Effect of Malaria and Schistosoma mansoni Coinfection on Selected Biochemical Profiles among Patients Attending Selected Health Institutions at Dembiya, Northwest Ethiopia

Background Malaria and schistosomiasis are infectious diseases that cause biochemical abnormalities. Malaria and Schistosoma mansoni coinfection causes exacerbations of health consequences and comorbidities. The study area is found in Ethiopia, where coinfection of malaria and S. mansoni is common. However, there is limited data on the biochemical profiles of patients coinfected with malaria and S. mansoni schistosomiasis in the study area. Hence, this study aimed to assess the effect of malaria and S. mansoni schistosomiasis coinfection on selected biochemical profiles. Methods An institutional-based comparative cross-sectional study was conducted from March 30 to August 10, 2022. Using a convenient sampling technique, 70 participants (35 cases and 35 controls) were enrolled in the study. Schistosoma mansoni was detected in stool samples using the wet mount and the Kato Katz method. To detect Plasmodium, both thick and thin blood films were prepared and stained with Giemsa. The blood sample was processed for the analysis of biochemical profiles. All data were analyzed using SPSS version 25. A p value of less than 0.05 was considered statistically significant. Results The mean values of alanine aminotransferase and aspartate aminotransferase (37.1 U/L and 41.9 U/L, respectively) in coinfected participants were significantly higher than in the healthy control participants (17.4 U/L and 22.0 U/L, respectively) (p < 0.05). Also, the median values of creatinine, total bilirubin, and direct bilirubin (1.51 mg/dL, 2.35 mg/dL, and 0.91 mg/dL, respectively) in coinfected participants were significantly higher than in the healthy control participants (0.85 mg/dL, 0.42 mg/dL, and 0.12 mg/dL, respectively) (p < 0.05). However, median values of total protein (4.82 g/dL) and mean values of glucose (66.6 mg/dL) in coinfected participants were significantly lower than in the healthy control participants (total protein (7.64 g/dL) and glucose (91.9 mg/dL)) (p < 0.05). The results of biochemical profiles in healthy participants were significantly different from those with light, moderate, and heavy S. mansoni infection intensity in malaria and S. mansoni coinfection (p < 0.05). Schistosoma mansoni infection intensity had a positive correlation with biochemical profiles except for total protein and glucose, which correlated negatively in coinfected participants (p > 0.05). Conclusion Biochemical profiles in coinfection were significantly changed as compared to healthy individuals. As a result, biochemical profile tests should be utilized to monitor and manage coinfection-related problems, as well as to reduce coinfection-related morbidity and death.


Background
Malaria is an infectious disease caused by protozoan parasites of the genus Plasmodium and transmitted by female Anopheles mosquitoes [1].Plasmodium falciparum and P. vivax are the main cause of malaria infection worldwide [2].According to the World Health Organization (WHO, 2023), malaria caused 249 million cases in 85 malaria endemic countries in 2022 and an increase of 5 million cases compared with 2021 in the world [3].
Malaria parasites go through a hepatocyte developmental stage.Sporozoites produced from the salivary gland of a mosquito must efectively target and penetrate hepatocytes [4].Tese parasites replicate in the red blood cells of their human host following an initial replication phase in the liver.Tese parasite erythrocytic replication cycles result in the typical disease symptoms, including fever, anemia, and eventually lead to organ failure and patient death [5].Sequestration of erythrocytes with mature forms of the parasite in the deep vascular beds of vital organs is the major pathologic hallmark of severe malaria.Plasmodium falciparum malaria frequently causes life-threatening complications such as cerebral malaria, renal failure, hepatic dysfunction, jaundice, abnormal bleeding, and severe anemia [6].
Furthermore, malaria induces biochemical changes within the host [7].Sporozoites invade hepatocytes in the liver stage, causing organ congestion, sinusoidal blockage, and cellular infammation.Hepatocyte changes can result in the leakage of parenchyma and membrane enzymes into the general circulation [8].Due to this, malaria causes biochemical abnormalities such as high bilirubin, elevated aspartate aminotransferase (AST), elevated alanine aminotransferase (ALT), and high creatinine, which increase the risk of disease complications [9].Malaria also afects almost all organ systems, but the most dangerous complication of severe malaria is acute kidney disease [10].Acute renal failure is characterized by pathological diseases in the kidneys that cause anemia, jaundice, hypoglycemia, acidosis, and coma [11].Te existence of hemoglobinopathies, nutritional status, demographic factors, and the degree of malaria immunity can all afect the physicochemical properties of red blood cells that have been infected with malaria parasites [12,13].
Schistosomiasis is an infection caused by a digenetic intravascular parasite that inhabits the venous portal mesenteric system in humans [14].Schistosoma mansoni, S. haematobium, and S. japonicum are the species that infect human beings among the Schistosoma species [15].Schistosomiasis, the second-most common tropical disease in the sub-Saharan region, continues to cause signifcant morbidity and mortality in developing countries [16].Based on World Health Organization (WHO, 2020), it caused 24 000 deaths and 2.5 million disability adjusted life years [17].
Te type of Schistosoma species and the severity of infection both infuence the clinical presentation and pathology of schistosomiasis [18].Te mesenteric plexus is a habitat for S. mansoni and leads to intestinal or hepatosplenic schistosomiasis which afects the intestine, liver, and spleen [16].Schistosoma mansoni infection's pathophysiological efects are mostly related to the development of granulomas brought on by the host cell-mediated immune response to soluble antigens released by parasite eggs trapped in the hepatic and intestinal vessels of infected hosts [14].Tese egg-induced granulomas cause liver failure, which leads to protein synthesis impairment and increment in ALT and AST levels [19].
Furthermore, schistosomiasis causes stunted growth, cognitive impairment, anemia, impaired aerobic capacity, and death [16].Te level and duration of exposure, the intensity of the infection, concurrent infections, nutritional status, parasite strain, and genetic predisposition are some of the factors that can infuence the clinical symptoms and severity of schistosomiasis disease [20].
Malaria and S. mansoni infections cause public health and socioeconomic development challenges [21].And also, morbidity and mortality related to malaria and S. mansoni infection remain a major concern in the world.Coinfection of these parasites is frequent in sub-Saharan Africa (SSA), where over 90% of these diseases occur due to a large geographic overlap [22][23][24].Both diseases have been associated with poverty, and factors that contribute to their spread include a low socioeconomic status, inadequate sanitation, limited access to potable water, a lack of education, and a lack of awareness [25,26].
In coinfection of malaria and S. mansoni, alters the balance between T helper cell 1 (T1) and T2 immune responses and lowering malaria immunological control.Schistosome eggs have immunomodulatory potential by inducing the alternative activation of macrophages and regulatory T-cell expansion.Tis may reduce the T1 response, but it may increase T2 immune responses, increasing the risk of early clinical malaria.And also, acute infection with schistosomes increases the levels of T1 cytokines, which increases the severity of malaria disease [27][28][29][30][31].In addition, this immunologic response alteration may result in a reduction of the efectiveness of malaria treatment in malaria and S. mansoni coinfection [32].However, research on the efect of coinfection with malaria and S. mansoni on biochemical profle is still limited.Studying malaria and S. mansoni coinfection's efect on biochemical profle is important to reducing diferent problems like impaired protein synthesis and liver fbrosis which are related to those coinfections.Also, well-informed alterations in biochemical profle in malaria and S. mansoni coinfection enable the clinician to establish reliable diagnosis and therapeutic interventions.Terefore, the current study assessed the efects of malaria and S. mansoni coinfection on biochemical profle.Te study populations were malaria and S. mansoni coinfected and healthy control participants.Women who were pregnant, people who had multiple intestinal parasite infections, people who were receiving antiretroviral therapy, people who had a history of chronic diseases like hypertension, cardiac disease, diabetes mellitus, and chronic renal disease, people who were positive for hepatitis B and hepatitis C viruses, smokers, and people who used alcohol excessively were excluded from the study.

Sample Size Determination and Sampling Technique.
Te sample size was determined based on rules of thumb that have been recommended by van Voorhis and Morgan: 30 study subjects per group are required to detect real diferences, which leads to about 80% power [33].Tus, 70 study participants (35 infected by both malaria and S. mansoni and 35 healthy participants; sex and age match control) were enrolled in the study.A convenient sampling technique was used to select study participants.All clinically suspected individuals for malaria and/or S. mansoni infection and those who fulflled the inclusion criteria and presented themselves to Dembiya selected health institutions outpatient department (OPD) were enrolled in the case group.Also, participants who were microscopically positive for both malaria and S. mansoni and gave blood and stool samples and whose age is fve years and above were enrolled in the study as study participants.All health-seeking individuals attending the voluntary council and testing (VCT) clinic of Dembiya selected health institutions and who fulflled the inclusion criteria were enrolled in the control group.In addition, participants who were microscopically negative for both malaria and S. mansoni and gave blood and stool samples, and whose age is fve years and above were enrolled in the study as healthy control participants.

Data Collection Procedures
2.3.1.Questionnaire Survey.Sociodemographic characteristics of study participants were collected using a semistructured questionnaire prepared in Amharic language.Te questionnaire was initially written in English language and translated into Amharic language.Sociodemographic data was collected by trained medical laboratory personnel and nurses.Trained clinicians who work at Dembiya selected health institutions OPD and VCT clinics assessed the clinical information and patient history.After identifying individuals who were eligible for the study, the volunteer study participants were linked to medical laboratory personnel for blood and stool sample collection.

Sample Collection and Laboratory Examination
2.4.1.Microscopic Detection of Plasmodium.Six microliters and 2 μl of capillary blood were collected and placed separately on a tiny glass slide for preparing thick and thin blood flms, respectively, by experienced laboratory technicians.Both thick and thin, were prepared and air dried.Absolute methanol was used to fx thin blood flms, and both flms were stained for 10 minutes with a 10% Giemsa working solution.An experienced malaria microscopist read both thin and thick blood flms with a 100x objective lens and examined 100 microscopic felds to rule out the presence or absence of malaria parasites.A trained malaria microscopist confrmed the disparity fndings [34].

Microscopic Detection of Schistosomes.
Each study participant provided a single stool specimen measuring approximately 1 g.Te sample was collected in a clean, dry, and leak-proof container labeled with a unique identifcation number.Each stool specimen was analyzed using the direct wet mount technique, followed by Kato-Katz slides prepared on a template containing 41.7 mg.Eggs counted for S. mansoni were recorded and afterward changed into eggs per Gram (EPG) of stool by multiplying by a factor of 24 [35].Finally, infection intensity was classifed as light (1-99 EPG), moderate (100-399 EPG), and heavy (400 and above EPG) using WHO standards [36].

Blood Sample Collection and Examination.
Tree milliliters (3 ml) of venous blood were collected by experienced blood collectors using a sterile disposable plastic syringe after cleaning the venous puncture site with 70% alcohol.Te collected blood sample was transferred into test tubes.Te collected venous blood was transferred into a nonanticoagulated tube and allowed to clot on the bench top.Blood was centrifuged at 2,500 revolutions per minute for four minutes, and serum was separated and stored in an Eppendorf tube at −20 °C until processed.Ten, serum was analyzed using Fully Auto Chemistry Analyzer (COBAS C 311) for serum levels of ALT, AST, creatinine, glucose, total bilirubin, direct bilirubin, and total protein [37].

Data Quality Control. Data collectors took appropriate
training to maintain data quality.Quality control was performed by re-reading all slides by an expert laboratory technologist to ensure the accuracy of Plasmodium and Schistosoma detection.Standard operating procedures and manufacturer instructions were strictly followed throughout the procedures, and all reagents were stored and prepared according to the manufacturer's instructions.
2.6.Data Management and Analysis.Data were coded and entered into the EpiData (v3.1) statistical software, and exported to Statistical Package for the Social Sciences (SPSS) version 25 for analysis.Te homogeneity of variance was checked using Levene's statistics.Te skewness, kurtosis, and Shapiro-Wilk normality tests were used for checking the distribution of continuous variables, and it revealed that ALT, AST, and glucose were normally distributed and total protein, creatinine, total bilirubin, and direct bilirubin were not normally distributed for each group.One-way analysis of variance (ANOVA), independent t-test, and post hoc Tukey Honest Signifcant Difference (HSD) tests were used for the comparison of normally distributed biochemical profles between groups.Biochemical profle values of ALT, AST, and glucose were normally distributed.Nonparametric tests, Kruskal-Wallis H, and Mann-Whitney U tests were used for comparison of abnormally distributed biochemical profles between groups.Biochemical profle values like creatinine, total bilirubin, direct bilirubin, and total protein were abnormally distributed.Te correlation of variables was assessed using Pearson and Spearman's rank correlation analysis techniques.For each group, the result was provided as the mean and standard deviation (SD) for normally distributed data.Similarly, the result was provided as the median and interquartile range (IQR) for abnormally distributed data for each group.A p value of less than 0.05 was considered statistically signifcant in all statistical analyses.

Intensity of Schistosoma mansoni Infection.
Te mean of the EPGs of stool from malaria and S. mansoni-coinfected participants was 224.9.Te means of the EPG in males and females were 246.3 and 184.0 in malaria and S. mansonicoinfected participants, respectively.From a total of 35 malaria and S. mansoni-coinfected participants 10 (28.5%), 22 (62.9%), and 3 (8.6%)had light, moderate, and heavy S. mansoni infection intensities, respectively (Figure 1).

Biochemical Profles across
One-way ANOVA revealed that the mean (SD) values of ALT, AST, and glucose showed a signifcant diference among healthy controls, light, moderate, and heavy S. mansoni-infected participants (p < 0.05 in each).Likewise, the Kruskal-Wallis H-test also revealed that the median (IQR) of creatinine, total bilirubin, direct bilirubin, and total protein showed signifcant diferences among healthy controls, light, moderate, and heavy S. mansoni-infected participants (p < 0.05 in each).Te mean values of ALTand AST were signifcantly lowered, but glucose was signifcantly higher in healthy control participants compared to those with light, moderate, and heavy S. mansoni infection intensity (p < 0.05).Te median values of creatinine, total bilirubin, and direct bilirubin were signifcantly lowered, but total protein was signifcantly higher in healthy participants compared to those with light, moderate, and heavy S. mansoni infection intensity in malaria and S. mansoni coinfection (p < 0.05).Total bilirubin was also signifcantly higher in patients with heavy S. mansoni infection compared to those with moderate S. mansoni infection (p < 0.05).However, the mean values of ALT, AST, and glucose were not signifcantly diferent between light and moderate, light and heavy, and moderate and heavy S. mansoni infections.Also, creatinine, direct bilirubin, and total protein median values did not difer signifcantly between light and moderate, light and heavy, and moderate and heavy S. mansoni infection (p > 0.05) (Table 6).the number of S. mansoni EPG of stool had been nonsignifcantly and positively correlated with biochemical profles (ALT and AST) (Pearson correlation coefcients r � 0.115 and 0.088, respectively; p > 0.05).However, glucose was nonsignifcant, and negatively correlated with the number of S. mansoni EPG of stool in malaria and S. mansoni coinfected participants (Pearson correlation coefcient r � −0.068; p > 0.05).Similarly, Spearman's rank order correlation analysis showed that the number of S. mansoni EPG of stool had been nonsignifcantly and positively correlated with biochemical profles (creatinine, total bilirubin, and direct bilirubin) (Spearman's rho correlation coefcient r � 0.136, 0.65, and 0.055, respectively; p > 0.05) in malaria and S. mansoni-coinfected participants.But the total protein of malaria and S. mansoni-coinfected participants was nonsignifcantly and negatively correlated with the number of S. mansoni EPG of stool (Spearman's rho correlation coefcient r � −0.096; p > 0.05) (Table 7).

Discussion
Malaria and S. mansoni infections cause public health and socioeconomic development challenges [21].Te present study aimed to investigate the efects of malaria and S. mansoni coinfection on the biochemical profles at Dembiya Selected Health Institutions, Northwest Ethiopia.
Te study found that participants with both malaria and S. mansoni coinfection had signifcantly higher mean values of ALT and AST, as well as median values of creatinine, total bilirubin, and direct bilirubin compared to the healthy control group.Conversely, participants who were coinfected with S. mansoni and malaria had mean glucose levels and median total protein levels that were considerably lower than those of healthy control participants.Tis fnding was similar to studies conducted in Ethiopia that found significantly higher values of ALT, AST, direct bilirubin, and lower values of glucose and total protein in the S. mansoni monoinfected participants as compared to healthy participants [39,40].In addition, this fnding was similar with a study conducted in Western Kenya that found signifcantly higher median value of ALT in the malaria and S. mansonicoinfected participants as compared to healthy participants [23].Tis fnding was also consistent with a study conducted in Yemen, which found signifcantly higher levels of AST, ALT, total bilirubin, and direct bilirubin in malaria patients compared to healthy individuals [9].A study carried out in India provided additional evidence similar to this fnding [41].Tis could be due to the coinfection of malaria and S. mansoni, which can cause more severe and chronically debilitating morbidity than a single parasite infection.Tis may cause a high degree of intravascular hemolysis of parasitized red blood cells, which leads to a high level of bilirubin in malaria and S. mansoni-coinfected participants [42].Also, this elevation of ALT, AST, total bilirubin, and direct bilirubin might be due to dysfunction of the liver in malaria and S. mansoni coinfection.Malaria and S. mansoni infection may cause jaundice and hepatomegaly, which lead to an elevation of liver enzymes and bilirubin [43][44][45][46].Low levels of glucose and total protein might be due to glucose utilization and impaired protein synthesis in malaria and S. mansoni coinfection [47].However, this fnding was in disagreement with studies conducted in Nigeria [48] and India [49], which found signifcantly higher values of ALT, AST, creatinine, total bilirubin, direct bilirubin, and lower levels of total protein in the healthy control group as compared to the malaria mono-infected group.Tis variation might be due to the diference in study participants, the intensity of infection, level and duration of exposure, concurrent infections, nutritional status, parasite strain, genetic predisposition, or the combined malaria and S. mansoni infection efect.
Additionally, in this study, the mean values of ALT and AST and the median values of creatinine, total bilirubin, and direct bilirubin were signifcantly lower in healthy control participants compared to those with light, moderate, and heavy S. mansoni infection intensity in malaria and S. mansoni coinfection.However, median values of total protein and mean values of glucose were signifcantly higher in healthy control participants compared to those with light, moderate, and heavy S. mansoni infection intensity in malaria and S. mansoni coinfection (p < 0.05).Correspondingly, the median value of total bilirubin was significantly elevated in heavy S. mansoni infection intensity compared with those with moderate S. mansoni infection intensity in malaria and S. mansoni coinfection.Tis fnding was comparable with a study conducted in Ethiopia [39].Tis imbalance in biochemical profles might be due to the impairment of organs like the liver and kidney as a result of the progression and increment of S. mansoni intensity.Since organ-specifc morbidity is brought on by the accumulation of S. mansoni eggs.Te severity of hepatomegaly usually correlates with the intensity of infection and can be exacerbated by chronic exposure to Plasmodium species [50][51][52].
Furthermore, in this study, fndings were confrmed by Pearson correlation analysis, where the number of S. mansoni EPG of stool had a positive correlation with biochemical profles (ALT and AST) and a negative correlation with glucose of malaria and S. mansoni-coinfected participants.In addition, these fndings were confrmed by Spearman's rank order correlation analysis, where the number of S. mansoni EPG in stool had a positive correlation with biochemical profles (creatinine, total bilirubin, and direct bilirubin) and a negative correlation with the total protein in malaria and S. mansoni-coinfected participants.Te possible scientifc reason for this correlation was due to schistosomiasis pathology, which often positively correlates with the intensity of the infection, as indicated by excreted egg counts.Tis might be due to the major causes of schistosomiasis complications caused by eggs, which may lodged within the host organs such as the liver, spleen, and kidneys, they leading to abnormal biochemical profles [53].

Journal of Tropical Medicine
Tis study had limitations.In this study, participants who were fve years of age and older were recruited, excluding children who were under fve years of age and pregnant women.Also, we did not enumerate parasite infections by molecular techniques such as polymerase chain reaction; we only determined parasite density by microscopy technique.

Conclusions and Recommendations
In the current study, biochemical profles in malaria and S. mansoni-coinfected participants were signifcantly altered compared to healthy control participants.Since mean values of ALT and AST were higher, but glucose was lower in malaria and S. mansoni-coinfected participants compared to healthy participants.In addition, the median values of total bilirubin, direct bilirubin, and creatinine were higher, but the total protein was lower in malaria and S. mansoni-coinfected participants compared to healthy participants.
Te mean values of ALT, AST, glucose, and the median values of creatinine, total bilirubin, direct bilirubin, and total protein in healthy participants were signifcantly diferent from those with light, moderate, and heavy S. mansoni infection intensity among malaria and S. mansoni-coinfected participants.Schistosoma mansoni infection intensity had a positive correlation with biochemical profles except for total protein and glucose, which correlated negatively in coinfected participants.
Terefore, screening patients for biochemical profle changes has the greatest role in treating malaria and S. mansoni-coinfected patients.Also, assessing biochemical profle changes in patients with malaria and S. mansoni coinfection is an important step toward reducing malaria and S. mansoni coinfection associated morbidity and mortality.Similarly, assessing biochemical profles is important for the diagnosis of infections and to monitor the progression of the disease.In general, further studies need to be conducted to elucidate the possible alteration of biochemical profles as a consequence of malaria and S. mansoni coinfection in diferent epidemiological settings, which include children under the age of 5 years and pregnant women.Finally, we would like to recommend that patients be screened and treated for malaria and S. mansoni coinfectionassociated biochemical profle abnormalities to prevent biochemical disorders.

Figure 1 :
Figure 1: Intensity of S. mansoni infection in malaria and S. mansoni coinfected participants at Dembiya selected health institutions, 2022.

Table 1 :
Sociodemographic characteristics of the study participants at Dembiya selected health institutions, 2022.

Table 2 :
Independent t test for ALT, AST, and glucose among study participants at Dembiya selected health institutions, 2022.

Table 3 :
Comparison of biochemical profles among study participants at Dembiya selected health institutions, 2022.

Table 4 :
Comparison of ALT, AST, and glucose among study participants at Dembiya selected health institutions, 2022.

Table 5 :
Biochemical profles at diferent level of Schistosoma mansoni infection intensity among study participants at Dembiya selected health institutions, 2022.

Table 6 :
Multiple pairwise comparisons of biochemical profles among study participants at Dembiya selected health institutions, 2022.

Table 7 :
Correlation of S. mansoni infection intensity with biochemical profles among malaria and S. mansoni coinfected participants at Dembiya selected health institutions, 2022.