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TITLE: 1 The severity of Plasmodium falciparum infection is associated with transcript levels of 2 var genes encoding EPCR-binding PfEMP1 3 4 Sixbert I. Mkumbaye*1, Christian W. Wang*2, Eric Lyimo3, Jakob S. Jespersen2, 5 Alphaxard Manjurano3, Jacklin Mosha3, Reginald A. Kavishe1, Steven B. Mwakalinga1, 6 Daniel TR. Minja4, John P. Lusingu4, Thor G. Theander2 and Thomas Lavstsen2#. 7 8 1) Kilimanjaro Christian Medical University College and Kilimanjaro Clinical Research 9 Institute, Moshi, Tanzania 10 2) Centre for Medical Parasitology, Department of International Health, Immunology & 11 Microbiology, University of Copenhagen and Department of Infectious Diseases, 12 Rigshospitalet, Copenhagen, Denmark 13 3) National Institute for Medical Research, Mwanza Research Centre, Mwanza, Tanzania 14 4) National Institute for Medical Research, Tanga Research Centre, Tanga, Tanzania 15 16 Running head: Pf var gene expression in patients 17 # Address correspondence to: Thomas Lavstsen, [email protected] 18 * Equal contributors. S.I.M and C.W.W contributed equally to this work. 19 20 21

IAI Accepted Manuscript Posted Online 30 January 2017Infect. Immun. doi:10.1128/IAI.00841-16Copyright © 2017 American Society for Microbiology. All Rights Reserved.

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ABSTRACT 22 By attaching infected erythrocytes to the vascular lining, Plasmodium falciparum parasites 23 leave blood circulation and avoid splenic clearance. This sequestration is central to 24 pathogenesis. Severe malaria is associated with parasites expressing an antigenically distinct 25 subset of P. falciparum erythrocyte membrane protein 1 (PfEMP1) mediating the binding to 26 endothelial receptors. Previous studies indicate that PfEMP1, with so-called CIDRα1 domains 27 capable of binding endothelial protein C receptor (EPCR), constitute the PfEMP1 subset 28 associated with severe paediatric malaria. To analyse the relative importance of different 29 subtypes of CIDRα1 domains, we compared pfemp1 transcripts in children with severe 30 malaria (including 9 fatal and 114 surviving cases), children hospitalised with uncomplicated 31 malaria (N=42), children with mild malaria not requiring hospitalisation (N=10) and children 32 with parasiteamia and no ongoing fever (N=12). High levels of transcripts encoding EPCR-33 binding PfEMP1 were found in patients with symptomatic infections and the abundance of 34 these transcripts increased with disease severity. The CIDRα1 subtype transcript composition 35 varied markedly between patients, and none of the subtypes were dominant. Transcript level 36 analyses targeting other domain types indicated that subtypes of DBLβ or DBLζ domains 37 might mediate binding phenomena that, in conjunction with EPCR binding, could contribute 38 to pathogenesis. The observations strengthen the rationale for targeting PfEMP1-EPCR 39 interaction by vaccines and adjunctive therapies. Interventions should target the EPCR 40 binding of all CIDRα1 subtypes. 41 42

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INTRODUCTION 43 Based on simple clinical observations, malaria patients can be divided into a smaller group 44 with severe manifestations and a much larger group with uncomplicated disease (1). In areas 45 of Africa with high to moderate malaria transmission, severe malaria is almost only seen in 46 children below five years of age (2). At the first entry point for healthcare, the majority of 47 African children diagnosed with Plasmodium falciparum malaria suffer an uncomplicated 48 febrile disease and are treated as outpatients. However, based on the initial assessment, the 49 examining physician hospitalise some patients with more manifest symptoms. These children 50 are not well, but based on simple triage, they can be further divided into a large group with 51 uncomplicated disease, which, upon correct treatment, will be very likely to survive the 52 disease, and a smaller group with severe disease, where a proportion will die despite 53 administration of what is currently considered optimal care (3). It is estimated that around 10 54 million children suffer severe malaria every year and that 5-10% of these children die (4) . 55 Even though most children in malaria-endemic areas are expected to experience several bouts 56 of malaria during childhood, only one to three of these are likely to cause severe illness, and 57 these usually occur early in life (5). This epidemiological picture has spurred the hypothesis 58 that the parasites causing severe malaria are phenotypically different from those causing 59 uncomplicated disease, and that children acquire immunity to severe malaria by mounting an 60 antibody response to the parasite proteins that convey the phenotype associated with severe 61 outcomes (6-9). 62 P. falciparum parasites depend on evading splenic destruction by anchoring infected 63 erythrocytes to endothelial cells. The sequestration of parasites in host capillaries drives 64 malaria pathogenesis, and immuno-epidemiological studies have indicated that an 65 antigenically restricted subset of the polymorphic P. falciparum erythrocyte membrane 66

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protein 1 (PfEMP1) adhesins are associated with life-threating infections experienced during 67 childhood in malaria-endemic regions (7, 8). PfEMP1 are expressed on the surface of 68 erythrocytes infected with the late blood stage parasites (trophozoites) where they mediate 69 attachment to receptors on the vascular lining (10), allowing the infected cells to avoid 70 circulation and passage though the spleen, where they are destroyed. 71 PfEMP1 are encoded by var genes. Each parasite genome harbours about 60 variants (11-13), 72 but each parasite expresses only one var gene at a time (14). Although PfEMP1 sequences are 73 extremely diverse, their domain architectures are highly organised, and all parasites carry 74 similar PfEMP1 repertoires which appear to bestow all parasites with the same fundamental 75 repertoire of human receptor specificities (15). The large multi-domain PfEMP1 proteins 76 consist of 2-9 Duffy binding like (DBL) and cysteine-rich inter-domain region (CIDR) domains, 77 which based on sequence similarity, can be further sub-divided into different groups (16, 17). 78 A single distinct group of PfEMP1, VAR2CSA, binds parasites to receptors in the placenta and 79 is a known virulence factor for pregnancy malaria (18, 19). It has proven more challenging to 80 characterise the PfEMP1 types or traits linked to parasites causing severe paediatric malaria. 81 Early studies implicated the so-called group A and B var gene variants (Fig. 1), which separate 82 from each other by chromosomal orientation and encoding of distinct N-terminal domains 83 (group A DBLα1 vs. group B DBLα0/2 domains) (20-24). Subsequent studies specified that 84 severe malaria is associated with parasites expressing the group B var gene subset encoding 85 domain cassette (DC) 8 (has DBLα2 domains) and group A var genes including those encoding 86 DC13 (25, 26). These PfEMP1 types were found to share binding phenotype (27) as both DC8 87 and DC13 bind endothelial protein C receptor (EPCR) via their CIDRα1.1 and CIDRα1.4 88 domains, respectively. Recently, the crystal structure of the CIDRα1-EPCR interaction was 89 solved, and a more precise description of which CIDRα1 subclasses bind EPCR was obtained 90

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(28), showing that most CIDRα1 domain variants bind EPCR. Jespersen et al. 2016 (29), 91 reporting near full-length sequence annotation of var transcripts in 44 patients, found that 92 CIDRα1 was the only domain type that was common between different PfEMP1 variants 93 expressed by severe malaria isolates. Parasites expressing DC13 PfEMP1 were recently shown 94 to bind both EPCR and ICAM-1 (30) suggesting that other PfEMP1 traits such as binding to 95 ICAM-1 in combination with EPCR binding could increase the risk of developing specific 96 syndromes. Similarly, PfEMP1 with a C-terminal domain cassette (DC) 6 containing DBLγ14-97 DBLζ5-DBLε4 domains (31) was linked to hospitalisation of P. falciparum infected Indian 98 adults. These studies included relatively few patients, and it is possible that the var type 99 quantification by sequence tags or by primers with limited sequence variant coverage has 100 overestimated these PfEMP1 traits or underestimated others. 101 102 Here, we show by quantitative PCR and a new, more comprehensive, set of var type specific 103 primers than previously used (25, 26), that parasites from Tanzanian children hospitalised 104 with malaria and diagnosed with either severe malarial anaemia, cerebral malaria or no 105 severe complication (uncomplicated malaria) were all characterised by high transcript levels 106 of genes encoding DC8 var genes and the subset of group A var genes encoding EPCR-binding 107 PfEMP1. Furthermore, severe disease was, in a few cases, associated with an increased level of 108 transcripts encoding specific subsets DBLβ or DBLζ domains. 109 110 MATERIAL AND METHODS 111 Sample collection 112 Samples were collected from 187 children who were blood-smear positive for P. falciparum. 113 The children were enrolled after obtaining informed consent from a parent or legally 114

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acceptable guardian. Of the 187 children, 165 were admitted to either Korogwe District 115 Hospital in north east Tanzania (N=110) or Magu District Hospital in the north west (N=55). 116 Children were clinically evaluated by study clinicians, and a blood sample was collected for 117 diagnostic and research purposes, after which treatment was instigated according to the 118 national guidelines. Samples were collected in 2013 and 2014. Blood samples from 22 non-119 hospitalised children living in Korogwe district who had mild malaria or malaria not 120 accompanied by fever were also included in the study. These children were recruited as part 121 of cross-sectional surveys in a village in 2007 and 2008. The study received ethical clearance 122 and approval from the National Health Research Ethics Committee in Tanzania (reference no. 123 NIMR/HQ/R.8c/Vol.II/436). 124 125 Primer design 126 The sequence diversity of the different PfEMP1 domain classes differs from the relatively 127 clear division of CIDRα1 domains into a few distinct subclasses to no particular subgrouping 128 of DBLδ domains (17). For this reason, design of informative primers was only possible for 129 the best-defined domain subclasses (Fig. S1). To maximise coverage while maintaining 130 specificity for regions encoding specific domain subclasses, primers were designed based on 131 full-length DBL and CIDR domain encoding sequences from seven P. falciparum genomes (17) 132 and 226 Illumina whole-genome sequenced P. falciparum field isolates (28, 32) (Fig. S1). 133 A particular effort was made to secure good coverage of group A var genes, in order to resolve 134 which group A and DC8 var gene subclasses are associated with severe malaria. Group A 135 PfEMP1 are characterised by having NTSA-DBLα1 and CIDRα1, -β, -γ or -δ domains, where the 136 NTSA sequences exhibits very little sub-grouping, and subclass of DBLα1 variants are largely 137 predicted by the subgrouping of the following CIDR domain (17, 25). Group A CIDRα1.4-7 138

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domains bind EPCR, whereas group A CIDRα1.2/3 domains are found in the var1 139 pseudogenes, which do not bind EPCR (28). The function of CIDRδ and the more diverse 140 CIDRβ and -γ domains is unknown, albeit suggested to be associated with rosetting (15). 141 Group B and C PfEMP1 have DBLα0 and CD36-binding CIDRα2-6 domains, apart from the 142 atypical DC8 type group B PfEMP1 which carry the DBLα2-CIDRα1.1/8 domains (EPCR-143 binding CIDR). Good coverage primers (Fig. S1 and S2) were successfully designed for all 144 CIDRα1 and N-terminal CIDRδ domain subclasses, although the diversity of CIDRα2-6 and N-145 terminal CIDRβ and -γ domains was difficult to capture. 146 The unusually conserved PfEMP1 variants, VAR2CSA (binding placental chondroitin sulphate 147 A (CSA)) and VAR3, (unknown binding specificity) were targeted by specific DBL primers. 148 Good coverage was also achieved for primers targeting C-terminal DBLε and DBLζ domains. 149 ICAM-1 binding has been mapped to group A DBLβ1/3 and group B and C DBLβ5 domains. 150 Primer sets with limited coverage but good specificity for DBLβ5 and two subsets of DBLβ1/3 151 (DBLb1/3-1 and DBLb1/3-2) were designed. For all primer sets, amplification efficiencies 152 >94% were ascertained by quantitative PCR (qPCR) measurements of serial 10-fold dilutions 153 of 3D7, HB3 and IT4/FCR3 gDNA, and predicted size or absence of PCR amplicons were 154 validated by gel electrophoresis. The specificity and coverage of each primer set was 155 evaluated in silico using USEARCH (the “search_pcr”) (33) against full length DBL and CIDR 156 domains extracted from the 233 genome sequence data, calling targets by allowing up to 2 157 mismatches in each primer. The result was manually parsed to remove hits with 3’ terminus 158 mismatches to either primer and to remove all but one (best) hit to each domain/contig 159 (multiple reports of an amplicon from the same locus may be generated when degenerate 160 primers are employed). These criteria were previously found to give good estimates of target 161 amplification (25). 162

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In summary, good specificity and coverage were achieved for primer sets capturing 163 transcripts encoding subclasses of CIDRα1 as well as CIDRδ, VAR2CSA and VAR3. The 164 coverage for var sequences encoding DBLζ and DBLε domains was also good, while the 165 coverage for CIDRγ, CD36-binding CIDRα2-6 and DBLβ was low. 166 167 Parasite RNA and qPCR 168 Erythrocytes (50–100 μL) pelleted by centrifugation from venous blood samples were 169 completely dissolved in 1 mL TRIzol® reagent (Invitrogen) and stored in liquid nitrogen, dry 170 ice or -80 °C until RNA purification. Total RNA was extracted, DNase treated (DNase 1, Sigma-171 Aldrich), and verified with no residual genomic DNA by qPCR using primers endogenous 172 housekeeping gene seryl-tRNA synthetase, before being reverse-transcribed (Superscript II, 173 Invitrogen) as described in (25). qPCR analyses were performed using QuantiTect SYBR Green 174 PCR master mix (Qiagen). Mastermix was distributed into primer-loaded tubes for qPCR 175 performed in 20-μL reactions using with the Rotorgene thermal cycler system (Corbett 176 Research) and the cycling conditions from (25). 177 Var transcript abundances were determined in relation to the averaged transcript abundance 178 of endogenous housekeeping genes seryl-tRNA synthetase and aldolase (ΔCtvar_primer = 179 Ctvar_primer − Ctaverage_control primers). ΔCtvar_primer values were translated into Transcript Units (Tu= 180 2 (5-ΔCt)) (25), where low abundant transcripts with ΔCt values >5 were all assigned to ΔCt=5 181 (Tu= 1). 182 For some primer sets, the reported transcript abundance was summarised by adding the 183 reported Transcript Unit for each primer set and subtracting the value 1 for each additional 184 value added (subtracting was done to assure that the summarised Tu was =1 if no signal was 185 detected for any of the transcripts summarised). 186

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Due to the differences in sequence type coverage between primer sets, and the unknown 187 sequence diversity of the targeted genes in each sample leading to variations in primer set 188 sensitivity between samples, the exact estimates of the relative expression of var gene types 189 within individual samples cannot be made. However, reported transcript levels can be used to 190 make rough assessments of the relative transcript abundances of different var types within a 191 patient or in patient groups (25). The transcript abundance of 16 was chosen across primers 192 to reflect a high level of transcripts and to ease figure interpretation. This threshold was 193 chosen based on the var2csa transcript level measured in parasites from pregnant women, 194 and knowledge about transcript levels in cultured parasite lines selected to predominantly 195 express a PfEMP1 protein. 196 197 Statistical Analyses 198 Quantitative comparisons of transcript levels were done separately for each primer set or 199 groups of primers between patient groups using Kruskal-Wallis rank sum test or Wilcoxon 200 rank sum test using Stata Statistical Software. Fisher’s exact test was used to test proportions 201 of patients with high level (Tu>16) of selected transcripts. 202 There was no statistically significant difference in the transcript abundance measured in 203 Korogwe and Magu (data not shown). Hence data from the two sites were pooled. 204 205 206

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RESULTS 207 Clinical characteristics of patients 208 One hundred and sixty-five Tanzanian children admitted to hospital with P. falciparum 209 malaria and 22 P. falciparum-positive children identified by positive rapid diagnostic test 210 during cross-sectional village surveys were included in the study. Children were classified as 211 having severe malaria if the haemoglobin level (Hb) was <5g/dl, if the Blantyre coma score 212 was <3, if there were clinical signs of respiratory distress or the parasitaemia was >200.000 213 parasites/μl (Table 1, Fig. 2). Children admitted to hospital with Hb>8.0 g/dl, a Blantyre coma 214 score of 5 and parasitaemia <200.000 parasites/μl were categorised as having uncomplicated 215 malaria. Children with severe malaria were divided into non-overlapping groups of those with 216 a Blantyre coma score<3 and Hb>5 g/dl (cerebral malaria), those with Hb<5 g/dl and a 217 Blantyre coma score=5 (severe anaemia) and those with overlapping symptomatology or 218 severity signs other than coma or low Hb (Fig. 2). Village children were divided into febrile 219 (<37.5 °C) and afebrile children. All children received prompt treatment and care, and as a 220 result, mortality was 0% among the children from the village and hospitalised children with 221 uncomplicated malaria and 7.3% among those with severe disease (Table 1). The hospital 222 studies were conducted at Magu District Hospital on the shore of Lake Victoria and Korogwe 223 District Hospital, 100 km from the Indian Ocean coastline. These sites are in separate 224 ecological zones 650 km apart. 225 226 Malaria patients have a high level of transcripts encoding PfEMP1 predicted to bind EPCR and 227 increasing levels are associated with increased disease severity. 228 The median transcript level reported for each primer set and the summarised transcript level 229 for combinations of primer sets targeting same main domain class (e.g. all EPCR-binding 230

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CIDRα1) were stratified according to clinical presentation (Table 2). The most striking 231 differences were found for the level of transcripts encoding CIDRα1 domains, which increased 232 with disease severity (median level of transcripts for CIDRα1 of all subtypes combined: 1, 12, 233 49 and 62 for village malaria without fever, village malaria with fever, uncomplicated hospital 234 malaria and severe hospital malaria, respectively; P=0.0001 Kruskal-Wallis rank test). For all 235 CIDRα1 primers but CIDRα1.5 and CIDRα1.6, there was a statistically significant association 236 between disease severity and transcript level (P<0.001 for all comparisons, Kruskal-Wallis 237 rank test). There was considerable heterogeneity within groups, and when comparing 238 between two disease outcomes (Table 2, Fig. S3) the difference in transcript level reached 239 statistical significance for only some of the CIDRα1 subclasses (e.g. CIDRα1.1 was higher in 240 village malaria with fever than in village malaria without fever (P=0.021), CIDRα1.1 was 241 higher in severe hospital malaria than in uncomplicated hospital malaria (P=0.004), 242 CIDRα1.4/6 higher in uncomplicated hospital malaria than in village malaria with fever 243 (P=0.012). 244 245 The assay allowed only crude comparisons between transcript levels within patients or 246 patient groups. However, the data did indicate that, within hospitalised patient groups, the 247 CIDRα1 encoding transcripts were generally found at higher levels than transcripts encoding 248 the other main group A var types characterised by encoding CIDRδ, domains. In hospitalised 249 patients, the abundance of transcripts encoding EPCR-binding domains of DC8 (i.e. CIDRα1.1 250 and CIDRα1.8: “CIDRα1.DC8”) was roughly similar to the abundance of transcripts encoding 251 group A EPCR-binding domains (CIDRα1.4-7: CIDRα1.A)(Table 2). Var2csa was found at high 252 levels in 25.1% of the patients across all patient groups, but with no statistically significant 253 relation to severity. 254

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The subclass of the N-terminal DBLα domain is predictive of the adjacent CIDR class. For this 255 reason, primers targeting loci encoding the 3´end of DBLα domains can to a certain degree be 256 used to infer expression of PfEMP1 with specific receptor binding phenotypes. 257 The ”DBLa2/1.1/2/4/7” primer set reports transcripts encoding CIDRα1 domains predicted 258 to bind EPCR (both DC8 and group A genes), whereas the ”DBLa1.5/6/8” primer set primarily 259 detects transcripts of group A genes encoding N-terminal CIDRβ/γ/δ domains predicted not 260 to bind EPCR. The DBLα1all primer set reports transcripts of most group A genes encoding N-261 terminal CIDRα1/β/γ/δ domains predicted not to bind CD36. Transcript levels reported by 262 these DBLα domain primer sets confirmed the results obtained with the CIDR primers in the 263 hospitalised children, showing a higher abundance of transcripts encoding EPCR-binding 264 PfEMP1 compared to transcripts encoding other group A DBLα1.5/6/8 domains. 265 266 Var expression patterns in patients with respect to mid and C-terminal PfEMP1 domain classes. 267 DBLβ, -γ and -δ domain classes each cover a broad sequence variation and include few well-268 defined subclasses, which makes it difficult to design subclass-specific primers that retain 269 target coverage. However, a few distinct loci were identified in a subset of sequences encoding 270 DBLβ1/3 and DBLβ5 domains, including some sequence variants previously associated with 271 ICAM-1 binding (30, 34, 35). Primers targeting these loci did not report significant different 272 levels between patients with severe and uncomplicated malaria. 273 The only C-terminal sequence trait unique to group A PfEMP1 is domain cassette 5 (DC5). DC5 274 is found in about 15% of group A PfEMP1 and has previously been associated with parasites 275 binding to PECAM1 (36). The median transcript abundance was low in all patient groups. 276 Twelve primer sets targeting all different DBLζ subclasses and most DBLε subclasses not 277 associated with VAR1, VAR2CSA or VAR3 were applied (Fig. S1, Table 2). DBLζ and DBLε 278

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domains are most often found together and form different domain cassettes (DC1-3, 6, 7, 9-279 12). When transcript levels were summarised for all DBLζ primers, median transcript levels 280 increased with increasing severity of patient group. However, most DBLζ subclass primers 281 reported low transcript levels, which did not differ significantly between patient categories 282 (data not shown). Although the median levels were low, a small proportion of patients with 283 severe outcomes had transcripts targeted by the DBLz2a or DBLz3 primers, resulting in a 284 significant difference in the medial DBLz2a or DBLz3 between children with severe malaria 285 and uncomplicated malaria (Table 2, Figure S3). The DBLz3 primers reported fewer 286 transcripts in villagers with fever than in those without fever (P=0.0009). When transcript 287 levels were summarised for all DBLε primers, the median transcript level was higher in the 288 hospitalised children than in the children from the village. 289 290 The var transcript profiles in patients with severe anaemia or cerebral malaria and in those who 291 died are largely similar 292 Transcript profiles were compared between patients with either severe anaemia or cerebral 293 malaria (Table 2). For nearly all primers, the median transcript levels were comparable 294 between the two groups of patients, the only exception was a higher level of transcripts 295 reported in cerebral malaria patients with the DBLb1/3-1 primer set (P=0.039). The median 296 transcript level was low in both patient groups, and the observed difference reflected that 297 about 25% of the patients with cerebral malaria had high levels of these transcripts (Fig. S4, 298 Table S1). 299 There was no statistically significant difference between the transcript levels in those who 300 died compared to those who survived a complicated malaria episode. Among those who died, 301 the median transcript level of genes encoding EPCR-binding PfEMP1 and particularly those 302

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belonging to group A was high, but there was considerable variation in transcript level within 303 this patient group (Table 2). 304 305 CIDRα1 transcript profiles differs considerably between patients but not according to patient 306 group 307 The primer sets used to measure transcripts encoding the six subclasses of EPCR-binding 308 domains (CIDRα1.1, CIDRα1.4-8) had a similar good coverage and specificity (Fig. S1): 309 Therefore, the transcript abundances reported with these primers within a patient can be 310 compared with caution. The transcript pattern varied markedly between patients. In most 311 patients, several different CIDRα1 types were expressed, but the dominant CIDRα1 subtype 312 varied from patient to patient. There was no association between disease outcome and 313 relative abundance of different CIDRα1 subtypes. The CIDRα1 subtype transcription patterns 314 are illustrated in 36 randomly selected patients (Fig. 3). Panels A-C showing six patients with 315 parasiteamia without fever, six with mild malaria and six hospitalised with uncomplicated 316 malaria illustrate the increasing levels of CIDRα1 subtype transcripts with increasing disease 317 severity. Panels D-E showing six patients with severe anaemia, six with cerebral malaria and 318 six patients who died illustrate that the majority of patients with severe disease had high 319 transcript abundances of several CIDRα1 subtypes and that none of the subtypes were 320 particularly dominant. 321

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DISCUSSION 322 Here, we took advantage of the improved resolution of var sequence diversity gained through 323 var genes extracted from over 200 recently sequenced P. falciparum genomes to design a new 324 set of primers for quantitative PCR transcript analysis of var subclasses. These primers 325 allowed an unprecedented sensitivity and specificity in detecting and quantifying var 326 transcripts encoding conserved sequence traits. In particular, var transcripts encoding group 327 A PfEMP1 as well as transcripts encoding DBLε or DBLζ domains were well covered. 328 Moreover, the coverage of specific DBLβ domains was improved. In general, the primers are 329 predicted to underestimate the expression level of the targeted traits. This was particularly 330 true for the primers targeting genes encoding CD36-binding CIDRα2-6, for which coverage 331 was estimated to be 17%. In addition to the improved primer set, this study included a higher 332 number of malaria patients and a broader spectrum of disease outcomes than in previous 333 studies employing qPCR (25, 26, 31). Patient groups included children with parasiteamia 334 without fever, children with mild malaria who could be treated in the village, children who 335 required hospitalisation, children with severe disease manifestations and children with a fatal 336 infection outcome. Our study is based on the hypothesis that the PfEMP1 parasite phenotype 337 is a determinant for disease outcome. Other factors relating to the patient (e.g. host genotype, 338 health-seeking behaviour) and parasite (e.g. drug resistance phenotype) will also contribute 339 to infection outcome. It should also be borne in mind that disease categorisation was based on 340 clinical presentation at diagnosis. Thus, parasites with a pathogenic phenotype may be 341 detected in patients with mild symptoms who are diagnosed early. On the other hand, 342 parasites with a non-pathogenic phenotype may be detected in patients, where the symptoms 343 are not caused by P. falciparum but competing pathologies. A recent study from Malawi 344 showed that more than 20% of patients classified as having cerebral malaria on clinical 345

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criteria similar to those used in the current study were, upon autopsy, concluded to have died 346 from causes other than malaria (37). Despite these inherent limitations, the median CIDRα1 347 transcript levels increased significantly with disease severity, being 50-100 times higher in 348 those with severe disease than in those with parasiteamia without fever. Unexpectedly, even 349 patients with mild fever symptoms had increased expression of diverse but, in particular, 350 EPCR-binding PfEMP1, compared to asymptomatic P. falciparum infected individuals. This 351 could imply that disease onset is associated with increased expression of, in particular, EPCR-352 binding PfEMP1; however, patient numbers were low, and more extensive studies are 353 required to confirm this hypothesis. The transcript levels in children with severe malaria 354 were similar to or exceeded the var2csa transcript level (encoding the PfEMP1 binding 355 parasites in placenta) in parasites isolated from pregnant women (38, 39). The var2csa 356 transcript levels were relatively high in one of four children regardless of disease severity. 357 This could reflect that VAR2CSA was expressed on the infected erythrocytes of these children. 358 However, the var2csa gene is unique among var genes in containing an upstream open 359 reading frame, which can repress translation of transcripts (40). 360 361 In line with previous observations (25, 26, 41), DC8 and group A PfEMP1 were highly 362 expressed in most hospitalised children and the abundance of transcripts encoding DC8 363 PfEMP1 was statistically significantly higher in patients with severe malaria compared to 364 those with uncomplicated disease. In contrast to previous reports, the difference in 365 abundance of transcripts encoding DC13 (containing CIDRα1.4) in patients with severe 366 malaria or uncomplicated disease did not reach statistical significance. However, EPCR 367 binding group B (i.e. DC8) and group A PfEMP1 were estimated to be expressed at similar 368 levels in severe malaria patients. Moreover, transcripts for all EPCR-binding CIDRα1 369

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subclasses were found highly expressed in individual patients and, in many, there were high 370 levels of transcripts encoding several CIDRα1 subclasses. Thus development of severe malaria 371 symptoms appears to be associated with EPCR-binding CIDRα1 in general. This is in line with 372 the observation that most CIDRα1 variants bind EPCR with high affinity (28), and implies that 373 a vaccine protecting children against severe malaria should target all or most CIDRα1 374 subtypes predicted to bind EPCR. 375 376 Due to the poor sensitivity in the detection of genes encoding CIDRα2-6 or CIDRβ/γ, the 377 abundance of these transcripts might have been underestimated. However, a recent study by 378 Jespersen et al (29), which did not rely on specific var gene primers, showed that genes 379 encoding CIDRα2-6 or CIDRβ/γ containing PfEMP1 were not dominating var transcript 380 profiles in patients with severe malaria. In the present study, the coverage of primers 381 targeting genes encoding group A N-terminal CIDRδ domains was high and, in agreement with 382 Jespersen et al, the data showed that these transcripts were highly expressed in only a few 383 patients with severe malaria. Together with early studies (20-24) linking group A PfEMP1 384 with severe disease, these studies suggest that parasites binding to CD36, or unknown 385 receptors through other group A-linked N-terminal CIDRβ/γ/δ subclasses, do not commonly 386 precipitate severe malaria symptoms in young children. 387 388 In support of the suggested additive pathogenic effect of ICAM-1 binding (30), primers 389 targeting group A DBLβ domains, of which some have been shown to bind ICAM-1, reported 390 elevated transcript levels in severe malaria patients, in particular among those with cerebral 391 complications. The median transcript levels were low and a minority of cerebral patients 392 (24%) exhibited a high expression of genes coding PfEMP1 predicted to bind EPCR and ICAM-393

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1. Both CD36- and EPCR-binding PfEMP1 can bind ICAM-1 (30, 34, 35, 42), but ICAM-1 and 394 CD36 binding does not appear to be a common phenotype for parasites causing severe 395 malaria (29). Thus, EPCR binding by CIDRα1 domains appears to be required for development 396 of severe symptoms, but ICAM-1 binding and other host receptor interactions may, in some 397 individuals (43), act in concert to strengthen cytoadhesion and aggravate disease. It is 398 conceivable that this notion also explains the inconsistent association of expression of diverse 399 DBLζ variants with both uncomplicated (DBLζ4 (25)) and severe (DBLζ2a and DBLζ3, this 400 study) paediatric malaria and severe malaria in adults (DBLζ5) (31). 401 It has previously been reported that var expression differed between cerebral malaria 402 patients with differing histopathology (44), however, the current study could not address this. 403 Future studies with better resolution of cerebral manifestations, e.g. qualified by examination 404 for retinopathy (45) are required to elucidate the suggested increased risk of developing 405 cerebral complications when parasites can bind both EPCR and ICAM-1. However, our results 406 suggest that circumstances relating to the patient, such as expression of EPCR in different 407 parts of the vasculature, regulation of the local inflammatory response or prior immune 408 priming may be more important determinants of disease manifestation than which subtype of 409 EPCR-binding PfEMP1 gene is being expressed. 410 411 In around one of five of the severe malaria patients in the present study, none of the used 412 primers reported high levels of var transcripts (here defined at Tu>16). Although most 413 primers used have a target-coverage below 100%, this could reflect that these patients 414 suffered from infection with parasites expressing CD36-binding PfEMP1 or that the symptoms 415 of some of these patients were not caused by the malaria parasites. 416 417

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Rosetting is a PfEMP1-mediated parasite phenotype previously associated with severe 418 malaria (46-48). Rosetting appears to be mediated by some group A DBLα1 domains (49-51), 419 but has also been suggested to be mediated by RIFIN (52) and STEVOR proteins (53). As the 420 PfEMP1 trait involved in rosetting phenotype is yet to be resolved and cannot be predicted 421 from domain subgrouping or amino acid sequence, this study cannot predict whether or to 422 what degree, for example, DBLα1 domains adjacent to the EPCR-binding CIDRα1 domains 423 contributed to pathology by mediating rosetting. 424 425 This study in combination with previous observations from other studies of var transcription 426 (25, 26, 29, 31) suggests that expression of EPCR-binding PfEMP1 is the overarching parasite 427 phenotype associated with the development of symptomatic and severe malaria and that its 428 clinical relevance is stable across various confounding host or environmental factors. In line 429 with this are the observations that parasites from children with severe disease and in vitro 430 adapted parasites expressing CIDRα1 PfEMP1 bind endothelial cells via EPCR (22, 54-56) and 431 that CIDRα1 domains bind EPCR with high affinity and the binding inhibits EPCR ability to 432 bind protein C, thereby potentially driving pathogenic endothelial inflammation (27, 28, 57-433 59). Finally, in malaria-endemic regions, IgG to EPCR-binding CIDRα1 domains are acquired 434 early in life and before antibodies to other classes of CIDR domains (56). Altogether, this 435 argues that a vaccine inducing IgG inhibiting the PfEMP1-EPCR interaction could reduce 436 severe disease and death due to P. falciparum, and even reduce the number of malaria 437 infections requiring hospitalisation. In addition, an adjunctive therapy aiming at alleviating 438 the potential damaging consequences directly associated with the PfEMP1-EPCR interaction 439 (60) may reduce the fatality rate or degree of sequelae for malaria patients. 440 441

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Acknowledgements 442 We are deeply grateful to the Tanzanian donors. 443 444 Funding information. 445 This work received financial support from Augustinus Fonden, Lundbeckfonden, Axel 446 Muusfeldts Fond, Grosserer L.F. Foghts Fond, Danish International Development Agency 447 (DANIDA) and the Danish Council for Independent Research (T1333-00220, 1331-00089B 448 and the Sapere Aude program DFF–4004-00624B). The funders had no role in study design, 449 data collection and interpretation, or the decision to submit the work for publication . 450 451 REFERENCES 452 453 1. Marsh K, Forster D, Waruiru C, Mwangi I, Winstanley M, Marsh V, Newton C, 454

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44. Tembo DL, Nyoni B, Murikoli RV, Mukaka M, Milner DA, Berriman M, Rogerson SJ, 615 Taylor TE, Molyneux ME, Mandala WL, Craig AG, Montgomery J. 2014. Differential 616 PfEMP1 expression is associated with cerebral malaria pathology. PLoS Pathog 617 10:e1004537. 618 45. Barrera V, Hiscott PS, Craig AG, White VA, Milner DA, Beare NA, MacCormick IJ, 619 Kamiza S, Taylor TE, Molyneux ME, Harding SP. 2015. Severity of retinopathy 620 parallels the degree of parasite sequestration in the eyes and brains of malawian 621 children with fatal cerebral malaria. J Infect Dis 211:1977-1986. 622 46. Carlson J, Helmby H, Hill AV, Brewster D, Greenwood BM, Wahlgren M. 1990. 623 Human cerebral malaria: association with erythrocyte rosetting and lack of anti-624 rosetting antibodies. Lancet 336:1457-1460. 625 47. Rowe A, Obeiro J, Newbold CI, Marsh K. 1995. Plasmodium falciparum rosetting is 626 associated with malaria severity in Kenya. Infect Immun 63:2323-2326. 627 48. Ockenhouse CF, Ho M, Tandon NN, Van Seventer GA, Shaw S, White NJ, Jamieson 628 GA, Chulay JD, Webster HK. 1991. Molecular basis of sequestration in severe and 629 uncomplicated Plasmodium falciparum malaria: differential adhesion of infected 630 erythrocytes to CD36 and ICAM-1. J Infect Dis 164:163-169. 631 49. Rowe JA, Moulds JM, Newbold CI, Miller LH. 1997. P. falciparum rosetting mediated 632 by a parasite-variant erythrocyte membrane protein and complement-receptor 1. 633 Nature 388:292-295. 634 50. Ghumra A, Semblat JP, Ataide R, Kifude C, Adams Y, Claessens A, Anong DN, Bull 635 PC, Fennell C, Arman M, Amambua-Ngwa A, Walther M, Conway DJ, Kassambara L, 636 Doumbo OK, Raza A, Rowe JA. 2012. Induction of strain-transcending antibodies 637 against Group A PfEMP1 surface antigens from virulent malaria parasites. PLoS Pathog 638 8:e1002665. 639 51. Angeletti D, Albrecht L, Blomqvist K, Quintana Mdel P, Akhter T, Bachle SM, 640 Sawyer A, Sandalova T, Achour A, Wahlgren M, Moll K. 2012. Plasmodium 641 falciparum rosetting epitopes converge in the SD3-loop of PfEMP1-DBL1alpha. PLoS 642 One 7:e50758. 643 52. Goel S, Palmkvist M, Moll K, Joannin N, Lara P, Akhouri RR, Moradi N, Ojemalm K, 644 Westman M, Angeletti D, Kjellin H, Lehtio J, Blixt O, Idestrom L, Gahmberg CG, 645 Storry JR, Hult AK, Olsson ML, von Heijne G, Nilsson I, Wahlgren M. 2015. RIFINs 646 are adhesins implicated in severe Plasmodium falciparum malaria. Nat Med 21:314-647 317. 648 53. Niang M, Bei AK, Madnani KG, Pelly S, Dankwa S, Kanjee U, Gunalan K, Amaladoss 649 A, Yeo KP, Bob NS, Malleret B, Duraisingh MT, Preiser PR. 2014. STEVOR is a 650 Plasmodium falciparum erythrocyte binding protein that mediates merozoite invasion 651 and rosetting. Cell Host Microbe 16:81-93. 652 54. Avril M, Tripathi AK, Brazier AJ, Andisi C, Janes JH, Soma VL, Sullivan DJ, Jr., Bull 653 PC, Stins MF, Smith JD. 2012. A restricted subset of var genes mediates adherence of 654 Plasmodium falciparum-infected erythrocytes to brain endothelial cells. Proc Natl Acad 655 Sci U S A 109:E1782-1790. 656 55. Claessens A, Adams Y, Ghumra A, Lindergard G, Buchan CC, Andisi C, Bull PC, Mok 657 S, Gupta AP, Wang CW, Turner L, Arman M, Raza A, Bozdech Z, Rowe JA. 2012. A 658 subset of group A-like var genes encodes the malaria parasite ligands for binding to 659 human brain endothelial cells. Proc Natl Acad Sci U S A 109:E1772-1781. 660

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56. Turner L, Lavstsen T, Mmbando BP, Wang CW, Magistrado PA, Vestergaard LS, 661 Ishengoma DS, Minja DT, Lusingu JP, Theander TG. 2015. IgG antibodies to 662 endothelial protein C receptor-binding cysteine-rich interdomain region domains of 663 Plasmodium falciparum erythrocyte membrane protein 1 are acquired early in life in 664 individuals exposed to malaria. Infect Immun 83:3096-3103. 665 57. Petersen JE, Bouwens EA, Tamayo I, Turner L, Wang CW, Stins M, Theander TG, 666 Hermida J, Mosnier LO, Lavstsen T. 2015. Protein C system defects inflicted by the 667 malaria parasite protein PfEMP1 can be overcome by a soluble EPCR variant. Thromb 668 Haemost 114. 669 58. Gillrie MR, Avril M, Brazier AJ, Davis SP, Stins MF, Smith JD, Ho M. 2015. Diverse 670 functional outcomes of Plasmodium falciparum ligation of EPCR: potential implications 671 for malarial pathogenesis. Cell Microbiol 17:1883-1899. 672 59. Sampath S, Brazier AJ, Avril M, Bernabeu M, Vigdorovich V, Mascarenhas A, 673 Gomes E, Sather DN, Esmon CT, Smith JD. 2015. Plasmodium falciparum adhesion 674 domains linked to severe malaria differ in blockade of endothelial protein C receptor. 675 Cell Microbiol 17:1868-1882. 676 60. Mosnier LO, Lavstsen T. 2016. The role of EPCR in the pathogenesis of severe malaria. 677 Thromb Res 141 Suppl 2:S46-49. 678 679 680

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Figure Legends. 681 Figure 1. Schematic presentation of typical PfEMP1 domain compositions. The N-terminal 682 “head structure” confers mutually exclusive receptor binding phenotypes: CSA (VAR2CSA), 683 EPCR (CIDRα1), CD36 (CIDRα2-6) and yet unknown phenotypes (CIDRδ/β/γ, VAR3). Group A 684 PfEMP1 are encoded by sub-telomeric genes transcribed towards the telomere. Group A 685 PfEMP1 include both EPCR binding and non-binding PfEMP1. Group B PfEMP1 are encoded by 686 telomeric genes transcribed towards the centromere, and include PfEMP1 binding EPCR 687 (DC8) and CD36. Group C PfEMP1 bind CD36 and are encoded by centromeric genes. 688 PfEMP1 typically have two to six domains C-terminal to the head structure. The subclass 689 composition of these domains varies, but in general follows the depicted order. C-terminal 690 domain subclass composition is generally unrelated to the division of the N-terminal head 691 structure, although some DBLβ sequences only occur in either group A or group B PfEMP1. 692 Most group A and DC8 group B PfEMP1 have four or more domains, whereas ~2/3 of the 693 remaining group B and the group C PfEMP1 have only DBLδ-CIDR tandem domains. See Rask 694 et al. 2010 (17) for detailed description of PfEMP1 diversity and domain architecture. The 695 estimated proportion of var genes of each group or with indicated domain classes is given. 696 697 Figure 2: Malaria patients enrolled in the study. Samples were collected in villages or at 698 District hospitals in Korogwe or Magu. Children were categorised into non-overlapping 699 groups. At the Hospital this included cerebral malaria (CM), severe anemia (SA) and those 700 with overlapping syndromes and/or other signs linked to severity (respiratory distress and 701 hyperparasiteamia). Boxes in red are categories presented in the Tables 1 and 2. 702 703

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Figure 3. Examples of levels (Tu) of var transcripts encoding different CIDRα1 subclasses in 704 six children with parasiteamia without on-going fever (panel A), six children with mild 705 malaria not requiring hospitalisation (panel B), six children hospitalised with uncomplicated 706 malaria (Panel C), six children with in severe malarial anaemia (Panel D), six children with 707 cerebral malaria (Panel E), and six children who succumbed to the infection (Panel F). Bars 708 represent transcript levels reported by primer sets: CIDRa1.1 (C1, red); CIDRa1.8 (C8, 709 orange); CIDRa1.4/6a (c4, blue), CIDRa1.5 (C5, black); CIDRa1.6b (C6, grey); and CIDRa1.7 (c7, 710 green). Red line indicate Tu=16. 711

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Table 1. Clinical characteristics of patients Age Se

Te perature Parasite ia H Bla t re s ore

La tate Glu ose Mortalit

ears oC /μl g/dl ol/L ol/L Sample Collection Malaria out o e N Mea sd % Fe ale Media p -p Media p -p Mea sd Mea i -

a Mea sd Mea sd %

Village No fever . . . . - . . . - . . . . . - . d d .

Village With fever . . . . - . . . - . . . . . - . d d .

Hospital U o pli ated . . . . - . . . - . . . . . - . . . . . .

Hospital Severe . . . . - . . . - . . . . . - . . . . . .

Hospital Severe a e ia . . . . - . . . - . . . . . - . . . . . .

Hospital Cere ral alaria . . . . - . . . - . . . . . - . . . . . .

Hospital Died . . . . - . . . - . . . . . - . . . . . .

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Ta le . Media var t a s ipt le els Tu*, th a d th pe e tiles patie t g oup

*T a s ipt u it Tu , A Tu of o espo ds to the ea t a s ipt le el of the t o o t ol ge es. § Bold i di ate that the le el su a ize the Tu epo ted se e al p i e s.CIDRa .DC su a izes CIDR . a d . ; CIDRa . A su a izes CIDR . - ; CIDRa .all su a izes CIDRa .DC a d CIDRa .A. Itali s i di ate that the p i e dete ts t a s ipts p edi ted to e ode EPCR i di g PfEMP do ai s. $P alues al ulated usi g Wil o o a k su test, o l P alues < . a e sho . ^ The olu i di ates hi h if a PfEMP g oup is asso iated ith the ta geted se ue e. ^^ The olu i di ates the p edi ted hu a e epto i di g spe ifi it if a of the do ai t pe e oded the ta geted se ue e. Re epto s ICAM a d PECAM a e i di ted i a kets, as e ide e of the i di ated do ai s o fe i g i di g to these e epto s a e spo adi a d thus ot lea l li ked to the do ai t pe.

P i e s §

Ta get PfEMP Village No fe e

N=

Village With fe e

N=

P$ Village ith s

ithout fe e

Hospital U o pli ated

ala ia UM N=

P Village fe e

s UM

Hospital Se e e ala ia

SM N=

P UM s SM

Hospital Se e e a e ia

SA N=

Hospital Ce e al ala ia

CM N=

P SA s CM

Hospital Died

N=

G oup^

Bi di g ^^

CIDRa1.all A&BA EPCR . . - . . . - . . . - . . . . - . . . - . . . - . . . - . CIDRa1.DC8 BA EPCR . . - . . . - . . . . - . . . - . . . . - . . . - . . . - .

CIDRa1.1 BA EPCR . . - . . . - . . . . - . . . - . . . . - . . . - . . . - . CIDRa1. BA EPCR . . - . . . - . . . - . . . - . . . - . . . - . . . - .

CIDRa1.A A EPCR . . - . . . - . . . - . . . . - . . . - . . . - . . . - . CIDRa1. / a A EPCR . . - . . . - . . . - . . . . - . . . - . . . - . . . - .

CIDRa1. A EPCR . . - . . . - . . . - . . . . - . . . - . . . - . . . - . CIDRa1. b A EPCR . . - . . . - . . . - . . . - . . . - . . . - . . . - .

CIDRa1. A EPCR . . - . . . - . . . - . . . - . . . - . . . - . . . - . CIDRd A . . - . . . - . . . . - . . . - . . . - . . . - . . . - .

CIDRg . A . . - . . . - . . . - . . . - . . . - . . . - . . . - . CIDRa . / A CD . . - . . . - . . . . - . . . - . . . - . . . - . . . - .

DBLa all A . . - . . . - . . . - . . . . - . . . - . . . - . . . - . DBLa / . / / / A . . - . . . - . . . . - . . . . - . . . - . . . - . . . - .

DBLa . / / A . . - . . . - . . . . - . . . - . . . - . . . - . . . - . DBL / - A ICAM . . - . . . - . . . - . . . - . . . - . . . - . . . . - . DBL / - A ICAM . . - . . . - . . . . - . . . - . . . - . . . - . . . - .

DBL B ICAM . . - . . . - . . . - . . . - . . . - . . . - . . . - . DBLz_all . . - . . . - . . . . - . . . - . . . - . . . - . . . - .

DBLz a - . - . . - . . . - . . . . - . . . - . . . - . DBLz . - . - . . . - . . . . - . . . . - . . . - . . . - .

DBLe_all . . - . . . - . . . - . . . . - . . . - . . . - . . . - . DC A PECAM . . - . . . - . . . - . . . - . . . - . . . - . . . - .

a sa E CSA . . - . . . - . . . - . . . - . . . - . . . - . . . - . a A . . - . . . - . . . - . . . - . . . - . . . - . . . - .

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N-terminal head structure 38%

has 1-3 DBL / Group

A 10%

72%

21%

has 1-3 DBL /ε/ζ

Group

A 10%

DC8

4%

Group B &

C 72%

VAR2CSA

3%

VAR3

1%

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187 children

Village 22

Korogwe 110 Magu 55

Febrile 10 Non febrile 12

Severe 75 Uncomplicated 35

Overlapping/other 30 SA not CM 24 CM not SA 21

Hospital 165

Severe 48 Uncomplicated 7



Uncomplicated 42 Severe 123

Survived 114 Died 9

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2

32

512

2

32

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2

32

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2

32

512

2

32

512

2

32

512

A

2

32

512

2

32

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2

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2

32

512

2

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512

2

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B

C

D

E

F

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