MRP 8/14 as Marker for Plasmodium falciparum-induced Malaria Episodes in Individuals in a Holoendemic Area

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1 CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, July 1997, p Vol. 4, No X/97/$ Copyright 1997, American Society for Microbiology MRP 8/14 as Marker for Plasmodium falciparum-induced Malaria Episodes in Individuals in a Holoendemic Area G. BORDMANN, 1 * G. BURMEISTER, 2 S. SALADIN, 1 H. URASSA, 3 S. MWANKYUSYE, 3 N. WEISS, 1 AND M. TANNER 1 Swiss Tropical Institute, Basel, 1 and BMA Biomedical Ltd., Augst, 2 Switzerland, and Ifakara Centre, Ifakara, Tanzania 3 Received 4 December 1996/Returned for modification 20 March 1997/Accepted 28 April 1997 Presence of Plasmodium falciparum parasites in the peripheral blood of patients in a holoendemic area does not necessarily show that their illness is due to malaria. The aim of the present project was therefore to look for biological markers related to symptomatology or clinical events during a malaria episode. We focused our work on a complex of heterodimeric calcium-binding proteins secreted by stimulated neutrophils and monocytes, named MIF or myeloid-related proteins (MRP 8/14). In a longitudinal study including 51 adults from Ifakara, Tanzania (84.7% prevalence for P. falciparum in adults during the study), the level of MRP 8/14 in the serum was significantly related to the parasite load (Spearman correlation coefficient, 0.52; P < ). In the serum from children up to 6 years admitted at a health post the MRP 8/14 levels were closely related to parasitemia but also to fever episodes (Spearman correlation coefficients, and 0.736; P < and P < 0.001, respectively). Although not specific to malaria, the measurement of MRP 8/14 could be an additional tool in assessing malaria-related morbidity. Malaria is a major cause of mortality and morbidity in areas of high endemicity. A high proportion of individuals are infected with malarial parasites at any one time. Many of these infections especially in newborn infants and adults are asymptomatic; however, it is often difficult to discriminate between individuals presenting at a health post with a developing infection becoming an active episode and parasite carriers who are ill for other reasons (17). Reported clinical symptoms of fever, headache, and myalgia are not always related to a malaria crisis, and microscopic demonstration of parasites in Giemsastained blood smears is therefore not a suitable diagnosis of active malaria. In hyper- or holoendemic malarial areas, immunological markers may, however, provide useful indicators for active malaria episodes. Consequently, we investigated the kinetics of two calcium-binding proteins designated MIF (myeloid inflammatory protein) or MRP-8 and MRP-14 (myeloidrelated protein) of the S-100 protein family in relation to reported fever episodes, symptoms, and parasite load. Like other S-100 proteins (3), MRP-8 and MRP-14 are secreted by stimulated neutrophils and monocytes (6). They have a tendency to form intracellular 8/14 heterocomplexes depending on the amount of Ca 2 available. Zwaldo and colleagues (21) described in 1986 the presence of these two antigens in a subset of human monocytes found in the peripheral blood and inflammatory tissues only. They showed that the expression of the two calcium-binding proteins is associated with myeloid-cell differentiation during a chronic or acute inflammation (20, 22). The resulting complex 8/14 can be detected in cells as well as in biological fluids and seems to correlate positively with various human inflammatory disorders (19). Antimicrobial properties of MRP 8/14 have been demonstrated (18), but the physiological functions of this protein complex in acute or chronic inflammation remain unknown. Since malarial symptomatology is accompanied by inflammatory events relevant to * Corresponding author. Mailing address: Swiss Tropical Institute, Socinstrasse 57, CH-4051 Basel, Switzerland. Phone: Fax: host defense at the start of an episode, the aim of this study was to investigate variations in MRP 8/14 during a malaria episode in order to determine whether these myeloid-related proteins can be used as an episode marker in a population exposed to high malaria transmission. MATERIALS AND METHODS Study area. This study was carried out with employees of the Ifakara Centre and with children from the St. Francis Hospital in Ifakara, in southern Tanzania. The area is holoendemic for malaria with absence of seasonal variation in parasitemia (16). The prevalence for Plasmodium falciparum was 84.7% for the adults included in this study. Investigated population and sampling. (i) Longitudinal survey of adults. Fiftyone healthy adult employees, 11 females and 40 males aged from 16 to 64 years, at the Ifakara Centre consented to participate in the study. Participants were monitored for 4 weeks, and a total of 204 samples were collected. The subjects were invited once a week to answer a clinical questionnaire about symptoms and antimalarial drug consumption. After examination by a physician, the axillary temperature was measured, 2 ml of venous blood was taken from each participant, and parasitemia and parasite species were determined on thick and thin blood smears. After centrifugation the serum samples were aliquoted in 200- l portions and frozen at 70 C until use. Suspected clinical malaria was treated according to the Tanzanian Drug Programme guidelines. A malaria attack was defined by a parasitemia equal or greater than 10 4 parasitized erythrocytes/ l of blood (based on a normal erythrocyte number of / l of blood, which corresponds to a parasitemia of 0.2%) and an axillary temperature 37.7 C. (ii) Children at a hospital health post. Fifty-eight children (36 females and 22 males) aged from 9 months to 6 years admitted to St. Francis Hospital with a presumptive malarial episode were studied. At hospital admission mothers, or in a few cases relatives, who presented the child to the health post were invited to participate in the study. Oral detailed information on aim and procedures was given by the Tanzanian practitioner, followed by a copy of the consent letter in Kiswahili. Thin and thick blood films were obtained by finger prick bleeding, and 0.5 to 1 ml of blood was collected from each child into a Microtainer tube. After centrifugation the serum samples were aliquoted in 100- l portions and frozen at 70 C until use. As for the adults, a malaria attack was defined as having a parasite density equal to or greater than 0.2% and an axillary temperature of 37.7 C. Appropriate therapy (chloroquine syrup or tablets) was administered by the medical doctor from the hospital according to the Tanzanian Drug Programme guidelines. Controls. (i) Adults. Six sera from west- and east-african adult individuals (two females and four males) living in Europe for more than 4 years with a reciprocal immunofluorescent-antibody titers to parasitized erythrocytes lower than 1:80 served as negative controls. (ii) Children. Five serum samples from healthy west- and east-african children aged 4 to 7 years, born in Europe and not previously exposed to malaria, were 435

2 436 BORDMANN ET AL. CLIN. DIAGN. LAB. IMMUNOL. FIG. 1. Distribution of MRP 8/14 levels and parasitemia in 204 samples from 51 adults with a parasitemia ranging between negative and 1% and axillary temperatures between 36.8 and 39.2 C. The MRP 8/14 values, measured in serum, were higher than 10,000 ng/ml in all the five clinical malaria cases. included as negative references. All control subjects were outpatients of the Swiss Tropical Institute. Detailed information on the aim and procedures of the study was given to the participant adults and parents of the children before enrollment. Laboratory investigations. (i) Thin and thick blood films. Giemsa-stained thick blood films were checked and declared negative if no parasite was found per 200 scanned leukocytes. The plasmodium species and the parasite densities were determined on the thin film. (ii) MRP 8/14 measurement by enzyme immunoassay. Levels of MRP 8/14 were measured by a commercial sandwich immunoassay according to the protocol of the manufacturer (BMA Biomedicals AG, Augst, Switzerland). All samples were carried out in duplicate. Duplicates were rejected and the samples were tested again if the difference between the two values exceeded 15%. RESULTS MRP 8/14 concentrations in sera from adults. MRP 8/14 levels in serum were measured in 204 samples from 51 adults (Fig. 1). Thirty-one of the 204 samples were negative for parasites, and the body temperatures for these individuals were below 37.7 C (Table 1). In this group, the geometric mean of measured MRP 8/14 was 2,140 ng/ml, with individual values ranging between 700 and 4,700 ng/ml. In a further 158 samples parasitemia was less than 0.2%. In 11 fever cases in this group, MRP 8/14 levels were between 600 and 4,750 ng/ml (Fig. 2a). During this follow-up of 51 adults, five clinical malaria cases with a parasitemia of 0.5% or above and symptomatology like headache, nausea, and myalgia were detected. The parasite load was equal to or greater than 0.2% in 15 samples with a peak of 1% in one case (Fig. 2b). Temperatures above 37.7 C (maximum, 39.4 C at episode climax) were detected 13 times, and the measured amount of MRP 8/14 ranged from 5,250 to 32,200 mg/ml in this high-parasite-load group. The physician reported 11 consultations of eight people complaining about symptoms including headache, myalgia, or nausea, but the Parasitemia TABLE 1. MRP 8/14 levels in 51 adults samples (%) fever cases a (%) MRP 8/14 (ng/ml) Negative 31 (15.2) 0 2,140 (700 4,700) 0.2% 158 (77.4) 11 (7) 3,420 (600 4,750) 0.2% 15 (7.3) 13 (86.6) 13,580 (5,250 32,200) Nonexposed controls 6 0 1,000 (620 1,430) a 37.7 C. measured axillary temperatures were always below 37.7 C and the parasitemia was less than 0.2%. MRP 8/14 measurement in this group ranged between 910 and 2,910 ng/ml. Treated with salicylic acid (aspirin), the symptoms of these people disappeared within 3 days without antimalarial therapy, and these reported complaints seemed not to be related to malaria. Temperatures of 37.7 C or above were measured only 11 times in adults with parasite loads below 0.2%. The difference between the MRP 8/14 levels in 31 aparasitemic subjects (geometric mean, 2,140 ng/ml) and those in individuals with parasitemia below 0.2% (geometric mean, 3,420 ng/ml) was not significant. All MRP 8/14 values of individuals with a parasitemia of 0.2% exceeded those of the other two groups, and the values in the 31 aparasitemic individuals were significantly higher than the MRP 8/14 concentrations in the nonexposed adults (geometric mean, 1,000 ng/ml). Overall, the Spearman correlation coefficient between parasite density and MRP 8/14 level in these samples was 0.52 (P ), and that between temperature and MRP 8/14 was (P 0.10). MRP 8/14 measurement in samples from children. Parasites were found in all samples of the cross-sectional cohort study (Table 2; Fig. 3). Fifteen samples from the 58 individuals with low parasitemia ( 0.5%) had a geometric mean of 4,230 ng of MRP 8/14 per ml, and six fever cases (40%) above 37.7 C were measured in this group. Forty-three samples were related to 31 fever cases with a parasitemia of 0.5% and higher, and the measured MRP 8/14 levels ranged between 9,850 and 33,480 ng/ml (geometric mean, 18,560 ng/ml). The MRP 8/14 mean of the group with high parasitemia ( 0.5%) was more than four times higher than that of the group with low ( 0.5%) parasitemia (Fig. 4), and fever was detected in 72.1% of the children whereas a 40% incidence of fever was found in subjects with low parasitemia. All measured MRP 8/14 values in exposed children were higher (ranging between 950 and 1,990 ng/ml) than 1,370 ng/ml, the mean for five samples from nonexposed children. Overall, the Spearman correlation coefficient between parasite density and MRP 8/14 level in all these samples was (P ), and that between temperature and MRP 8/14 was (P ) (Table 2). DISCUSSION The objective assessment of a malarial episode in individuals living in areas of high endemicity at admission to a hospital

3 VOL. 4, 1997 MRP 8/14 AS MARKER FOR MALARIA 437 Downloaded from FIG. 2. (a) Samples with an MRP 8/14 value reaching 10,000 ng/ml were not found in the low-parasitemia ( 0.2%) or aparasitemic group despite the 11 fever cases with an axillary temperature above 37.7 C. (b) In contrast, in the group with high parasitemia ( 0.2%) 11 samples with MRP 8/14 values above 10,000 ng/ml of serum corresponded to 11 fever cases above 37.7 C. Parasitemia TABLE 2. MRP 8/14 levels in 58 children participants (%) fever cases a MRP 8/14 (ng/ml) Negative % 15 (25.9) 6 (40) 4,230 (1,200 8,340) 0.5% 43 (74.1) 31 (72.1) 18,560 (9,850 33,480) Nonexposed controls 5 0 1,370 (950 1,990) a 37.7 C. remains unsatisfactory. Severity of an episode can be assessed by evaluating and combining a panel of criteria including fever and parasite load (14). Parasite load and fever in a semiimmune population remain unsatisfactory markers for severity. Several other laboratory markers, like acute-phase proteins or various cytokines and cytokine receptors have also been studied (2, 4, 7, 8). Since high parasitemia may occur in these children without causing morbidity, the parasite load must be considered an insufficient morbidity marker. It is now well established that malarial fever is triggered by endogenous pyrogens like interleukins 1 and 6 and tumor necrosis factor (TNF) alpha (1, 11). These factors are mainly genetically restricted and correlate only partially with malaria severity (9, 12). The low concentrations of serum cytokines often coupled to a short half-life make measurements difficult or inadequate in a clinical setting. The study reported here was focused on the level in serum of a protein complex which is related to inflammatory events but whose essential functions remain unknown. Serum MRP 8/14 levels fluctuate during a malarial episode and are positively related to the parasite load as well as to the fever episode (15). The relationship between fever and MRP level was more pronounced in adults than children, who are more likely to suffer non-malaria-related temperature peaks (5, 15, 20). On the other hand, parasite tolerance without symptoms differs between age groups and implies different parasitemia thresholds. Our cross-sectional study on children, based on admission to the health post for presumptive malaria, precluded any access to nonparasitized samples and unfortunately excluded a comparison to nonmalarial diseases. In addition, ethical reasons did not allow monitoring of MRP levels in children during and after therapy. Hence, several questions remain open. The kinetics and fluctuation of MRP 8/14 during a malarial episode as well as the influence of on May 7, 2018 by guest

4 438 BORDMANN ET AL. CLIN. DIAGN. LAB. IMMUNOL. FIG. 3. All MRP 8/14 values measured for the samples of the group with low parasitemia ( 0.5%) were below 10,000 ng/ml. consumption of antimalarials and/or of analgesic drugs on MRP production remain unexplored. It would be interesting to know whether the MRP 8/14 levels, which were always higher in aparasitemic adult subjects from an endemic area than in corresponding nonexposed individuals, correlate with liver dysfunction during the sporozoite infestation. A chronic inflammation of splenic tissues which is often seen in individuals from malaria-endemic regions could also be considered as another Downloaded from on May 7, 2018 by guest FIG. 4. In our study the malaria prevalence in children was 100%; only 25.9% of the children had a parasitemia below 0.5%. All measured MRP 8/14 levels were below 10,000 ng/ml despite the fact that six fever cases were detected in this group (a), whereas 72.1% of the group with a high parasite load ( 0.5%) had a body temperature of 37.7 C or higher and only one sample was measured below 10,000 ng of MRP 8/14 per ml (b).

5 VOL. 4, 1997 MRP 8/14 AS MARKER FOR MALARIA 439 hypothesis to explain these differences. Measurements of MRP 8/14 have physiological relevance as they are a direct measure of inflammatory events. Despite strong correlation between measured MRP 8/14 levels and parasitemia or fever, this molecule is not specifically related to malarial etiology and we cannot exclude interference induced by concomitant infections. On the other hand, very little is known about whether MRP receptors would be able to modulate the level of circulating MRP. In addition, whether MRP 8/14 production and severity of inflammatory events correlate with parasite and/or host genotype is still unknown. Evaluation of biological criteria for diagnosing clinical malaria among semi-immune subjects from endemic areas remains difficult (15), and a malaria case definition is perhaps of limited utility for diagnostic purposes or in health facility environmental surveys, but a sensitive case definition can be achieved by excluding patients with clear alternative diagnoses. With the enormous prevalence in children from the Ifakara region (16), the definition of a malaria case in children becomes ambiguous. However, in conclusion, our data support the idea that, limited to field studies focused on the perceived burden of illness, sequential measurements of MRP 8/14, together with other nonspecific malaria markers, like the two soluble TNF receptors stnf-r1 and stnf-r2 (10, 13), could provide new tools as malaria episode markers. ACKNOWLEDGMENTS We thank T. Smith for statistical analysis and D. Charlwood and G. Pluschke for critical reading of the manuscript. 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ACUTE PHASE REACTANT PROTEINS IN NIGERIAN ADULTS WITH MALARIA INFECTION. Nguepi JP 1, Amaefula, ET 2

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