Analysis of Pro-Inflammatory Cytokines Response Among Typhoid Patients Co-Infection with Plasmodium falciparum In Khartoum State -Sudan

Ibrahim Mohammed Ibrahim Elsayed¹, Abdulrahman A. M. Bashir¹,

Qutoof Hashim Taha¹, Salah Marajan², Danya. H. Taha²*

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

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

*Correspondence: daniataha78@gmail.com;

DOI: https://doi.org/10.71431/IJRPAS.2026.5610

INTRODUCTION

Salmonella enterica is a Gram-negative, facultative intracellular bacterium responsible for millions of infections annually, particularly in low-resource settings. Among its numerous serovars, S. Typhi and S. Paratyphi are the primary agents of enteric (typhoid) fever in humans, while non-typhoidal salmonellae (NTS) such as S. Typhimurium and S. Enteritidis are more commonly associated with bacteremia and gastroenteritis [1,2].

The infection typically occurs via ingestion of contaminated food or water and leads to systemic dissemination through macrophage invasion and immune evasion.

A hallmark of typhoid fever is its complex interaction with the host immune system. Pro-inflammatory cytokines—especially IL-1β, IL-6, IL-8, and TNF-α—play critical roles in mediating immune responses and disease severity. While these cytokines are essential for controlling intracellular pathogens, excessive production can contribute to sepsis and organ damage [3].

Malaria, caused by Plasmodium species, particularly P. falciparum, is another major public health threat in endemic areas. Malaria diagnosis primarily relies on microscopy and rapid diagnostic tests, but molecular tools like PCR offer higher sensitivity in detecting low parasitemia. Similar to typhoid, malaria also induces strong pro-inflammatory responses, with IL-6 and IL-8 implicated in severe disease manifestations such as cerebral malaria and anemia [4] [5] [6] [7] [8] .

Importantly, co-infection with Salmonella and Plasmodium is not uncommon in tropical regions and can complicate diagnosis and treatment. Both pathogens manipulate the host immune response, and co-infection may result in either additive inflammation or immune suppression. However, the precise cytokine dynamics in co-infected individuals remain poorly understood.

Rationale

Typhoid fever triggers an immune response involving pro-inflammatory cytokines such as IL-6 and IL-8. However, in cases of co-infection with Plasmodium falciparum, this response may be altered. While pro-inflammatory cytokines play a clear role in malaria pathogenesis and parasite clearance, their impact in the context of Salmonella infection, particularly in co-infection settings, remains unclear. Understanding how these cytokines behave in co-infected patients is essential to uncover potential immune modulation and its effect on disease progression and severity

MATERIAL AND METHODS

Specimen Preparation

Venous blood specimens were collected from each participant under strict aseptic conditions. Two samples (3 mL each) were obtained: one into an ethylenediaminetetraacetic acid (EDTA) tube and the other into a plain, non-additive tube. All specimens were immediately labeled with a unique laboratory identification number and transported promptly to the laboratory for subsequent analysis.

For the detection of malaria parasites, a drop of whole blood was placed on a clean glass slide using a Pasteur pipette. Both thick and thin blood smears were prepared following standard parasitological protocols. After air drying, the thin smear was fixed with absolute methanol, while the thick smear remained unfixed. Both smears were stained using 10% Giemsa solution (v/v) for 20 minutes, followed by microscopic examination under oil immersion to detect and identify Plasmodium species, with particular attention to Plasmodium falciparum.

For the detection of Salmonella bacteremia, blood samples were cultured on solid media including MacConkey agar and blood agar, and incubated aerobically. Presumptive colonies were further analyzed through a series of biochemical tests for species confirmation. These included:

Oxidase test, Indole production, Carbohydrate fermentation using Triple Sugar Iron (TSI) agar, Citrate utilization test, Urease test.

All microbiological procedures were carried out in accordance with established clinical microbiology standards to ensure accurate identification of Salmonella spp.

The Widal test was conducted using the rapid slide agglutination method to detect Salmonella O and H antigens. Positive reactions were confirmed by tube agglutination, with titers >1:80 considered significant, based on the manufacturer’s guidelines.

IL-6 and IL-8 levels in cell-free supernatants were quantified in duplicate using a sandwich ELISA (ELISA MAX™️ Deluxe Set, BioLegend, USA) according to the manufacturer’s protocol. For IL-6, a top standard of 500 pg/mL was serially diluted two-fold to 7.8 pg/mL. IL-8 standards were prepared by reconstituting the lyophilized stock (85 ng/mL) to a 500 pg/mL working standard, followed by serial dilutions to 15.6 pg/mL. Assay diluent served as the zero standard. Optical density was read at 450 nm, and cytokine concentrations were determined from standard curves.

ELISA Procedure

Cytokine levels were measured using a sandwich ELISA kit (BioLegend, USA) following the manufacturer’s protocol. Plates were coated overnight at 4°C with capture antibody, blocked for 1 hour at room temperature, and incubated with 100 µL of standards or samples for 2 hours. Detection antibody, avidin-enzyme, and TMB/H₂O₂ substrate were added sequentially. The reaction was stopped with 2N H₂SO₄, and optical density was read at 450 nm using a microplate reader (Thermo Lab Systems, Finland). Cytokine concentrations were determined from standard curves based on mean OD values.

Ethical Considerations

Ethical approval was obtained from the National University, and permission was granted by hospital administrations. Informed consent was obtained from all participants.

Study Population

The study included patients diagnosed with typhoid fever, Plasmodium falciparum malaria, co-infected patients, and healthy individuals.

Inclusion criteria

 Patients with confirmed typhoid fever co-infected with P. falciparum, who had not received antimicrobial or antimalarial treatment prior to sampling.

Exclusion criteria

 Patients who had received any treatment (antibiotics or antimalarial) before enrollment.

RESULT

Demographic Characteristics of Study Participants

A total of 140 peripheral blood samples were collected from patients diagnosed with typhoid fever (bacteriologically confirmed), malaria (microscopically confirmed), and those co-infected with both diseases.

Participants were recruited from various hospitals and clinical centers across Khartoum State, Sudan. All samples were re-tested and confirmed at the Department of Medical Laboratory Sciences, National University, Sudan.

The participants included 91 (65%) males and 49 (35%) females [Figure 3.1]. Age distribution was 7% (10–20 years), 18% (21–30 years), 29% (31–40 years), 43% (41–50 years), and 3% (≥51 years). Most resided in Khartoum (45%), followed by Bahri (33%), Omdurman (18%), and other states (4%).

Partiacipant Classification

Blood samples from 66 bacteriologically confirmed typhoid patients were microscopically examined for P. falciparum, revealing 25 (38%) co-infections. Among 140 participants, 35 (25%) had malaria, with 28 infected by P. falciparum and 7 by P. vivax.

Additionally, 39 healthy controls tested negative for both malaria parasites and showed insignificant antibody titers against S. enterica.

Participant Grouping and Cytokine Quantification

Participants were categorized into four clinical groups based on laboratory findings and clinical status: typhoid patients (n=41), Plasmodium falciparum-infected patients (n=28), typhoid-malaria co-infected patients (n=25), and healthy controls negative for both typhoid and malaria (n=39).

Serum concentrations of pro-inflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) were quantified via ELISA to determine their levels (pg/ml) across these groups.

To evaluate the inflammatory response in typhoid patients co-infected with P. falciparum, mean IL-6 and IL-8 levels following whole blood stimulation with Salmonella and malaria antigens were compared among all study groups relative to healthy controls.

 Plasma IL-6 Concentrations Among Study Groups

Significant differences were observed in mean plasma IL-6 concentrations across all groups (p < 0.0001). Typhoid patients stimulated with Salmonella antigen exhibited a markedly elevated mean IL-6 level (133.3 pg/ml) compared to healthy controls (37.6 pg/ml; 95% CI: 88.04–103.3; p = 0.0062) (Figure 3.4). Conversely, typhoid and P. falciparum co-infected patients showed a significantly lower mean IL-6 concentration (17.02 pg/ml) than healthy controls (37.6 pg/ml; 95% CI: 12.04–21.3; p = 0.0064) Additionally, P. falciparum mono-infected patients had significantly higher mean IL-6 levels (107 pg/ml) relative to controls (37.6 pg/ml; 95% CI: 42.04–45.3; p = 0.0078).

 Plasma IL-8 Concentrations Among Study Groups

Plasma IL-8 levels exhibited considerable variability across clinical groups and healthy controls, ranging from below the assay detection limit to increases exceeding 100-fold. Within the typhoid patient group, IL-8 levels showed strong inter-correlation regardless of malaria co-infection, reflecting notable fluctuations during typhoid infection.

Typhoid patients demonstrated a significantly elevated IL-8 response compared to healthy controls, with mean concentrations of 165 ± 7.1 pg/ml versus 17 ± 5.9 pg/ml, respectively (p = 0.0067) (Figure 3.5). In contrast, typhoid patients co-infected with P. falciparum showed a non-significant decrease in IL-8 levels compared to controls (15 ± 7.1 pg/ml vs. 17 ± 5.9 pg/ml; p = 0.861).

Patients infected solely with P. falciparum exhibited significantly higher IL-8 concentrations (184 ± 5.1 pg/ml) relative to healthy controls (17 ± 5.9 pg/ml; p = 0.0076).

Figure 1: The mean IL-6 concentration across all participants

Figure 2: The mean IL-8 concentration across all participants

DISCUSSION

Previous studies provide contrasting findings. Keuter et al. (2015) observed elevated IL-6 and IL-8 in typhoid fever but reduced cytokine production in the acute phase, suggesting immune modulation. Akter et al. (2013) reported higher levels of IL-1, IL-6, and TNF-α in co-infected patients, while Waad Hadi (2021) confirmed significant elevation of IL-6 and IL-8 in typhoid patients compared to controls. Ndoricyimpaye et al. (2022) found that IL-6 and other pro-inflammatory markers were significantly higher in severe malaria, while regulatory cytokines were more expressed in mild cases.

These findings highlight the need to further explore cytokine behavior in typhoid–malaria co-infection, especially in endemic areas like Sudan, to better understand disease mechanisms and guide clinical management.

CONCLUSION

This study found that IL-6 and IL-8 levels were significantly elevated in typhoid and malaria mono-infections compared to healthy controls. However, co-infected patients showed a marked reduction in IL-6 and a non-significant decline in IL-8, suggesting that P. falciparum co-infection may suppress the inflammatory response typically seen in typhoid fever.

Recommendations

Conduct larger studies to confirm these findings and explore cytokine dynamics in co-infections.

Include cytokine profiling in future research and diagnostic approaches for febrile illnesses in endemic areas.

REFERENCE

1.      Ochman H, Groisman EA. The origin and evolution of species differences inEscherichia coliandSalmonella typhimurium. EXS1994;69: 479–493

2.      Fierer J, Guiney DG. Diverse virulence traits underlying different clinical outcomes ofSalmonellainfection.JClinInvest2001;107:775–780.

3.      Mohammed, Omnia Yousif Elemam. "Antimicrobial Sensitivity of Salmonellatyphi and Salmonella paratyphi Isolated at Wad Medani, Gezira State and Stak laboratories, Khartoum State-Sudan." PhD diss., University of Gezira, 2017.

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

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

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

7.      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.

8.      R.E. Coleman, N.J. Maneechai, N. Rachaphaew, C. Kumpitak, R.S. Miller, V. 18. Soyseng, K. Thimasarn, J. Sattabongkot, Comparison of field and expert laboratory microscopy for active surveillance for asymptomatic plasmodium falciparum and plasmodium vivax in Western Thailand, Am. J. Trop. Med. Hyg. 67 (2002) 141–144.