Univariate analysis of 18 individual biomarkers‡ among malaria-negative patients for all three countries (A–C)
| (A) Brazil AUROC* (CI), N | |||
| Electronic | Strict | Loose | |
| Haematological biomarkers | |||
| Lymphocyte count | 0.67 (0.59 to 0.74), 257 | 0.66 (0.59 to 0.72), 408 | 0.66 (0.6 to 0.72), 442 |
| Neutrophil count | 0.77 (0.7 to 0.84), 257 | 0.8 (0.74 to 0.86), 408 | 0.79 (0.73 to 0.84), 442 |
| RBC count | 0.61 (0.52 to 0.69), 258 | 0.58 (0.51 to 0.65), 408 | 0.58 (0.51 to 0.64), 442 |
| WBC count | 0.81 (0.75 to 0.87), 257 | 0.83 (0.77 to 0.88), 408 | 0.82 (0.77 to 0.87), 442 |
| Protein biomarkers | |||
| AGP | 0.59 (0.51 to 0.68), 252 | 0.54 (0.47 to 0.61), 402 | 0.52 (0.46 to 0.59), 434 |
| Chitinase 3-like 1 | 0.58 (0.5 to 0.66), 246 | 0.54 (0.47 to 0.6), 394 | 0.55 (0.49 to 0.61), 424 |
| CRP† | 0.61 (0.52 to 0.69), 259 | 0.61 (0.54 to 0.68), 412 | 0.62 (0.55 to 0.68), 446 |
| IP-10/IP-10/CRG-2 | 0.6 (0.52 to 0.68), 252 | 0.53 (0.46 to 0.59), 402 | 0.53 (0.47 to 0.59), 434 |
| Galectin-9 | 0.63 (0.55 to 0.71), 252 | 0.56 (0.49 to 0.63), 401 | 0.57 (0.5 to 0.63), 433 |
| hCC2 | 0.51 (0.43 to 0.6), 244 | 0.51 (0.44 to 0.58), 392 | 0.52 (0.46 to 0.59), 424 |
| HBP‡ | 0.67 (0.52 to 0.81), 113 | 0.68 (0.55 to 0.8), 144 | 0.64 (0.51 to 0.76), 151 |
| HPTGN | 0.48 (0.4 to 0.57), 248 | 0.51 (0.44 to 0.58), 398 | 0.51 (0.45 to 0.58), 430 |
| IL-4 | 0.58 (0.5 to 0.65), 249 | 0.53 (0.47 to 0.59), 398 | 0.54 (0.48 to 0.59), 429 |
| IL-6 | 0.49 (0.43 to 0.54), 247 | 0.49 (0.44 to 0.54), 395 | 0.48 (0.43 to 0.52), 426 |
| LBP | 0.58 (0.5 to 0.66), 248 | 0.54 (0.48 to 0.61), 397 | 0.52 (0.46 to 0.58), 429 |
| Lipocalin-2/NGAL | 0.49 (0.41 to 0.57), 249 | 0.51 (0.44 to 0.57), 396 | 0.51 (0.44 to 0.57), 428 |
| sPLA/Lp-PLA2 | 0.54 (0.46 to 0.62), 252 | 0.53 (0.46 to 0.59), 402 | 0.52 (0.45 to 0.58), 434 |
| TRAIL | 0.56 (0.49 to 0.64), 252 | 0.53 (0.47 to 0.59), 402 | 0.53 (0.48 to 0.59), 434 |
|
(B) Gabon
AUROC** (CI), N | |||
| Electronic | Strict | Loose | |
| Haematological biomarkers | |||
| Lymphocyte count | 0.58 (0.45 to 0.71), 81 | 0.52 (0.4 to 0.63), 167 | 0.55 (0.45 to 0.65), 222 |
| Neutrophil count | 0.78 (0.66 to 0.89), 80 | 0.72 (0.62 to 0.83), 165 | 0.67 (0.57 to 0.77), 219 |
| RBC count | 0.55 (0.41 to 0.68), 81 | 0.52 (0.41 to 0.63), 167 | 0.53 (0.43 to 0.63), 222 |
| WBC count | 0.67 (0.54 to 0.79), 81 | 0.6 (0.48 to 0.72), 167 | 0.61 (0.5 to 0.71), 222 |
| Protein biomarkers | |||
| AGP | 0.77 (0.65 to 0.9), 80 | 0.7 (0.59 to 0.82), 163 | 0.65 (0.55 to 0.75), 220 |
| Chitinase 3-like 1 | 0.6 (0.46 to 0.74), 79 | 0.6 (0.48 to 0.72), 162 | 0.62 (0.52 to 0.72), 217 |
| CRP† | 0.71 (0.59 to 0.82), 81 | 0.65 (0.55 to 0.75), 167 | 0.63 (0.53 to 0.72), 224 |
| IP-10/IP-10/CRG-2 | 0.6 (0.48 to 0.73), 80 | 0.51 (0.4 to 0.62), 164 | 0.52 (0.43 to 0.62), 221 |
| Galectin-9 | 0.7 (0.58 to 0.83), 80 | 0.6 (0.48 to 0.71), 163 | 0.54 (0.43 to 0.64), 219 |
| hCC2 | 0.55 (0.41 to 0.69), 77 | 0.52 (0.4 to 0.64), 159 | 0.51 (0.41 to 0.61), 216 |
| HBP‡ | |||
| HPTGN | 0.64 (0.5 to 0.78), 77 | 0.62 (0.51 to 0.74), 159 | 0.55 (0.45 to 0.66), 214 |
| IL-4 | 0.46 (0.4 to 0.52), 79 | 0.49 (0.45 to 0.53), 163 | 0.51 (0.47 to 0.55), 220 |
| IL-6 | 0.51 (0.47 to 0.55), 80 | 0.51 (0.48 to 0.55), 164 | 0.51 (0.47 to 0.55), 221 |
| LBP | 0.69 (0.56 to 0.83), 78 | 0.67 (0.55 to 0.78), 160 | 0.6 (0.5 to 0.71), 217 |
| Lipocalin-2/NGAL | 0.67 (0.54 to 0.8), 79 | 0.6 (0.49 to 0.72), 163 | 0.58 (0.48 to 0.68), 219 |
| sPLA/Lp-PLA2 | 0.58 (0.44 to 0.71), 80 | 0.54 (0.43 to 0.65), 164 | 0.58 (0.48 to 0.68), 221 |
| TRAIL | 0.5 (0.5 to 0.5), 74 | 0.5 (0.49 to 0.5), 156 | 0.49 (0.48 to 0.5), 212 |
| (C) Malawi AUROC** (CI), N | |||
| Electronic | Strict | Loose | |
| Haematological biomarkers | |||
| Lymphocyte count | 0.56 (0.47 to 0.66), 154 | 0.51 (0.45 to 0.58), 303 | 0.52 (0.47 to 0.58), 461 |
| Neutrophil count | 0.67 (0.58 to 0.77), 143 | 0.73 (0.67 to 0.79), 273 | 0.7 (0.65 to 0.76), 414 |
| RBC count | 0.46 (0.36 to 0.56), 155 | 0.53 (0.46 to 0.59), 305 | 0.56 (0.5 to 0.61), 463 |
| WBC count | 0.69 (0.6 to 0.78), 155 | 0.72 (0.66 to 0.78), 304 | 0.68 (0.63 to 0.73), 461 |
| Protein biomarkers | |||
| AGP | 0.56 (0.46 to 0.66), 158 | 0.54 (0.48 to 0.6), 309 | 0.54 (0.49 to 0.59), 466 |
| Chitinase 3-like 1 | 0.49 (0.39 to 0.59), 155 | 0.5 (0.43 to 0.56), 304 | 0.5 (0.44 to 0.55), 462 |
| CRP† | 0.55 (0.45 to 0.65), 156 | 0.6 (0.54 to 0.67), 305 | 0.58 (0.53 to 0.63), 462 |
| IP-10/IP-10/CRG-2 | 0.66 (0.56 to 0.75), 158 | 0.6 (0.53 to 0.66), 309 | 0.61 (0.56 to 0.66), 466 |
| Galectin-9 | 0.71 (0.62 to 0.8), 158 | 0.61 (0.55 to 0.67), 309 | 0.63 (0.57 to 0.68), 466 |
| hCC2 | 0.59 (0.49 to 0.69), 158 | 0.55 (0.49 to 0.62), 309 | 0.55 (0.5 to 0.6), 466 |
| HBP‡ | 0.53 (0.39 to 0.68), 63 | 0.55 (0.44 to 0.66), 106 | 0.52 (0.41 to 0.63), 124 |
| HPTGN | 0.54 (0.45 to 0.64), 157 | 0.51 (0.45 to 0.58), 307 | 0.51 (0.46 to 0.57), 464 |
| IL-4 | 0.48 (0.4 to 0.57), 157 | 0.48 (0.42 to 0.53), 306 | 0.47 (0.42 to 0.51), 463 |
| IL-6 | 0.56 (0.47 to 0.65), 158 | 0.61 (0.55 to 0.67), 307 | 0.59 (0.54 to 0.64), 465 |
| LBP | 0.52 (0.42 to 0.61), 157 | 0.54 (0.47 to 0.61), 267 | 0.53 (0.47 to 0.59), 394 |
| Lipocalin-2/NGAL | 0.56 (0.46 to 0.66), 156 | 0.65 (0.59 to 0.72), 265 | 0.61 (0.56 to 0.67), 392 |
| sPLA/Lp-PLA2 | 0.58 (0.47 to 0.68), 158 | 0.55 (0.49 to 0.61), 308 | 0.56 (0.51 to 0.61), 466 |
| TRAIL | 0.61 (0.51 to 0.71), 157 | 0.62 (0.56 to 0.68), 306 | 0.62 (0.57 to 0.67), 463 |
Common biomarkers such as CRP and haematological biomarkers were included for reference. In this context, we defined performance as follows: dark blue (AUROC≥0.7), light blue (AUROC>0.65 and <0.7), orange (AUROC 0.6–0.65) and red (AUROC<0.6)
*AUROC has a value between 0 and 1, where 1 corresponds to an effect classifier, 0.5–1 that assigns classes randomly.
†CRP was measured with a NycoCard device.
‡Freeze-thaw experiments to evaluate the stability of the biomarkers after five cycles (referred to as ‘treated’) were performed with Luminex 9-plexes and 2-plexes. Three samples each were freeze-thawed up to six times and compared with samples after the first thawing (referred to as ‘untreated’; biomarkers were considered stable with 80–120% recovery). Samples were analysed in triplicate and showed good stability up to five freeze-thaw cycles for all analytes showing acceptable results, except for the C2 and C4b biomarkers (C2: 2/3 (66.7%) samples were stable; C4b: two samples failed the sixth freeze-thaw cycle). As a result, these biomarkers were excluded as they would never be suitable as the basis of a diagnostic test.
§HBP was evaluated in a small group of patients in Malawi and Brazil; however, HBP did not show promise and was not evaluated further.
AGP, A-1-acid glycoprotein; AUROC, area under the receiver operating characteristic; CRP, C-reactive protein; HBP, heparin-binding protein; IL-4, interleukin-4; IP-10, gamma-induced protein 10; LBP, lipopolysaccharide binding protein; NGAL, neutrophil gelatinase-associated lipocalin; RBC, red blood cell; TRAIL, TNF-related apoptosis-inducing ligand; WBC, white blood cell.