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The spread of Plasmodium falciparum isolates carrying mutations in the kelch13 (Pfkelch13) gene associated with artemisinin resistance (PfART-R) in southeast Asia threatens malaria control and elimination efforts. Emergence of PfART-R in Africa would result in a major public health problem. In this systematic review, we investigate the frequency and spatial distribution of Pfkelch13 mutants in Africa, including mutants linked to PfART-R in southeast Asia. Seven databases were searched (PubMed, Embase, Scopus, African Journal Online, African Index Medicus, Bioline, and Web of Science) for relevant articles about polymorphisms of the Pfkelch13 gene in Africa before January, 2019. Following PRISMA guidelines, 53 studies that sequenced the Pfkelch13 gene of 23 100 sample isolates in 41 sub-Saharan African countries were included. The Pfkelch13 sequence was highly polymorphic (292 alleles, including 255 in the Pfkelch13-propeller domain) but with mutations occurring at very low relative frequencies. Non-synonymous mutations were found in only 626 isolates (2·7%) from west, central, and east Africa. According to WHO, nine different mutations linked to PfART-R in southeast Asia (Phe446Ile, Cys469Tyr, Met476Ile, Arg515Lys, Ser522Cys, Pro553Leu, Val568Gly, Pro574Leu, and Ala675Val) were detected, mainly in east Africa. Several other Pfkelch13 mutations, such as those structurally similar to southeast Asia PfART-R mutations, were also identified, but their relevance for drug resistance is still unknown. This systematic review shows that Africa, thought to not have established PfART-R, reported resistance-related mutants in the past 5 years. Surveillance using PfART-R molecular markers can provide valuable decision-making information to sustain the effectiveness of artemisinin in Africa.

Background Translating genomic technologies into healthcare applications for the malaria parasite Plasmodium falciparum has been limited by the technical and logistical difficulties of obtaining high quality clinical samples from the field. Sampling by dried blood spot (DBS) finger-pricks can be performed safely and efficiently with minimal resource and storage requirements compared with venous blood (VB). Here, the use of selective whole genome amplification (sWGA) to sequence the P. falciparum genome from clinical DBS samples was evaluated, and the results compared with current methods that use leucodepleted VB. Methods Parasite DNA with high (>95%) human DNA contamination was selectively amplified by Phi29 polymerase using short oligonucleotide probes of 8–12 mers as primers. These primers were selected on the basis of their differential frequency of binding the desired ( P. falciparum DNA) and contaminating (human) genomes. Results Using sWGA method, clinical samples from 156 malaria patients, including 120 paired samples for head-to-head comparison of DBS and leucodepleted VB were sequenced. Greater than 18-fold enrichment of P. falciparum DNA was achieved from DBS extracts. The parasitaemia threshold to achieve >5× coverage for 50% of the genome was 0.03% (40 parasites per 200 white blood cells). Over 99% SNP concordance between VB and DBS samples was achieved after excluding missing calls. Conclusion The sWGA methods described here provide a reliable and scalable way of generating P. falciparum genome sequence data from DBS samples. The current data indicate that it will be possible to get good quality sequence on most if not all drug resistance loci from the majority of symptomatic malaria patients. This technique overcomes a major limiting factor in P. falciparum genome sequencing from field samples, and paves the way for large-scale epidemiological applications.

DNA samples derived from vertebrate skin, bodily cavities and body fluids contain both host and microbial DNA; the latter often present as a minor component. Consequently, DNA sequencing of a microbiome sample frequently yields reads originating from the microbe(s) of interest, but with a vast excess of host genome-derived reads. In this study, we used a methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA based on differences in CpG methylation density. MBD fused to the Fc region of a human antibody (MBD-Fc) binds strongly to protein A paramagnetic beads, forming an effective one-step enrichment complex that was used to remove human or fish host DNA from bacterial and protistan DNA for subsequent sequencing and analysis. We report enrichment of DNA samples from human saliva, human blood, a mock malaria-infected blood sample and a black molly fish. When reads were mapped to reference genomes, sequence reads aligning to host genomes decreased 50-fold, while bacterial and Plasmodium DNA sequences reads increased 8-11.5-fold. The Shannon-Wiener diversity index was calculated for 149 bacterial species in saliva before and after enrichment. Unenriched saliva had an index of 4.72, while the enriched sample had an index of 4.80. The similarity of these indices demonstrates that bacterial species diversity and relative phylotype abundance remain conserved in enriched samples. Enrichment using the MBD-Fc method holds promise for targeted microbiome sequence analysis across a broad range of sample types.

Plasmodium parasite–specific antibodies are critical for protection against malaria, yet the development of long-lived and effective humoral immunity against Plasmodium takes many years and multiple rounds of infection and cure. Here, we report that the rapid development of short-lived plasmablasts during experimental malaria unexpectedly hindered parasite control by impeding germinal center responses. Metabolic hyperactivity of plasmablasts resulted in nutrient deprivation of the germinal center reaction, limiting the generation of memory B cell and long-lived plasma cell responses. Therapeutic administration of a single amino acid to experimentally infected mice was sufficient to overcome the metabolic constraints imposed by plasmablasts and enhanced parasite clearance and the formation of protective humoral immune memory responses. Thus, our studies not only challenge the current model describing the role and function of blood-stage Plasmodium -induced plasmablasts but they also reveal new targets and strategies to improve anti- Plasmodium humoral immunity. Early humoral responses to malaria fail to induce durable protective antibodies. Butler and colleagues report that low-affinity, short-lived plasmablasts become nutrient sinks for glutamine and starve germinal center B and T cells, thereby reducing the generation of high-affinity B cells and long-lived plasma cells and memory B cells.

Background. Rapid diagnostic tests (RDTs) account for more than two-thirds of malaria diagnoses in Africa. Deletions of the Plasmodium falciparum hrp2 (pfhrp2) gene cause false-negative RDT results and have never been investigated on a national level. Spread of pfhrp2-deleted P. falciparum mutants, resistant to detection by HRP2-based RDTs, would represent a serious threat to malaria elimination efforts. Methods. Using a nationally representative cross-sectional study of 7,137 children under five years of age from the Democratic Republic of Congo (DRC), we tested 783 subjects with RDT-/PCR+ results using PCR assays to detect and confirm deletions of the pfhrp2 gene. Spatial and population genetic analyses were employed to examine the distribution and evolution of these parasites. Results. We identified 149 pfhrp2-deleted parasites, representing 6.4% of all P. falciparum infections country-wide (95% confidence interval 5.1–8.0%). Bayesian spatial analyses identified statistically significant clustering of pfhrp2 deletions near Kinshasa and Kivu. Population genetic analysis revealed significant genetic differentiation between wild-type and pfhrp2-deleted parasite populations (GST = .046, p ≤ .00001). Conclusions. Pfhrp2-deleted P. falciparum is a common cause of RDT-/PCR+ malaria among asymptomatic children in the DRC and appears to be clustered within select communities. Surveillance for these deletions is needed, and alternatives to HRP2-specific RDTs may be necessary.

Abstract Background To better understand transmission dynamics, we characterized Plasmodium falciparum genetic diversity in Eswatini, where transmission is low and sustained by importation. Methods Twenty-six P. falciparum microsatellites were genotyped in 66% of confirmed cases (2014–2016; N = 582). Population and within-host diversity were used to characterize differences between imported and locally acquired infections. Logistic regression was used to assess the added value of diversity metrics to classify imported and local infections beyond epidemiology data alone. Results Parasite population in Eswatini was highly diverse (expected heterozygosity [HE] = 0.75) and complex: 67% polyclonal infections, mean multiplicity of infection (MOI) 2.2, and mean within-host infection fixation index (FWS) 0.84. Imported cases had comparable diversity to local cases but exhibited higher MOI (2.4 vs 2.0; P = .004) and lower mean FWS (0.82 vs 0.85; P = .03). Addition of MOI and FWS to multivariate analyses did not increase discrimination between imported and local infections. Conclusions In contrast to the common perception that P. falciparum diversity declines with decreasing transmission intensity, Eswatini isolates exhibited high parasite diversity consistent with high rates of malaria importation and limited local transmission. Estimates of malaria transmission intensity from genetic data need to consider the effect of importation, especially as countries near elimination. In contrast to the commonly held perception that P. falciparum diversity declines with decreasing transmission intensity, infections from Eswatini exhibited high parasite diversity consistent with high rates of malaria importation and limited local transmission.

Plasmodium falciparum histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) are exclusively recommended for malaria diagnosis in Uganda; however, their functionality can be affected by parasite-related factors that have not been investigated in field settings. Using a cross-sectional design, we analysed 219 RDT-/microscopy+ and 140 RDT+/microscopy+ dried blood spots obtained from symptomatic children aged 2-10 years from 48 districts in Uganda between 2017 and 2019. We aimed to investigate parasite-related factors contributing to false RDT results by molecular characterization of parasite isolates. ArcGIS software was used to map the geographical distribution of parasites. Statistical analysis was performed using chi-square or Fisher's exact tests, with P ≤ 0.05 indicating significance. Odds ratios (ORs) were used to assess associations, while logistic regression was performed to explore possible factors associated with false RDT results. The presence of parasite DNA was confirmed in 92.5% (332/359) of the blood samples. The levels of agreement between the HRP2 RDT and PCR assay results in the (RDT+/microscopy+) and (RDT-/microscopy+) sample subsets were 97.8% (137/140) and 10.9% (24/219), respectively. Factors associated with false-negative RDT results in the (RDT-/microscopy+) samples were parasite density (<1,000/μl), pfhrp2/3 gene deletion and non-P. falciparum species (aOR 2.65, 95% CI: 1.62-4.38, P = 0.001; aOR 4.4, 95% CI 1.72-13.66, P = 0.004; and aOR 18.65, 95% CI: 5.3-38.7, P = 0.001, respectively). Overall, gene deletion and non-P. falciparum species contributed to 12.3% (24/195) and 19.0% (37/195) of false-negative RDT results, respectively. Of the false-negative RDTs results, 80.0% (156/195) were from subjects with low-density infections (< 25 parasites per 200 WBCs or <1,000/μl). This is the first evaluation and report of the contributions of pfhrp2/3 gene deletion, non-P. falciparum species, and low-density infections to false-negative RDT results under field conditions in Uganda. In view of these findings, the use of HRP2 RDTs should be reconsidered; possibly, switching to combination RDTs that target alternative antigens, particularly in affected areas, may be beneficial. Future evaluations should consider larger and more representative surveys covering other regions of Uganda.

Blastocystis is a common enteric protist colonizing probably more than 1 billion people with a large variety of non-human hosts. Remarkable genetic diversity has been observed, leading to the subdivision of the genus into multiple subtypes (ST), some of which are exclusively found in non-human hosts. The aim of this study was to determine the distribution of Blastocystis STs/18S alleles in symptomatic (abdominal pain, anal pruritus, diarrhea, headache, nauseas and/or vomit) and asymptomatic children from nine geographical regions of Colombia. A total of 2026 fecal samples were collected as part of a national survey to estimate the frequency of intestinal parasites in children. A set of 256 samples that were Blastocystis positive was finally selected. The samples were submitted to DNA extraction, Real Time PCR and sequencing using Blastocystis-specific primers targeting the small subunit rRNA gene for ST identification. DNA of Ascaris lumbricoides (16.4%), Trichuris trichiura (8.2%), hookworms (Necator americanus/Ancylostoma duodenale) (7.3%), Giardia duodenalis (23.1%), Entamoeba complex (82%), Entamoeba coli (55%), Hymenolepis nana (0.8%), Endolimax nana (33.2%) and Neobalantidium coli (2.7%) was detected in the Blastocystis-positive samples. We detected ST1 (21.4%), ST2 (19.5%), ST3 (55.5%), ST4 (0.8%), ST6 (2%) and ST7 (0.8%); alleles 1, 2, 4, 81, 82 and 83 for ST1; alleles 9, 11, 12, 15, 67, 71 and 73 for ST2; alleles 34, 36, 38, 45, 49, 55, 134 and 128 for ST3; allele 42 for ST4; allele 122 for ST6, and allele 142 for ST7. Further studies implementing high-resolution molecular markers are necessary to understand the dynamics of Blastocystis transmission and the role of this Stramenopila in health and disease.

Accurate quantification of parasite density in the human host is essential for understanding the biology and pathology of malaria. Semi-quantitative molecular methods are widely applied, but the need for an external standard curve makes it difficult to compare parasite density estimates across studies. Droplet digital PCR (ddPCR) allows direct quantification without the need for a standard curve. ddPCR was used to diagnose and quantify P. falciparum and P. vivax in clinical patients as well as in asymptomatic samples. ddPCR yielded highly reproducible measurements across the range of parasite densities observed in humans, and showed higher sensitivity than qPCR to diagnose P. falciparum , and equal sensitivity for P. vivax . Correspondence in quantification was very high (>0.95) between qPCR and ddPCR. Quantification between technical replicates by ddPCR differed 1.5–1.7-fold, compared to 2.4–6.2-fold by qPCR. ddPCR facilitates parasite quantification for studies where absolute densities are required, and will increase comparability of results reported from different laboratories.

Human infections due to the monkey malaria parasite Plasmodium knowlesi are increasingly being reported from Malaysia. The parasite causes high parasitaemia, severe and fatal malaria in humans thus there is a need for urgent measures for its control. The MSP4 is a potential vaccine candidate, which is well studied in Plasmodium falciparum and Plasmodium vivax; however, no study has been conducted in the orthologous gene of P. knowlesi. In this study, we investigated the level of polymorphisms, haplotypes, natural selection and population structure of full-length pkmsp4 in 32 clinical samples from Malaysian Borneo along with 4 lab-adapted strains. We found low levels of polymorphism across the gene with exon I showing higher diversity than the exon II. The C- terminal epidermal growth factor (EGF) domains and GPI-anchored region within exon II were mostly conserved with only 2 non-synonymous substitutions. Although 21 amino acid haplotypes were found, the frequency of mutation at the majority of the polymorphic positions was low. We found evidence of negative selection at the exon II of the gene indicating existence of functional constraints. Phylogenetic haplotype network analysis identified shared haplotypes and indicated geographical clustering of samples originating from Peninsular Malaysia and Malaysian Borneo. High population differentiation values were observed within parasite populations originating from Malaysian Borneo (Kapit, Sarikei and Betong) and laboratory-adapted strains obtained from Peninsular Malaysia and Philippines indicating distinct population structure. This is the first study to genetically characterize the full-length msp4 gene from clinical isolates of P. knowlesi from Malaysia and thus would be very useful for future rational vaccine studies. Further studies with higher number of samples and functional characterization of the protein will be necessary.