Analysis of pro inflammatory cytokinse response among hepatitis B patients co-infected with Plasmodium falciparum in Khartoum state, Sudan

Qutoof H. Taha¹, Amna B Hamadnaallah², Danya H Taha², Hind H Taha³, Wahaj M. Mohammed⁴, Abdelhakam H Ali⁵, Salah Marajan⁵, Abdulbasit Faraj Hadhr⁶

1. The National University, Khartoum, Sudan, Faculty of Medical Laboratory Science, Department of Parasitology.

2. The National University, Khartoum, Sudan, Faculty of Medical Laboratory Science, Department of Microbiology.

2. Sudan University of Science and Technology, Khartoum, Sudan, Faculty of Pharmacy, Department of Pharmacognosy.

3. University of Kordofan, Al-Ubayyid, Sudan, Faculty of Medicine, Department of Obstetrics and Gynecology.

4. Shendi University, Shendi, Sudan, Faculty of Medical Laboratory Science, Department of Parasitology.

5. University of Al Butana, Gezira, Sudan, Faculty of Medical Laboratory Science, Department of Microbiology.

5. The National University, Khartoum, Sudan, Faculty of Medical Laboratory Science, Department of Microbiology.

6. Oassar International University, Libya, Saraj Ealem Institute of Medical Science, Libya.

*Correspondence: daniataha78@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2026.5308      

INTRODUCTION   

                Malaria remains  global public health threat , transmitted through the bite of an infected Anopheles mosquito. There are  400  species knowns , about 60 are vectors ,  and 30 of these are of major importance. Malaria parasites are eukaryotic single-celled microorganisms that belong to the genus Plasmodium [1].  Over 100 Plasmodium species exist, only four—P. falciparum, P. vivax, P. ovale, and P. malariae—infect humans under natural conditions [2]. These four species differ morphologically, immunologically, in their geographical distribution, relapse patterns, and drug responses. P. falciparum is the agent of severe, potentially fatal malaria and is the principal cause of malaria deaths in young children in Africa [3].According to the World Health Organization, it causes more than 1 million deaths from approximately 300 to 500 million infections annually [2,4]. Although newer techniques exist [3,5], manual microscopy examination of blood smears (invented in the late 19th century) remains the “gold standard” for malaria diagnosis [4,6]. However, diagnosis by microscopy requires special training and considerable expertise [7]. P. falciparum accounts for over 90% of global malaria mortality, making it a continuing public health priority [9]. In 2018, an estimated 228 million malaria cases and 405,000 deaths were recorded, predominantly affecting children under five years of age [10]. Malaria is endemic in over 90 countries and affects nearly 40% of the world’s population. Cases of imported malaria have also been increasingly reported in non-endemic regions such as North America and Europe, often linked to travel [11].In parallel, hepatitis B virus (HBV) infection is another major global health issue. HBV is a DNA virus of the Hepadnaviridae family that infects hepatocytes and circulates through the bloodstream [12]. Over two billion individuals worldwide have been infected with HBV, with 350 million identified as chronic carriers [13–15]. Of these, around 25% are at risk of developing severe liver complications such as cirrhosis and hepatocellular carcinoma (HCC) [16]. HBV transmission occurs through exposure to infected blood and bodily fluids [17]. All HBsAg-positive individuals are infectious, particularly those also expressing HBeAg due to higher viral loads.

A study in Ghana reported significantly elevated IL-10 levels in pregnant women co-infected with chronic hepatitis B (CHB) and malaria compared to those with CHB alone and healthy controls, suggesting a heightened anti-inflammatory response in co-infected individuals [18].

In Nigeria, elevated serum levels of interferon-gamma (IFN-γ) were observed in HBV-infected pregnant women compared to uninfected controls, while IL-10 levels were reduced but not statistically significant. IL-6 levels were significantly higher in infected women (81.0±26.1 pg/mL) [19]. Another Nigerian study among blood donors found a 40.67% co-infection rate of malaria and hepatitis B, with statistically significant gender differences (P<0.05). Younger age groups (18–32 years) showed higher prevalence rates, with males more frequently affected than females [19].

Iranian researchers examined Th1/Th2 cytokines in chronic HBV patients and found significantly decreased IL-4 levels compared to healthy controls. Similar findings were reported by Monsalve-De Castillo et al. [20], who observed reduced IL-4 levels during both acute and convalescent phases of HBV infection. These reductions may result from IL-10-mediated autoregulatory mechanisms. Contrarily, other studies found elevated IL-4 and IL-5 during acute, self-limiting HBV infections [21], and a positive correlation between viral load and circulating IL-4 and IL-6 in chronic HBV patients [23].

Experimental studies in HBV transgenic mice showed that IFN-γ produced by HBV-specific cytotoxic T lymphocytes (CTLs) could inhibit viral replication through nitric oxide (NO)-mediated mechanisms [22,23]. Further studies demonstrated that inflammatory cytokines like IFN-α and IFN-γ can eliminate HBV replication during co-infections with other viruses, such as lymphocytic choriomeningitis virus and cytomegalovirus [24]. These findings support the role of cytokine-dependent, noncytopathic antiviral responses in viral clearance during acute hepatitis [22]. In marine experimental study Valerie Pasquetto et al reported  that IFN-γ produced by hepatitis produced by hepatitis B surface antigen–specific CTLs is sufficient to inhibit HBV replication in the liver of HBV transgenic mice (23), and that this process is mediated by nitric oxide (NO) (24). They  have also demonstrated that HBV replication can be abolished in these mice in response to inflammatory cytokines, especially IFN-α and IFN-γ that are produced in the liver during lymphocytic choriomeningitis virus, murine cytomegalovirus, and adenovirus (25) infections.

MATERIAL AND METHOD

Study area and population

This study was conducted in bashaer and soba in Khartoum state Sudan 2023 from January to April 2023 it  was conducted on patients infected by malaria and patients infected by hepatitis B and patients co-infected by malaria and hepatitis B. Study groups were randomly selected based on simple random sampling technique

 Ethical Approval: All procedures were performed in accordance with the ethical standards of the Ministry of Health Research Ethics Committee and the National University Ethics Committee guidelines Khartoum, Sudan.

Sample collection and examination

Blood samples were collected from patients presenting with acute malaria symptoms. Three (3) mls of venous blood was collected in EDTA blood collection tube. From which 3 drops were placed on whateman filter paper to get dry blood spot. Immediately the blood tubes were centrifuged, plasma was separated and transformed to 1.5 ml Eppendorf for subsequent immuno-assay. Packed cells and plasma were kept at -20°C, while dry blood spots were kept at room temperature till the time of analysisThick and thin blood smears were made together in one slide. The smears were allowed to air dry.then stain with giemsa stain

Examination of blood by immunochromatochroghey test(ICT) for HBsAg: Rapid HBsAg Immunochromatograhy diagnostic test (ICT) was performed according to the manufacturer's instructions (SD South Korea?). In brief, 5µl blood was placed into the sample well. Following to this, 3 drops of buffer were added and the result was read after 15 minutes to detect and identify the malaria spp.

Detection of HB virus surface antigen: HBs Ag, were detected using commercial captured ELISA methods as described by the supplier (Diasource, Belgium) Samples and reagents were brought to room temperature. In each plate one well was used as blank, three wells were used for the negative control, and two well for positive controls supplied in the kit. The rest of the wells were used for the samples. Fifty micro liters (50µl) of positive control, negative control, and samples were added into designated wells. Then( 50µl) of Anti HBs.Peroxidase solution was added into each well except the blank well . Then the plates were covered with plate cover and incubate for 60 min at 37ºc. The plates were washed 5 times with diluted washing buffer (1:20) (PBS +1% tween 20). A 100µl of substrate was added (equal volume of chromogen solution (A&B) and incubated for 15min at 37ºc. The reaction was stopped by addition of 100µl stop solution (2NH2SO4). The absorbance was measured using microplate reader within 15min at 450nm with reference 630nm; Positive samples will be identified according to the cut off value calculated from positive and negative control in the kit.

Cytokines Measurement: Concentration of 6,IL-12) cytokines (IFN--10-, TNF- α, IL-1ß, IL-5,IL were measured in aliquots of cell-free supernatants by a sandwich ELISA using same set of reagents provided by ( Biolegends ELISA MAX Deluxe ). The (Biolegand TM) set of cytokines contains the components necessary to develop ELISA for natural or recombinant cytokines in serum, plasma and cell culture supernatants. The assay of all cytokines measured was similar in the procedure, the difference is confined to the concentration of standards. Standards Preparation:

RESULT

The gender ratio of the participants was 106(65%) males and 45(35%) females [Figure3.1]. The age distribution of the participants was 14% (10-20 year), 35% (21-30years) 33% (31-40 years) 14% (41-50years) and years). GENDER DISTRIBUTION Female 35% 4% (51 and above)

Figure 3.1: Distribution of the participants according to the gender

Most of the participants were resident in Khartoum (41%) followed by omdurman (33%), Bahari(20%) and from other states (6%).

Grouping of the participants:

Blood from sixty six (70) virologically confirmed hepatitis patients were examined microscopically for the presence of malaria parasite. 33(47.1%) out of 70  HBsAg positive patients were found co-infected with P.falciprum parasite. Of the 151 participants 34 (22.6%) patients were microscopically confirmed as having malaria of whom 26 participants individuals were collected and tested for HBV and malari parasites.  All of healthy controls showed no were infected with P.falciparum and 8 with P.vavax. Blood from 46 healthy malaria parasite and insignificant antibody titre for  HBV (Figure 3.2).  

Figure.3.2: Distribution of the participants according to their laboratory investigation and  the clinical status.

After being  classified into 4 different clinical  groups , cytokines ( interleukin-10 (IL10) and  interferon-gamma (IFN-γ)) concentrations were measured using ELISA techniques to obtain their concentration pg/ml in different study group,  hepatitis patients (n=37), P.falciparum infected  patients (n=26) , typhoid and malaria coinfected patients (n= 33) and healthy control ( HBV negative , malaria negative (n=46) (Figure3.3). To measure the IL-10 and (IFN-γ) in HBV patients who were co-infected with  falciparum malaria,  the mean of IL-10 and IFN-γ  cytokines level were compared in all study groups with mean of  these cytokines level of healthy controls after the whole blood of the participants  being stimulated with HBV and malaria antigens.

IL-10 concentrations in plasma of the studied groups:

There were statistically significant different between the means of IL-10 concentration in all groups (P value < 0.0021). 

Difference in IL‑10 levels between HBV patients and controls. 

The mean IL-10 concentration in the  supernatant of whole blood of the  HBV patients- stimulated with HBV antigen- (121.1 pg/ml) was significantly lower than  the healthy controls (41.6 pg/ml) (95% CI 88.04 to 103.3) ,p value =0.0043) Figure 3.4. 

Difference in IL‑10 levels between falciparum malaria and HBV co-infected patients and controls:

Among HBV and P. falciparum co-infected patients, the mean of IL-10 concentration (17.02pg/ml) was significantly lower than the healthy controls (33.6 pg/ml (95% CI 12.04 to 21.3) (p value < 0.0064) Figure 3.4 

Difference in IL‑10 levels between falciparum malaria patients and controls: The mean of IL-10 concentration of P.falciparum infected patients (107 pg/ml) was significantly higher than the mean  IL-10 in healthy controls( 42.6 pg/ml) (95 % CI: 52.06 to  63.04)(P value: 0.0018) Figure 3.3.

Figure 3.3: The mean IL-10 concentration across all participants.

Overall IFN-γ  response:

 Overall there was a statistically significant difference between the means of IFN-γ  levels across  all groups (P value < 0.0046). 

IFN-γ  levels in HBV patients compared with healthy controls:

The mean IFN-γ  concentration in the supernatant of whole blood of the  hepatitis patients-stimulated with HBV antigen (133.3±3.9pg/ml) was significantly higher than  the healthy controls (34.6±2.1 pg/ml (p.value =0.0074) Figure 3.4. 

IFN-γ levels in HBV and P.falciparum co-infected patients and controls:

In the HBV patients with falciparum  co-infection compared with the healthy controls, there was  significant elevation in the levels of IFN-γ with mean concentration (51±2.1) p. value 0.0091) Figure 3.4.

 IFN-γ levels in P. falciparum infected patients compared with healthy controls:

The mean IFN-γ concentration in  P.falciparum- infected patients  was  significantly lower compared  to  the mean IFN-γ concentration in the  controls and they showed  statistically significant difference between(31.2±1.9pg/ml and 66.3±3P.value 0.00814) respectively.  Figure 3.4.

Figure 3.4: The mean IFN-γ concentration across all participants.

DISCUSSION 

Chronic hepatitis b infection is an issue of significant public health relevance. Malaria and HBV share a common intra-hepatic niche, and each may independently cause liver function test abnormalities [27]. Immunologically, both pathogen may also overlap, as each is observed to mainly trigger T helper type1(Th1) cytokine responses 28. It is therefore logical that infected individuals with Malaria+CHB maintained variable levels of cytokines. Participants in this study were classified into three different clinical  groups  all of the participants are chronic hepatitis b patient  some were infected with P.falciparum, other with p.vivax, still others infected by both parasites(mixed), cytokines ( interleukin-10 (IL10) and  interferon-gamma (IFN-γ) concentrations were measured using ELISA techniques to obtain their concentration pg/ml in each study group.  Relative to the control group , the increased serum levels of these cytokines in the Malaria or CHB groups have been previously reported [28]. In particular, elevated levels of IL-10 among the P.falciparum =CHB group is a common finding amongst african [28], and as such, the cytokine has been implicated in the immunopathology of  malaria [29].. The synergistic elevation in levels of the IL-10 in the Falciparum Malaria+CHB group may be attributed to immune factors that limit the infections at the liver-stage. Particularly, natural killer (NK) and natural killer T (NKT) cells which are abundantly available in the liver, interact with pathogens and initiate liver-stage cell-mediated immunity[, 30]. For HBV infections, NK cells contribute to liver inflammation by tumor necrosis factor– related apoptosis-inducing ligand (TRAIL)-mediated death of hepatocytes, which is nonantigen– specific, and can be switched on by a milieu of cytokines [31]. Therefore, it is possible that, in individuals  with falciparum Malaria+CHB, cytokines released in response to P. falciparum could further activate the apoptosis of HBV-infected hepatocytes, and exacerbate liver damage.While peripheral levels of TNF-α, IL-1β and IL-6 were similar in individual with Malaria andCHB, the pro-inflammatory cytokines were significantly increased in those with falciparum Malaria+CHB, which further suggests a possible additive effect of the infections. The correlation analysis with the Falciparum Malaria+CHB group suggests that increased pro-inflammatory cytokine levels as a necessary immune response against malaria helped in reducing HBV intensity. This corroborates with studies indicating that P. falciparum malaria modulates viremia in chronic hepatitis B virus infection [32, 33]. IL-10 and IL-4 are key anti-inflammatory cytokines that regulate the activities of pro-inflammatory cytokines responses. Thus, the diminished peripheral levels of IL-10 in the ndividuals with Falciparum Malaria+CHB may suggest susceptibility to cytokine imbalance (towards Th1)(34).

CONCLUSION

Taken together the findings suggests that Malaria+CHB could aggravate inflammatory cytokine responses and increase susceptibility to liver injury among infected individuals  in endemic settings.

CONSENT TO PARTICIPATE  

All participants involved in this study provided their informed consent prior to participation. The purpose, procedures, risks, and benefits of the study were clearly explained to each participant. Participation was entirely voluntary, and participants were informed of their right to withdraw at any time without consequence. Written informed consent was obtained in accordance with the ethical standards of the institutional.

ETHICAL APPROVAL

 All procedures were performed in accordance with the ethical standards of the Ministry of Health Research Ethics Committee and the National University Ethics Committee guidelines Khartoum , Sudan.

FUNDING DECLARATION

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

 REFERENCES:

1.        Snow RW, Korenkromp EL &Gouws E (2004) Pediat-ric mortality in Africa:Plasmodiumfalciparummalariaas a cause or risk.Am J Trop Med Hyg71(Suppl. 2),16–24.

2.        E. Korenromp, J. Miller, B. Nahlen, T. Wardlaw M. Young, World Malaria Report 2005, Technical Report, World Health Organization, Geneva, 2005.

3.        T. Hanscheid, Current strategies to avoid misdiagnosis of malaria, Clin. Microbiol. Infect. 9 (2003) 497–504.

4.        WHO, Basic Malaria Microscopy. Part I. Learner’s Guide, World Health Organization, 1991.

5.        M.M. Kettelhut, P.L. Chiodini, H. Edwards, A. Moody, External quality assessment schemes raise standards: evidence from the UKNEQAS parasitology subschemes, J. Clin. Pathol. 56 (2003) 927–932.

6.        Snow RW. Global malaria eradication and the importance of Plasmodium falciparum epidemiology in Africa. BMC Med. 2015 Feb 03;13:23. [Abstract]

7.        Garcia LS. Malaria. Clin Lab Med. 2010 Mar;30(1):93-129. [Abstract]

8.        Breman JG. Eradicating malaria. SciProg. 2009;92(Pt 1):1-38. [Abstract]

9.        Shepardcw, simard ep, finelli l, fiore ae, bell bp. Hepatitis b virus infection: epidemiology andvaccination. Epidemiol rev. 2006; 28(1): 112 – 25.

10.    Poland ga. Hepatitis b immunization in health care workers dealing  with vaccine non-response.Am j prev med 1998;15(1):73_7.  

11.    .Helsl j, castrova j, benes c, novotna l, spkowitz ka, dehovitz

12.    Control of occupational hepatitis b among healthcare workers in the Czech 1982 to 1995                                         

13.    Infect control hosp epidemiol 2000; 21(5): 343-6.

14.    zukerman .jn, zukerman aj. Current topics in hepatitis. J infect 2000; 41(2): 130–6

15.    Franco E, Bagnato B, Marino MG, Meleleo C, Serino L, Zaratti L. Hepatitis B : Epidemiology and prevention in developing countries. World J hepatol. 2012;4(3):74–80.DOI10.4254-wjh.v4.i3.74PubMed:22489259

16.    Cdc. Hepatitis b faqs for the public | division of  viral  hepatitis | cdc. 2016[cited2018jan9].Availaple from:https://www.cdc.gov/hepatitis/hepatitis b/bfaq.htm.

17.    Nguyen mh, keeffe eb. Are hepatitis b e antigen (hbeag)-positive chronic  hepatitis b and  hbeag negative  chronic  hepatitis b  distinct diseases? Clin infect dis 2008. Nov;47(10):1312-1314. 10.1086\59257.

18.    Anabire NG, Aryee PA, Abdul-Karim A, Quaye O, Awandare GA, Helegbe GK. (2019). Impact of malaria and hepatitis B co-infection on clinical and cytokine profiles among pregnant women. PLOS ONE, 14(4): e0215550. https://doi.org/10.1371/journal.pone.021555

19.     (Paulyn Tracy Aernan, Terdzungwe Thaddaeus Sar, Simon Hiifan Torkula, Prevalence of Plasmodia and hepatitis B virus co-infection in blood donors at Bishop Murray Medical Centre, Makurdi, Benue State, Nigeria, Asian Pacific Journal of Tropical Medicine, Volume 4, Issue 3, 2011,.)

20.    Monsalve-De Castillo F, Romero TA, Estevez J, Costa LL, Atencio R, Porto L, et al. Concentrations of cytokines, soluble interleukin-2 receptor, and soluble CD30 in sera of patients with hepatitis B virus infection during acute and convalescent phases. Clin Diagn Lab Immunol. 2002;9(6):1372-5. [PubMed ID: 12414777]. https://doi.org/10.1128/CDLI.9.6.1372-1375.2002.

21.    Penna A, Del Prete G, Cavalli A, Bertoletti A, D'Elios MM, Sorrentino R, et al. Predominant T-helper 1 cytokine profile of hepatitis B virus nucleocapsid-specific T cells in acute self-limited hepatitis B. Hepatology. 1997;25(4):1022-7.[PubMed ID: 9096614]. https://doi.org/10.1002/hep.510250438.

22.    Pasquetto V, Guidotti LG, Kakimi K, Tsuji M, Chisari FV. Host-virus interactions during malaria infection in hepatitis B virus transgenic mice. J Exp Med. 2000 Aug 21;192(4):529-36. doi: 10.1084/jem.192.4.529. PMID: 10952722; PMCID: PMC2193244.

23.    Mor G, Cardenas I, Abrahams V, and Guller S. Inflammation and pregnancy: the role of the immune system at the implantation site. Annals of the New York Academy of Sciences. 2011; 1221(1):80–7.

24.    Eldaim N, and Elbadawi E. The effect of malaria on biochemical liver function parameters in Sudanese pregnant women. Journal of Physiobiochemical Metabolism. 2012

25.    Mahtab M-A, Rahman S, Khan M, Mamun A, and Afroz S. Etiology of fulminant hepatic failure: experiencefrom a tertiary hospital in Bangladesh. Hepatobiliary Pancreat Dis Int. 2008; 7(2):161–4. PMID:18397851

26.    Ifudu O, and Fowler A. Hepatitis B virus infection and the response to erythropoietin in end-stage renal disease. ASAIO journal. 2001; 47(5):569–72. PMID: 11575840

27.     Elefsiniotisa IS, Brokalakia H, Argyropoulosa E, Magaziotou I, Derdemezib A, Mihasa C, and Tsoumakasb K. Evaluation of liver enzymes in asymptomatic chronic hepatitis B virus infected pregnant women. Annals of Gastroenterology: Quarterly Publication of the Hellenic Society of Gastroenterology. 2013; 26(1):59.

28.     Nmorsi O, Isaac C, Ohaneme B, and Obiazi H. Pro–inflammatory cytokines profiles in Nigerian pregnant women infected with Plasmodium falciparum malaria. Asian Pacific Journal of Tropical Medicine.2010; 3(9):731–3.

29.     Suguitan AL Jr, Cadigan TJ, Nguyen TA, Zhou A, Leke RJ, Metenou S, Thuita L, Megnekou R, Fogako  J, Leke RG, and Taylor DW. Malaria-associated cytokine changes in the placenta of women with preterm

30.    deliveries in Yaounde, Cameroon. The American journal of tropical medicine and hygiene. 2003 Dec 1; 69(6):574–81. PMID: 14740871

31.    Holz LE, Fernandez-Ruiz D, and Heath WR. Protective immunity to liver-stage malaria. Clinical & translational immunology. 2016 Oct; 5(10):e105

32.    Dunn C, Brunetto M, Reynolds G, Christophides T, Kennedy PT, Lampertico P, Das A, Lopes AR, Borrow P, Williams K, and Humphreys E. Cytokines induced during chronic hepatitis B virus infection promotea pathway for NK cell–mediated liver damage. 2007; 204(3):667–80. https://doi.org/10.1084/jem. 20061287 PMID: 17353365

33.    Pasquetto V, Guidotti LG, Kakimi K, Tsuji M, and Chisari FV. Host–virus interactions during malaria infection in hepatitis B virus transgenic mice. The Journal of experimental medicine. 2000; 192(4):529– 36. PMID: 10952722

34.    Brown AE, Mongkolsirichaikul D, Innis B, Snitbhan R, and Webster HK. Falciparum malaria modulates viremia in chronic hepatitis B virus infection. Journal of Infectious Diseases. 1992; 166(6):1465–6. PMID: 1431272