Original Scientific Article ACUTE PHASE REACTANT PROTEINS IN NIGERIAN ADULTS WITH MALARIA INFECTION Nguepi JP 1, Amaefula, ET 2 1 Departments of Medical Laboratory Science and 2 Orthopaedics and Traumatology, Niger Delta University Wilberforce Island, Bayelsa State Nigeria. ABSTRACT An increased level of Acute Phase Reactant Proteins (APRP) including C- reactive protein (CRP) and antistreptolysin O (ASO) are known markers of acute phase reaction. Our main focus was to measure the levels of these Acute Phase Reactant Proteins ( APRP) in malaria patients before, during, and after treatment with a view to interpreting the level as either markers or predictors of disease severity and /or prognosis. CRP and ASO were measured in hundred (100) patients (pre and post treatment) together with apparently healthy individuals.thin and thick blood smears were made for each sample and Giemsa-stained, the film and the tubes for quantitation blood count were examined under the microscope for malaria parasite. Our results showed that the level of CRP was statistically significantly increased (P< 0.05) in both pre and post treated states compared with the controls. This trend was also observed with ASO titre( P<0.05 ), but reduced in the post treatment samples.there were no significant differences in the value of pre and post treatment levels in both CRP and ASO titre respectively.we therefore concluded that the increase in levels of CRP is associated with malaria infection. Key Words: Malaria, reactant, acute phaseantistreptrolysin. Address for communication Amaefula, ET; Department of Orthopaedics and Traumatology, Niger Delta University Wilberforce Island, Bayelsa State Nigeria E-mail: amaetemples@yahoo.com Niger Delta Medical Journal Vol. 1 Issue 2 July December 2015 Page 128
INTRODUCTION Despite extensive studies on Plasmodiumfalciparium, malaria remains the most important public health problem in the tropics 1 ; especially in sub saharan Africa where about 90 percent of the 500 million clinical cases result in the death of almost 3 million people each year 2. In Nigeria,it has been estimated that malaria is the commonest cause of out patient hospital attendance in children and adults and the commomest cause of work absenteeism 3 Approximately about 40 percent of the world population is at risk ofcontacting malaria 4. Malaria infections are complicated by many inflammatory responses which may enhance cell to cell interaction and host-derived factors such as cytokines. Parasites products either directly damage host tissue, or more importantly, stimulate the overproduction of host cytokines 5. Acute phase reaction is a fast unspecific and highly complex reaction of the animal organism to injury and infection. Patients infected with malaria parasite are marked with elevated C-reactive protein 6. This has been attributed to inflammation that leads to tissue damage. Most acute phase reactant proteins are synthesized by hepatocytes in which transcription is controlled by cytokines. CRP is an acute phase protein that serves as an early marker of inflammation or infection.nalk et al 7 reported that there is an increase in level of CRP in the sera of malaria patients. Moderate amounts of cytokines such as tumour necrosis factor- alpha TNF-α.γ-interferon and interleukin-1 are necessary for the human host to fight invading micro-organism but the overinduction of cytokines can be very harmful to the host.tumour necrosis factor can cause fever and disturbance of the immune response 8. Evidence suggests that the protein (Acute Phase Protein) is secreted by the liver primarily in hepatocytes following inflammation. C- reactive protein acts as one of the main diagnostic markers for inflammation 8.Studies have shown that CRP protein could be used for the assessment of general well being of humans and animals 9. In this study, we investigated CRP and ASO levelsin both pre and post treatment of acute malaria infection. MATERIALS AND MEHODS Niger Delta Medical Journal Vol. 1 Issue 2 July December 2015 Page 129
Patients A total number of 100 subjects who attended the Premier Specialist Medical Centre in Victoria Island Lagos Nigeriawere recruited for this study. The diagnosis of malaria was made using Quantitative Blood Count (QBC) and Giemsa stained blood film in fifty (50) malaria infected patients, and fifty (50) apparently healthy individuals at pre and post treatments. CRPand ASO were also measured in these individuals. Methods: Venous blood (4.5ml) was drawn from a vein at theanticubital fossa of each of the 50 patientsand 50 controls subjects into EDTA and plain container before and after treatment. A 65µl of blood from EDTA samples was filled by capillary action into QBC tubes for malaria parasite.thick and thin blood film were made for each sample using Giemsa-stain according to standard technique. Thin and thick smears were prepared by placing 3 drops of blood respectively onto clean slides which were then Giemsa-stained 10. Prepared slides were read using a x 100 oil immersion lens by experienced microscopist, and any discordance in results was resolved by a second microscopist.the clotted blood from the plain sample tubes was centrifuged at 200 rpm for 10 minutes and clear serum was obtained.the immuno-turbidimetric test was used for detection of CRP and ASO in the serum by goat antihuman antibodies usingbiosystem latex for both qualitative and quantitative analysis. Statistics: Data were expressed as mean, standard error of the mean (SEM) and P values less than 0.05 were considered statistically significant. Student s t-test was used for evaluation of the result, while Quantitative SPSS version 17 computer software was used to obtain the descriptive statistics. Results: Table 1 showed the percentage of antistreptolysin (ASO) and C-reactive protein (CRP) in malaria patients. Table 2 showed the mean (± the SEM)ofCRP and ASO in the malaria patients and the controls. Table 3 showedthe p-values of CRP and ASO in the malaria patients and the controls. The level of CRP was significantly increased (P<0.05) in both pre- and post-treatment states respectively compared with the controls. Thistrend was also observed with ASO titres (P<0.05) when compared with the controls.however, comparisons between pre- and post-treatment exhibited no significant differences in both parameters. DISCUSSION Niger Delta Medical Journal Vol. 1 Issue 2 July December 2015 Page 130
Pannen et al 11 suggested that measurement of acute phase proteins can be of diagnostic or prognostic importance under certain clinical conditions, while Hurt et al 12 proposed CRP as a tool in malaria epidemiology studies to measure fevers. The increase in serum CRP concentration in patients with manifestations of severe malaria than in the uncomplication type is similar to that of other African authors 12. The authors documented an association of severe malaria with high circulating levels of inflammatory cytokines such as tumour necrosis factor- alpha, interleukin-1 and interleukin-6. These cytokines responsible for acute illness also causes stimulation, production and sustained levels of CRP. The higher level of mean serum CRP concentration between the patients and controls in the various groups reinforces the effect of malaria antigen in the induction of CRP production 13. In this study we measured the levels of the acute phase proteins, andour results showed a statistically significant increase (P<0.05 respectively) in the level of CRP and ASO in both pre and post treatment states of malaria infection compared with controls. However, there was no significant difference in the mean value of post-treatment level of ASO, compared with the controls (P<0.05). Ourstudy agrees with similar work of Gillespie et al., 14 whoobserved that CRP tend to increase inplasmodium infection and even higher in severe cases, but this decreases with treatment. Our findings differ from previous reports in that the sample size of subjects before and after treatment was larger than has ever been reported in this part of the world.crp is a more reliable index of acute response to most disease conditions, hence this knowledge may be invaluable in monitoring degree of infection and/or responses to treatment since the concentration of CRP seems to diminish with effective treatment. ASO is also a marker of streptococcus infection and has been linked with many acute phase conditions too. Our results has proven that malaria may as well induce ASO production. Most ASO positive serology may not indicate streptococcal infection except with a very high titre. We conclude therefore that positive serological reactions to CRP and ASO titre could be associated with malaria infection and will be beneficial as diagnostic and prognostic tools in treatment of malaria. REFERENCES 1 AdegunJA.,Adegboyega, JA. andawosusi AO. Knowledge and the preventive strategies Niger Delta Medical Journal Vol. 1 Issue 2 July December 2015 Page 131
of Malaria among Migrant Farmers in Ado- Ekiti Local Government Area of Ekiti State, Nigeria Am. J. Sci. Ind. Res., 2011, 2(6): 883-889 2 World Health Organisation Africa malaria report, world health organization bulletin, 20021-4 3 Salako LA, Malaria and its control in Nigeria.Nigeria board of trustees, Nigeria National Merit award Lagos 1993.1-61. 4 Cox F. 2002, History of human Parasitological Clinmicro boil rev 15, 41: 545-612. 5 Jakobsen PH, Bate CA, Taverne J, Playfair JH (1995). "Malaria: toxins, cytokines and disease." Parasite Immunol. 17(5): 22 3-231 6 Hurt N, Smith T, Tanner M, Mwankusye S, Bordmann G, Weiss NA, Teuscher T. Evaluation of C-reactive protein and haptoglobin as malaria episode markers in an area of high transmission in Africa. Trans R Soc Trop Med Hyg. 1994;88:182 6. 7 Nalk,P., Vollen A (1994). Serum C reactive protein levels and falciparum malaria.trans. Roy Trop Med 78: 812-813 8 Wright JP, Young GO, Tigler-Wybrandi N. Predictors of acute relapse of Crohn disease. A laboratory and clinical study. Dig Dis Sci. 1987;32:164 170.3 9. Carolyn Cray, Julia Zaias, Norman H Altman,Acute Phase Response in Animals: A Review Comp Med. 2009 Dec; 59(6): 517 526 10 World Health Organisation (2010) Basic Malaria Microscopy.Part 1. Learner,s Guide Geneva 11 Pannen BHJ., Robotham, JL ( 1995). The acute phase response.new Horiz. 12: 183-197 12 Hurt N., Smith,T., Tanner M., Mwakusye,S., Bordmann G., Weiss NA. Evolution of C- reactive protein and haptoglobin as malaria episodes markers in area of high transmission in Africa Trans Soc Trop Med 88: 182-186 13 HaghighiL.( 1969) C-reactive protein in malaria. J ClinPathol 22: (4) 430-432 14 Gillespie SH, Dow C, Raynes JG, Behrens RH, Chiodini PL, Mcadam, KPW J.(1991) Measurement of acute phase proteins for assessing severity of Plasmodium falciparum malaria.j ClinPathol 44: 228-238 Niger Delta Medical Journal Vol. 1 Issue 2 July December 2015 Page 132
TABLE 1: Mean Percentage Valuesof ASO and CRP in Pre- and Post-treatment Malaria Patients CRP ASO Control(n = 50) 2 ±0.0 2.24 ± 0.13 Pre-treatment(n = 50) 3.14 ±0.22 3.07± 0.19 Post- treatment(n = 50) 3.15 ± 0.47 2.64 ±0.36 CRP= C - Reactive Protein; ASO= Antistreptolysin O TABLE 2: The mean (± S.E.M ) of CRP and ASOof Pre- and Post-treatments Malaria Patients CRP ASO n = 50 n = 50 TOTAL POSITIVE 21.0 28.0 PRE-TREATMENT 42.0 56.0 TOTAL POSITIVE 22.0 25.0 POST TREATMENT 44.0 50.0 Table 3: The P-values of CRP and ASO in Malaria Patients and Controls Niger Delta Medical Journal Vol. 1 Issue 2 July December 2015 Page 133
Parameters Compared P- Values CRPaVs CRP P > 0.05 CRPaVs CCRP P< 0.05 CRPbVs CCRP P< 0.05 ASOaVs ASO P > 0.05 ASOaVs CASO P < 0.05 ASObVs CASO P > 0.05 KEY: CRPa : C - Reactive Protein before treatment CRPb : C - Reactive Protein after treatment ASOa:Anti-Streptolysin O ASOb:Anti-Streptolysin O CCRP :ControlC - Reactive Protein CASO : Control Anti-Streptolysin O a b : Before : After Niger Delta Medical Journal Vol. 1 Issue 2 July December 2015 Page 134
3 Titre (mg/l) 2 1 0 Controls (CRP) CRP (a) CRP (b) C-reactive protein (CRP) before and after treatment in Malaria infection a- Before treatment b- After treatment Niger Delta Medical Journal Vol. 1 Issue 2 July December 2015 Page 135
3 Titre (Iu/ml) 2 1 0 Controls (ASO) ASO (a) ASO (b) Antistreptolysin O (ASO) before and after treatment in Malaria infection a- Before treatment b- After treatment Niger Delta Medical Journal Vol. 1 Issue 2 July December 2015 Page 136