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Plasmodium falciparum with the histidine rich protein 2 gene (pfhrp2) deleted from its genome can escape diagnosis by HRP2-based rapid diagnostic tests (HRP2-RDTs). The World Health Organization (WHO) recommends switching to a non-HRP2 RDT for P. falciparum clinical case diagnosis when pfhrp2 deletion prevalence causes ≥ 5% of RDTs to return false negative results. Tanzania is a country of heterogenous P. falciparum transmission, with some regions approaching elimination and others at varying levels of control. In concordance with the current recommended WHO pfhrp2 deletion surveillance strategy, 100 health facilities encompassing 10 regions of Tanzania enrolled malaria-suspected patients between February and July 2021. Of 7863 persons of all ages enrolled and providing RDT result and blood sample, 3777 (48.0%) were positive by the national RDT testing for Plasmodium lactate dehydrogenase (pLDH) and/or HRP2. A second RDT testing specifically for the P. falciparum LDH (Pf-pLDH) antigen found 95 persons (2.5% of all RDT positives) were positive, though negative by the national RDT for HRP2, and were selected for pfhrp2 and pfhrp3 (pfhrp2/3) genotyping. Multiplex antigen detection by laboratory bead assay found 135/7847 (1.7%) of all blood samples positive for Plasmodium antigens but very low or no HRP2, and these were selected for genotyping as well. Of the samples selected for genotyping based on RDT or laboratory multiplex result, 158 were P. falciparum DNA positive, and 140 had sufficient DNA to be genotyped for pfhrp2/3. Most of these (125/140) were found to be pfhrp2+/pfhrp3+, with smaller numbers deleted for only pfhrp2 (n = 9) or only pfhrp3 (n = 6). No dual pfhrp2/3 deleted parasites were observed. This survey found that parasites with these gene deletions are rare in Tanzania, and estimated that 0.24% (95% confidence interval: 0.08% to 0.39%) of false-negative HRP2-RDTs for symptomatic persons were due to pfhrp2 deletions in this 2021 Tanzania survey. These data provide evidence for HRP2-based diagnostics as currently accurate for P. falciparum diagnosis in Tanzania., (© 2024. The Author(s).)

Naturally occurring human infections by zoonotic Plasmodium species have been documented for P. knowlesi, P. cynomolgi, P. simium, P. simiovale, P. inui, P. inui-like, P. coatneyi, and P. brasilianum. Accurate detection of each species is complicated by their morphological similarities with other Plasmodium species. PCR-based assays offer a solution but require prior knowledge of adequate genomic targets that can distinguish the species. While whole genomes have been published for P. knowlesi, P. cynomolgi, P. simium, and P. inui, no complete genome for P. brasilianum has been available. Previously, we reported a draft genome for P. brasilianum, and here we report the completed genome for P. brasilianum. The genome is 31.4 Mb in size and comprises 14 chromosomes, the mitochondrial genome, the apicoplast genome, and 29 unplaced contigs. The chromosomes consist of 98.4% nucleotide sites that are identical to the P. malariae genome, the closest evolutionarily related species hypothesized to be the same species as P. brasilianum, with 41,125 non-synonymous SNPs (0.0722% of genome) identified between the two genomes. Furthermore, P. brasilianum had 4864 (82.1%) genes that share 80% or higher sequence similarity with 4970 (75.5%) P. malariae genes. This was demonstrated by the nearly identical genomic organization and multiple sequence alignments for the merozoite surface proteins msp3 and msp7. We observed a distinction in the repeat lengths of the circumsporozoite protein (CSP) gene sequences between P. brasilianum and P. malariae. Our results demonstrate a 97.3% pairwise identity between the P. brasilianum and the P. malariae genomes. These findings highlight the phylogenetic proximity of these two species, suggesting that P. malariae and P. brasilianum are strains of the same species, but this could not be fully evaluated with only a single genomic sequence for each species., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

Microscopy is the gold standard for malaria epidemiology, but laboratory and point-of-care (POC) tests detecting parasite antigen, DNA, and human antibodies against malaria have expanded this capacity. The island nation of Haiti is endemic for Plasmodium falciparum (Pf) malaria, though at a low national prevalence and heterogenous geospatial distribution. In 2015 and 2016, serosurveys were performed of children (ages 6-7 years) sampled in schools in Saut d'Eau commune (n = 1,230) and Grand Anse department (n = 1,664) of Haiti. Children received malaria antigen rapid diagnostic test and provided a filter paper blood sample for further laboratory analysis of the Pf histidine-rich protein 2 (HRP2) antigen, Pf DNA, and anti-Pf IgG antibodies. Prevalence of Pf infection ranged from 0.0-16.7% in 53 Saut d'Eau schools, and 0.0-23.8% in 56 Grand Anse schools. Anti-Pf antibody carriage exceeded 80% of students in some schools from both study sites. Geospatial prediction ellipses were created to indicate clustering of positive tests within the survey areas and overlay of all prediction ellipses for the different types of data revealed regions with high likelihood of active and ongoing Pf malaria transmission. The geospatial utilization of different types of Pf data can provide high confidence for spatial epidemiology of the parasite.

As malaria control programmes concentrate their efforts towards malaria elimination a better understanding of malaria transmission patterns at fine spatial resolution units becomes necessary. Defining spatial units that consider transmission heterogeneity, human movement and migration will help to set up achievable malaria elimination milestones and guide the creation of efficient operational administrative control units. Using a combination of genetic and epidemiological data we defined a malaria transmission unit as the area contributing 95% of malaria cases diagnosed at the catchment facility located in the town of Guapi in the South Pacific Coast of Colombia. We provide data showing that P. falciparum malaria transmission is heterogeneous in time and space and analysed, using topological data analysis, the spatial connectivity, at the micro epidemiological level, between parasite populations circulating within the unit. To illustrate the necessity to evaluate the efficacy of malaria control measures within the transmission unit in order to increase the efficiency of the malaria control effort, we provide information on the size of the asymptomatic reservoir, the nature of parasite genotypes associated with drug resistance as well as the frequency of the Pfhrp2/3 deletion associated with false negatives when using Rapid Diagnostic Tests.

Commercial malaria rapid diagnostic tests (RDTs) detect P. falciparum histidine rich protein 2 (PfHRP2) and cross react with PfHRP3, a structural homologue. Here, we analysed natural variations in PfHRP2 and PfHRP3 sequences from Indian isolates and correlated these variations with RDT reactivity. A total 1392 P. falciparum positive samples collected from eight endemic states were PCR amplified for Pfhrp2 and Pfhrp3 genes and were sequenced. The deduced protein sequences were analysed for repeat variations and correlated with RDT reactivity. Out of 1392 PCR amplified samples, a single sample was Pfhrp2 negative and two samples were Pfhrp3 negative. Complete Pfhrp2 and Pfhrp3 sequences were obtained for 769 samples and 750 samples, respectively. A total of 16 distinct repeat motifs were observed for Pfhrp2 and 11 for Pfhrp3, including some new repeat types. No correlation was found between variations in the size of Pfhrp2 repeat types 2 and 7, nor between any combinations of repeat motifs, and performance of a commercial RDT at low parasite densities. The findings suggest that sequence diversity in Pfhrp2 and Pfhrp3 genes in Indian isolates is not likely to negatively influence performance of currently used PfHRP2 RDTs.

Isothermal nucleic acid amplification assays such as the loop mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to amplify the DNA. To further facilitate the use of LAMP assays in remote settings, simpler sample preparation methods and lyophilized reagents are required. The performance of a commercial malaria LAMP assay (Illumigene Malaria LAMP) was evaluated using two sample preparation workflows (simple filtration prep (SFP)) and gravity-driven filtration prep (GFP)) and pre-dispensed lyophilized reagents. Laboratory and clinical samples were tested in a field laboratory in Senegal and the results independently confirmed in a reference laboratory in the U.S.A. The Illumigene Malaria LAMP assay was easily implemented in the clinical laboratory and gave similar results to a real-time PCR reference test with limits of detection of ≤2.0 parasites/μl depending on the sample preparation method used. This assay reliably detected Plasmodium sp. parasites in a simple low-tech format, providing a much needed alternative to the more complex molecular tests for malaria diagnosis., Competing Interests: All the supplies and reagents (including the LAMP kits) for this evaluation were provided by Meridian Inc. Meridian also covered the field operational costs for this evaluation.

The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times.

Understanding the spatial clustering of Plasmodium falciparum populations can assist efforts to contain drug-resistant parasites and maintain the efficacy of future drugs. We sequenced single nucleotide polymorphisms (SNPs) in the dihydropteroate synthase gene (dhps) associated with sulfadoxine resistance and 5 microsatellite loci flanking dhps in order to investigate the genetic backgrounds, genetic relatedness, and geographic clustering of falciparum parasites in the Democratic Republic of the Congo (DRC). Resistant haplotypes were clustered into subpopulations: one in the northeast DRC, and the other in the balance of the DRC. Network and clonal lineage analyses of the flanking microsatellites indicate that geographically-distinct mutant dhps haplotypes derive from separate lineages. The DRC is therefore a watershed for haplotypes associated with sulfadoxine resistance. Given the importance of central Africa as a corridor for the spread of antimalarial resistance, the identification of the mechanisms of this transit can inform future policies to contain drug-resistant parasite strains.