Genotyping Plasmodium falciparum gametocytes using amplicon deep sequencing.

BACKGROUND: Understanding the dynamics of gametocyte production in polyclonal Plasmodium falciparum infections requires a genotyping method that detects distinct gametocyte clones and estimates their relative frequencies. Here, a marker was identified and evaluated to genotype P. falciparum mature gametocytes using amplicon deep sequencing. METHODS: A data set of polymorphic regions of the P. falciparum genome was mined to identify a gametocyte genotyping marker. To assess marker resolution, the number of unique haplotypes in the marker region was estimated from 95 Malawian P. falciparum whole genome sequences. Specificity of the marker for detection of mature gametocytes was evaluated using reverse transcription-polymerase chain reaction of RNA extracted from NF54 mature gametocytes and rings from a non-gametocyte-producing strain of P. falciparum. Amplicon deep sequencing was performed on experimental mixtures of mature gametocytes from two distinct parasite clones, as well as gametocyte-positive P. falciparum field isolates to evaluate the quantitative ability and determine the limit of detection of the genotyping approach. RESULTS: A 400 bp region of the pfs230 gene was identified as a gametocyte genotyping marker. A larger number of unique haplotypes was observed at the pfs230 marker (34) compared to the sera-2 (18) and ama-1 (14) markers in field isolates from Malawi. RNA and DNA genotyping accurately estimated gametocyte and total parasite clone frequencies when evaluating agreement between expected and observed haplotype frequencies in gametocyte mixtures, with concordance correlation coefficients of 0.97 [95% CI: 0.92-0.99] and 0.92 [95% CI: 0.83-0.97], respectively. The detection limit of the genotyping method for male gametocytes was 0.41 pfmget transcripts/µl [95% CI: 0.28-0.72] and for female gametocytes was 1.98 ccp4 transcripts/µl [95% CI: 1.35-3.68]. CONCLUSIONS: A region of the pfs230 gene was identified as a marker to genotype P. falciparum gametocytes. Amplicon deep sequencing of this marker can be used to estimate the number and relative frequency of parasite clones among mature gametocytes within P. falciparum infections. This gametocyte genotyping marker will be an important tool for studies aimed at understanding dynamics of gametocyte production in polyclonal P. falciparum infections.

Submicroscopic malaria infection is not associated with fever in cross-sectional studies in Malawi.

BACKGROUND: Submicroscopic Plasmodium falciparum infections are widespread in many areas. However, the contribution of these infections to symptomatic malaria is not well understood. This study evaluated whether participants with submicroscopic P. falciparum infections have higher prevalence of fever than uninfected participants in southern Malawi. METHODS: A total of 16,650 children and adults were enrolled in the course of six cross-sectional surveys during the dry season (October-November) and after the rainy season (April-May) between 2012 and 2014 in three districts in southern Malawi. Demographic and socioeconomic data were collected in conjunction with data on clinical histories, use of malaria preventive measures, and anti-malarial medication taken within 2 weeks of the survey. Axillary temperatures were measured, and blood samples were collected for P. falciparum detection by microscopy and PCR. Participants without malaria parasites detected on microscopy but with a positive PCR for P. falciparum were defined as having submicroscopic infection. Fever was defined as having any one of: reported fever in the past 2 weeks, reported fever in the past 48 h, or a temperature of ≥ 37.5 °C measured at the time of interview. RESULTS: Submicroscopic P. falciparum infections and fever were both detected in 9% of the study population. In the final analysis adjusted for clustering within household and enumeration area, having submicroscopic P. falciparum infection was associated with reduced odds of fever in the dry season (odds ratio = 0.52; 95% CI 0.33-0.82); the association in the rainy season did not achieve statistical significance (odds ratio = 1.20; 95% CI 0.91-1.59). The association between submicroscopic infection and fever was consistent across all age groups. When the definition of fever was limited to temperature of ≥ 37.5 °C measured at the time of interview, the association was not statistically significant in either the rainy or dry season. CONCLUSIONS: In this series of cross-sectional studies in southern Malawi, submicroscopic P. falciparum infection was not associated with increased risk of fever. Submicroscopic detection of the malaria parasite is important in efforts to decrease transmission but is not essential for the clinical recognition of malaria disease.

Comparison of msp genotyping and a 24 SNP molecular assay for differentiating Plasmodium falciparum recrudescence from reinfection.

BACKGROUND: Current World Health Organization guidelines for conducting anti-malarial drug efficacy clinical trials recommend genotyping Plasmodium falciparum genes msp1 and msp2 to distinguish recrudescence from reinfection. A more recently developed potential alternative to this method is a molecular genotyping assay based on a panel of 24 single nucleotide polymorphism (SNP) markers. METHODS: Performance parameters of these two genotyping methods were compared using data from two recently completed drug efficacy trials. Blood samples from two anti-malarial therapeutic trials were analysed by both msp genotyping and the 24 SNP assay. Additionally, to conserve time and resources, the statistical program R was used to select the most informative SNPs for a set of unrelated Malawian samples to develop a truncated SNP-based assay for the region surrounding Blantyre, Malawi. The ability of this truncated assay to distinguish reinfection from recrudescence when compared to the full 24 SNP assay was then analysed using data from the therapeutic trials. RESULTS: A total of 360 samples were analysed; 66 for concordance of msp and SNP barcoding methodologies, and 294 for assessing the most informative of the 24 SNP markers. SNP genotyping performed comparably to msp genotyping, with only one case of disagreement among the 50 interpretable results, where the SNP assay identified the sample as reinfection and the msp typing as recrudescence. Furthermore, SNP typing was more robust; only 6% of samples were uninterpretable by SNP typing, compared to 19.7% when msp genotyping was used. For discriminating reinfection from recrudescence, a truncated 6 SNP assay was found to perform at 95.1% the accuracy of the full 24 SNP bar code. CONCLUSIONS: The use of SNP analysis has similar sensitivity to the standard msp genotyping in determining recrudescence from reinfection. Although more expensive, SNP typing is faster and less work intensive. Limiting the assay to those SNPs most informative in the geographical region of interest may further decrease the workload and the cost, making this technique a feasible and affordable alternative in drug efficacy trials.

Cerebrospinal fluid Plasmodium falciparum histidine-rich protein-2 in pediatric cerebral malaria.

BACKGROUND: Cerebral malaria (CM) causes a rapidly developing coma, and remains a major contributor to morbidity and mortality in malaria-endemic regions. This study sought to determine the relationship between cerebrospinal fluid (CSF) Plasmodium falciparum histidine rich protein-2 (PfHRP-2) and clinical, laboratory and radiographic features in a cohort of children with retinopathy-positive CM. METHODS: Patients included in the study were admitted (2009-2013) to the Pediatric Research Ward (Queen Elizabeth Central Hospital, Blantyre, Malawi) meeting World Health Organization criteria for CM with findings of malarial retinopathy. Enzyme-linked immunosorbent assay was used to determine plasma and CSF PfHRP-2 levels. Wilcoxon rank-sum tests and multivariable logistic regression analysis assessed the association of clinical and radiographic characteristics with the primary outcome of death during hospitalization. RESULTS: In this cohort of 94 patients, median age was 44 (interquartile range 29-62) months, 53 (56.4%) patients were male, 6 (7%) were HIV-infected, and 10 (11%) died during hospitalization. Elevated concentrations of plasma lactate (p = 0.005) and CSF PfHRP-2 (p = 0.04) were significantly associated with death. On multivariable analysis, higher PfHRP-2 in the CSF was associated with death (odds ratio 9.00, 95% confidence interval 1.44-56.42) while plasma PfHRP-2 was not (odds ratio 2.05, 95% confidence interval 0.45-9.35). CONCLUSIONS: Elevation of CSF, but not plasma PfHRP-2, is associated with death in this paediatric CM cohort. PfHRP-2 egress into the CSF may represent alteration of blood brain barrier permeability related to the sequestration of parasitized erythrocytes in the cerebral microvasculature.

Subtle changes in Plasmodium falciparum infection complexity following enhanced intervention in Malawi.

With support from the Global Fund, the United States President's Malaria Initiative (PMI) and other cooperating partners, Malawi is implementing a comprehensive malaria control programme involving indoor residual spraying in targeted districts, universal coverage with insecticide-treated bed nets, use of rapid diagnostic tests to confirm the clinical diagnosis of malaria and use of the highly effective artemisinin-based combination therapy, artemether-lumefantrine (AL), as the first-line treatment for malaria. We genotyped 24 genome-wide single nucleotide polymorphisms (SNPs) in Plasmodium falciparum infections (n=316) sampled from a single location in Malawi before (2006 and 2007) and after enhanced intervention (2008 and 2012). The SNP data generated were used to examine temporal changes in the proportion of multiple-genotype infections (MIs), mean number of heterozygous SNPs within MIs, parasite genetic diversity (expected heterozygosity and genotypic richness), multilocus linkage disequilibrium and effective population size (N(e)). While the proportion of MIs, expected heterozygosity, genotypic richness, multilocus linkage disequilibrium and Ne were unchanged over time, the mean number (±standard deviation) of heterozygous SNPs within MIs decreased significantly (p=0.01) from 9(±1) in 2006 to 7(±1) in 2012. These findings indicate that the genetic diversity of P. falciparum malaria parasites in this area remains high, suggesting that only subtle gains, if any, have been made in reducing malaria transmission. Continued surveillance is required to evaluate the impact of malaria control interventions in this area and the rest of Malawi, and to better target control interventions.

Human cerebral malaria and Plasmodium falciparum genotypes in Malawi.

BACKGROUND: Cerebral malaria, a severe form of Plasmodium falciparum infection, is an important cause of mortality in sub-Saharan African children. A Taqman 24 Single Nucleotide Polymorphisms (SNP) molecular barcode assay was developed for use in laboratory parasites which estimates genotype number and identifies the predominant genotype. METHODS: The 24 SNP assay was used to determine predominant genotypes in blood and tissues from autopsy and clinical patients with cerebral malaria. RESULTS: Single genotypes were shared between the peripheral blood, the brain, and other tissues of cerebral malaria patients, while malaria-infected patients who died of non-malarial causes had mixed genetic signatures in tissues examined. Children with retinopathy-positive cerebral malaria had significantly less complex infections than those without retinopathy (OR = 3.7, 95% CI [1.51-9.10]).The complexity of infections significantly decreased over the malaria season in retinopathy-positive patients compared to retinopathy-negative patients. CONCLUSIONS: Cerebral malaria patients harbour a single or small set of predominant parasites; patients with incidental parasitaemia sustain infections involving diverse genotypes. Limited diversity in the peripheral blood of cerebral malaria patients and correlation with tissues supports peripheral blood samples as appropriate for genome-wide association studies of parasite determinants of pathogenicity.