mechanisms. Methods: Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a village located 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F1 adult progeny. Bioassays were performed to assess resistance profile to insecticides. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance and Plasmodium infection rates were investigated. Results: Anopheles funestus s.s. was most abundant species in Elende (85%) followed by Anopheles gambiae s.s. (15%) with both having a similar sporozoite rate. Both species exhibited high levels of resistance to pyrethroids (<40% mortality). An. gambiae s.s. was also resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate. An. funestus s.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119S ace-1 resistance allele (15%) were detected in An. funestus s.s. and An. gambiae s.s., respectively. Furthermore, the high pyrethroid/DDT resistances in An. gambiae s.s. corresponded with an increase frequency of 1014F kdr allele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression of CYP6P5, CYP6P9a and CYP6P9b. Conclusion: The resistance to multiple insecticide classes observed in these vector populations alongside the high Plasmodium sporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas

Pyronaridine–artesunate real-world safety, tolerability, and effectiveness in malaria patients in 5 African countries: A single-arm, open-label, cohort event monitoring study

BACKGROUND In Phase II/III randomized controlled clinical trials for the treatment of acute uncomplicated malaria, pyronaridine-artesunate demonstrated high efficacy and a safety profile consistent with that of comparators, except that asymptomatic, mainly mild-to-moderate transient increases in liver aminotransferases were reported for some patients. Hepatic safety, tolerability, and effectiveness have not been previously assessed under real-world conditions in Africa. METHODS AND FINDINGS This single-arm, open-label, cohort event monitoring study was conducted at 6 health centers in Cameroon, Democratic Republic of Congo, Gabon, Ivory Coast, and Republic of Congo between June 2017 and April 2019. The trial protocol as closely as possible resembled real-world clinical practice for the treatment of malaria at the centers. Eligible patients were adults or children of either sex, weighing at least 5 kg, with acute uncomplicated malaria who did not have contraindications for pyronaridine-artesunate treatment as per the summary of product characteristics. Patients received fixed-dose pyronaridine-artesunate once daily for 3 days, dosed by body weight, without regard to food intake. A tablet formulation was used in adults and adolescents and a pediatric granule formulation in children and infants under 20 kg body weight. The primary outcome was the hepatic event incidence, defined as the appearance of the clinical signs and symptoms of hepatotoxicity confirmed by a >2× rise in alanine aminotransferase/aspartate aminotransferase (ALT/AST) versus baseline in patients with baseline ALT/AST >2× the upper limit of normal (ULN). As a secondary outcome, this was assessed in patients with ALT/AST >2× ULN prior to treatment versus a matched cohort of patients with normal baseline ALT/AST. The safety population comprised 7,154 patients, of mean age 13.9 years (standard deviation (SD) 14.6), around half of whom were male (3,569 [49.9%]). Patients experienced 8,560 malaria episodes; 158 occurred in patients with baseline ALT/AST elevations >2×ULN. No protocol-defined hepatic events occurred following pyronaridine-artesunate treatment of malaria patients with or without baseline hepatic dysfunction. Thus, no cohort comparison could be undertaken. Also, as postbaseline clinical chemistry was only performed where clinically indicated, postbaseline ALT/AST levels were not systematically assessed for all patients. Adverse events of any cause occurred in 20.8% (1,490/7,154) of patients, most frequently pyrexia (5.1% [366/7,154]) and vomiting (4.2% [303/7,154]). Adjusting for Plasmodium falciparum reinfection, clinical effectiveness at day 28 was 98.6% ([7,369/7,746] 95% confidence interval (CI) 98.3 to 98.9) in the per-protocol population. There was no indication that comorbidities or malnutrition adversely affected outcomes. The key study limitation was that postbaseline clinical biochemistry was only evaluated when clinically indicated. CONCLUSIONS Pyronaridine-artesunate had good tolerability and effectiveness in a representative African population under conditions similar to everyday clinical practice. These findings support pyronaridine-artesunate as an operationally useful addition to the management of acute uncomplicated malaria. TRIAL REGISTRATION ClinicalTrials.gov NCT03201770

Multiple insecticide resistance and Plasmodium infection in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon

Background: Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance, Plasmodium infection rate, insecticide resistance profiles and underlying resistance mechanisms. Methods: Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a locality situated 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F 1 adults. Bioassays were performed to assess resistance profile to the four insecticides classes. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance and Plasmodium infection rates were investigated. Results: Anopheles funestus s.s. was the most abundant species in Elende (85%) followed by Anopheles gambiae s.s. (15%) with both having similar sporozoite rate. Both species exhibited high levels of resistance to the pyrethroids, permethrin and deltamethrin (&lt;40% mortality). An. gambiae s.s. was resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate. An. funestus s.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119S ace-1 resistance allele (15%) were detected in An. funestus s.s. and An. gambiae s.s., respectively. Furthermore, the high pyrethroid/DDT resistances in An. gambiae corresponded with an increase frequency of 1014F kdr allele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression of CYP6P5, CYP6P9a and CYP6P9b. Conclusion: The resistance to multiple insecticide classes observed in these vector populations alongside the significant Plasmodium sporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas

Plasmodium falciparum infection in febrile Congolese children: prevalence of clinical malaria ten years after introduction of Artemisinin-combination therapies

dataset used in the paper