REVISED VERSION (R1) SUPPLEMENTARY INFORMATION MARKED UP COPY (ICME-D-16-00245)
Imported Falciparum Malaria in Adults: Host and Parasite-related Factors Associated with Severity: The French Prospective Multicenter PALUREA Cohort Study
Correspondence should be addressed to :
Fabrice Bruneel, Service de Reanimation, Centre Hospitalier de Versailles, 177 rue de Versailles, 78157 Le Chesnay, France
Phone: +33139639 058 Fax: +33139638688 E-mail:
Supplementary Methods
Data collected
Standard data collected included demographic data; previous medical history; country of malaria acquisition; malaria chemoprophylaxis; clinical, laboratory, and imaging findings; treatments given in ICU and hospital; and vital status at ICU and hospital discharge. Acute illness severity was assessed at ICU admission using the Glasgow Coma Scale (GCS) score [1], Simplified Acute Physiology Score II (SAPS II) [2], and Sequential Organ Failure Assessment (SOFA) score [3].
Parasitological studies
Ex vivo antimalarial susceptibility assay
Chloroquine, doxycycline, dihydroartemisinine, and quinine were purchased from Sigma-Aldrich Company (Saint-Louis, MO); mono-desethyl-amodiaquine was obtained from the World Health Organization (Geneva, Switzerland). Mefloquine was purchased from Roche (Paris, France) and lumefantrine from Novartis Pharma (Basel, Switzerland). Atovaquone was from GlaxoSmithKline (Evreux, France). Antimalarial drug stock solutions were prepared in appropriate solvents; whereas dilutions were made in water, except for lumefantrine, which was diluted in ethanol; and atovaquone, which was diluted in methanol. The dilutions were distributed and dried in 96-well tissue culture plates in the following concentration ranges: quinine in ethanol, 25-3200 nM; mono-desethyl-amodiaquine in water, 7.5-1920 nM; mefloquine in methanol, 3.2-400 nM; dihydroartemisinine in water, 0.25-64 nM; lumefantrine in ethanol, 1.25-320 nM; chloroquine in water, 5-3200 nM; atovaquone in methanol, 5-384 nM; and doxycycline in water, 0.1-502 mM.
The reference laboratory P. falciparum clones 3D7 (Africa) and W2 (Indochina) were used to control each batch of plates. For each drug tested, three control wells were drug-free, and each concentration was studied in duplicate or triplicate. Infected erythrocytes were washed and cultured in drug-coated plates for 42 hours before freezing, as described elsewhere [4]. Briefly, after washes, blood samples were resuspended in 1.5% hematocrit in RPMI 1640 medium (Gibco, InVitrogen Life Technologies, Cergy Pontoise, France), buffered with 25 mM HEPES (Gibco, InVitrogen Life Technologies), and 25 mM NaHCO3 (Sigma-Aldrich, Saint-Louis, MO), and supplemented with 10% human serum (Abcys SA, Paris, France). If parasitemia exceeded 1%, infected erythrocytes were diluted to 0.5%-1% with uninfected erythrocytes (human blood type O+). Parasite growth was assessed by adding 1 mCi of tritiated hypoxanthine with a specific activity of 14.1 Ci/mmol (Perkin-Elmer, Courtaboeuf, France) to each well at time zero. The plates were incubated for 42 hours under controlled atmospheric conditions consisting of 5% O2, 5% CO2, and 85% N2 at 37°C; frozen at -20°C; and thawed to lyse the erythrocytes. The content of each well was collected on standard filter microplates (Unifilter GF/B; Perkin-Elmer) and washed using a cell harvester (Filter-Mate Cell Harvester; Perkin-Elmer). Filter microplates were dried, and 25 mL of scintillation cocktail (Microscint O; Perkin-Elmer) was placed in each well. Radioactivity incorporated into nucleotides by the parasites was measured using a scintillation counter (Top Count; Perkin-Elmer). The drug concentration inhibiting 50% of parasite growth (IC50) was defined as the drug concentration associated with incorporation of 50% of the tritiated hypoxanthine by the parasite in the drug-free control wells. The IC50 values were determined using non-linear regression analysis of log-based dose-response curves.
*Parasite DNA extraction
Parasite DNA was extracted from venous blood for molecular analysis using the QIAamp DNA Mini Kit, Qiagen® (Venlo, The Netherlands) before 2008 and the MagNA Pure LC DNA Isolation Kit I, Roche Diagnostics (Basel, Switzerland), after 2008, according to the manufacturer’s recommendations.
*pfcrt76 single-nucleotide polymorphisms (SNPs)
This resistance marker is associated with chloroquine resistance. A 201-nucleotide fragment of the pfcrt gene (containing codon 76) was amplified by polymerase chain reaction (PCR) using CRT 76 sense 5’-TTT AGG TGG AGG TTC TTG TC-3’ and CRT 76 antisense 5’- AAT AAA GTT GTG AGT TTC GGA 3’ primers (Eurogentec, Seraing, Belgium). The reaction mixture for PCR amplifications included 2 mL of genomic DNA, 2.5 mL of 10X reaction buffer (Applied Biosystems, Foster City, CA), 0.75 mM of each primer, 200 mM of a deoxynucleoside triphosphate mixture (dGTP, sATP, dTTP, and dCTP), 4 mM of MgCl2, and 1 unit of AmpliTaq Gold DNA (Applied Biosystems) in a final volume of 25 mL. The thermal cycler (ICycler Thermal Cycler, BioRad, Marnes-la-Coquette, France) was programmed as follows: initial incubation at 95°C for 7 min; 40 cycles at 95°C for 40 s, 59°C for 40 s, and 72°C for 40 s; and a final 1-min elongation step at 72°C. The PCR products were loaded onto 1.5% agarose gel containing 0.5 mg/mL of ethidium bromide.
*pfdhfr single-nucleotide polymorphism (SNP)
The pfdhfr gene polymorphism is associated with antifolate resistance. A 119-nucleotide fragment of the dhfr gene (containing codon 108) was amplified by PCR using DHFR 108 sense 5’-TGT GGA TAA TGT AAA TGA TAT GCC-3’ and DHFR 108 antisense 5’- CAT TTA TCC TAT TGC TTA AAG GTT-3’ primers (Eurogentec). The reaction mixture for PCR amplifications included 2 mL of genomic DNA, 2.5 mL of 10X reaction buffer (Applied Biosystems), 0.75 mM of each primer, 200 mM of a deoxynucleoside triphosphate mixture (dGTP, sATP, dTTP, and dCTP), 6 mM of MgCl2 and 1.25 units of AmpliTaq Gold DNA (Applied Biosystems) in a final volume of 25 mL. The thermal cycler (ICycler Thermal Cycler, BioRad) was programmed as follows: initial incubation at 95°C for 5 min; 40 cycles at 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min; and a final 1-min elongation step at 72°C. The PCR products were loaded onto 1.5% agarose gel containing 0.5 mg/mL ethidium bromide.
A 116-nucleotide fragment of the dhfr gene (containing codons 51 and 59) was amplified by PCR using DHFR 51-59 sense 5’-CAC TTT AGA GGT CTA GGA AAT AAA GGA -3’ and DHFR 51-59 antisense 5’- TCA ATT TTT CAT ATT TTG ATT CAT TCA C-3’ primers (Eurogentec). The reaction mixture for PCR amplifications included 2 mL of genomic DNA, 2.5 mL of 10X reaction buffer (Applied Biosystems), 0.45 mM of each primer, 200 mM of a deoxynucleoside triphosphate mixture (dGTP, sATP, dTTP, and dCTP), 1.6 mM MgCl2, and 1.25 units of AmpliTaq Gold DNA (Applied Biosystems) in a final volume of 25 mL. The thermal cycler (ICycler Thermal Cycler, BioRad) was programmed as follows: initial incubation at 95°C for 7 min; 40 cycles at 94°C for 45 s, 56°C for 40 s, and 72°C for 45 s; and a final 5-min elongation step at 72°C. The PCR products were loaded onto 1.5% agarose gel containing 0.5 mg/mL ethidium bromide.
Restriction fragment length polymorphisms (RFLPs)
The genotypes of pfcrt K76T and pfdhfr S108N codons were determined by analyzing RFLPs of PCR products using appropriate restriction enzymes purchased from Ozyme (Saint Quentin en Yvelines, France). For pfcrt K76T, 10 mL of the PCR product was incubated with ApoI restriction enzymes in a total volume of 30 mL according to the manufacturer’s instructions. For dhfr S108N, 6 mL of the PCR product was incubated with BsrI restriction enzymes in a total volume of 20 mL according to the manufacturer’s instructions. The digestion products were resolved on 2-2.5% agarose gels (Invitrogen, Life Technologies,). DNA from laboratory P. falciparum strains (3D7 and W3) were used as controls for PCR and digestion.
Sequencing of the pfdhfr gene
The genotypes of the pfdhfr N51I and C59R codons were determined by sequencing. The 116-pb PCR product was used as a template for additional nested PCR amplification with the DHFR 51-59 primers. PCR products were purified using the QIAquick PCR purification Kit (Qiagen) and sequenced using an ABI PRISM Big Dye Terminator Cycle sequencing Kit (Applied Biosystems) in an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), following the manufacturer’s protocol.
Clonal analysis
The number of genotypes within isolates was determined for each patient using a previously published fragment analysis method [5]. Briefly, the method was based on the polymorphism of the gene encoding the merozoite surface protein-2. PCR amplification of part of the merozoite surface protein-2 gene (msp-2) with a primer pair including a 5’ sense primer labeled with fluorescein was followed by capillary gel electrophoresis to separate alleles having different base-pair numbers. Each genotype was characterized by the size and area under the curve of the peak corresponding to its msp-2 PCR products measured during capillary electrophoresis. The description of each isolate included the number of msp-2 genotypes and the size of the corresponding PCR products within the isolate. Block 3 of the msp-2 domain was amplified on 2 mL of DNA extract in 50 mL of reaction mixture containing 15 pmoL of each primer (5’-GAA GGT AAT TAA AAC ATT GTC-3’ [sense] and 5’-GAC ACC TCG TCG TTG TAG GGA G-3’ [antisense]) (Eurogentec), buffer (15 mM Tris-HCL [pH 8.0], 50 mM of KCl, and 6 mM of MgCl2), 200 mM of dNTP, and 1.25 U of AmpliTaq Gold DNA (Applied Biosystems). The thermal cycling conditions were as follows: 7 min at 95°C, followed by 40 cycles of 30 s at 95°C, 30 s at 42°C, and 30 s at 72°C. The amplification products were then processed in an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) to count and quantify fluorescent fragments as previously described [5].
PfHRP2 quantification
One blood sample (5 mL on lithium heparinate) was collected on the ICU admission day (D0) then rapidly centrifuged (10 000 rpm for 15 min) to allow plasma collection in a cryotube, which was stored immediately at -80°C before transport to the reference laboratory (Parasitology Department and French Malaria Reference Center of the Bichat-Claude Bernard Teaching Hospital) for plasma PfHRP-2 assay using the malaria Ag Celisa kit (Cellabs, Sydney, New South Wales, Australia), following the manufacturer’s instructions. Briefly, microwells were precoated with anti-P. falciparum monoclonal capture antibody. Plasma samples in serial 10-fold dilutions together with positive and negative controls (100 mL) were added to the wells and incubated for 1 h at room temperature. Anti-falciparum antibody conjugate (diluted 1:200) was added and incubated for 1 h at room temperature. Finally, tetramethyl benzidine substrate was added, and the sample was incubated for 15 min at room temperature. The reaction was stopped by adding 2M sulfuric acid, and the results were read spectrophotometrically at 450 nm on a Sunrise PR 3100 plate reader (BioRad). Concentrations were derived from standard curves established using purified PfHRP2 standard (provided by Cellabs) diluted in RPMI 1640. Samples with optical densities outside the linear part of the curve were retested at increasing dilutions until an accurate concentration was determined.
Host biomarkers and hemoglobin electrophoresis
sTREM-1
Briefly, samples were incubated in microwell plates coated with a monoclonal antibody specific for TREM-1. After washing to eliminate unbound substances, an enzyme-linked polyclonal antibody specific for TREM-1 was added to the wells. Further washing was performed to eliminate unbound conjugate and a substrate solution was added to the wells. Color development was stopped and the optical density of each well was determined within 30 min. using a microplate reader (Sunrise, Tecan, Männedorf, Switzerland) set at 450 nm, with a wavelength correction set at 540 nm. In our experiments, the minimum detectable concentration of sTREM-1 was 67 pg/mL.
Hemoglobin electrophoresis
As recommended by the French Society of Clinical Biology [6], we used at least two different assay techniques to identify the Hb variants. One technique was high-performance liquid chromatography, a widely used method for Hb-variant detection. The retention time and peak shape are very helpful for identifying many Hb variants. The other method used to differentiate Hb variants was cellular acetate electrophoresis at alkaline pH. Finally, we confirmed the presence of the Hb S variant by performing a sickle solubility test.
Statistical analysis
A classification and regression tree (CART) was built to identify the factors most strongly associated with VSM. The CART included the biomarkers selected by previous univariate logistic regression, ethnic origin, and co-infection on D0-D1. The following rules were used: at least 30 cases for parent nodes and 10 for child nodes; 10 cross-validations; and a cost complexity factor of 0.01. The CARTs were built using the “rpart” package, which uses R software v.2.13 (The R Project for Statistical Computing at http://cran.r-project.org/web/packages/rpart/index.html).
Supplementary Results
Plasma PfHRP2 level was assayed in 128/139 UM patients and 130/155 SM patients. Determination of the number of clonal P. falciparum populations per patient and assessment of antimalarial drug resistance using molecular techniques were possible in 139/140 (99%) UM patients and in 148/155 (95%) SM patients. Antimalarial drug resistance evaluated based on the IC50 of the main antimalarials was available for only 43% to 66% of the patients overall.
Supplementary References
1. Teasdale G, Jennett B (1974) Assessment of coma and impaired consciousness. A practical scale. Lancet ii: 81-84.
2. Le Gall JR, Lemeshow S, Saulnier F (1993). A new acute simplified score (SAPS II) based on a European/North American multicenter study. JAMA 270: 2957-2963.
3. Vincent JL, Moreno R, Takala J, Willatts S, De Mendonça A, Bruining H et al (1996) The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction/failure. Intensive Care Med 22:707-710.
4. Le Bras J, Deloron P (1983) In vitro study of drug sensitivity of Plasmodium falciparum: evaluation of a new semi-micro test. Am J Trop Med Hyg 32:447-451.
5. Jafari S, Le Bras J, Bouchaud O, Durand R (2004) Plasmodium falciparum clonal population dynamics during malaria treatment. J Infect Dis 189:195-203.
6. Bardakdjian-Michau J, Dhondt JL, Ducrocq R, Galactéros F, Guyard A, Huchet FX et al (2003) Good practices for the study of hemoglobin. Ann Biol Clin (Paris) 61:401-409.
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Supplementary Table 1. Criteria for severe malaria according to the modified 2000 World Health Organization definition (see *, † and §)
Clinical criteriaImpaired consciousness: Glasgow Coma Scale score <11* and/or ≥ two convulsions§
Respiratory distress: requirement for noninvasive and/or endotracheal mechanical ventilation or spontaneous breathing with PaO2 <60 mm Hg (if FiO2 ≥0.21)†, and/or respiratory rate >32/min*
Circulatory collapse: systolic blood pressure <80 mm Hg despite adequate volume repletion
Abnormal bleeding
Jaundice: clinical jaundice or bilirubin >50 mmol/L
Macroscopic hemoglobinuria: if unequivocally related to acute malaria (patients with blackwater fever are not included)
Laboratory criteria
Severe anemia: hemoglobin <5 g/dL
Hypoglycemia: blood glucose <2.2 mmol/L
Acidemia (pH <7.35) or acidosis (serum bicarbonate <15 mmol/L)
Hyperlactatemia: arterial lactate >5 mmol/L
Hyperparasitemia ³4%
Renal impairment: serum creatinine >265 µmol/L or blood urea nitrogen >17 mmol/L*
* Glasgow Coma Scale score criterion of 11 instead of 9 ; respiratory rate >32/minute, and blood urea nitrogen >17 mmol/L are the modifications according to reference [9].