1. Introduction

Malaria remains a major public health concern in several tropical countries in Sub-Saharan African countries [1,2]. In the last twenty years, Angola has witnessed significant growth in malaria interventions, accompanied by cross-border initiatives and regional endeavours across Southern Africa [3]. According to the National Director of Public Health, in Angola, malaria continues to be a serious public health problem and is the leading cause of illness and death. In 2022, around 9,211,346 cases of malaria were reported, which constituted an increase of 0.4% compared to 2021, on the other hand, there was a 10% reduction in the number of deaths from malaria, compared to the previous year [1]. Angola has been implementing robust strategies aligned with the National Malaria Control Program with the ambitious objective of eliminating malaria by 2030, however complex factors threaten the sustainability of this progress, including environmental diversity, transmission intensities, and mosquito vectors, as well as the internal displacement of the population and the emergence of resistance to drug and insecticide therapies are some of the challenges faced in Angola Added to this is the emergence of new epidemiological frameworks that end up making it difficult to define priorities thus, continuous support is needed in the form of traditional malaria control methods, as well as support to implement new techniques to accelerate the decreasing trends in malaria burden [3].

Plasmodium infection is normally multiorganic and affects many organs [4]. The liver is the main organ affected during Plasmodium infection since part of the parasitic cycle takes place in this organ, and this organ plays a large role in the normal functioning of organisms among them the uptake, metabolism, phagocytosis and elimination of microorganisms and endotoxins [5-7]. Also is responsible for generating effective immunoreactivity against microorganisms and generating tolerance to avoid immunoreactivity with “proper” and harmless substances such as dietary compounds [8,9]. In malaria infection, the liver plays a protective role against blood stages of plasmodium, since it is more critical than the spleen in mediating the suppressive effects of testosterone on malaria resistance, which results in differences between men and women in terms of severity of malaria [10].

Previous studies concluded that patients with P. falciparum, P. vivax, P. malariae and P. ovale infections had elevated serum alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (Gamma GT), bilirubin, and hypoalbuminemia [11-13]. Despite Angola's malaria-endemic status, there has been no published research exploring the dynamics of liver function in the adult population during Plasmodium falciparum infection. In this study, we investigated the dynamics of liver function among patients with malaria in Luanda, the capital city of Angola.

2. Methods

2.1 Study design and setting

A cross-sectional study was performed with 199 patients hospitalized due to malaria in Josina Machel, a tertiary Hospital, from March to August 2023. The patients were invited and freely consented to participate in the study. The study was approved by the scientific council of the Institute of Health Sciences of Agostinho Neto University (118/GD/ICISA/UAN/2021) and by the clinical management of Josina Machel Hospital (36/DPC/HJM/2023).

2.2 Sample collection and laboratory testing

Malaria diagnoses were performed by Josina Machel Hospital professionals using rapid malaria antigen tests (SD-Bioline Malaria AG Pf/PAN) and confirmed with the microscopy technique of direct visualization of the parasite by Giemsa-stained peripheral blood thickening. Patients who presented parasitemia less than 50p/mm³ were classified as low parasitemia level, between 50 - 1000 p/mm³ as moderate level and patients who presented parasitemia above 1000 p/mm³ were classified as high parasitemia. An estimated volume of 3 mL of whole blood was collected in a tube containing EDTA. Biochemical screening was performed with the automatic biochemical analyzer Cobas C111 (Roche), using the AST (Roche, Germany), ALT (Roche, Germany), Gama GT (Roche, Germany), and Bilirubin (Roche, Germany) detection kits, for evaluation of possible liver damage resulting from malaria disease. All laboratory processing as well as the interpretation of the results were carried out according to the manufacturer's instructions. ABO/RH blood group phenotypes were characterized (Lorne Laboratories Limited, UK), following the manufacturer's instructions.

2.3 Statistical analysis

The data obtained in this study were analyzed using SPSS v29 (IBM SPSS Statistics, USA). Absolute and relative frequencies were presented as descriptive analysis. The chi-square (X²) test was used to assess the relationship between categorical variables. In addition, independent samples t-test and One-Way ANOVA were calculated for samples with normal data distribution. All reported p-values are two-tailed and deemed significant when p<0.05.

3. Results

3.1 Demographic profile and liver function

The demographic profile and liver function among patients with malaria are presented in Table 1. The predominant demographic characteristics of the 199 participants enrolled in the study were age group between 20–30 years old (45.2%, 90/199), female gender (61.8%, 123/199), patients from urbanized areas (53.8%, 107/199), average education level (43.2%, 86/199) and employed (75.4%, 150/199). Overall, mean liver function indices were as follows, ALT (47.0 ± 120), AST (83.1 ± 115), Gamma GT (72.6 ± 124), and Bilirubin (233 ± 3230). Although no statistical significance was observed, higher mean levels of ALT (62.3 ± 170) and AST (94.2 ± 113) were observed in patients aged over 40 years, while higher Gamma GT and Bilirubin levels were observed in patients aged between 20 and 30 years. Likewise, all mean liver function values, such as ALT (47.0 ± 120), AST (85.8 ± 95.7), Gama GT (75.6 ± 133), and Bilirubin (440 ± 4444), were elevated in residents from urbanized regions. The distribution of ALT means according to education level was statistically significant (p=0.038). Unliterate patients had higher AST (87.9 ± 92.3) and lower bilirubin (0.32 ± 0.33) mean values, while patients with high educational levels had higher values for AST (132 ± 318) or gamma GT (89.2 ± 52.5) and lower values for AST (64.3 ± 49.3). Patients who reported some type of work categorized as employees, were those with the highest average values for ALT (52.0 ± 136), AST (85.7 ± 124), and Bilirubin (309 ± 3720), while the unemployed presented higher average values for gamma GT Range (104 ± 187).

Note: Bold number means that results were statistically significant for independent samples t-test and/or One-Way ANOVA (p<0.05).

Table 1: Demographic profile and liver function among patients with malaria in Luanda, Angola.

Table 2: Biological factors related to liver function profile among patients with malaria in Luanda, Angola.

3.2 Biological factors related to liver function

The predominant clinical data in the studied population were as follows, about 54% (108/199) of the patients had moderate disease, and 39% used Artesunato (39.7%, 79/199) or Coarten (38.7%, 77/199), respectively. Blood group O (46%, 91/199) and positive Rh (96%, 190/199) factors were predominant. About 71% (141/199) of the studied population had low parasitemia. Biological factors related to liver function profile among patients with malaria are presented in Table 2. A decrease in mean ALT (68.7 to 50.7) values was observed from non-severe to severe patients while we observed increases in AST (56.6 to 134), gamma GT (51.2 to 99.0), and Bilirubin (0.72 to 1029) from non-severe to severe patients. The increase in mean AST values from non-severe to severe patients was statistically significant (p=0.002). Regarding the effects of antimalarials on liver function, we observed higher mean ALT values in patients who used Artimeter (85.2 ± 240), while higher mean values of AST (111 ± 145) and Gamma GT (96.3 ± 165) were observed in patients treated with Artesunate. Higher mean Bilirubin (603 ± 5209) values have been observed in patients treated with Coarten. Differences in mean AST values depending on the antimalarial used were statistically significant (p=0.022). Despite the low frequency, group AB presented high mean AST and gamma GT values. High ALT values were observed in group B, while high Bilirubin values were observed in group O. The differences in the mean values of gamma GT depending on the blood groups were statistically significant (p=0.006). Although no statistical significance was observed (p>0.05), all elevated values of ALT (48.5 ± 122), AST (85.9 ± 117), gamma GT (74.9 ± 126) and Bilirubin (245 ± 3307) were observed in patients with positive RH factor.

4. Discussion

Malaria continues to be a major health problem in tropical countries [1,2]. Liver dysfunction has long been known as a major clinical feature of patients with severe malaria [6,13,14]. To the best of our knowledge, this is one of the first studies that conduct a laboratory evaluation of changes in liver biochemical indices among patients infected with P. falciparum and treated with artemisinin derivatives in patients with different levels of malaria disease severity in Luanda, the capital city of Angola. Abnormalities in liver function values have been observed very frequently, especially in regions endemic to P. falciparum [5,13]. Also, a previous study concluded that patients with P. vivax, P. malariae and P. ovale infections had slightly elevated serum bilirubin, aminotransferase and alkaline phosphatase levels, and hypoalbuminemia, however, these abnormalities returned to normal within a few weeks after treatment with therapies based on artemisinin derivatives [11]. When we examine indices of liver function of the present studied population, we observed certain variations in the overall mean levels of ALT, AST, Gamma GT and bilirubin that might represent clinical importance for the management of

 patients with malaria in Angola. Specifically, elevated levels of ALT and AST were observed among older patients, while higher levels of gamma GT and bilirubin manifest among individuals aged 20 to 30 years. Furthermore, residents of urbanized regions have elevated levels of all studied markers of liver function, accentuating the potential influence of demographic factors on the liver health of patients with malaria. It is also worth mentioning that the influence of educational level and professional status on liver function parameters reveals interesting associations. Patients with lower levels of education have elevated levels of AST, while those with higher levels of education have elevated levels of AST and Gamma GT. Similarly, employed individuals have elevated levels of ALT, AST and bilirubin, highlighting the potential impact of occupational factors on liver function (Table 1).

Regarding biological factors, clinical data reveal significant relationships between AST and Gamma GT levels with disease severity/use of antimalarial agents and different blood groups (p<0.05). The increase in parasite load was not related to abnormalities in liver function values, which is similar to what was observed in previous studies that revealed that liver function abnormalities were not related to the grade of parasitaemia, fever, duration of the illness, nutritional status of the patient or associated medical problems [15]. Similar to other studies, we also identified a relatively large proportion of cases with elevated transaminases after treatment with Artimeter, Artesunate or Coarten in patients with naturally acquired malaria [5]. High levels of liver function indices were observed in the studied population, even during treatment, which could represent the hepatic effects associated with malaria after hemolysis (Table 2). Blood group O and Rh-positive are dominant and are in line with previous studies carried out in Angola, which show that these are prevalent in the Angolan population [16,17]. The influence of blood groups on liver function disorders highlights the complex interplay between genetic predispositions and liver health, with statistical significance observed in GT gamma levels across ABO blood groups. Furthermore, our findings reveal elevated liver function markers among Rh-positive individuals, although without statistical significance.

This study has potential limitations that should be considered. Studies have documented that abnormal liver function profiles in malaria patients return to normal within a few weeks of treatment [7],  however, this was not observed in this study due to the cross-sectional nature of the study. However, future prospective studies should be carried out in Angola, to evaluate the liver function of individuals two or more weeks after infection with P. falciparum, or another species treated with artemisinin derivatives. Even so, our findings present the patterns of variations in liver function indices in patients with P. falciparum treated with different antimalarials, whether Artimeter, Artesunate or Coarten in Angola. Further investigation may clarify other contributing either demographic or biological factors to abnormalities in liver profiles among patients with malaria in Angola.

5. Conclusion

In summary, this study elucidates the role of demographic and biological determinants on liver function in patients with malaria, highlighting the multifactorial determinants that shape liver health in this vulnerable population. Our findings not only improve our understanding of malaria-associated histopathology but also highlight the need for personalized interventions that address demographic and biological determinants to mitigate liver morbidity in malaria-endemic regions. Further studies may clarify other contributing demographic or biological factors to abnormal liver profiles among patients with malaria in Angola.

Acknowledgements

The authors would like to thank the patients with malaria for their participation and the clinical staff from the Josina Machel Hospital for institutional support.

Conflict of interest statement

The authors declare no conflicts of interest.

Finding

This project was funded by the European Union and the African Union through the 2022 ARISE-PP-13 Project, which the African Academy of Sciences coordinates. Also, this research was funded by the Fundação Calouste Gulbenkian (FCG), under the ENVOLVE Ciência PALOP program that funded the HITOLA project (Number 250466), AREF (AREF-312-CRUZ-F-C0931|AREF-312-SEBA-S-C1029), Science and Technology Development Project (PDCT) within the scope of the MUTHIVAO project (Number 36 MESCTI/PDCT/2022), FCT MARVEL (PTDC/SAU-PUB/4018/2021), FCT GHTM-UID/04413/2020 and LA-REAL-LA/P/0117/2020.

Authors' contributions

Conceptualization: ENMS and CSS. Data curation: ENMS and CSS. Formal analysis: ENMS and CSS. Investigation: CSS, EC, VM, MN, IP, CF, LC, and ENMS. Supervision: ENMS. Validation: ENMS and CSS. Writing—original draft: ENMS and CSS. Writing—review and editing: CSS, EC, and ENMS. All authors have seen and approved the submitted version of this manuscript.

ORCID ID

Cruz S. Sebastião - https://orcid.org/0000-0003-1232-0119

Euclides Nenga Manuel Sacomboio - https://orcid.org/0000-0002-2341-9133

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