Dual CRISPR/Cas13a Cascade Strand Displacement-Triggered Transcription for Point-of-Care Detection of Plasmodium in Asymptomatic Malaria.

Asymptomatic infections of Plasmodium parasites are major obstacles to malaria control and elimination. A sensitive, specific, and user-friendly method is urgently needed for point-of-care (POC) Plasmodium diagnostics in asymptomatic malaria, especially in resource-limited settings. In this work, we present a POC method (termed Cas13a-SDT) based on the cascade sequence recognition and signal amplification of dual Cas13a trans-cleavage and strand displacement-triggered transcription (SDT). Cas13a-SDT not only achieves exceptional specificity in discriminating the target RNA from nontarget RNAs with any cross-interaction but also meets the sensitivity criterion set by the World Health Organization (WHO) for effective malaria detection. Remarkably, this novel method was successfully applied to screen malaria in asymptomatic infections from clinical samples. The proposed method provides a user-friendly and visually interpretable output mode while maintaining high accuracy and reliability comparable to RT-PCR. These excellent features demonstrate the significant potential of Cas13a-SDT for POC diagnosis of Plasmodium infections, laying a vital foundation for advancing malaria control and elimination efforts.

Exploring mosquito abundance and Plasmodium infection through nested-PCR: implications for disease surveillance and control.

The Plasmodium is responsible for malaria which poses a major health threat, globally. This study is based on the estimation of the relative abundance of mosquitoes, and finding out the correlations of meteorological parameters (temperature, humidity and rainfall) with the abundance of mosquitoes. In addition, this study also focused on the use of nested PCR (species-specific nucleotide sequences of 18S rRNA genes) to explore the Plasmodium spp. in female Anopheles. In the current study, the percentage relative abundance of Culex mosquitoes was 57.65% and Anopheles 42.34% among the study areas. In addition, the highest number of mosquitoes was found in March in district Mandi Bahauddin at 21 °C (Tmax = 27, Tmin = 15) average temperature, 69% average relative humidity and 131 mm rainfall, and these climatic factors were found to affect the abundance of the mosquitoes, directly or indirectly. Molecular analysis showed that overall, 41.3% of the female Anopheles pools were positive for genus Plasmodium. Among species, the prevalence of Plasmodium (P.) vivax (78.1%) was significantly higher than P. falciparum (21.9%). This study will be helpful in the estimation of future risk of mosquito-borne diseases along with population dynamic of mosquitoes to enhance the effectiveness of vector surveillance and control programs.

Dynamics of Plasmodium species and genotype diversity in adults with asymptomatic infection in Gabon.

OBJECTIVES: We investigated the diversity and dynamics of Plasmodium infection in serially collected samples from asymptomatic participants of a clinical trial assessing the efficacy and safety of ivermectin in Gabon. We checked whether the baseline sample reflected the P. falciparum genotype and Plasmodium species diversity seen over 7 days of follow-up. METHODS: Blood samples were collected at inclusion, every 8 hours until hour 72, daily until day 7, and on day 14. Plasmodium species was determined by qPCR and pfmsp1 length polymorphism was assessed for P. falciparum genotyping. RESULTS: In 17/48 (35%) individuals, all pfmsp1 genotypes identified during the assessed period were detected at baseline; in 31/48 (65%), new genotypes were found during follow-up. Additional sampling at hour 24 allowed the identification of all genotypes seen over 7 days in 50% of the individuals. Ivermectin did not impact the genotype dynamics. Mixed Plasmodium spp. infections were detected in 28/49 (57%) individuals at baseline, and detection of non-falciparum infections during follow-up varied. CONCLUSIONS: Our results reveal complex intra-host dynamics of P. falciparum genotypes and Plasmodium species and underscore the importance of serial sampling in clinical trials for antimalarial drugs with asymptomatically P. falciparum-infected individuals. This might allow a more accurate identification of genotypes in multiple infections, impacting the assessment of drug efficacy.

Prevalence and genetic diversity of simian malaria in wild macaque populations across Thailand: Implications for human health.

Over the past year, P. falciparum infections have declined in Thailand, yet nonhuman primate malaria infections have correspondingly increased, including Plasmodium knowlesi and P. cynomolgi. Nevertheless, little is known about simian malaria in its natural macaque hosts, Macaca mulatta and Macaca fascicularis. This study aims to address several research questions, including the prevalence and distribution of simian malaria in these two Thai wild macaque species, variations in infection between different macaque species and between M. fascicularis subspecies, and the genetic composition of these pathogens. Blood samples were collected from 82 M. mulatta and 690 M. fascicularis across 15 locations in Thailand, as well as two locations in Vietnam and Myanmar. We employed quantitative real-time PCR targeting the Plasmodium genus-specific 18S ribosomal RNA (rRNA) gene to detect malaria infection, with a limit of detection set at 1,215.98 parasites per mL. We genotyped eight microsatellite markers, and the P. cynomolgi dihydrofolate reductase gene (DHFR) was sequenced (N = 29). In total, 100 of 772 samples (13 %) tested positive for malaria, including 45 (13 %) for P. cynomolgi, 37 (13 %) for P. inui, 16 (5 %) for P. coatneyi, and 2 (0.25 %) for Hepatocystis sp. in Saraburi, central and Ranong, southern Thailand. Notably, simian malaria infection was observed exclusively in M. fascicularis and not in M. mulatta (P = 0.0002). Particularly, P. cynomolgi was detected in 21.7 % (45/207) of M. f. fascicularis living in Wat Tham Phrapothisat, Saraburi Province. The infection with simian malaria was statistically different between M. fascicularis and M. mulatta (P = 0.0002) but not within M. fascicularis subspecies (P = 0.78). A haplotype network analysis revealed that P. cynomolgi shares a lineage with reference strains obtained from macaques. No mutation in the predicted binding pocket of PcyDHFR to pyrimethamine was observed. This study reveals a significant prevalence of simian malaria infection in M. fascicularis. The clonal genotypes of P. cynomolgi suggest in-reservoir breeding. These findings raise concerns about the potential spread of nonhuman primate malaria to humans and underscore the need for preventive measures.

Confirmatory Assay for Laboratory Diagnosis of Malaria Using Molecular Approach.

BACKGROUND: Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite. METHODS: Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18 S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel. RESULTS AND CONCLUSION: The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination.

Malaria Prevalence in Asymptomatic and Symptomatic Children Living in Rural, Semi-Urban and Urban Areas in Eastern Gabon.

BACKGROUND: Malaria remains a major public health issue in the world despite a decline in the disease burden. However, though symptomatic malaria is diagnosed and treated, asymptomatic infections remain poorly known and support transmission. This study assessed the prevalence of symptomatic and asymptomatic Plasmodium spp. infections in three areas in Gabon to monitor and evaluate the impact of malaria. METHODS AND RESULTS: A cross-sectional study was conducted in three areas of Gabon. Febrile and afebrile children aged 6 months to 15 years were included in this study. Malaria prevalence was determined by microscopy of and using rapid diagnostic test (RDT). Plasmodium spp. species were identified by PCR according to the Snounou method. The data were recorded in Excel, and the statistical analyses were performed using the software R version R 64 × 3.5.0. A total of 2381(333 asymptomatic and 107 symptomatic) children were included. The overall prevalence of malaria was 40% (952/2381), with the majority (77% symptomatic and 98% asymptomatic) of infections caused by Plasmodium falciparum. A high prevalence of malaria was found in infected children in rural and semi-rural areas. In these two areas, a higher prevalence of Plasmodium malariae was observed in asymptomatic. Furthermore, mixed infections were more prevalent in asymptomatic children than in symptomatic. CONCLUSION: This study showed that the prevalence of Plasmodium spp. infection varied according to the regions. The main species was Plasmodium falciparum, but in asymptomatic children the prevalence of Plasmodium malariae was high in rural areas. To help fight malaria more effectively asymptomatic infections should be taken into account and treated.

Evaluation of Malaria Microscopy Diagnostic Performance at 40 Public Health Facilities in Abidjan, Côte d'Ivoire in 2020.

PURPOSE: Microscopic diagnosis of Giemsa-stained thick and thin blood films remained the gold standard laboratory method for the diagnosis of malaria. In this context, we felt it was important to conduct this evaluation with 40 public medical biology laboratories (MBLs) in the Abidjan 1 health region that perform blood parasitology tests to improve their implementation process. METHODS: This descriptive and analytical study took place in July 2020 and involved participating laboratories (PLs) from the public sector in Abidjan. A set of 3 blood smear slides of variable parasite densities (PDs) with assigned values (AVs) of parasite densities and assigned Plasmodium species was used. The criterion for establishing the parasite density compliance interval was assigned values of ± 25%, and the performance rates were compared to the 80% recommended by the WHO for the African region. RESULTS: Nearly a quarter (11/40) of the participating laboratories had a compliance rate greater than 80%, including 10 with a performance of 100% for the ability to identify parasites. Regarding identifying plasmodial species, a concordance rate of 100% was obtained for slide 1 for Plasmodium falciparum, while this rate was 20% for slide 2 for Plasmodium ovale. For parasite densities < 200/µl, 87.5% of the participating laboratories (PLs) had a performance rate lower than 80%, while 95% of these PLs had a performance rate higher than 80% for parasitaemia > 2000/µl. CONCLUSIONS: There is a need to strengthen adapted to low parasitaemia, to improve the biological confirmation of malaria in Côte d'Ivoire.

Investigation of Intestinal and Blood Parasites in People Returning to Turkey with a History of Traveling Abroad During the Pandemic

Objective: To investigate intestinal and blood parasites in people who have a history of traveling abroad during the Coronavirus disease-2019 pandemic and returning to Turkey. Methods: In this study, 104 patients with gastrointestinal system and/or fever complaints who had traveled abroad during the pandemic period and returned to Turkey were included. Parasitic agents were investigated by taking blood and stool samples from the patients. Additionally, urine samples were obtained from patients with hematuria or dysuria with the suspicion of schistosomiasis. A direct microscopic examination, the Crypto-Giardia immunochromatographic test, and ELISA methods were used in the examination of the stool samples. In order to detect Plasmodium species, blood samples were examined by preparing both the rapid diagnostic test and thick drop and thin smear preparations. Results: One or more parasite species were detected in 38 (38.5%) of 104 patients included in the study. While intestinal parasites were detected in 16 (32%) of 50 patients who traveled to Iran and 16 (33.3%) of 48 patients who traveled to Northern Iraq, blood parasites were not found. Schistosoma mansoni was detected in all 5 of the patients with a history of traveling to Sudan. Plasmodium falciparum was detected in 1 patient who traveled to the African continent. Conclusion: It is vital to take precautions to prevent parasitic diseases, such as malaria and schistosomiasis, during travels to African countries. During travels to neighboring countries of Turkey, such as Northern Iraq and Iran, hygiene should be paid attention to, so as to prevent contracting intestinal parasitic diseases. In addition, it was concluded that people who plan to travel abroad should have information about the endemic parasitic diseases of the country that they are going to.

Characterising co-infections with Plasmodium spp., Mansonella perstans or Loa loa in asymptomatic children, adults and elderly people living on Bioko Island using nucleic acids extracted from malaria rapid diagnostic tests.

BACKGROUND: Regular and comprehensive epidemiological surveys of the filarial nematodes Mansonella perstans and Loa loa in children, adolescents and adults living across Bioko Island, Equatorial Guinea are lacking. We aimed to demonstrate that blood retained on malaria rapid diagnostic tests, commonly deployed for malaria surveys, could be used as a source of nucleic acids for molecular based detection of M. perstans and L. loa. We wanted to determine the positivity rate and distribution of filarial nematodes across different age groups and geographical areas as well as to understand level of co-infections with malaria in an asymptomatic population. METHODOLOGY: M. perstans, L. loa and Plasmodium spp. parasites were monitored by qPCR in a cross-sectional study using DNA extracted from a subset malaria rapid diagnostic tests (mRDTs) collected during the annual malaria indicator survey conducted on Bioko Island in 2018. PRINCIPAL FINDINGS: We identified DNA specific for the two filarial nematodes investigated among 8.2% (263) of the 3214 RDTs screened. Positivity rates of M. perstans and L. loa were 6.6% and 1.5%, respectively. M. perstans infection were more prominent in male (10.5%) compared to female (3.9%) survey participants. M. perstans parasite density and positivity rate was higher among older people and the population living in rural areas. The socio-economic status of participants strongly influenced the infection rate with people belonging to the lowest socio-economic quintile more than 3 and 5 times more likely to be L. loa and M. perstans infected, respectively. No increased risk of being co-infected with Plasmodium spp. parasites was observed among the different age groups. CONCLUSIONS/SIGNIFICANCE: We found otherwise asymptomatic individuals were infected with M. perstans and L. loa. Our study demonstrates that employing mRDTs probed with blood for malaria testing represents a promising, future tool to preserve and ship NAs at room temperature to laboratories for molecular, high-throughput diagnosis and genotyping of blood-dwelling nematode filarial infections. Using this approach, asymptomatic populations can be reached and surveyed for infectious diseases beyond malaria.

Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant.

Aims: Genetically encoded green fluorescent protein (GFP)-based redox biosensors are widely used to monitor specific and dynamic redox processes in living cells. Over the last few years, various biosensors for a variety of applications were engineered and enhanced to match the organism and cellular environments, which should be investigated. In this context, the unicellular intraerythrocytic parasite Plasmodium, the causative agent of malaria, represents a challenge, as the small size of the organism results in weak fluorescence signals that complicate precise measurements, especially for cell compartment-specific observations. To address this, we have functionally and structurally characterized an enhanced redox biosensor superfolder roGFP2 (sfroGFP2). Results: SfroGFP2 retains roGFP2-like behavior, yet with improved fluorescence intensity (FI) in cellulo. SfroGFP2-based redox biosensors are pH insensitive in a physiological pH range and show midpoint potentials comparable with roGFP2-based redox biosensors. Using crystallography and rigidity theory, we identified the superfolding mutations as being responsible for improved structural stability of the biosensor in a redox-sensitive environment, thus explaining the improved FI in cellulo. Innovation: This work provides insight into the structure and function of GFP-based redox biosensors. It describes an improved redox biosensor (sfroGFP2) suitable for measuring oxidizing effects within small cells where applicability of other redox sensor variants is limited. Conclusion: Improved structural stability of sfroGFP2 gives rise to increased FI in cellulo. Fusion to hGrx1 (human glutaredoxin-1) provides the hitherto most suitable biosensor for measuring oxidizing effects in Plasmodium. This sensor is of major interest for studying glutathione redox changes in small cells, as well as subcellular compartments in general. Antioxid. Redox Signal. 37, 1-18.

Nationwide molecular surveillance of three Plasmodium species harboured by symptomatic malaria patients living in Ghana.

BACKGROUND: Clinical presentations of malaria in Ghana are primarily caused by infections containing microscopic densities of Plasmodium falciparum, with a minor contribution from Plasmodium malariae and Plasmodium ovale. However, infections containing submicroscopic parasite densities can result in clinical disease. In this study, we used PCR to determine the prevalence of three human malaria parasite species harboured by suspected malaria patients attending healthcare facilities across the country. METHODS: Archived dried blood spots on filter paper that had been prepared from whole blood collected from 5260 patients with suspected malaria attending healthcare facilities across the country in 2018 were used as experimental material. Plasmodium species-specific PCR was performed on DNA extracted from the dried blood spots. Demographic data and microscopy data for the subset of samples tested were available from the original study on these specimens. RESULTS: The overall frequency of P. falciparum, P. malariae and P. ovale detected by PCR was 74.9, 1.4 and 0.9%, respectively. Of the suspected symptomatic P. falciparum malaria cases, 33.5% contained submicroscopic densities of parasites. For all regions, molecular diagnosis of P. falciparum, P. malariae and P. ovale was significantly higher than diagnosis using microscopy: up to 98.7% (75/76) of P. malariae and 97.8% (45/46) of P. ovale infections detected by PCR were missed by microscopy. CONCLUSION: Plasmodium malariae and P. ovale contributed to clinical malaria infections, with children aged between 5 and 15 years harbouring a higher frequency of P. falciparum and P. ovale, whilst P. malariae was more predominant in individuals aged between 10 and 20 years. More sensitive point-of-care tools are needed to detect the presence of low-density (submicroscopic) Plasmodium infections, which may be responsible for symptomatic infections.

Automated liquid handling robot for rapid lateral flow assay development.

The lateral flow assay (LFA) is one of the most popular technologies on the point-of-care diagnostics market due to its low cost and ease of use, with applications ranging from pregnancy to environmental toxins to infectious disease. While the use of these tests is relatively straightforward, significant development time and effort are required to create tests that are both sensitive and specific. Workflows to guide the LFA development process exist but moving from target selection to an LFA that is ready for field testing can be labor intensive, resource heavy, and time consuming. To reduce the cost and the duration of the LFA development process, we introduce a novel development platform centered on the flexibility, speed, and throughput of an automated robotic liquid handling system. The system comprises LFA-specific hardware and software that enable large optimization experiments with discrete and continuous variables such as antibody pair selection or reagent concentration. Initial validation of the platform was demonstrated during development of a malaria LFA but was readily expanded to encompass development of SARS-CoV-2 and Mycobacterium tuberculosis LFAs. The validity of the platform, where optimization experiments are run directly on LFAs rather than in solution, was based on a direct comparison between the robotic system and a more traditional ELISA-like method. By minimizing hands-on time, maximizing experiment size, and enabling improved reproducibility, the robotic system improved the quality and quantity of LFA assay development efforts.

A threshold-free approach with age-dependency for estimating malaria seroprevalence.

BACKGROUND: In malaria serology analysis, the standard approach to obtain seroprevalence, i.e the proportion of seropositive individuals in a population, is based on a threshold which is used to classify individuals as seropositive or seronegative. The choice of this threshold is often arbitrary and is based on methods that ignore the age-dependency of the antibody distribution. METHODS: Using cross-sectional antibody data from the Western Kenyan Highlands, this paper introduces a novel approach that has three main advantages over the current threshold-based approach: it avoids the use of thresholds; it accounts for the age dependency of malaria antibodies; and it allows us to propagate the uncertainty from the classification of individuals into seropositive and seronegative when estimating seroprevalence. The reversible catalytic model is used as an example for illustrating how to propagate this uncertainty into the parameter estimates of the model. RESULTS: This paper finds that accounting for age-dependency leads to a better fit to the data than the standard approach which uses a single threshold across all ages. Additionally, the paper also finds that the proposed threshold-free approach is more robust against the selection of different age-groups when estimating seroprevalence. CONCLUSION: The novel threshold-free approach presented in this paper provides a statistically principled and more objective approach to estimating malaria seroprevalence. The introduced statistical framework also provides a means to compare results across studies which may use different age ranges for the estimation of seroprevalence.

Cross-border malaria in the triple border region between Brazil, Venezuela and Guyana.

The state of Roraima, in Brazil, has recently seen an increase in the number of reported Plasmodium falciparum infections believed to be imported from neighboring countries. The objective of this study was to determine the prevalence of Plasmodium species among patients attending malaria health posts in Roraima and quantify the infections attributable to imported malaria. This cross-sectional case study was carried out between March 2016 and September 2018. Study participants were recruited as they exited the malaria health post. Information about residence, occupation and travel history was collected using a questionnaire. A dried blood spot was collected and used for malaria diagnosis by PCR. A total of 1222 patients were enrolled. Of the 80% Plasmodium positive samples, 50% were P. falciparum, 34% P. vivax, 8% mixed P. falciparum/P. vivax and 0.2% mixed P. falciparum/P. ovale infections and 8% tested positive for Plasmodium, but the species could not be identified. 80% of the malaria patients likely acquired infections in Venezuela and the remaining 20% acquired in Guyana, Brazil, Suriname and French Guyana. 50% of the study participants reported to be working in a mine. Results from this study support the hypothesis that imported malaria contribute to the bulk of malaria diagnosed in Roraima. These findings are in keeping with previous findings and should be considered when developing malaria control interventions.

Presence and potential distribution of malaria-infected New World primates of Costa Rica.

BACKGROUND: In South and Central America, Plasmodium malariae/Plasmodium brasilianum, Plasmodium vivax, Plasmodium simium, and Plasmodium falciparum has been reported in New World primates (NWP). Specifically in Costa Rica, the presence of monkeys positive to P. malariae/P brasilianum has been identified in both captivity and in the wild. The aim of the present study was to determine the presence of P. brasilianum, P. falciparum, and P. vivax, and the potential distribution of these parasites-infecting NWP from Costa Rica. METHODS: The locations with PCR (Polymerase Chain Reaction) positive results and bioclimatic predictors were used to construct ecological niche models based on a modelling environment that uses the Maxent algorithm, named kuenm, capable to manage diverse settings to better estimate the potential distributions and uncertainty indices of the potential distribution. RESULTS: PCR analysis for the Plasmodium presence was conducted in 384 samples of four primates (Howler monkey [n = 130], White-face monkey [n = 132], Squirrel monkey [n = 50], and red spider monkey [n = 72]), from across Costa Rica. Three Plasmodium species were detected in all primate species (P. falciparum, P. malariae/P. brasilianum, and P. vivax). Overall, the infection prevalence was 8.9%, but each Plasmodium species ranged 2.1-3.4%. The niche model approach showed that the Pacific and the Atlantic coastal regions of Costa Rica presented suitable climatic conditions for parasite infections. However, the central pacific coast has a more trustable prediction for malaria in primates. CONCLUSIONS: The results indicate that the regions with higher suitability for Plasmodium transmission in NWP coincide with regions where most human cases have been reported. These regions were also previously identified as areas with high suitability for vector species, suggesting that enzootic and epizootic cycles occur.

Pediatric Malaria: Global and North American Perspectives.

Malaria is a leading cause of death in children less than 5 years of age globally, and a common cause of fever in the returning North American traveler. New tools in the fight against malaria have been developed over the past decades: potent artemisinin derivatives; rapid diagnostic tests; long-lasting insecticidal bed nets; and a new vaccine, RTS,S/AS01. Thwarting these advances, parasite and Anopheles vector resistance are emerging. In the meantime, clinicians will continue to see malaria among febrile travelers from the tropics. Early recognition, diagnosis, and treatment can be lifesaving, but rely on the vigilance of frontline clinicians.

C-reactive protein as an early biomarker for malaria infection and monitoring of malaria severity: a meta-analysis.

This study investigated whether C-reactive protein (CRP) can be used as a marker for the early detection and monitoring of malaria severity. Potentially relevant studies were searched in Medline (PubMed), Scopus, and Web of Science. Differences in CRP between (1) severe malaria and uncomplicated malaria, (2) uncomplicated malaria and asymptomatic malaria, (3) uncomplicated malaria and febrile/healthy controls, and (4) asymptomatic malaria and febrile/healthy controls were estimated using random-effects models. Twenty-nine studies were included for meta-analysis. The results of meta-analysis demonstrated higher mean CRP levels in (1) patients with severe malaria compared with uncomplicated malaria (p < 0.001, standard mean difference [SMD]: 1.52, 95% confidence interval [CI]: 0.91-2.12, I2: 95.1%), (2) patients with uncomplicated malaria than in those with asymptomatic malaria (p: 0.001, SMD: 1.65, 95% CI: 0.67-2.62, I2: 96.7%), (3) patients with uncomplicated malaria compared with febrile/healthy controls (p < 0.001, SMD: 2.38, 95% CI: 1.37-3.40, I2: 98.5%), and (4) patients with asymptomatic malaria compared with febrile/healthy controls (p < 0.001, SMD: 2.55, 95% CI: 1.60-3.50, I2: 99.2%). This study demonstrated CRP levels are a biomarker for the early detection and monitoring of malaria severity.

Evaluation of the interaction between insecticide resistance-associated genes and malaria transmission in Anopheles gambiae sensu lato in central Côte d'Ivoire.

BACKGROUND: There is evidence that the knockdown resistance gene (Kdr) L1014F and acetylcholinesterase-1 gene (Ace-1R) G119S mutations involved in pyrethroid and carbamate resistance in Anopheles gambiae influence malaria transmission in sub-Saharan Africa. This is likely due to changes in the behaviour, life history and vector competence and capacity of An. gambiae. In the present study, performed as part of a two-arm cluster randomized controlled trial evaluating the impact of household screening plus a novel insecticide delivery system (In2Care Eave Tubes), we investigated the distribution of insecticide target site mutations and their association with infection status in wild An. gambiae sensu lato (s.l.) populations. METHODS: Mosquitoes were captured in 40 villages around Bouaké by human landing catch from May 2017 to April 2019. Randomly selected samples of An. gambiae s.l. that were infected or not infected with Plasmodium sp. were identified to species and then genotyped for Kdr L1014F and Ace-1R G119S mutations using quantitative polymerase chain reaction assays. The frequencies of the two alleles were compared between Anopheles coluzzii and Anopheles gambiae and then between infected and uninfected groups for each species. RESULTS: The presence of An. gambiae (49%) and An. coluzzii (51%) was confirmed in Bouaké. Individuals of both species infected with Plasmodium parasites were found. Over the study period, the average frequency of the Kdr L1014F and Ace-1R G119S mutations did not vary significantly between study arms. However, the frequencies of the Kdr L1014F and Ace-1R G119S resistance alleles were significantly higher in An. gambiae than in An. coluzzii [odds ratio (95% confidence interval): 59.64 (30.81-131.63) for Kdr, and 2.79 (2.17-3.60) for Ace-1R]. For both species, there were no significant differences in Kdr L1014F or Ace-1R G119S genotypic and allelic frequency distributions between infected and uninfected specimens (P > 0.05). CONCLUSIONS: Either alone or in combination, Kdr L1014F and Ace-1R G119S showed no significant association with Plasmodium infection in wild An. gambiae and An. coluzzii, demonstrating the similar competence of these species for Plasmodium transmission in Bouaké. Additional factors including behavioural and environmental ones that influence vector competence in natural populations, and those other than allele measurements (metabolic resistance factors) that contribute to resistance, should be considered when establishing the existence of a link between insecticide resistance and vector competence.

Prevalence of Plasmodium spp. in symptomatic BaAka Pygmies inhabiting the rural Dzanga Sangha region of the Central African Republic.

INTRODUCTION: Malaria remains a diagnostic and therapeutic challenge in many endemic regions of sub-Saharan Africa. It is one of the most important causes of morbidity and mortality, especially in children <5 years. Plasmodium falciparum is responsible for the majority of severe malaria cases in sub-Saharan Africa, but is not the exclusive one. OBJECTIVE: The objective of the study was to assess the prevalence of Plasmodium spp. in BaAka Pygmies with clinical symptoms of malaria, and define the percentage distribution of infections caused by species other than P. falciparum in order to assess the need for diversification of malaria treatment protocols. MATERIAL AND METHODS: The study was conducted during the dry and rainy seasons in 2018 and involved a group of 540 symptomatic BaAka Pygmies, patients of both genders, aged 1-75-years-old. Two diagnostic methods for detecting Plasmodium in the bloodstream were used: RDTs targeting HRP2-protein specific for P. falciparum, and PCR assays aimed at detecting P. falciparum, P. vivax, P. ovale, P. malariae species. RESULTS: Only 40.5% of symptomatic patients tested with RDTs for P. falciparum infections were positive. Molecular tests (PCR) confirmed P. falciparum in 94.8% of the samples and also revealed the genetic material of P. malariae (11.1%), P. ovale (9.8%), and P. vivax (0.7%). BaAka Pygmies aged <5 years of age dominated in patients with positive results; the common clinical symptoms reported by the sick individuals were fever, shivers and fatigue. CONCLUSIONS: The study suggests the need for introducing accurate diagnostic methods for the diagnosis of malaria and the revision of malaria treatment protocols. Assessment of the Pfhrp2/Pfhrp3 deletions is necessary for evaluating malaria epidemiology in Central Africa.

Rediscovery of Plasmodium (Huffia) huffi (Apicomplexa, Haemosporida): a lost lineage from toucans.

Literature data on toucans haemosporidians are scarce and all reports come from investigations in Brazil. Muniz et al. (Rev Bras Malariol 3: 339-356, Muniz et al., Rev Bras Malariol 3:339-356, 1951) and Muniz and Soares (Rev Bras Malar 611-617, Muniz J, Soares R de RL (1954) Nota sôbre um parasita do gênero Plasmodium encontrado no Ramphastos toco Müller, 1776, "Tucano-Açu", e diferente do Plasmodium huffi: Plasmodium pinottii n. sp. Rev Bras Malar 611 - 617.) described two Plasmodium species, P. huffi and P. pinottii, in Ramphastos toco. Later, Manwell and Sessler (J Protozol 18: 570-574, Manwell and Sessler, Malaria Parasites of Toucans J Protozol 18:570-574, 1971) established a new subspecies, P. nucleophilum toucani. In the last review on avian haemosporidians, Valkiunas (Valkiunas, Avian malaria parasites and other haemosporidia, CRC Press, New York, 2005) highlighted that P. huffi was insufficiently characterized, considering it a lost lineage. Also, the original description of P. huffi was considered insufficiently clear, due to a possible co-infection of the toucan hosts with a Novyella-like species. Here, we redescribed the species Plasmodium (Huffia) huffi based on morphological and molecular data, which were found in two toucan species, Ramphastos toco and Pteroglossus aracari from Brazil. Morphological features of the specimens are markedly the same as the original description. In R. toco, we observe two individuals infected, one infected only with P. huffi and one co-infected with P. huffi and the Novyella-like species, as observed in the original description. Also, we observe one R. toco infected only with the Novyella-like species, identified by morphological and molecular data as Plasmodium (Novyella) nucleophilum nucleophilum. In this way, it was possible to redescribe Plasmodium huffi in detail, without the doubt characters observed in the original description. Moreover, by applying species delimitation algorithms to a large Plasmodium phylogeny, we were able to identify new possible hosts for P. huffi and extend its geographic distribution to include North America.