Evaluating blood culture collection practice in children hospitalized with acute illness at a tertiary hospital in Malawi.

BACKGROUND: Blood culture collection practice in low-resource settings where routine blood culture collection is available has not been previously described. METHODOLOGY: We conducted a secondary descriptive analysis of children aged 2-23 months enrolled in the Malawi Childhood Acute Illness and Nutrition (CHAIN) study, stratified by whether an admission blood culture had been undertaken and by nutritional status. Chi-square test was used to compare the differences between groups. RESULTS: A total of 347 children were included, of whom 161 (46%) had a blood culture collected. Children who had a blood culture collected, compared to those who did not, were more likely to present with sepsis (43% vs. 20%, p < 0.001), gastroenteritis (43% vs. 26%, p < 0.001), fever (86% vs. 73%, p = 0.004), and with poor feeding/weight loss (30% vs. 18%, p = 0.008). In addition, hospital stay in those who had a blood culture was, on average, 2 days longer (p = 0.019). No difference in mortality was observed between those who did and did not have a blood culture obtained. CONCLUSION: Blood culture collection was more frequent in children with sepsis and gastroenteritis, but was not associated with mortality. In low-resource settings, developing criteria for blood culture based on risk factors rather than clinician judgement may better utilize the existing resources.

Blood culture is key to investigating bloodstream infections, but in-hospital decisions to perform blood culture in a low-resource setting have not been previously described. We linked blood culture data to the Childhood Acute Illness and Nutrition (CHAIN) cohort at a Malawi tertiary hospital and compared clinical characteristics and outcomes of children between those who did and did not have a blood culture done on admission. Of those hospitalized, 46% of the children had a blood culture collected at admission. Only 3% of blood cultures had significant growth of pathogenic bacteria. There were significant differences in nutritional status, presenting symptoms, clinical diagnoses and hospital length of stay between those who received blood culture collection on admission and those who did not, but there was no difference in mortality. Clinical judgement used to determine blood culture collection may not best identify children most at risk.

The presence of enteropathy in HIV infected children on antiretroviral therapy in Malawi.

BACKGROUND: Undernutrition and malnutrition in children in low- and middle-income countries contribute to high mortality rates. Stunting, a prevalent form of malnutrition, is associated with educational and productivity losses. Environmental enteric dysfunction (EED) and human immunodeficiency virus (HIV) infection worsen these conditions. This study seeks to investigate the presence of enteropathy using EED fecal biomarkers in HIV-infected children who are stable on antiretroviral therapy (ART) across various nutritional statuses. By understanding the interplay between EED, HIV, and nutritional status, this study aims to provide insights that can inform targeted interventions to optimize nutritional outcomes in HIV infected children. METHODS/PRINCIPAL FINDINGS: This study evaluated the levels of alpha-1-antitrypsin, calprotectin and myeloperoxidase in frozen fecal samples from 61 HIV infected (mean age 9.16 ±3.08 years) and 31 HIV uninfected (6.65 ±3.41 years) children in Malawi. Anthropometric measurements and clinical data were collected. The height-for-age z-score (-1.66 vs -1.27, p = 0.040) and BMI-for-age z-score (-0.36 vs 0.01, p = 0.037) were lower in HIV infected children. Enzyme-linked immunosorbent assays were used to measure biomarker concentrations. Statistical tests were applied to compare biomarker levels based on HIV status and anthropometric parameters. Myeloperoxidase, alpha-1-antitrypsin, and calprotectin concentrations did not differ between HIV infected and HIV uninfected children of different age groups. In HIV infected children from 5-15 years, there is no difference in biomarker concentration between the stunted and non-stunted groups. CONCLUSION/SIGNIFICANCE: Our study found a higher prevalence of stunting in HIV infected children compared to uninfected children, but no significant differences in biomarker concentrations. This suggests no causal relationship between enteropathy and stunting in HIV infected children. These results contribute to the understanding of growth impairment in HIV infected children and emphasize the need for further research, particularly a longitudinal, biopsy-controlled study.

Hepatic mitochondrial and peroxisomal alterations in acutely ill malnourished Malawian children: A postmortem cohort study.

Objectives: To describe and compare liver mitochondrial and peroxisomal histopathology by nutritional status in children who died following hospitalization for acute illness in Malawi. Methods: Liver tissue was collected using Minimally Invasive Tissue Sampling from eleven children under-five years old who died during hospitalization and were either non-wasted (n = 4), severely wasted (n = 4) or had edematous malnutrition (n = 3). Histology was assessed on hematoxylin and eosin stained slides. Mitochondrial and peroxisomal ultrastructural features were characterized using electron microscopy (EM) and immunofluorescence (IF). Results: Hepatic steatosis was present in 50 % of non-wasted and severely wasted children and all children with edematous malnutrition. Edematous malnutrition was associated with 56 % and 45 % fewer mitochondria than severe wasting (p < 0.001) and no wasting (p = 0.006), respectively, and abnormal mitochondrial morphology compared to severe wasting (p = 0.002) and no wasting (p = 0.035). Peroxisomal abundance was reduced in edematous malnutrition compared to severe wasting (p = 0.005), but did not differ from no-wasting. Conclusion: Edematous malnutrition is associated with reduced abundance and altered morphology of hepatic mitochondria and peroxisomes. Interventions targeting improvements in hepatic metabolic function may be beneficial in improving metabolism and reducing mortality in children with severe malnutrition, particularly in those with nutritional edema.

Trajectories of resting energy expenditure and performance of predictive equations in children hospitalized with an acute illness and malnutrition: a longitudinal study.

There is scarce data on energy expenditure in ill children with different degrees of malnutrition. This study aimed to determine resting energy expenditure (REE) trajectories in hospitalized malnourished children during and after hospitalization. We followed a cohort of children in Bangladesh and Malawi (2-23 months) with: no wasting (NW); moderate wasting (MW), severe wasting (SW), or edematous malnutrition (EM). REE was measured by indirect calorimetry at admission, discharge, 14-and-45-days post-discharge. 125 children (NW, n = 23; MW, n = 29; SW, n = 51; EM, n = 22), median age 9 (IQR 6, 14) months, provided 401 REE measurements. At admission, the REE of children with NW and MW was 67 (95% CI [58, 75]) and 70 (95% CI [63, 76]) kcal/kg/day, respectively, while REE in children with SW was higher, 79 kcal/kg/day (95% CI [74, 84], p = 0.018), than NW. REE in these groups was stable over time. In children with EM, REE increased from admission to discharge (65 kcal/kg/day, 95% CI [56, 73]) to 79 (95% CI [72, 86], p = 0.0014) and was stable hereafter. Predictive equations underestimated REE in 92% of participants at all time points. Recommended feeding targets during the acute phase of illness in severely malnourished children exceeded REE. Acutely ill malnourished children are at risk of being overfed when implementing current international guidelines.

Co-transmission of Related Malaria Parasite Lineages Shapes Within-Host Parasite Diversity.

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.

Intra-host dynamics of co-infecting parasite genotypes in asymptomatic malaria patients.

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 <83.3%) and were deemed to have primarily arisen from superinfection (inoculation of unrelated parasite haplotypes through multiple mosquito bites). The rest were related at the half-sib level or greater and were deemed to have been inoculated into individual human hosts via parasite co-transmission from single mosquito bites. These findings add further to the growing weight of evidence indicating that a single blood sample poorly captures within-host parasite diversity and underscore the importance of repeated blood sampling to accurately capture within-host parasite ecology. Our data also demonstrate a more pronounced role for parasite co-transmission in generating within-host parasite diversity in high transmission settings than previously assumed. Taken together, these findings have important implications for understanding the evolution of drug resistance, malaria transmission, parasite virulence, allocation of gametocyte sex ratios and acquisition of malaria immunity.

High-Resolution Single-Cell Sequencing of Malaria Parasites.

Single-cell genomics is a powerful tool for determining the genetic architecture of complex communities of unicellular organisms. In areas of high transmission, malaria patients are often challenged by the activities of multiple Plasmodium falciparum lineages, which can potentiate pathology, spread drug resistance loci, and also complicate most genetic analysis. Single-cell sequencing of P. falciparum would be key to understanding infection complexity, though efforts are hampered by the extreme nucleotide composition of its genome (∼80% AT-rich). To counter the low coverage achieved in previous studies, we targeted DNA-rich late-stage parasites by Fluorescence-Activated Cell Sorting and whole genome sequencing. Our method routinely generates accurate, near-complete capture of the 23 Mb P. falciparum genome (mean breadth of coverage 90.7%) at high efficiency. Data from 48 single-cell genomes derived from a polyclonal infection sampled in Chikhwawa, Malawi allowed for unambiguous determination of haplotype diversity and recent meiotic events, information that will aid public health efforts.