Status of selected biochemical and coagulation profiles and platelet count in malaria and malaria-Schistosoma mansoni co-infection among patients attending at Dembiya selected Health Institutions, Northwest Ethiopia

Malaria and schistosomiasis are infectious diseases that cause coagulation disorders, biochemical abnormalities, and thrombocytopenia. Malaria and Schistosoma mansoni co-infection cause exacerbations of health consequences and co-morbidities.This study aimed to compare the effect of malaria and Schistosoma mansoni co-infection and malaria infection on selected biochemical and coagulation profiles, and platelet count. An institutional-based comparative cross-sectional study was conducted from March 30 to August 10, 2022. A total of 70 individuals were enrolled in the study using a convenient sampling technique. Wet mount and Kato Katz techniques were conducted to detect Schistosoma mansoni in a stool sample. Blood films were prepared for the detection of plasmodium. The data was coded and entered into EpiData version 3.1 before being analyzed with SPSS version 25. An independent t test was used during data analysis. A P-value of less than 0.05 was considered statistically significant. The mean [SD] of alanine aminotransferase, aspartate aminotransferase, creatinine, total bilirubin, and direct bilirubin in the co-infected was higher than in malaria infected participants. However, the mean of total protein and glucose in co-infected was lower than in the malaria infected participants. The mean of prothrombin time, international normalization ratio, and activated partial thromboplastin time in co-infected was significantly higher, while the platelet count was lower compared to malaria infected participants. Biochemical and coagulation profiles, and platelet count status in co-infection were changed compared to malaria infected participants. Therefore, biochemical and coagulation profiles and platelet count tests should be used to monitor and manage co-infection related complications and to reduce co-infection associated morbidity and mortality.


Questionnaire survey
Socio-demographic characteristics of study participant were collected using a semi-structured questionnaire prepared in Amharic language.The questionnaire was initially written in English language and translated to Amharic language.Socio-demographic data was collected by principal investigator (PI), trained Medical Laboratory Personnel and Nurses.Trained clinicians who work at Dembiya selected health institutions Outpatient department (OPD) assessed the clinical information and patient history.Following identifying individuals who were eligible for the study, then the volunteer study participants were linked to Medical Laboratory Personnel for blood and stool samples collection.

Sample collection and laboratory examination
Microscopic detection of plasmodium On a microscopic glass slide, 6 micro liter and 2 micro liter of capillary blood were placed separately for preparing thick and thin blood films, respectively.So blood films, both thick and thin, were prepared and air dried.Absolute methanol was used to fix thin blood films, and both films were stained for 10 min with a 10% Geimsa working solution.
An experienced malaria microscopist and PI read both thin and thick blood films with a 100 × objective lens and examine100 microscopic field's to rule out the absence or presence of the malaria parasites.The discrepancy results were confirmed by another experienced malaria microscopist.Malaria parasitemia was determined in thick blood films along with 200 white blood cells.It was calculated by using the formula:Parasite/μL = Parasite counted/200WBC × Total white blood cells count.Parasitaemia was classified into three categories according to the number of parasites/μl of blood, low (< 1000), moderate (1000-9999) and high (≥ 10,000) 33 .

Microscopic detection of schistosome
Single stool specimen of about one gram was collected from each study participant.The sample was collected in a clean, dry, and leak-proof container with a unique identification number.Each stool specimen was first examined using the direct wet mount technique, and then Kato-Katz slides prepared on a template containing 41.7 mg of stool.Eggs counted for S. mansoni were recorded and later converted into eggs per gram (EPG) of stool, multiplying by a factor of 24 34 .Finally, infection intensity (light (1-99 EPG), moderate (100-399 EPG), and heavy (≥ 400 EPG)) was classified according to WHO criteria 35 .

Blood sample collection for biochemical and coagulation profiles and platelet count examination
Seven milli litre of venous blood was collected by blood collectors and PI using a sterile disposable plastic syringe after cleaning the venous puncture site with 70% alcohol.The collected blood sample was transferred into three test tubes.The first 2.7 ml of the collected blood sample was placed into 3.2 % sodium citrate anticoagulant test tube.For PT and APTT analysis, platelet-poor plasma was prepared by centrifuging at 1500 revolution per minute for 15 min 34 .Then plasma was separated and stored in Eppendorf tube at − 20 °C until processed.The coagulation profiles (PT, APTT, and INR) were done at Felege Hiwot Compressive Specialized Hospital Laboratory by using Semi-Auto Coagulation Analyzer (HumaClot Duo Plus Human, Germany) 36 .
The 2 ml of blood was transferred into ethylene diamine tetra-acetic acid (EDTA) test tube for platelet count.Platelet count was determined using Fully Auto Hematology Analyzer 37 .
The remaining venous blood was transferred into nonanticoagulated tube and allowed to clot at bench top.Then, blood centrifuged at 2500 revolutions per minute for four minute and serum was separated and stored in Eppendorf tube at − 20 °C until processed.Then, serum was analyzed by using Fully Auto Chemistry Analyzer 38 for serum level of ALT, AST, creatinine, glucose, total bilirubin, direct bilirubin, and total protein 38 .
Serological tests.Immune-chromatographic assay was used to determine hepatitis B virus and hepatitis C virus to exclude individuals who were positive for these diseases 34 .
Urine collection and HCG examination.Urine was collected from all women whose age is 15-49 years in the study using a clean urine cup and a urine human chorionic gonadotropin (HCG) test was performed for both the cases and the controls using a rapid chromatographic immunoassay test strip to exclude pregnant women 39 .

Data quality control
Data collectors took appropriate training in order to maintain data quality.Quality control was performed by re-reading all slides by an expert laboratory technologist, to ensure the accuracy of the detection of Plasmodium and Schistosoma which was conducted by laboratory technologists.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.

Data management and analysis
Data was coded and entered into the EpiData (v3.1) statistical software, and exported to the statistical package of social science (SPSS) version 25 for analysis.The homogeneity of variance was checked by using Levene's statistics.The Skewness, kurtosis, and Shapiro-Wilk normality tests were used for checking the distribution of continuous variables, and it revealed that some data were normally distributed and some data were not normally distributed for each group.Independent t test Difference test was used for comparison of normally distributed biochemical and coagulation profiles, and platelet count between groups.For each group, the result was provided as the mean and SD for normally distributed data.A p-value of less than 0.05 was considered statistically significant in all statistical analyses.

Ethics approval and consent to participate
This study was conducted after ethical approval was obtained from research and ethics committee of School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar.Moreover, letter of support was submitted to the Dembiya Primary Hospital, Chuahit Health Center, and Abrija Health Center.Before starting the actual data collection, permission was obtained from the Hospital and Health Centers Chief Executive Officer or health facilities and the Administrator.Additionally, after explaining the purpose, benefits, and the possible risks of the study, written informed consent from the age of 16 and above and/or assent from those less than 16 years old study participants along with written informed consent from their respective parents/ caregiver/guardians was obtained.And also, written informed consent from illiterate study participants was obtained from their respective parents/guardians.All laboratory results were kept confidential.Since those were stored in a file using codes without study participants name.Apparently, those positive for parasites and with biochemical and coagulation profiles, and platelet count abnormality were linked to the hospital and health centers for appropriate treatment and management.

Result
Socio-demographic characteristics of study participants

Intensity of malaria parasitaemia
The overall mean of malaria parasitemia on S. mansoni and malaria coinfected group was 10,891.9.The mean of parasitemia of malaria in males and females were 8784.2 and 14,931.8 in S. mansoni and malaria co-infected group, respectively.From a total of 35 S. mansoni and malaria coinfected group 4 (11.4%),18 (51.4%),and 13 (37.1%)were due to low, moderate, and high malaria parasitemia infection, respectively.The overall mean of malaria parasitemia on malaria infected group was 8539.4.The mean of parasitemia of malaria in males and females were 7866.7 and 9251.8 malaria infected group, respectively.And also, from a total of 35 malaria infected group 3 (8.6%),24 (68.6%), and 8 (22.9%) were due to low, moderate, and high malaria parasitemia infection, respectively (Table 2).

Coagulation profiles and platelet count among study participants
The percentages of prolonged PT, INR, APTT, and low platelet count were higher in malaria and S. mansoni co-infected participants than in malaria infected participants.
The coagulation profiles like PT, INR, APTT, and platelet count were normally distributed; a parametric test (Independent t test) was used to compare the mean difference of these coagulation profiles between cases and controls.As a result, an independent t test revealed higher mean values for PT, INR, and APTT but a lower mean value of platelet count in malaria and S. mansoni co-infected participants than in malaria infected participants.The mean of PT, INR, and APTT in the age 5-14 year co-infected individuals were higher than other age groups.Also, the mean of platelet count was low in the age 5-14 year group compared to other age groups (Tables 11  and 12).

Discussion
Malaria and schistosomiasis co-infections are common, especially in Africa 40 .The co-infection of malaria and schistosomiasis has a significant impact, including exacerbated health consequences and co-morbidities.
In this study, the mean of malaria parasitemia was higher in malaria and S. mansoni co-infection compared to malaria infection.This finding was similar to a study conducted in Western Ethiopia in 2016 by Mebrate Dufera, that found the mean of malaria parasitemia was higher in malaria and S. mansoni co-infection compared to malaria mono-infection 41 .However, this finding was in deviation from a study conducted in Northwest Ethiopia which found higher mean malaria parasitemia in malaria monoinfection than malaria and S. mansoni co-infection 42 .The variation could be explained by variation in the intensity of schistosomiasis co-infection, exposure levels, and duration, and immunity status of the study participants, which would affect malaria parasitemia.
Also, in this study the percentages of elevated ALT, AST, total bilirubin , direct bilirubin, and creatinine in the age 5-14 year malaria and S. mansoni co-infected individuals were higher than other age groups (15-24 years, 25-34 years, 35-44 years, and > 44 years).In addition, the percentages of lowered total protein and glucose were high in the age 5-14 year group compared to other age groups (15-24 years, 25-34 years, 35-44 years, Further study need to be conducted to elucidate the possible alteration of biochemical and coagulation profiles and platelet count consequences of malaria and S. mansoni co-infection in different epidemiological settings which includes children under the age of 5 years and pregnant women.Finally, we would like to recommend that patients be screened for malaria and S. mansoni co-infection-associated biochemical and coagulation profiles, and platelet count abnormalities to prevent biochemical and coagulation disorders. https://doi.org/10.1038/s41598-024-56529-wwww.nature.com/scientificreports/ https://doi.org/10.1038/s41598-024-56529-w

Figure 2 .
Figure 2. Prevalence of abnormal coagulation profiles and platelet count of study participants at Dembiya selected health institutions, 2022.

Table 1 .
). Socio-demographic characteristics of the study participants at Dembiya selected health institutions, 2022.

Table 2 .
Intensity of malaria parasitaemia among study participants at Dembiya selected health institutions, 2022.Prevalence of abnormal biochemical profiles of study participants at Dembiya selected health institutions, 2022.

Table 3 .
Prevalence of abnormal biochemical profiles of malaria and S. mansoni co-infected study participants at Dembiya selected health institutions, 2022.N number.

Table 4 .
Prevalence of abnormal biochemical profiles of malaria infected study participants at Dembiya selected health institutions, 2022.N = number.

Table 5 .
Comparison of biochemical profiles among study participants at Dembiya selected health institutions, 2022.

Table 6 .
Age, gender, and infection status based comparison of biochemical profiles among malaria and S. mansoni co-infected study participants at Dembiya selected health institutions, 2022.

Table 7 .
Age, gender, and infection status based comparison of biochemical profiles among malaria infected study participants at Dembiya selected health institutions, 2022.

Table 8 .
Prevalence of abnormal coagulation profiles and platelet count of malaria and S. mansoni co-infected study participants at Dembiya selected health institutions, 2022.N number.

Table 9 .
Prevalence of abnormal coagulation profiles and platelet count of malaria infected study participants at Dembiya selected health institutions, 2022.N number.

Table 10 .
Comparison of coagulation profiles and platelet count among study participants at Dembiya selected health institutions, 2022.
ProfilesMalaria mono-infected participants malaria and S. mansoni co-infected participants p value

Table 11 .
Age, gender, and infection status based comparison of coagulation profiles and platelet count among malaria and S. mansoni co-infected study participants at Dembiya selected health institutions, 2022.

Table 12 .
Age, gender, and infection status based comparison of coagulation profiles and platelet count among malaria infected study participants at Dembiya selected health institutions, 2022.