Plasma cell-free DNA predicts pediatric cerebral malaria severity
Iset Medina Vera, Anne Kessler, Li-Min Ting, Visopo Harawa, Thomas Keller, Dylan Allen, Madi Njie, McKenze Moss, Monica Soko, Ajisa Ahmadu, Innocent Kadwala, Stephen Ray, Tonney S. Nyirenda, Wilson L. Mandala, Terrie E. Taylor, Stephen J. Rogerson, Karl B. Seydel, Kami Kim
Iset Medina Vera, Anne Kessler, Li-Min Ting, Visopo Harawa, Thomas Keller, Dylan Allen, Madi Njie, McKenze Moss, Monica Soko, Ajisa Ahmadu, Innocent Kadwala, Stephen Ray, Tonney S. Nyirenda, Wilson L. Mandala, Terrie E. Taylor, Stephen J. Rogerson, Karl B. Seydel, Kami Kim
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Clinical Research and Public Health Infectious disease Inflammation

Plasma cell-free DNA predicts pediatric cerebral malaria severity

Abstract

BACKGROUND Prediction of adverse outcomes in cerebral malaria (CM) is difficult. We hypothesized that cell-free DNA (cfDNA) levels would facilitate identification of severe and potentially fatal CM cases.METHODS In this retrospective study, plasma from Malawian children with CM (n = 134), uncomplicated malaria (UM, n = 77), and healthy controls (HC, n = 60) was assayed for cfDNA using a fluorescence assay. Host and parasite cfDNA was measured by quantitative PCR. Immune markers were determined by ELISA, Luminex, or cytometric bead array.RESULTS Total cfDNA increased with malaria severity (HC versus UM, P < 0.001; HC versus CM, P < 0.0001; UM versus CM, P < 0.0001), was elevated in retinopathy-positive (Ret+) CM relative to Ret– CM (7.66 versus 5.47 ng/μL, P = 0.027), and differentiated Ret+ fatal cases from survivors (AUC 0.779; P < 0.001). cfDNA levels in patients with non–malarial febrile illness (NMF, P = 0.25) and non–malarial coma (NMC, P = 0.99) were comparable with UM. Host DNA, rather than parasite DNA, was the major cfDNA contributor (UM, 268 versus 67 pg/μL; CM, 2824 versus 463 pg/μL). Host and parasite cfDNA distinguished CM by retinopathy (host, AUC 0.715, P = 0.0001; parasite, AUC 0.745, P = 0.0001), but only host cfDNA distinguished fatal cases (AUC 0.715, P = 0.0001). Total cfDNA correlated with neutrophil markers IL-8 (rs = 0.433, P < 0.0001) and myeloperoxidase (rs = 0.683, P < 0.0001).CONCLUSION Quantifying plasma cfDNA is a simple assay useful in identifying children at risk for fatal outcome and has promise as a point-of-care assay. Elevated cfDNA suggests a link with host inflammatory pathways in fatal CM.FUNDING NIH NCATS (AK), Burroughs-Wellcome (AK), and National Health and Medical Research Council of Australia (SJR).

Authors

Iset Medina Vera, Anne Kessler, Li-Min Ting, Visopo Harawa, Thomas Keller, Dylan Allen, Madi Njie, McKenze Moss, Monica Soko, Ajisa Ahmadu, Innocent Kadwala, Stephen Ray, Tonney S. Nyirenda, Wilson L. Mandala, Terrie E. Taylor, Stephen J. Rogerson, Karl B. Seydel, Kami Kim

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Figure 2

Total plasma cfDNA levels are elevated with malaria severity.

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Total plasma cfDNA levels are elevated with malaria severity. (A–C) Tota...
(A–C) Total plasma cfDNA levels (ng/μL) for clinical and control groups as measured by fluorometry. (A) Healthy controls (HC, n = 60) versus uncomplicated malaria (UM, n = 77) versus cerebral malaria (CM, n = 134). (B) CM classified as Ret– CM (n = 30) versus Ret+ CM (n = 104). (C) Survivors (Ret– CM, n = 22; Ret+ CM, n = 91) versus fatal cases (Ret– CM, n = 7; Ret+ CM, n = 13). (D) Total cfDNA levels in UM (n = 30) or CM (n = 67) convalescence from acute infection to 30-day follow-up (30d FU). (E) Total serum cfDNA levels of healthy controls (HC, n = 33), non–malaria febrile (NMF, n = 40), uncomplicated malaria (UM, n = 47), and non–malaria coma (NMC, n = 49) clinical groups. Shown are median levels ± IQR; statistical significance was determined by Mann-Whitney U test (B), Kruskal-Wallis test for multiple comparisons (A, C, and E), or Wilcoxon signed rank test for paired comparisons (D). P < 0.05 was considered significant.