Supplementary Appendix

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1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Gonçalves BP, Huang C-Y, Morrison R, et al. Parasite burden and severity of malaria in Tanzanian children. N Engl J Med 2014;370: DOI: /NEJMoa

2 Supplementary Appendix Parasite Burden and Severity of Malaria in Tanzanian Children Bronner P. Gonçalves 1,2, Chiung-Yu Huang 3, Robert Morrison 4, Sarah Holte 5, Edward Kabyemela 6, D. Rebecca Prevots 2, Michal Fried 1,4,6, Patrick E. Duffy 1,4,6 1 Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, NIH 2 Laboratory of Clinical Infectious Diseases Epidemiology Unit, National Institute of Allergy and Infectious Diseases, NIH 3 Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, NIH 4 Seattle Biomedical Research Institute, Seattle, Washington 5 Fred Hutchinson Cancer Research Center, Seattle, Washington 6 MOMS Project, Muheza Designated District Hospital, Muheza, Tanzania 1

3 Supplementary Appendix Table of Contents Exclusion Criteria 4 Duration of follow-up 4 Placental Malaria 4 Treatment regimen 5 Plasmodium falciparum-specific histidine rich protein 2 quantification assay 6 Determination of red blood cell disorders 7 Statistical Analysis 7 Severe Malaria rate 9 Multiple infections per child 10 Moderately Severe Malaria 10 Author s Contributions 11 Figure S1 12 Figure S2 15 Figure S3 16 Figure S4 17 Figure S5 18 Figure S6 19 Table S1 20 Table S2 21 Table S3 22 Table S4 22 2

4 Table S5 23 Table S6 24 Table S7 25 Table S8 26 Table S9 27 Supplementary references 28 3

5 Supplementary Appendix Exclusion criteria 1045 mothers (1075 children) gave consent to participate in this study. Enrolled children excluded from the analyses in this manuscript included 18 twin pairs (36 children), 1 triplet set (3 children), 4 stillbirths, 11 early neonatal deaths, and 92 neonates who were not examined beyond 4 weeks of age because their families moved out of the study area. Infants with any evidence of HIV infection were also excluded (n = 41 total) as well as 6 infants with sickle cell disease. Duration of follow-up 688/882 (78%) children were followed for at least one year, which is the time window when the majority of severe malaria events occur; 449/882 (51%) were followed for at least 2 years and 203/882 (23%) were followed for at least 3 years. 680 children had at least one visit in 2006, when the study was closed. Placental Malaria Placental malaria was defined as the detection of at least one parasite in a blood slide. Placental blood samples were obtained by mechanically pressing full-thickness placental tissue just after delivery. 4

6 Treatment regimen Amodiaquine was used for first-line treatment of uncomplicated malaria (84.6%). Around the time our study ended, amodiaquine was reported by others to fail in % of treated clinical episodes during 28 days of followup in this area 1. In our study, parasites were detected within 2 weeks after 994/3,822 (26.0%) amodiaquine treatments, and within 4 weeks in 2,017/3,822 (52.3%). Treatment failure events were rarely associated with severe disease (N=7 (0.7% of 994 treatment failures) and N=6 (0.6% of 1023 treatment failures) for an interval from day 4 up to day 14 after treatment initiation and from day 15 up to day 28, respectively). Severe malaria episodes were treated with quinine, other than 17 severe anemia episodes and 2 episodes of severe respiratory distress, which were treated with amodiaquine. High density infections, regardless of their clinical presentation, were primarily treated with quinine. Neither artemisinin-containing combination therapies nor chloroquine were used for treatment in the cohort, as they were not recommended by the Tanzanian Ministry of Health and Social Welfare during the study period. Treatment regimens did not change during study period. 17 children with severe malaria received blood transfusion, when clinicians concluded the child was in distress related to anemia. 27 children with hemoglobin levels <5g/dl who did not have signs or symptoms of distress, did not receive blood transfusion; their hemoglobin levels improved after treatment with quinine (n=10) or amodiaquine (n=16). Statistical analyses of severe malaria risk factors were repeated after excluding 4 episodes with possible misclassification (2 severe anemia episodes with improperly processed samples and 2 episodes of respiratory distress with clinical signs of bronchopneumonia) and the 27 episodes of severe 5

7 anemia that did not receive blood transfusion: statistical models yielded similar results with the exception of transmission season (effect disappeared) and HbAS (protective effect was stronger); Paired comparisons also presented similar results, except the comparison between PfHRP-2 levels during high density infection before or after a severe malaria episode vs. during severe malaria episode, which was not significant (p=0.07, N=27) even though fold difference was similar (2.73 (95% CI, )). After excluding these cases, a similar proportion of children (54/82) who developed severe malaria also developed a prior or subsequent high density infection with only mild symptoms. Plasmodium falciparum-specific histidine rich protein 2 quantification assay P. falciparum-specific histidine rich protein 2 (PfHRP-2) plasma concentrations were determined during infection in all available samples (N=1108) from the cohort, which included 475 samples from the children who suffered severe malaria. Samples were tested using a doublesite sandwich ELISA. Plates were washed 3 times with PBS/Tween (0.05%, 200 μl/well) between all incubations. Plates were coated overnight at 4 ºC with mouse anti-hrp2 IgM antibody (cat# MPFM-55A, Immunology Consultants Laboratories, Inc, Newberg, OR, USA) at 1 μg/ ml in PBS (100ul/well). 2% bovine serum albumin (BSA) solution in PBS (200 μl/well) was used for blocking at room temperature for 2 hours. Individual plasma samples were diluted 1:10 and tested in triplicate for 1 hour at room temperature. Mouse anti-hrp2 IgG antibody (cat# MPFG-55P, Immunology Consultants Laboratories, Inc, Newberg, OR, USA) was diluted to 0.2 μg/ ml in a solution of 2% BSA, 1% Tween 20 in PBS, transferred to ELISA plate (100 μl/well), and incubated at room temperature for 1 hour. 100 μl/well of TMB substrate (KPL, Inc., Gaithersburg, MD, USA) was added, and incubated for 5 min in the dark. 100 μl/well of 6

8 TMB stop solution (KPL, Inc., Gaithersburg, MD, USA) was added to terminate the reaction. Results were measured spectrophotometrically at 450 nm. Positive and negative controls were used in each plate. Standard curve was established using purified PfHRP-2 (Immunology Consultants Laboratories, Inc, Newberg, OR, USA) in PBS. The assay had a detection limit (defined by average levels + 2 standard deviations of 20 uninfected US volunteers) of 0.42 ng/ml (samples were diluted 1:10). The assay detected HRP2 in 91.9% of samples tested, and logtransformed PfHRP2 levels correlated significantly with log-transformed parasite density (R=0.36, p<0.001), similar to earlier studies 2. Determination of red blood cell disorders Hemoglobin type (HbAA, HbAS and HbSS) was determined by cellulose acetate paper electrophoresis according to the manufacturer s instructions (Helena Laboratories, Beaumont, Texas, USA). Genotyping for α-thalassemia was done according to the protocol described by Chong et al 3. Statistical Analysis For the survival analysis, univariate and multivariate Cox proportional hazards models were fitted. The event variables analyzed separately were time to the first severe malaria episode, time to the first moderately severe malaria episode, and time to the first severe or moderately severe malaria episode. Variables included in these analyses were: village of residence, bed net use, sickle cell trait, alpha-thalassemia genotype, transmission season, birth season, gender, and a single variable combining status with respect to placental malaria and maternal parity. Transmission season was defined as high between May and October. There were four residential 7

9 areas in the study, defined by proximity to nearest village or town center: Muheza Township, Magila, Mkanyageni and Bwembwera. All variables included in Cox models were assessed for conformity to the proportional hazards assumption using the statistic based on the Schoenfeld residuals, and if the variable did not meet the assumption, the multivariate model was stratified on this variable. Variables that had a p-value <0.20 in the univariate analysis and those that are biologically related to the outcome were included in a multivariate Cox regression, to adjust for possible confounding. To account for possible heterogeneity in exposure, analysis including only individuals that were infected during the follow-up was also performed and similar results were obtained (data not shown). Another model was used to analyze the effect of independent variables on the risk of developing severe malaria during an infection: GEE method. In this analysis, each visit with a positive blood smear was included as an observation. To avoid violation of the assumption of noninformative number of observations per child, all variables that had an effect on the risk of parasitemia were included regardless of their level of statistical significance in the GEE model. An exchangeable working correlation structure was used in GEE, and robust standard errors were estimated. A similar analysis was performed with the following variables as the dependent variable: moderately severe malaria, moderately severe or severe malaria, and parasite density (logtransformed). For all these different models, except the model with parasite density as dependent variable, a logit link function was used. Because the results obtained using GEE model to study the risk of developing severe malaria during parasitemia were similar to those obtained by Cox models, only results from survival analysis are shown. 8

10 To estimate and compare rates of severe malaria and high parasite density by age strata, Poisson regression model was used in combination with GEE to account for correlation due to repeated measures for each child. Number of events (severe malaria episodes or high parasite density infections) in each stratum was regressed on an indicator variable whose value depended on whether the event was severe malaria or high parasite density infection. Indicator variables for each of the 4 age strata (0-24 months, months, months, and 96 or more months) were also generated. An offset of log follow-up time in each stratum was used. The estimated coefficients of the main effects were used to calculate rate ratios and confidence intervals for the comparison of severe malaria to high density infection rates within each age strata, and for comparisons of severe malaria and of high density infection in each age strata relative to the referent week stratum. Data analyses were conducted using STATA version 11.1 (Stata Corporation, College Station, Texas, United States) and R (R Foundation for Statistical Computing, Vienna, Austria). Severe Malaria rate The overall incidence rate of severe malaria in this study was 0.05 (95% CI, ) episodes per child-year, which is the same as the minimum annual rates estimated for under-five children in other East African communities with stable malaria transmission, such as Kilifi, Kenya and Ifakara, Tanzania 4,5. Those earlier hospital-based studies had key differences to our prospective longitudinal cohort study that would lead to their relative underestimation of severe malaria rates: up to 2/3 of childhood deaths around Kilifi, Kenya occurred outside the hospital 4, suggesting that many severe malaria events were also not captured by passive detection; 9

11 incidences in those studies were calculated over the first 4 years of life, rather than the first 2 years of life on average in our study when the incidence of severe malaria is highest (Fig. 2b). Thus, the true rate of severe malaria in the Kilifi and Ifakara populations was probably substantially higher than the rate observed in our cohort, likely explained by the intensive followup and treatment included in our longitudinal study design. Multiple infections per child The number of visits with positive blood smear or of independent infections per child varied substantially. While 167 children were never infected during follow-up, 119/882 (13.5%) developed at least 10 independent infections. These children had longer follow-up (P<0.001), lived more often in Magila and Bwembwera and less often in Muheza, and used bed nets less frequently (41.1% vs. 66.7%). Moderately Severe Malaria 132 (15%) children developed at least one episode of moderately severe malaria during followup (total of 155 episodes). 74.8% of these episodes prompted hospitalization. Only 5/132 children experienced more than 2 episodes of moderately severe malaria. Most children with moderately severe malaria presented with a single clinical condition (Supplementary Appendix Fig. S4). Combining moderately severe malaria and severe malaria events yielded an epidemiological pattern similar to severe malaria alone: most children developed severe or moderately severe 10

12 symptoms after being previously exposed to the parasite (Supplementary Appendix Fig. S3). Factors affecting time to first severe malaria episode had similar effects on time to first moderately severe malaria episode (Supplementary Appendix Table S2). In addition, alphathalassemia significantly reduced risk of moderately severe malaria. Moderately severe malaria and severe malaria combined as a single outcome yielded similar results to their individual analyses. GEE models that examined severe or moderately severe malaria risk at the time of any infection were developed and similar results were obtained (data not shown). Authors Contributions: PD and MF designed the study. PD, MF, EK and RM were involved in data acquisition. PD, BG, MF, CH, RM, SH and DP contributed to analysis and/or interpretation of data. PD, MF and BG wrote the first draft of this manuscript. BG, CH, RM, SH, EK, DP, MF and PD reviewed and approved the final version of this manuscript. 11

13 Parasite density (per 200 WBC) FIGURES (SUPPLEMENTARY APPENDIX) Figure S1. Parasite density by clinical severity and age: (a) Distribution of independent infections by parasite density and age, with clinical severity indicated (all children included); (b) Distribution of independent infections by parasite density, age and clinical severity in children who developed severe malaria during follow-up (N=102); (c) Distribution of PfHRP-2 levels (log-transformed log10(x+1), where x represents PfHRP-2 levels in ng/ml) by age and clinical severity in children who developed severe malaria during follow-up (all available samples from these children were measured, N=475) (a) Mild/Asymptomatic Infection Moderately Severe Malaria Severe Malaria Age (in weeks) 12

14 Parasite density (per 200 WBC) (b) Mild/Asymptomatic Infection Moderately Severe Malaria Severe Malaria Age (in weeks) 13

15 HRP-2 (log-transformed) (c) 6 Mild/Asymptomatic Infection Moderately Severe Malaria Severe Malaria Age (in weeks) 14

16 Parasite density (per 200 WBC) Figure S2. Parasite density by clinical severity (all parasitemias) (whiskers represent 5th and 95th percentiles). In a logistic GEE model assessing the risk of developing severe malaria during infection, the proportional reduction in entropy due to parasite density as continuous and categorical covariate were 3.2 and 7.6%, respectively 6. (Severe Malaria=SM; Moderately Severe Malaria=Moderately SM) SM Moderately SM Mild Asymptomatic 15

17 Risk of Severe/Moderately Malaria Figure S3. Risk of developing severe malaria or moderately severe malaria during first and subsequent infections (y-axis represents risk; x-axis represents infection, arranged according to order of occurrence). Numbers over each bar represent the number of severe malaria or moderately severe malaria episodes used to calculate risk for each infection (number of children with severe malaria or moderately severe malaria at that particular infection/ total number of children that had at least the indicated number of infections) /715 41/501 31/ /276 12/213 8/120 5/86 3/47 1/25 5/169 1/62 1/ Order of Infections 16

18 Figure S4. Children with moderately severe malaria usually present with discrete syndromes (N=155). 17

19 Parasite density (per 200 WBC) Figure S5. Geometric mean of parasitemia and its 95% confidence interval by age (in 4-week intervals) (all parasitemias were included in this analysis) Age (in weeks) 18

20 Figure S6. Mild and severe malaria episodes (blue dots = Mild or asymptomatic episodes; red dots = Severe malaria episodes). Children are ordered in the y-axis by age at the time of their first severe malaria episode, or by age at first parasitemia if they did not experience severe malaria. 19

21 TABLES (SUPPLEMENTARY APPENDIX) Table S1. Age, parasite density and PfHRP-2 levels by syndrome during severe malaria episodes. Clinical Syndromes Age (in weeks)* Parasite density ( per 200 WBC)* HRP-2 (in ng/ml)** n=7 Convulsion 45.7 ( ) ( ) 1007 ( ,242) n=25 Severe Anemia 33.7 ( ) 724 ( ) 509 ( ) n=19 Respiratory Distress 47.4 ( ) 524 ( ) 2999 ( ) n=15 Prostration 45.5 ( ) 990 ( ) 1046 ( ) n=8 Multiple Syndromes 37.3 ( ) 3208 ( ) 7319 ( ,972) * Median (Q1-Q3) **Geometric Mean (95%CI) 20

22 Table S2. Multivariate Cox model on the time to first moderately severe malaria and on the time to first severe or moderately severe malaria. Moderately Severe Malaria Moderately Severe Malaria + Severe Malaria * Variables Hazard Ratio P Hazard Ratio P Sickle cell genotype Hemoglobin AS vs. AA 0.42[ ] [ ] Bed Net usage vs. no Bed Net usage 0.55[ ] Male vs. Female 1.30[ ] [ ] 0.87 Village Magila Ref - Ref - Bwembwera 1.22[ ] [ ] 0.64 Mkanyageni 1.05[ ] [ ] 0.06 Muheza Township 0.59[ ] [ ] High Transmission season vs. Low Transmission Season 2.06[ ] < [ ] Parity and Placental Malaria interaction First delivery/no placental malaria Ref - Ref - Second or later delivery/no placental malaria 1.19[ ] [ ] 0.54 First delivery/placental Malaria 0.79[ ] [ ] 0.53 Second or later delivery/ Placental Malaria 2.72[ ] [ ] Alpha-Thalassemia a2/a2 Ref - Ref - a2/a [ ] [ ] 0.15 a3.7/a [ ] [ ] 0.58 *Model was stratified on bed net use since it did not meet proportional hazards assumption 21

23 Table S3. High density infection (HDI) and severe malaria (SM) rates and their rate ratio by age Rate ratio (HDI to Age (weeks) HDI rate* SM rate* SM) p-value (0.96,2.70) (1.69,3.03) < (4.03,7.72) < (7.81,23.63) <0.001 * episodes per child per 24 weeks 95% Confidence Interval Table S4. High density infection and Severe Malaria rate changes by age Severe Malaria High density infection Age Rate ratio p- Rate ratio (weeks) (95%CI)* value (95%CI)* p-value NA 1 NA (0.31,0.69) < (0.91,1.43) (0.08,0.31) < (0.75,1.27) 0.88 *baseline weeks 22

24 Table S5. Total number of severe malaria, moderately severe malaria (SM) or severe malaria/moderately severe malaria (SM/MSM) episodes per child Number of episodes Severe Malaria Moderately SM SM/MSM

25 Table S6. Recurrence pattern of severe malaria episodes. Only children with 2 or more severe malaria episodes are included in this table (N=15). Chi ld 1st SM 2nd SM 3rd SM 4th SM Age at 1 st SM (weeks) Δ Age (2nd - 1st)(weeks) Δ Age (3rd - 2nd)(weeks) Δ Age (4th - 3rd)(weeks) 1 P P A A P P P A R A A R C A A A A A P R+P A H P P A+P R R R P R R A C P R R+P A Severe anemia C Convulsion H Hypoglycemia Respiratory R Distress P Prostration 24

26 Table S7. Number of PfHRP-2 measurements per child Number of visits with PfHRP-2 measured Number of children (%) 1 42 (16%) 2 52 (19.8%) 3 41 (15.6%) 4 22 (8.4%) 5 33 (12.5%) 6 14 (5.3%) 7 18 (6.8%) 8 16 (6.1%) 9 12 (4.6%) 10 8 (3%) 11 3 (1.1%) 13 1 (0.4%) 14 1 (0.4%) 25

27 Table S8. Characteristics of children with at least one HRP-2 measurement (N=263) Characteristic Population Number of Infants 263 N (%) Maternal parity First delivery 74 (28.1) Second delivery 54 (20.5) Third or subsequent delivery 135 (51.3) Residence area Magila 58 (22%) Muheza Township 74 (28.1%) Mkanyageni 67 (25.5%) Bwembwera 64 (24.3%) Bed net usage 122 (49.2) Sex Male 133 (50.6) Female 130 (49.4) Transmission season at birth High 118 (44.9) Low 145 (55.1) Sickle cell trait 25 (9.6) Mean (SD) Duration of follow-up (weeks) 137 (38.3) Mean age at visits with PfHRP-2 (weeks) 64.2 (36.6) Median (Q1-Q3) Parasitemia at visits with PfHRP-2 (per 200 WBCs) 1268 ( ) 26

28 Table S9. Baseline characteristics of children followed for at least 1 year and children followed for less than one year Characteristic Follow-up < 1 year Follow-up>1 year Number of Infants Maternal Characteristics Mean (SD) Mean (SD) Maternal age (years) 24.9 (6.1) 26.4 (6.3) N (%) N (%) Maternal parity First delivery 73 (37.6) 181 (26.3) Second delivery 44 (22.7) 157 (22.8) Third or subsequent delivery 77 (39.7) 350 (50.9) Residence area Magila 21 (10.8) 123 (17.9) Muheza Township 92 (47.4) 308 (44.8) Mkanyageni 42 (21.6) 146 (21.2) Bwembwera 39 (20.1) 111 (16.1) Bed net usage 76 (68.5) 379 (61.6) Infant Characteristics Mean (SD) Mean (SD) Birth weight (Kg) 3.2 (0.43) 3.2 (0.43) N (%) N (%) Sex Male 105 (54.1) 352 (51.2) Female 89 (45.9) 336 (48.8) Transmission season at birth High 129 (66.5) 303 (44) Low 65 (33.5) 385 (56) Sickle cell trait 37 (20.3) 105 (15.5) Postneonatal Mortality 27 (13.9) 8 (1.2) 27

29 Supplementary References 1. Lemnge M, Alifrangis M, Kafuye MY, et al. High reinfection rate and treatment failures in children treated with amodiaquine for falciparum malaria in Muheza villages, Northeastern Tanzania. Am J Trop Med Hyg 2006;75: Manning L, Laman M, Stanisic D, et al. Plasma Plasmodium falciparum histidine-rich protein-2 concentrations do not reflect severity of malaria in Papua new guinean children. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America;52: Chong SS, Boehm CD, Higgs DR, Cutting GR. Single-tube multiplex-pcr screen for common deletional determinants of alpha-thalassemia. Blood 2000;95: Snow RW, Schellenberg JR, Peshu N, et al. Periodicity and space-time clustering of severe childhood malaria on the coast of Kenya. Trans R Soc Trop Med Hyg 1993;87: Snow RW, Bastos de Azevedo I, Lowe BS, et al. Severe childhood malaria in two areas of markedly different falciparum transmission in east Africa. Acta Trop 1994;57: Zheng B. Summarizing the goodness of fit of generalized linear models for longitudinal data. Statistics in medicine 2000;19: