Low antibodies against Plasmodium falciparum and imbalanced pro-inflammatory cytokines are associated with severe malaria in Mozambican children: a case–control study

The area of study was located in the Manhiça District, southern Mozambique. Detailed descriptions of the area have been reported elsewhere [24]. Briefly, Manhiça is characterized by a perennial malaria transmission with some seasonality and of moderate intensity, mostly attributable to P. falciparum. The majority of SM cases occur in children under five years of age. Prostration, ARD and SA are the most common clinical presentations, with coma being infrequent and slightly shifted to older children [23].

Between April and November 2006, a sex and age (+/−3 months) matched case–control study was conducted, with a primary aim of characterizing the cytoadhesion phenotypes of P. falciparum isolates [25]. Children under five years of age attending the Manhiça District Hospital with a clinical diagnosis of P. falciparum malaria were recruited after written informed consent was given by their parents or guardians. Clinical malaria was defined as the presence of fever (axillary temperature ≥37.5 °C) with an asexual P. falciparum parasitaemia of ≥500/μL by microscopic examination of Giemsa-stained blood smears; this definition of malaria has a sensitivity and specificity of >90% in children from Manhiça [26]. Cases were children presenting with clinical malaria and at least one of the following definitions of SM [27]: CM (Blantyre Coma Score ≤2), SA (packed cell volume <15% or hemoglobin <5 g/dL), ARD (lactate >5 mM and/or chest indrawing or deep breathing), prostration (inability to sit or breastfeed in children old enough to do so), hypoglycaemia (blood glucose <2.2 mM) and MS (≥2 convulsions in the preceding 24 h) . Controls were outpatient children with malaria not showing any of the mentioned signs of severity and able to take oral medication (UM group). All patients were reviewed by the study pediatrician to confirm that malaria was the sole or principal cause of the disease. Children with positive bacteraemia were excluded from the study. Malnutrition was defined as the presence of marasmus or kwashiorkor by clinical examination or as a mid-upper arm circumference <12.5 cm in children >12 months of age. SM patients were admitted and treated with intravenous quinine until able to switch to oral therapy, while UM controls were treated following Mozambican national guidelines at that time (artesunate plus sulphadoxine-pyrimethamine). The study was approved by the National Mozambican and the Hospital Clinic of Barcelona Ethics Review Committees.

Before treatment, peripheral blood was taken by venipuncture into a tube containing lithium heparin, and 2 drops of blood were spotted onto filter paper (Schleicher & Schuell; n° 903^TM). Biochemical determinations (alanine aminotransferase, bilirubin and creatinine) and full blood counts were performed using Vitros DT60 and Sysmex Kx21 analyzers, respectively. Lactate was determined using Lactate Pro® (Fact Canada) at the bed side. After centrifugation, plasma was stored at −80 °C, erythrocyte pellets were Buffy-coat depleted, washed 3 times in phosphate-buffered saline (PBS) and cryopreserved in glycerolyte solution.

DNA was extracted from a 50 ml blood drop onto filter paper with QIAamp DNA Mini Kit (Qiagen), and resuspended in 150 ml of water. Five ml of DNA samples, tested in triplicate, were used to measure parasite density by a real-time quantitative PCR (qPCR) targeting the P. falciparum 18 S ribosomal RNA gene, as described elsewhere [28]. Multiplicity of infection (MOI) was determined by nested PCR-typing of the polymorphic regions of msp-1 and msp-2 genes [29], and estimated as the highest number of msp-1 or msp-2 alleles in the sample.

IgG and IgM antibodies were measured against the recombinant proteins merozoite surface protein 1 (MSP-1₁₉, 19 kD fragment, 3D7 [30]), erythrocyte binding antigen 175 (EBA-175, F2 region, CAMP [31]), apical membrane antigen 1 (AMA-1, full ectodomain, 3D7 [32]) and a Duffy binding like alpha (DBLα) domain from a Pf EMP1 involved in rosetting through adhesion to complement receptor 1 (R29var1 minimal domain, [33]), all produced at the International Centre for Genetic Engineering and Biotechnology (New Delhi, India). High-binding 96-well microplates (Nunc Maxisorp) were coated with 200 ng/well of recombinant protein in 0.05 M carbonate-bicarbonate buffer. Plates were washed with 0.05% Tween-20 in PBS (PBS-Tween), blocked with 2% bovine serum albumin (BSA) in PBS-Tween for 8 h at 4 °C and washed again. One hundred μl of plasma at 1/200 dilution along with positive (pool from 8 hyper-immune Mozambican adults) and negative controls (9 unexposed Europeans) were added in duplicate to wells. After incubation overnight at 4 °C, peroxidase-conjugated goat anti-human IgG or IgM secondary antibodies (Sigma) were added at 1/30000 and 1/2000, respectively. After 1 h of incubation and washing, 100 μl/well of a phosphate solution with 0.012% of H₂O₂ substrate and o-phenylendiamine chromagen were added for 5 min and the colorimetric reaction was stopped with 25 μl/well of H₂SO₄. Specific reactivity of plasmas was obtained as optical density (OD) values measured at 492 nm (Multiskan EX, Labsystems) and normalized by dividing OD of each sample by the OD of the positive control run in each plate.

Five paediatric isolates (three from SM patients [MOZ1, MOZ2 and MOZ4] and two from UM patients [MOZ3 and MOZ5]) and two placental isolates from O blood group individuals [25, 34], were tested for antibody recognition. MOZ2 was adapted to in vitro culture, whereas the remaining isolates were used in antibody measurements without in vitro expansion. IgG were also measured against four laboratory clones with different receptor binding phenotypes: R29 (rosetting, [35]), ITG_ICAM (ICAM-1, [36]), FCR3_CSA (CSA, [37]) and E8B_CD36 (CD36, [38]). The study samples, plus negative and positive controls, were tested blindly in a single assay against each parasite. Briefly, cryopreserved ring-stage parasites were thawed in a sorbitol gradient and cultured to late trophozoites following standard methods. Cells were washed three times in PBS and resuspended at 1% of haematocrit and 1-5% parasitaemia in 1% BSA-PBS solution. Five μl of plasma were added to 95 μl/well of erythrocyte suspension, incubated for 30 min at room temperature and stained for 30 min with 100 μl of polyclonal rabbit anti-human IgG (DakoCytomation) at 1/200. Subsequently, cells were incubated with 100 μl of AlexaFluor®-conjugated donkey anti-rabbit IgG (Invitrogen) diluted at 1/1000 and 10 mg/mL ethidium bromide for 30 min in darkness. Samples were washed three times with PBS-BSA between incubations. Data from 1000 positive events was acquired with a Becton-Dickinson FACSCalibur flow cytometer. Reactivity against IEs surface antigens was expressed as the difference between the mean fluorescence intensity (MFI) of IEs and the MFI of uninfected red blood cells.

Concentrations (pg/mL) of IL-12p70, IL-2, IFN-γ, IL-4, IL-5, IL-10, IL-8, IL-6, IL-1β, TNF, TNF-β and TGF-β1 in plasma were measured using fluorescent bead immunoassays (Human Th1/Th2 11plex and Human TGF-β1 FlowCytomix Simplex kits, Bender MedSystems, Austria) following manufacturer’s instructions. Beads fluorescence was acquired with a Becton-Dickinson FACSCanto II and analysed in FlowCytomix Pro2.2.1 software (Bender MedSystems). Concentration of each analyte was obtained by extrapolating fluorescence intensity to a 7-point dilution standard curve supplied by the manufacturer. Any value below the limits of detection was given a value of half the detection limit for that cytokine.

Prevalence of recognition of parasites and recombinant proteins by antibodies in children was considered positive if MFI or normalized OD values were above the mean of the negative controls plus three standard deviations for each antigen. Spearman’s rank correlations were performed to evaluate correlations between immunological parameters. Comparisons between matched case–control pairs for categorical variables were done using McNemar´s chi-squared, and reported as the number of pairs with a divergent result between SM and UM. Continuous variables were analysed using Sign test, and reported as the median difference between SM and UM values. Children were stratified by SM clinical presentations, and compared to their matched controls. Adjustment for multiple comparisons was done by Monte Carlo permutation tests with 1000 random permutations [39]. Associations between immune responses and parasite qPCR densities were assessed by multivariate linear regression with log-transformed parasite density as the outcome and log-transformed antibodies or cytokines as independent variables. All data collected were analysed using Stata version 10.0 (Stata Corporation). P-values <0.05 were considered statistically significant.

Among 142 children recruited, four tested negative for P. falciparum by qPCR and were excluded together with their matched pairs. Therefore, 134 children (67 case–control pairs) were finally analysed. The clinical characteristics of the patients are summarized in Table 1. Prostration, ARD, SA and MS were the most prevalent symptoms of severity, whereas hypoglycaemia and CM were only described in five and three children, respectively. Forty-four (65%) children presented with two or more SM criteria. The 23 cases with a single criterion of severity distributed as follows: 13 prostration, 6 MS, 3 SA and 1 ARD. Three SM patients died yielding a case-fatality rate of 2%, and two were transferred to Maputo Central Hospital. Parasite densities by qPCR were higher in SM cases than in paired controls, but this difference was not statistically significant (P = 0.087). Children with SM or UM did not differ in malnutrition, indirect clinical indicators of AIDS (oral candidiasis) or in the number of days with clinical symptoms prior to recruitment. Pre-treatment with an anti-malarial was only reported for one patient with SM. Previous history of SM was more common among children with SM (11 [16%]) than in children with UM (3 [4%]; divergent pairs = 13 [18%], P = 0.037).

Prevalence and levels of IgG against DBLα were significantly lower in children with SM compared to their matched controls (P = 0.032 and P < 0.001, respectively), whereas no differences were found for IgG against merozoite antigens (Figure 1A). In contrast, IgG levels against P. falciparum lysate were significantly higher in children with SM than in matched controls (P = 0.004; Figure 1B). Recognition of antigens on the surface of IEs did not differ between SM and UM children for any of the parasites tested (Table 2). Overall, IgG prevalence was highest for R29 and E8B_CD36 IEs, and lowest for CSA binding IEs (FCR3_CSA) and placental isolates. A similar seroprevalence was found for IEs isolated from SM patients (MOZ1, MOZ2, MOZ4) or UM patients (MOZ3, MOZ5; Table 2). The number of IgM responders against MSP-1₁₉ and AMA-1 was lower in SM (P = 0.038 and P = 0.024, respectively), and a similar but not significant trend was found for DBLα (Figure 2A). Likewise, anti-MSP-1₁₉ and AMA-1 IgM levels were lower in SM compared to their UM pairs (P = 0.050 and P = 0.047, respectively). In general, different antibody responses correlated weakly (Additional file 1), with the exception of IgG against EBA-175 and MSP-1₁₉ (Spearman’s rho = 0.8635, P < 0.001) and IgM against EBA-175 and AMA-1 (Spearman’s rho = 0.8595, P < 0.001).

A stratified analysis by SM clinical presentations showed that levels of IgG against DBLα were lower both for SA (P = 0.031), ARD (P = 0.015) and prostration (P = 0.002) compared to their respective matched controls (see Additional file 2). Similar to the main analysis, no differences were found for other recombinant antigens or for IgG against IEs surface (data not shown). Levels of IgM were significantly lower in children with SA, ARD and prostration for at least one of the antigens tested. Immune responses in children with CM and hypoglycaemia could not be assessed separately due to the low prevalence of these groups (Table 1).

A linear regression model including all antibody responses showed that IgG against DBLα was the only antibody independently associated to lower P. falciparum densities. The proportional reduction of parasite densities per a two-fold increment in IgG levels was 0.27 when both SM and UM children were included in the analysis ([95% confidence interval [CI] 0.08, 0.85], P = 0.026) and 0.11 including only SM cases ([95% confidence interval [CI] 0.02, 0.72], P = 0.022), but the effect was not observed among UM controls (1.34 [95% CI 0.28, 6.50], P = 0.712).

Children with SM had significantly higher IL-6 concentrations in plasma than children with UM (P = 0.047, Figure 3A). IL-1β was more frequently detected in children with SM (P = 0.013), although no significant differences were found for IL-1β concentrations. Conversely, children with SM had lower levels of TGF-β1 and IL-12 (P < 0.001 and P = 0.039, respectively; Figure 3B and 3C), and lower prevalence of detectable IL-12 concentrations than matched UM controls (P = 0.011; Figure 3C). IL-5 and TNF-β were excluded from the statistical analysis since levels in most samples were below the limit of detection (109/134 [81%] for IL-5 and 132/134 [99%] for TNF-β). Overall, there were weak or moderate correlations within cytokine responses (Additional file 3), with the exception of IL-6 and IL-10 (Spearman’s rho = 0.8682, P < 0.001). No significant correlations were found between antibodies and cytokines.

Stratification by severe clinical presentations showed that children with ARD had higher levels of IL-10 (P = 0.032) and IL-6 (P = 0.002) and lower levels of TGF-β1 (P = 0.002) compared to their controls (Additional file 2). TGF-β1 concentrations were also reduced in the prostration group (P = 0.007).

IL-12 concentrations were associated with a reduction of parasite densities when all children were included in the analysis (0.69 [95% CI 0.54, 0.90], P = 0.006) and including only children with SM (0.62 [95% CI 0.44, 0.89], P = 0.010), but the association was not found in children with UM (0.88 [95% CI 0.58, 1.33], P = 0.528). On the other hand, higher concentrations of IL-10 were associated with an increase in parasite densities in all groups (all children: 2.44 [95% CI 1.58, 3.77], P < 0.001; SM cases: 2.59 [95% CI 1.37, 4.92], P = 0.004; UM controls: 2.62 [95% CI 1.38, 5.01], P = 0.004).