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BackgroundMalnutrition increases risk of acquiring infections but clinical characteristics and hospital outcomes among children in low resource settings with high rates of antimicrobial resistance have not been clearly described.AimOur main aim was to ascertain prevalence of bacteraemia in hospitalised children at Queen Elizabeth Central Hospital, Malawi.MethodsWe conducted a secondary analysis of a prospective study of children who had a blood culture collected during hospitalisation.ResultsOut of 175 children who had blood cultures collected during hospitalisation, 75 had severe acute malnutrition (SAM), 31 moderate acute malnutrition (MAM), and 69 no acute malnutrition (NAM). Twelve (7%) had bacteraemia (8 SAM, 1 MAM, 3 NAM) and seventeen (10%) died (14 SAM, 2 MAM, 1 NAM). Fever, vomiting and convulsions were least common in SAM (69%, 37%, 1%) compared to MAM (90%, 81%, 10%) and NAM (99%, 46%, 29%; pSalmonella Typhimurium (31%) and Escherichia coli (23%). High rates of bacterial resistance were noted to gentamicin (58%), a first-line antibiotic, and ceftriaxone (33%), a second-line antibiotic.ConclusionsMortality and bacteraemia rates are highest in hospitalised children with SAM. Despite this, SAM children do not present with typical clinical features, including fever, vomiting and convulsions. Given the high rate of antimicrobial resistance in this setting, a high index of infection clinical suspicion, awareness of local susceptibility patterns and evidence-based antibiotic guidelines are needed to optimise clinical care and antimicrobial stewardship.Lay SummaryMalnutrition increases the risk of having an infection but symptoms and hospital outcomes among children with malnutrition, in countries like Malawi with high rates of antimicrobial resistance, have not been clearly described. This study describes a study of children who had a blood culture collected during admission to Queen Elizabeth Central Hospital, Malawi. Of 175 children who had blood cultures collected, 12 (7%) had a bacteria found (‘bactaeriemia’) and 17 (10%) died. Fever, vomiting and convulsions were significantly less common in severe malnutrition compared to children with moderate malnutrition and those with no malnutrition. Mortality was significantly higher in those with bacteraemia than without. High rates of bacterial resistance were noted to first- and second-line antibiotics. Mortality and bacteraemia rates are highest in hospitalised children with SAM even though they do not present with typical features of bacteraemia.

Summary In high-transmission regions, we expect parasite lineages within complex malaria infections to be unrelated due to parasite inoculations from different mosquitoes. This project was designed to test this prediction. We generated 485 single-cell genome sequences from fifteen P. falciparum malaria patients from Chikhwawa, Malawi—an area of intense transmission. Patients harbored up to seventeen unique parasite lineages. Surprisingly, parasite lineages within infections tend to be closely related, suggesting that superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Both closely and distantly related parasites comprise an infection, suggesting sequential transmission of complex infections between multiple hosts. We identified tetrads and reconstructed parental haplotypes, which revealed the inbred ancestry of infections and non-Mendelian inheritance. Our analysis suggests strong barriers to secondary infection and outbreeding amongst malaria parasites from a high transmission setting, providing unexpected insights into the biology and transmission of malaria.

Malaria patients can carry one or more clonal lineage of the parasite, Plasmodium falciparum, but the composition of these infections cannot be directly inferred from bulk sequence data. Well-defined, complete haplotypes at single-cell resolution are ideal for describing within-host population structure and unambiguously determining parasite diversity, transmission dynamics and recent ancestry but have not been analyzed on a large scale. We generated 485 near-complete single-cell genome sequences isolated from fifteen P. falciparum patients from Chikhwawa, Malawi, an area of intense malaria transmission. Matched single-cell and bulk genomic analyses revealed patients harbored up to seventeen unique lineages. Estimation of parasite relatedness within patients suggests superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Our single-cell analysis indicates strong barriers to establishment of new infections in malaria-infected patients and allows high resolution dissection of intra-host variation in malaria parasites.

Malaria-infected individuals often harbor mixtures of genetically distinct parasite genotypes. We studied intra-host dynamics of parasite genotypes co-infecting asymptomatic adults in an area of intense malaria transmission in Chikhwawa, Malawi. Serial blood samples (5 ml) were collected over seven consecutive days from 25 adults with asymptomatic Plasmodium falciparum malaria and analyzed to determine whether a single peripheral blood sample accurately captures within-host parasite diversity. Blood samples from three of the participants were also analyzed by limiting dilution cloning and SNP genotyping of the parasite clones isolated to examine both the number and relatedness of co-infecting parasite haplotypes. We observed rapid turnover of co-infecting parasite genotypes in 88% of the individuals sampled (n = 22) such that the genetic composition of parasites infecting these individuals changed dramatically over the course of seven days of follow up. Nineteen of the 25 individuals sampled (76%) carried multiple parasite genotypes at baseline. Analysis of serial blood samples from three of the individuals revealed that they harbored 6, 12 and 17 distinct parasite haplotypes respectively. Approximately 70% of parasite haplotypes recovered from the three extensively sampled individuals were unrelated (proportion of shared alleles, • We examined if a single blood sample from a malaria-infected individual accurately captures within-host parasite diversity • We also examined patterns of kinship between co-infecting parasite haplotypes to infer malaria transmission dynamics • A single sample poorly captured within-host parasite diversity indicating the need for repeat sampling of infections • Single mosquito bites played a key role in generating within-host parasite diversity even in this high transmission area

Malaria patients can carry one or more clonal lineage of the parasite, Plasmodium falciparum. Well-defined, complete haplotypes at single-cell resolution are ideal for describing within-host population structure and unambiguously determining parasite diversity, transmission dynamics and recent ancestry but have not been analyzed on a large scale. We generated 485 near-complete single-cell genome sequences isolated from fifteen P. falciparum patients from Chikhwawa, Malawi, an area of intense malaria transmission. Matched single-cell and bulk genomic analyses revealed patients harbored up to seventeen unique lineages. Current statistical approaches were unable to accurately reconstruct infection composition from bulk sequence data. Analysis of parasite relatedness within patients suggests superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Our single-cell analysis indicates strong barriers to establishment of secondary infections, providing new insights into the biology and transmission of malaria.

Single-cell genomics is a powerful tool for determining the genetic structure of complex communities of unicellular organisms. Patients infected with the malaria-causing parasite,Plasmodium falciparum, often carry multiple, genetically distinct parasites. Little is known about the diversity and relatedness of these lineages. We have developed an improved single-cell genomics protocol to reconstruct individual haplotypes from infections, a necessary step in uncovering parasite ecology within the host. This approach captures singly-infected red blood cells (iRBCs) by fluorescence-activated cell sorting (FACS) prior to whole genome amplification (WGA) and whole genome sequencing (WGS). Here, we demonstrate that parasites in late cell cycle stages, which contain increased DNA content, are far superior templates for generating high quality genomic data. Targeting of these cells routinely generates near-complete capture of the 23MbP. falciparumgenome (mean breadth of coverage 90.7%) at high efficiency. We used this approach to analyze the genomes of 48 individual cells from a polyclonal malaria infection sampled in Chikhwawa, Malawi. This comprehensive dataset enabled high-resolution estimation of the clonality and the relatedness of parasite haplotypes within the infection, long-standing problems in malaria biology.