Acta Tropica, 52(1992)121 128

121

© 1992 Elsevier Science Publishers B.V. All rights reserved 0001-706X/92/$05.00 ACTROP 00242

Serum protein concentrations in Plasmodium f alciparum malaria W. Graninger a, F. Thalhammer a, U. Hollenstein a, G.M. Zotter b, P.G. K r e m s n e r b'c aDepartment of lnfi, ctious Diseases and Chemotherapy, University of Vienna, Vienna, Austria, bLandesinstitut fiir Tropenmedizin Berlin, Berlin, Germany and Clnstitutfiir Spezifische Prophylaxe und Tropenmedizin, University of Vienna, Vienna, Austria (Received 27 May 1991; revised version received 20 July 1992; accepted 4 August 1992)

In patients with uncomplicated Plasmodium Jalciparum infection cytokine-mediated serum protein levels of C-reactive protein (CRP), coeruloplasmin (COE), betaz-microglobulin (B2M), alphal-acid glycoprotein (AAG), alphal-antitrypsin (AAT), haptoglobin (HPT), prealbumin (PRE), retinol binding protein (RBP), albumin (ALB) and transferrin (TRF) were measured in an endemic area of the Amazonian rain forest. Semi-immune (SI) and nonimmune (NI) patients were investigated. In both patient groups the serum concentrations of CRP, COE and B2M were elevated on admission. In addition AAG and AAT concentrations were increased in NI patients compared to control subjects. Significantly lower serum concentrations of HPT, PRE, RBP, ALB and TRF were seen in both patient groups during the acute phase of the disease, and were more pronounced in NI patients. After a 28-day follow-up, AAT and B2M were normal in SI patients but HPT, AAT and B2M were still significantly altered in NI patients. Key words: Serum protein; Tumor necrosis factor; Interleukin-6; Plasmodiumfalciparum malaria; Malaria

Introduction Plasmodiumfalciparum malaria presents with fever, chills, hepatosplenomegaly, and sometimes complications such as cerebral, renal or pulmonal involvement (Phillips and Solomon, 1990). Many clinical manifestations have been attributed to mediators such as tumour necrosis factor (TNF), (Kwiatkowski et al., 1990; Beutler and Cerami, 1988b) and interleukin-6 (IL-6), (Grau et al., 1990; Kern et al., 1989), which were found to be elevated in blood of patients with P. falciparum malaria. Fever, induction of prostaglandin Ee-synthesis and lipoprotein lipase, activation of endothelium, neutrophils, lymphocytes and macrophages are modulated by TNF (Sullivan et al., 1988; Salyer et al., 1990). Destruction of invading organisms effected by these mechanisms is mediated. High serum concentrations of TNF at the time of admission are associated with high mortality in malaria (Scuderi et al., 1986; Grau et al., 1989). Cytokines also induce the synthesis of serum proteins (Perlmutter et al., 1986) such as C-reactive protein (CRP), alphal-antitrypsin (AAT), alphal-acid glycoprotein Correspondence to." W. Graninger, Department of Infectious Diseases and Chemotherapy, University of Vienna, Wfihringer Gfirtel 18-20, A-1090 Vienna, Austria.

122 (AAG), haptoglobin (HPT) and fibrinogen. In 17 nonimmune patients with acute P. falciparum malaria the serum concentrations of CRP and serum amyloid A were related to the severity of disease (Gillespie et al., 1991). While the determination of cytokine concentration in serum is rather sophisticated, the serum concentration of "acute phase proteins" can be determined easily. In a prospective trial we examined serum protein concentrations during acute P. falciparum malaria before and after treatment in semi-immune (SI) and nonimmune (NI) patients.

Materials and Methods

Thirty-seven patients from an outpatient clinic in Rio Branco, Acre, Brazil with untreated P. falciparum malaria proven by blood slide took part in the study. No clinical complications (e.g., cerebral malaria) or other infections were diagnosed. Twenty-eight patients were male and 9 patients were female; their ages ranged from 16 to 50 years (median 26 years). The detailed description of the history of the patients, their treatment of P. falciparum malaria, immune status and immune response is given in previous papers (Kremsner et al., 1989c, 1990a, 1990b). Patients were classified as semi-immune (SI, n = 15) if they had had three or more malaria attacks in the preceding two years. If they had suffered only two or less previous bouts of malaria during their life they were considered as nonimmune (NI, n = 22). Fifteen healthy, age- and sex-matched subjects from Rio Branco served as controls (CO). All participants in the study received oral clindamycin, 5 mg/kg body weight twice daily for five consecutive days (Kremsner et al., 1989c). Informed oral consent was obtained. Serum samples were drawn on admission (day 0), on day 5 and on day 28 after initiation of chemotherapy and stored at - 8 0 ° C until analysis. Parasite counts in thick blood smears were determined as described previously (Kremsner et al., 1989c). Serum concentrations of prealbumin (PRE), CRP, transferrin (TRF), AAG, HPT, coeruloplasmin (COE), AAT, albumin (ALB), and alpha2-macroglobulin (A2M) were measured by laser-nephelometry (Reimer et al., 1978; Fink et al., 1989), (Behring, Marburg, Germany). The detection limit for H P T was 20 rag/100 ml. RBP and HPX were determined by radial immundiffusion using commercially available agar plates (Partigen, Behring). Beta2-microglobulin (B2M) was measured by radioimmunoassay (RIA; flzMG - - Pharmacia Diagnostica, Uppsala, Sweden).

Statistical analysis Results were expressed as median serum protein concentration with corresponding 95% confidence intervals. Differences between groups were calculated using the Kruskal-Wallis test and the Mann-Whitney U-test. Values of day 0, day 5, and day 28 were analyzed by the Wilcoxon rank sum test for dependent samples.

Results

In all patients chemotherapy consisting of 5 mg of clindamycin/kg twice a day for 5 days eliminated the plasmodia within the first week. No symptoms were seen on

123 day 28 of the follow-up period. The parasitaemia rate of SI and NI patients is given in Table 1. The median serum concentrations of the proteins with corresponding 95% confidence intervals are shown in Table 2. Normal serum concentrations were found for HPX and A2M. Serum proteins which were lowered significantly at admission compared to the control group, were HPT, PRE, RBP, ALB in SI patients. In addition to these lower levels of T R F were found in NI patients. Serum concentrations of CRP, COE and B2M were found to be elevated in SI patients on day 0. In the NI group A A G and AAT were also elevated. Normalization of altered serum protein concentrations was seen within 28 days except for HPT, B2M and AAT. In SI, median H P T levels were 34 mg/100 ml on day 28, while this protein was undetectable in serum of all NI patients after the same time interval. In addition NI patients still had significantly altered values for B2M and AAT on day 28 compared to SI patients and controls. Of all the parameters studied, those which showed most pronounced alteration between days 0 and 5 in CO, SI and NI were PRE, AAT and AAG.

Discussion

Changes in the serum protein pattern have been described in a few reports of acute P. falciparum malaria (Gillespie et al., 1991; Migasena et al., 1978; Schelp et al., 1977, 1983; Naik and Voller, 1984; Mousa et al., 1973; Murphy et al., 1972). Hepatic protein synthesis is regulated by various cytokines. These cytokines exert a wide array of biological activities on a variety of cell types (Pfizenmacher et al., 1987; Beutler et al., 1988a). Monocytes and macrophages produce inflammatory cytokines such as TNF, IL-1 and IL-6 in response to microorganisms and microbial products. The role of T N F in the expression of the clinical features in septicaemia and malaria has been elucidated recently (Clark et al., 1987; Ferrante et al., 1990; Kremsner et al., 1989b). Among other effects, these cytokines switch on the hepatic synthesis of the socalled "acute phase proteins". Three groups of "acute phase proteins" (Alper, 1974) are distinguished according to the time sequence of their appearance in the serum: the earliest ones, CRP and serum amyloid A protein (group 1) may increase in amount more than 1000-fold within 6-10 hours of infection. The serum concentration of AAT, AAG, H P T and fibrinogen, proteins of the second group, increase 2-5 fold within 24 hours. The serum concentrations of COE, the complement factors C3 and C4 (group 3), double within 72 hours. AAT, AAG, CRP and COE have been found TABLE ! Rate of parasitaemia in semi-immuneand nonimmune patients The median number of asexual parasites/i,tl blood is noted (95% confidenceintervals). Parasitaemia

Semi-immune

Nonimmune

Day 0 Day 5 Day 28

1890 (390-4572) 0 (0-4) 0 (0-0)

3113 (1380-13284) 6 (0-45) 0 (0-0)

124 TABLE 2 Plasma protein levels on days 0, 5 and 28 (median and 95% confidence intervals) CO

SI

NI

PRE

(0) (5) (28)

32 31 30

(24-36) (16-38) (21 36)

21 a 23 27

(9-27) (16-26) (17-38)

17"'b 17~ 28

(14 19) (12-22) (25-35)

CRP

(0) (5) (28)

0 0 0

(0.0 0.5) (0.0 l.l) (0-0)

3.9" 0.8 0

(1.5-8.7) (0.5-1.2) (0 O)

4.7" 1.4 0

(2.8 5.7) (0.4-2.4) (0-0)

B2M

(0) (5) (28)

1.5 1.3 1.5

(1.2-1.8) (1.2-2.3) (1.2-1.6)

3.2a 2.1 a 1.8

(2.9 6.0) (1.6-2.8) (1.3 2.3)

5.0" 2.7 ~ 2.0 a

(2.9-6.7) (2.3-3.0) (1.7-2.6)

TRF

(0) (5) (28)

256 259 202

(196 272) (192-306) (156-273)

190 209" 232

(178-250) (185-231) (171-366)

185" 197a 215

(169-220) (172 233) (194-264)

AAT

(0) (5) (28)

209 205 184

(185 212) (187-243) (142-205)

252 265 209

(193-300) (244-299) (178-276)

297" 307" 234a

(279-327) (285 356) (215 255)

HPT

(0) (5) (28)

109 I07 115

(58-230) (75-246) (61-164)