Inflammatory cytokines as potential biomarkers for early diagnosis of severe malaria in children in Ghana

Globally, malaria is the most prevalent infectious disease affecting human health [1] and takes an estimated 435,000 lives each year, especially among young African children [2]. The 2022 world malaria report gave an estimation of 247 million cases and 619,000 deaths worldwide in 2021 [3]. Unfortunately, children aged under 5 years, remain the most vulnerable population affected by malaria, and accounts for 67% (274,000) of these deaths.

Generally, malaria is an uncomplicated febrile illness that resolves with time [2]. However, in approximately 1% to 2% of cases, malaria progresses to a severe disease that is lethal. This progression may be influenced by age, exposure and immune status [4], therefore, children under 5 years and the aged population in endemic areas, as well as pregnant women and HIV patients are at risk of severe malaria [5]. Severe malaria (SM) is a complex multi-system disorder [6] caused almost exclusively by Plasmodium falciparum [7]. SM is of paramount importance because its mortality rate remains unacceptably high at 58% from the year 2000 to 2019 [8].

Inflammatory cytokines have gained the interest of researchers in recent times as they have been implicated in the pathogenesis of many diseases despite their complex association with multiple regulatory pathways. Unregulated levels and a change in their expression patterns are said to have the potential as biomarkers for several diseases [9]. Several studies have identified and compared cytokine profiles in different disease and their levels are said to correlate with malaria disease severity and death [10‐13]. Inflammatory cytokines are categorized based on their effect on inflammation as pro-inflammatory, which include IL-1, IFN-γ, tumour necrosis factor (TNF), IL-12 and IL-18, or anti-inflammatory, which include IL-4, IL-10, IL-13 and transforming growth factor-beta (TGF-β) [10]. Pro-inflammatory cytokines promote inflammation and tissue damage [14], whereas anti-inflammatory cytokines prevent the damaging effects of pro-inflammatory cytokines by resolving inflammation and facilitating tissue repair [14, 15].

Progression of uncomplicated malaria to severe disease is therefore warranted by an imbalance in the levels of pro- and anti-inflammatory cytokines, that is, lower levels of anti-inflammatory cytokines compared to pro-inflammatory cytokines [16, 17]. Several studies have identified cytokine profiles during various clinical manifestations of malaria (SM and UM). For instance, increased levels of IL-12, IL-6, IL-10, IL-1β and IFN-γ have been identified during severe malaria whereas low levels of 1L-12p70, IL-10, IFN-α, and IFN-γ have been associated with SM in children [10, 13, 18, 19]. However, the diagnostic capability of these cytokines in differentiating between the disease states (SM and UM) in terms of cut-off values was not established.

This study aims to measure the levels of inflammatory cytokines among the various groups of participants (SM, UM and Febrile Controls), and compare the levels in each group to that of the SM group, establishing the degree of significance of the differences between the levels. Additionally, the diagnostic potential of these cytokines to differentiate between SM and UM cases was determined, with their corresponding cut-off values, sensitivities and specificities.

Inflammatory cytokines have been implicated in the pathogenesis of several diseases despite their complex association with multiple regulatory pathways. Unregulated levels and a change in their expression patterns, is said to have potential as biomarkers of several diseases [9, 21]. Malaria is no different as cytokines have been reported to contribute to its progression from uncomplicated to severe [11]. Studies have identified cytokine profiles able to differentiate the various clinical manifestations of malaria (SM and UM), although their diagnostic potential in terms of cut-off values has not yet been established. In this study we aimed to corroborate detection of these immune markers among malaria groups and also explore their disgnostic potential in discriminating severe malaria from umcomplicated malaria and other febrile conditions.

We observed that the levels of the pro-inflammatory cytokines: IL-1β, IL-6, IL-2, IL-12, IL-17A, Eotaxin/CCL-11, 1FN-γ, RANTES, MCP-1, IL-15, IL-1RA, IL-2R, IP-10, TNF, MIG, MIP-1α, MIP-1β, IL-7, IL-8 and anti-inflammatory cytokines: IL-4 and IL-10 differed significantly among malaria-infected groups and the control groups. This is consistent with findings that inflammatory cytokines are elevated in peripheral blood during malaria infection [22]. There was no statistically significant difference in the levels of the pro-inflammatory cytokines among the SM and UM groups, except for IL-17A which was significantly higher in the SM group compared to the UM group. IL-17A is secreted by immune cells and is responsible for the mediation of granulopoiesis, infiltration of neutrophils and recruitment of T lymphocytes into peripheral tissues during inflammation [23]. High levels of IL-17A have been implicated in severe malaria anemia, multiple organ dysfunction, including acute renal failure during severe malaria [23]. In other infections such as COVID-19, increased levels of IL-17A, among other pro-inflammatory cytokines, have been associated with very poor prognosis including multiple organ dysfunction and death [24]. Mbengue et al. [25] reported that there is a strong induction in pro-inflammatory cytokine production during severe malaria as a result of accumulation of high concentrations of toxic parasite components at sites of P. falciparum infected red blood cells sequestration. In agreement, we observed that even though there was no significant difference in the pro-inflammatory levels between the SM and UM, the levels in the SM group seem to increase as that of the UM group decrease especially with IL-1β. These results are in line with several other observations that pro-inflammatory cytokines promote inflammation and tissue damage, which may imply that their levels correlate with malaria disease severity and death [11, 14, 26].

GM-CSF, IL-5 and IFN-α, though pro-inflammatory cytokines, did not show any significant difference in their levels in the SM group compared to the UM and FC groups. This discrepancy may be explained by the fact that GM-CSF only stimulates the synthesis and release of TNF during malaria but its concentration remains the same either at the time of diagnosis or after treatment of malaria [27].

Anti-inflammatory cytokines are produced to prevent the damaging effects of pro-inflammatory cytokines [15], therefore, an imbalance in the levels of pro- and anti-inflammatory cytokines contribute significantly to the pathogenesis of severe malaria [16]. Progression of uncomplicated malaria to severe is hence warranted by lower levels of anti-inflammatory cytokines compared to pro-inflammatory cytokines. This plausibly explains why there was no significant difference in the levels of IL-10, IL-13 and IL-4 among the SM and UM groups whereas the pro-inflammatory cytokine, IL-17A, was found to be increased in the SM group.

It is of great significance that biomarkers of malaria disease severity be identified to inform critical clinical management decisions including whether a patient has, or is progressing to severe malaria and therefore needs to be admitted, or referred to a tertiary healthcare facility for aggressive case management [28]. The diagnostic abilities of the cytokines to differentiate SM from UM was determined using the ROC curve analysis which quantifies how accurately medical diagnostic tests can discriminate between two patient states: diseased and non-diseased (controls) [29]. The area under the curve (AUC) is an effective measure of sensitivity and specificity that describes the inherent validity of diagnostic tests (in this case cytokines). AUC takes on values from 0 to 1 (0–100%), where a value of 0 indicates the perfect inability of a cytokine to differentiate disease from control group and a value of 1 (100%) indicates a perfectly accurate test, meaning the cytokine is perfect in the differentiation of diseased patients from control group [30]. An AUC of 0.5 (50%) suggests no discriminative ability of the cytokine, 0.7–0.8 (70–80%) is considered moderate, 0.8–0.9 (80–90%) is considered excellent, and more than 0.9 (90%) is considered perfect. Using the SM group as the disease state and the UM group as the control, the results obtained showed that IL-1β and IL-17A (AUC = 72.7% and 82.4% respectively) are good biomarkers for the discrimination of SM from UM cases. The AUC values of the other cytokines fell within the 0.5–0.6 (50–60%) range and therefore, were considered unable to distinguish SM from UM patients.

A major limitation of the study was low sample size, which was mainly due to the COVID-19 pandemic, which restricted the rate of patients’ visits to the hospital. Also, is this study was in partial fulfilment of an undergraduate degree in Medical Laboratory Sciences in the University of Ghana, there were time limitations for data collection.

Increase in the levels of pro-inflammatory cytokines in the SM infected group compared to the UM and febrile control groups corresponds to increase in malaria disease severity. IL-17A is a good candidate biomarker for development of diagnostic tests for SM. IL-1β was also found to have good diagnostic potential and should be considered. This study augments the currently insufficient data on appropriate biomarkers for SM diagnosis.