Hematological and Biochemical changes in Schistosoma mansoni infected patients at Haik Primary Hospital, North-East Ethiopia: A comparative cross-sectional study.

BACKGROUND: Schistosomes are blood dwelling parasites that affect more than 260 million people globally, and over 800 million people are at risk of infection in 74 countries. It causes acute and chronic debilitating diseases. The parasite is reported to alter the hematological and biochemical parameters in humans. Therefore, this study was aimed to evaluate the hematological and biochemical changes in S. mansoni infected adult patients compared to apparently healthy controls.
METHODS: A comparative cross-sectional study was conducted at Haik Primary Hospital from February to April 2021. One hundred and eighty study participants consisting of 90 S. mansoni infected patients and 90 apparently healthy controls were recruited using systematic random sampling method. Socio-demographic characteristics and other variables were collected using questionnaires. Stool sample was examined microscopically to detect S. mansoni infection using direct wet mount and Kato Katz technique. In apparently healthy controls, S. mansoni infection was rule out using direct wet mount and Kato Katz technique. Moreover, the intensity of S. mansoni infection was assessed using Kato Katz technique. Blood sample was collected from each study participant to determine the hematological and biochemical profiles. Data were entered in to Epi Data version 3.1 and analyzed using SPSS version 26.0 software. Kolmogorov-Smirnov and Shapiro Wilk normality tests were done to assess the distribution of continuous variables. The Mann-Whitney U test and Kruskal Wallis H test was done to compare the differences among nonnormally distributed variables between S. mansoni infected patients and healthy controls. P-values <0.05 at 95%CI were considered as statistically significant. RESULT: The mean age (SD) of S. mansoni infected patients and apparently healthy controls was 30.33 (±12.26) and 31.2 (±12.85) years old, respectively. The prevalence of anemia, and thrombocytopenia among S. mansoni infected patients were 23.3% and 26.7%, respectively. Erythrocytic sedimentation rate (ESR) was significantly elevated among S. mansoni infected patients than apparently healthy controls. The median white blood cell count, red blood cell count, red blood cell indices, and platelet indices were significantly lower among S. mansoni infected patients compared to apparently healthy controls (P<0.05). On the other hand, the median eosinophil count was significantly elevated among S. mansoni infected patients compared to apparently healthy controls (P<0.05). This study also showed significantly elevated values of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and direct bilirubin and lower albumin, total cholesterol and triglycerides among S. mansoni infected patients compared to apparently healthy controls. Kruskal Wallis H test showed a significant difference in the median of most hematological and biochemical parameters between moderate and heavy intensity of infection with light intensity of infection and apparently healthy controls.
CONCLUSION: The findings of this study showed significantly altered hematological values and liver function tests among S. mansoni infected patients compared to apparently healthy controls. Therefore, screening of S. mansoni infected patients for various hematological and biochemical parameters and providing treatment to the underlying abnormalities is very crucial to avoid schistosomiasis associated morbidity and mortality.

Introduction

Schistosomiasis is one of the most common neglected tropical disease caused by the blood dwelling trematodes of the genus Schistosoma [1]. Three Schistosoma species (Schistosoma mansoni, Schistosoma hematobium, and Schistosoma japonicum) are responsible for the majority of Schistosomiasis cases in the World [2]. It is endemic in more than 74 countries with more than 260 million people infected and 800 million people being at risk of infection globally and of which, more than 90% of the cases occur in Africa [3,4]. Schistosomiasis is the second devastating parasitic disease following malaria [5]. Schistosomiasis is a major public health problem in sub-Saharan African countries with 120 million people infected and more than 150, 000 and 130, 000 died annually due to nonfunctioning kidney (from S. haematobium) and hematemesis (from S. mansoni), respectively [6,7].

Schistosoma mansoni infection is transmitted to humans through skin penetration by the freely swimming cercarial stage of the parasite during contact with contaminated water [8]. Several factors such as human population density, human contact with fresh water, presence or absence of piped water and sanitation facilities in the community, households, schools and health facilities, distance of snail habitat from human habitation, and snail control methods determine the persistent prevalence and intensity of schistosomiasis in a given ecological setting [2].

Clinically, Schistosomiasis results in acute, subacute and chronic clinical manifestations [9]. Schistosoma mansoni leads to gastrointestinal manifestations like nonspecific intermittent abdominal pain, diarrhea, and rectal bleeding, mucosal hyperplasia, pseudo-polyposis, and polyposis interspersed with normal bowel [10]. It also causes chronic hepatic schistosomiasis resulting from the host’s granulomatous cell-mediated immune response to the soluble egg antigen of S. mansoni, which leads to irreversible fibrosis and severe portal hypertension [11]. Most of schistosomiasis morbidity and mortality are due to hepatic and intestinal granulomatous inflammation induced by the tissue trapped worm egg [12]. In patients with chronic and advanced schistosomiasis, elevated level of liver function tests such as aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), direct bilirubin (DBL), total bilirubin (TB) and declined level of white blood cell (WBC), red blood cells (RBC) and platelets were reported [13].

Schistosoma mansoni is an intravascular parasite that alters the hematological profiles and causes different hematological abnormalities such as anemia, thrombocytopenia, low RBC count, decreased level of RBC indices, and low WBC count [14-16]. A study conducted in Western Burkina Faso found significantly lower Hemoglobin (Hgb), hematocrit (HCT), mean cell volume (MCV), mean corpuscular hemoglobin concentration (MCHC) and mean platelet volume (MPV) among S. mansoni infected patients compared to apparently health controls [15].

Studies also reported the impact of S. mansoni infection on different liver function tests. A review on hepatosplenic Schistosomiasis reported that markers of liver injury such as ALT, AST, ALP, and bilirubin, were significantly higher among S. mansoni infected patients than healthy controls [17]. A study conducted in Bahia, Brazil reported that geometric means of liver injury biomarkers in S. mansoni infected women had significantly higher levels of ALT (p < 0.01), AST (p < 0.01), and γ-GT (p < 0.01) compared to those in uninfected controls [18]. A study conducted in Egypt reported a highly significant elevation of serum ALT, AST, and ALP in S. mansoni infected patients [19].

In Ethiopia, schistosomiasis is a major public health problem with more than 5 million people infected and more than 37 million people at risk of infection [20]. A systematic review and meta-analysis reported 18.7% (95%CI: 14.7–23.5) pooled prevalence of S. mansoni infection in Ethiopian general population and 37.13% (95%CI: 30.02–44.24) among children [21,22]. A study conducted in Northwest Ethiopia reported significantly lower mean values of Hgb, RBC, MCHC, total protein (TP), total cholesterol (TC), and total WBC count among S. mansoni infected patients compared to healthy controls. On the other hand, this study found an elevated values of AST and ALT among S. mansoni infected patients [23].

Schistosoma mansoni infection is reported to be endemic in Haik town [24]. However, there was no study conducted in the area to evaluate the impact of S. mansoni infection on hematological and biochemical profiles of patients. Therefore, this study was aimed to assess the impact of S. mansoni infection on hematological and biochemical changes among adult S. mansoni patients attending Haik Primary Hospital, Haik town, North-East Ethiopia.

Methods and materials

Ethical approval and consent to participate

Ethical approval was obtained from the Research and Ethical Review Committee of the Wollo University, College of Medicine and Health Sciences with ethical review number “CMHS190/02/2021”. Written informed consent was obtained from each study participant. All information pertaining to patient biodata that could be potentially used to identify the patient such as the patient names, were converted in to codes. Access to the original patient data was allowed only to the principal investigator and to the hospitals’ medical laboratory technologists. Study participants with confirmed S. mansoni infection, underlying hematological, biochemical and platelet abnormalities were treated according to the hospital treatment guideline.

Study area

This study was conducted at Haik Primary Hospital. Haik Primary Hospital is found in Haik town, South Wollo Zone, Amhara Regional State, North-East Ethiopia, about 430 Km far from Addis Ababa, the capital city of Ethiopia (Fig 1). The town has a geographic coordinate of 13030.59”N latitude and 039028.849”E longitude. Its altitude is about 2200m above sea level and the area covers 447.8 km2 with a total population of 108,993. The major occupations of the inhabitants include trades, civil service, daily labor, and subsistence agriculture in the suburban villages. There are different water bodies in the district like Ankerca River, Logo Lake, Ardibo Lake, and Kette River which are used as major sources of irrigation activities and sanitation in the district. These rivers might be served as a habitat for the snail intermediate host of S. mansoni. Previously conducted studies indicated that S. mansoni is a public health problem and endemic in Haik town. A study done among school children reported the prevalence of S. mansoni infection to be 45%. [25].

Fig 1

Map of the study area.

The above maps were generated by ourselves using the ArcGIS version 10.8 software. The shape files of Ethiopia administrative regions, zones, woredas and towns were freely downloaded from a link https://africaopendata.org/dataset/ethiopia-shapefiles.

Study design and period

Health facility based comparative cross-sectional study was conducted at Haik Primary Hospital North-East Ethiopia, from February to April 2021.

Eligibility criteria

In this study, all S. mansoni infected adults (≥18 years old) visiting Haik Primary Hospital during the study period and who are willing to participate in the study were included. Apparently healthy controls (participants, who are negative for S. mansoni and other parasitic infections that are reported to alter the hematological and biochemical parameters of infected individuals) were recruited from healthy blood donors while donating blood at Dessie blood bank. However, Chronic alcohol drinkers, regular khat chewer, current cigarette smokers, viral hepatitis B and C patients, HIV/ADIS patients, pregnant women, anticoagulant or anti-aggregant drug users and chronic diseases apart from Schistosomiasis were carefully excluded. Patients were also excluded if they are presented with splenectomy, diabetes mellitus, hypertension, use hepatotoxic drugs, thrombocytopenic drugs, or drugs that change platelet function (such as acetylsalicylic acid), and chronic renal disease. Lactating mothers, and Patients with a history of malignancy were also excluded. Patients infected with other parasitic diseases which cause alteration of hematological and biochemical profiles were excluded. Both S. mansoni infected patients and healthy controls were screened for malaria, Hookworm infection, and tirichuriasis and those infected with these parasites were excluded.

Sample size and sampling technique

According to the rule of thumb recommended by van Voorhis and Morgan, 30 participants per group are required to detect real differences which could lead to 80% power [26]). In this study, more study participants have been recruited to increase the reliability of the study. Thus, a total of 180 study participants (90 S. mansoni infected adults and 90 apparently healthy controls) were enrolled in this study. A systematic random sampling technique was employed to recruit the study participants at Haik Primary Hospital based on the inclusion and exclusion criteria. While reviewing the hospital registration book, on average two S. mansoni infected adult are expected to be reported in the Hospital. Within 90 days of data collection period a total of 180 cases are expected to be reported. Then K value was calculated as 180/90 = 2. Finally, with lottery method sampling was begin in the second S. mansoni infected patient and the sampling process continued by systematically recruiting every other patient.

Data collection and Laboratory investigation

Socio-demographic characteristics and clinical data

Socio-demographic characteristics and other variables about the study participants were collected using pre-tested questionnaire by the investigators and trained data collectors. Clinical information and physical diagnosis of S. mansoni infected adults and apparently healthy controls were assessed by physicians and trained nurses at Haik Primary Hospital and Dessie blood bank, respectively.

Stool sample collection and processing

Stool sample was collected from each S. mansoni suspected patient visiting Haik Primary Hospital and healthy control at Dessie blood bank by a leak-proof container. Schistosoma mansoni was diagnosed by direct wet mount using normal saline (0.85% sodium chloride solution) and Kato–Katz technique at Haik Primary Hospital laboratory. Proportion of the stool sample of patients with confirmed S. mansoni infection was preserved with formalin and transported to Wollo university laboratory to determine the intensity of infection using the Kato–Katz technique.

Blood sample collection and processing

Following an aseptic technique, about 6 ml of venous blood was collected using a sterile disposable syringe from each study participant by experienced blood sample collectors at Haik primary hospital and Dessie blood bank. Then, about 3 ml of the collected blood was added into a EDTA anticoagulated test tube and used for complete blood count (CBC), while the remaining 3 ml of blood was dispensed into a serum separator tube for the biochemical test as described elsewhere [27,28].

Determination of hematological and biochemical profiles

Complete blood count was done using DIRUI BF 6500 automated hematology analyzer (Dirui Industrial CO. Ltd., P.R., China) within 2 hours of blood collection. The instrument works based on the principle of semiconductor laser flow cytometry combined with cytochemical staining, electrical impedance, and cyanide free colorimetry to characterize blood cells and hemoglobin (Hgb) according to Hgb cyanide free Hgb measurement method as described elsewhere [27]. The hematological parameters were classified as low, normal and high based on the established hematological reference range [29].

The liver function tests such as aspartate and alanine aminotransferases (AST and ALT), total bilirubin (TB), direct bilirubin (DB), total protein (TP), albumin protein (ALB), and alkaline phosphatase (ALP) were measured by DIRUI CS-T240 fully automated biochemistry analyzer (Dirui Industrial Co., Ltd. India). The results of the biochemical profiles were classified as low, normal, and high based on the locally established reference range for selected clinical chemistry parameters in Northern Ethiopia [28].

Statistical analysis

Data were entered to Epi Data version 3.1 and exported to statistical package for social science (SPSS) version 26.0 software for statistical analysis. Socio-demographic and other variables were presented in the form of frequency and percentage by tables. Kolmogorov-Smirnov and Shapiro Wilk normality tests were conducted to check the distribution of continuous variables. The continuous variables were s not normally distributed and expressed in the form of the median (interquartile range, IQR). Since the continuous variables were not normally distributed and the groups to be compared were four, the non-parametric Kruskal Wallis H test was used to compare the hematological and biochemical parameters between patients with light, moderate and heavy intensity of infection and health controls. On the other hand, the Mann-Whitney U test was used to compare differences among non-normally distributed variables between S. mansoni infected and non-infected study participants. Variables with a P-value of <0.05 at 95%CI were considered as statistically significant.

Quality assurance

The quality of the blood sample was maintained by collecting and processing it according to the standard operating procedures (SOPs) [27,30]. Samples were checked to the acceptable criteria like the absence of hemolysis and clotting, sample volume, collection time, and correct labeling. Safety and specimen handling procedures were strictly followed. The performance of the coagulometer was checked by the daily running of two-level controls (Normal and High). The performance of the hematology analyzer was checked by daily background checking. The quality of the Kato Katz was checked daily and 10% of the slides were randomly selected and reexamined by an experienced laboratory technologist who was blind to the first examination result.

Result

Socio-demographic characteristics of the study participants

In this study, a total of 180 study participants (90 S. mansoni infected adults consisting of 48 males and 42 females with age between 18 and 62 years old and 90 healthy controls consisting 49 males and 41 females with age between 19 and 54 years old) were involved. The mean age (SD) of S. mansoni infected participants and healthy controls was 30.33±12.26 and 31.2 ±12.85 years old, respectively. About 60% of S. mansoni infected study participants are urban residents and 48.9% of them were married. About 30% of the study participants were illiterate (Table 1).

Table 1

Socio-demographic characteristics, of S. mansoni infected study participants and healthy controls at Haik Primary Hospital, North-East Ethiopia, from February to April 2021.

Variable		S. mansoni positive (n = 90)N (%)	Control (n = 90)N (%)
Mean age and standard division		30.33±12.26	31.2 ±12.85
Sex	Male	48 (53.3)	49 (54.4)
	Female	42 (46.7)	41(45.6)
Residence	Urban	54 (60)	50 (55.6)
	Rural	36 (40)	40 (44.4)
Marital status	Single	43 (47.8)	40(44.4)
	Married	44 (48.9)	46(51.1)
	Divorce	0	4(4.4)
	Widowed	3 (3.3)	0
Educational status	Illiterate	27 (30)	28(31.1)
	Primary	24 (26.7)	22(24.4)
	Secondary	24 (26.7)	23(25.6)
	College and above	15 (19.7)	17(16.7)
Occupation	Student	27 (30)	27(30)
	Gov.t employee	9(10)	11(12.2)
	Farmer	12 (13.3)	13(14.4)
	House wife	27(30)	25(27.8)
	Private	15(19.7)	16(17.8)

Hematological profiles of S. mansoni infected patients

Out of 90 S. mansoni infected study participants, 33.3% of them had high WBC count and 26.7% of them had eosinophilia. On the other hand, the majority of the S. mansoni infected patients had normal basophil count (93.3%), monocyte count (83.3%), and lymphocyte counts (76.7%). Almost all of the S. mansoni infected study participants had low MCH (96.1%) and RDW (90%). About 26.7% of the study participants had low MCV. Thrombocytopenia was found in 26.7% of S. mansoni infected study participants (Table 2).

Table 2

Hematological profiles of S. mansoni infected study participants visiting Haik Primary Hospital, North-East Ethiopia, from February to April 2021.

Variable	S. mansoni infected adult (n = 90)
	Normal n (%)	Low n (%)	High n (%)	Reference range
				Male	Female
WBC (103μL)	15(50)	45(16.3)	30(33.3)	3.6–10.6	3.6–10.6
Neutrophile (103μL)	51(56.7)	21(23.3)	18(20)	1.7–7.5	1.7–7.5
Lymphocyte (103μL)	69(76.7)	18(20)	3(3.3)	1.0–3.2	1.0–3.2
Monocyte (103μL)	75(83.3)	12(13.3)	3(3.3)	0.1–1.3	0.1–1.3
Eosinophile (103μL)	66(73.3)	-	24(26.7)	0–0.3	0–0.3
Basophil(103μL)	84(93.3)	-	6(6.7)	0–0.2	0–0.2
RBC (106μL)	19(21.1)	71(78.9)	-	4.2–6.0	3.8–5.2
HCT (%)	60(66.7)	12(13.3)	18(20)	40–54	35–49
MCV (Fl)	66(73.3)	24(26.7)	-	80–100	80–100
MCH (pg)	3(3.3)	87(96.7)	-	26–34	26–34
MCHC (g/Dl)	39(43.3)	51(56.7)	-	32–36	32–36
RDW (%)	9(10)	81(90)	-	11.5–14.5	11.5–14.5
PT (103μL)	60(66.7)	24(26.7)	6(6.7)	150–450	150–450
MPV (Fl)	75(83.3)	15(16.7)	-	7.0–12.0	7.0–12.0
PDW (%)	42(46.7)	9(10)	39(43.3)	9.6–16	9.6–16
ESR (mm/h)	72(80%)	18(20%)	-	0–15	0–20

WBC: White blood cell; RBC: Red blood cell; PT: Platelet; Hgb: Hemoglobin; HCT: Hematocrit; MCV: Mean cell volume; RDW: Red cell distribution width; MCHC: Mean corpuscular hemoglobin concentration; MPV: Mean platelet volume; PDW: Platelet distribution width; ESR: Erythrocytic sedimentation rate.

Prevalence and severity of anemia among S. mansoni infected patients

The overall prevalence of anemia among S. mansoni infected patients was 23.3%. The prevalence was higher among females (28.6%) than among males (18.8%). Regarding severity, about 12.5% and 6.25% of males had mild and moderate anemia, respectively. Moreover, 21.4% and 7.14% of females had mild and moderate anemia (Fig 2). In majority of S. mansoni infected patients, the type of anemia observed was microcytic hypochromic that corresponds to low level of MCV and MCHC.

Fig 2

Severity of anemia among S. mansoni infected patients: The first chart indicate the severity amnemia among males while the last three chart indicate anemia severity in females.

The blue chart indicate the frequency and the red indicate the percent of anemia.

Biochemical profiles of S. mansoni infected adults and healthy controls

The levels of ALT (26.7%), AST (91.1%), DBIL (56.7%), and ALP (90%) were increased in S. mansoni infected patients. However, 15.6% and 6.6% of S. mansoni infected patients had low level of albumin and total protein, respectively. The level of TC and TG in S. mansoni infected patients and healthy controls were within the normal reference intervals. Almost all biochemical profiles in apparently healthy controls were within the normal reference range (Table 3).

Table 3

Biochemical profiles of S. mansoni infected adults and healthy controls at Haik Primary Hospital, North-East Ethiopia, from February to April 2021.

variables		S. mansoni infected adults (N = 90)	Healthy controls (N = 90)	Reference range
		Frequency (%)	Frequency (%)
ALT (IU/L)	Normal	66 (73.3)	85 (94.4)	5–23
	High	24(26.7)	5(5.6)
AST (IU/L)	Normal	8(8.9)	87(96.7)	14.2–34.9
	High	82(91.1)	3(3.3)
TP (g/dL)	Low	6(6.6)	0	6.09–7.85
	Normal	84(93.3)	90(100)
	High	0	0
ALB (g/dL)	Low	14(15.6)	1(1.1)	4.42–5.46
	Normal	76(84.4)	89(98.9)
	High	0	0
TBIL (mg/dL)	Normal	87(96)	90(100)	0.1–0.81
	High	3(3.3)	0
DBIL (mg/dL)	Normal	39(43.3)	88(97.8)	0.03–0.53
	High	51(56.7)	2(2.2)
ALP (IU/L)	Low	0	0	66–456
	Normal	9(10)	88(97.8)
	High	81(90)	2(2.2)
TG (mg/dL)	Normal	90	90(100)	<150 (Fasting)
	High	0	0	≥150 (Fasting)
TC (mg/dL)	Normal	90	90(100)	<150

ALT: Alanine aminotransferase; ALP: Alkaline phosphatase; AST: Aspartate aminotransferase; L: liter; mg: Milligram; dL: deciliter; IU: International Unit; TP: Total protein; ALB: Albumin; DBIL: Direct Bilirubin; TBIL: Total Bilirubin; TC: Total cholesterol; TG: Triglyceride.

Comparison of hematological and biochemical profiles between S. mansoni infected patients and apparently healthy controls

Schistosoma mansoni infected patients had significantly lower hematological values compared to the healthy controls (P <0.05). However, Eosinophile count was significantly higher among S. mansoni infected patients compared to healthy controls with a median [IQR] of 0.37 (0.29) ×103μL and 0.15(0.3) ×103μL, respectively (P<0.001). The Mann-Whitney U test showed significantly lower WBC count, RBC count, Hgb value, MCV, MCH, MCHC, RDW, MPV, PDW and ESR in S. mansoni infected adults compared to healthy controls (p<0.05) (Table 4).

Table 4

Comparison of hematological and biochemical profiles among S. mansoni infected adults and apparently healthy controls at Haik Primary Hospital, North-East Ethiopia from February to April 2021.

Variable	S. mansoni infected adult (n = 90)		Healthy controls (n = 90)		P-value
	Median (IQR)	95%CI	Median (IQR)	95%CI
WBC (103μL)	5.8 (3.9)	3.01–8.24	7.8 (4.8)	7.00–9.38	0.032
Neutrophile (103μL)	3.67 (5.2)	2.98–3.45	3.94(3.2)	3.89–4.02	0.052
Lymphocyte (103μL)	2.1 (1)	1.97–2.3	2.24(1.1)	2.05–2.35	0.046
Monocyte (103μL)	0.44 (0.26)	0.36–0.46	0.51(0.3)	0.48–0.61	0.061
Eosinophile (103μL)	0.37 (0.29)	0.29–0.42	0.15(0.3)	0.098–0.174	0.001
Basophil(103μL)	0.065 (.04)	0.055–0.07	0.064(0.03)	0.061–0.07	0.91
RBC (106μL)	3.8 (1.13)	3.12–4.02	5.8(1.13)	5.31–5.99	0.012
Hgb (g/dL)	14.2 (3.1)	10.64–18.24	15.42(6.4)	12.84–19.21	0.024
HCT (%)	42.6 (6.42)	33.51–45.98	46.31(9.4)	39.19–52.01	0.061
MCV (fL)	81.7 (7.9)	77.95–82.15	86.57(8.4)	80.97–90.00	< 0.001
MCH (pg)	24.6 (2.6)	24.2–25	28.82(3.42)	26.49–29.21	< 0.001
MCHC (g/dL)	30 (0.9)	26.85–30.24	33.4(2.6)	31.74–33.85	< 0.001
RDW (fL)	37.7 (6.4)	36.35–38.7	62.88(12.3)	59.41–64.20	< 0.001
PT (103μL)	244 (152)	230.5–258.5	277.0 (75)	262.0–279.32	0.215
MPV (fL)	6.05 (1.6)	5.55–6.4	15.6 (1.0)	14.63–16.01	< 0.001
PDW (fL)	17.6 (3.5)	17.1–18.4	9.7 (1.2)	9.41–9.89	< 0.001
ESR (mm)	15.3(5.8)	12.86–15.96	5.4(1.1)	4.87–5.76	<0.001
ALT (IU/L)	28(19)	26.00–32.95	20.23(16.21)	19.04–21.00	<0.04
AST (IU/L)	55.5(23)	52.5–60.00	24.41(17.64)	21.51–26.01	≤0.001
TP (g/dL)	7.35(1.1)	7.15–7.55	7.83(1.6)	7.57–9.12	0.215
ALB (g/dL)	3.25(0.7)	3.01–3.42	4.47(0.84)	3.85–4.73	0.036
TBIL (mg/dL)	0.44(0.3)	0.32–0.56	0.43(0.2)	0.31–0.48	0.45
DBIL (mg/dL)	0.26(0.18)	0.195–0.28	0.12(0.1)	0.098–0.25	0.02
ALP(IU/L)	425.3(355)	382.95–459.80	128(55.3)	118.07–156.91	≤0.001
TG (mg/dL)	31.6(18.6)	29.45–34.90	86.7(21.8)	52.71–94.56	0.026
TC (mg/dL)	82.75(21.8)	76.35–88.85	121(32.1)	115.98–142.58	0.004

The Mann-Whitney U test also showed significantly elevated values for AST, ALT, DBIL and ALP in S. mansoni infected patients compared to the control group (P-value 0.05). On the other hand, the TC, ALB and TG level were significantly decreased among S. mansoni infected patients compared to apparently healthy controls (P-value <0.05) (Table 4).

Intensity of infection, biochemical and hematological values

The overall median EPG of S. mansoni infected patients was 215 EPG. Of the 90 S. mansoni infected patients, 55.6% (95%CI: 44.4–65.6), 28.9% (95%CI: 20–38.9), and 15.6% (95%CI, 8.9–23.3) had light, moderate, and heavy intensity of infection respectively. The WBC and RBC count was significantly lower in patients with moderate and heavy intensity of infection compared to patients with light intensity of infection and apparently healthy controls (P<0.05). The value of Hgb decreases as the intensity of infection increase. However, this difference was not statistically significant (P>0.05). According to the Kruskal Wallis H test, the median WBC count, RBC count, MCV, RDW, MCHC, MCH, MPV, PDW, Hgb and ALB in patients with moderate and heavy intensity of infection were significantly lower than in patients with light intensity of infection, and in healthy controls (P<0.05). On the other hand, the values of AST, ALT, DBIL and ALP were significantly elevated among patients with moderate, and heavy intensity of infection compared to those with light intensity of infection, and healthy controls (P<0.05) (Table 5).

Table 5

Association of S. mansoni intensity of infection with hematological and biochemical profiles of patients and healthy controls.

Variable	Light (1–99 EPG)	Moderate (100–399 EPG)	Heavy (>400 EPG)	Healthy control (0 EPG)	P-value
	Median (IQR)	Median (IQR)	Median (IQR)	Median (IQR)
WBC (103μL)	7.69 (4.21)	3.42 (5.2)	2.19 (3.14)	7.8 (4.8)	0.001
Neutrophile (103μL)	3.74 (5.3)	3.21(2.1)	2.84 (1.8)	3.94(3.2)	0.21
Lymphocyte (103μL)	2.4 (0.98)	1.80 (0.55)	1.01 (0.42)	2.24(1.1)	0.08
Monocyte (103μL)	0.46 (0.18)	0.42 (0.1)	0.38 (0.09)	0.51(0.3)	0.16
Eosinophile (103μL)	0.32 (0.098)	0.38 (0.12)	0.43 (0.23)	0.15(0.3)	0.001
Basophil(103μL)	0.067 (0.04)	0.064 (0.04)	0.0635 (0.04)	0.064(0.03)	0.7
RBC (106μL)	4.02 (3.2)	3.61(2.41)	2.05 (1.02)	5.8(1.13)	<0.001
Hgb (g/dL)	16.58 (8.7)	13.21 (6.32)	11.63 (5.84)	15.42(6.4)	<0.001
HCT (%)	44.3 (22.3)	38.27 (22)	35.64 (21.4)	46.31(9.4)	<0.001
MCV (fL)	82.01 (18.6)	76.42 (18.3)	74.51 (18.01)	86.57(8.4)	< 0.001
MCH (pg)	26.25 (5.4)	24.03 (5.4)	22.38 (5.34)	28.82(3.42)	< 0.001
MCHC (g/dL)	31.71 (1.01)	29.31(1.2)	27.98 (0.98)	33.4(2.6)	< 0.001
RDW (fL)	38.81 (7.1)	37.02 (6.83)	36.31(6.84)	62.88(12.3)	< 0.01
PT (103μL)	252 (144)	238 (123)	236 (122.6)	277.0 (75)	0.051
MPV (fL)	6.39 (1.65)	5.81 (1.61)	5.49 (1.61)	15.6 (1.0)	< 0.001
PDW (fL)	18.39 (4.14)	17.4 (3.4)	17.01 (3.35)	9.7 (1.2)	< 0.001
ESR (mm)	16.01(5.98)	14.21 (5.01)	12.93 (3.17)	5.4(1.1)	<0.001
ALT (IU/L)	24.9 (18.02)	28.1 (19.01)	32.79 (20.3)	20.23(16.21)	<0.04
AST (IU/L)	46.44 (21.53)	49.23 (22.01)	59.37 (24.2)	24.41(17.64)	<0.001
TP (g/dL)	7.49 (1.03)	7.25 (1.01)	7.03 (0.98)	7.83(1.6)	0.06
ALB (g/dL)	3.45 (0.91)	3.14 (0.71)	3.00 (0.65)	4.47(0.84)	0.04
TBIL (mg/dL)	0.33 (0.023)	0.64 (0.1)	0.51 (0.14)	0.43(0.2)	0.21
DBIL (mg/dL)	0.15 (0.03)	0.24 (0.12)	0.28 (016)	0.12(0.1)	0.03
ALP(IU/L)	381.9 (265)	431.3 (344)	438.9 (356)	128(55.3)	<0.001
TG (mg/dL)	29.84 (16.2)	31.23(18.3)	33.21(18.6)	86.7(21.8)	0.045
TC (mg/dL)	80.49 (18.1)	82.35 (21.75)	84.19 (22.01)	121(32.1)	0.04

EPG: egg per-gram of stool

Discussion

Schistosoma mansoni is a blood-dwelling parasitic worm that alters the hematological and biochemical profiles in patients with S. mansoni infection [31]. The finding of this study showed that the median of WBC count was significantly lower in S. mansoni infected adult patients compared to apparently healthy controls (5.8×103μL vs 7.8×103μL) (P<0.032). This finding was supported by report from Sanja Town Northwest Ethiopia [23]. In addition, this finding was also supported by a study that reported decreased WBC count in 57% of S. mansoni infected patients [32]. The Eosinophil count was significantly higher among S. mansoni infected adults than in healthy controls. This was in agreement with a report from Brazil and Western Uganda [33,34]. Eosinophilia in S. mansoni infected patients is due to powerful defense reactions against the tissue migrating larvae during acute phase of infection, release of antigen from spontaneously dying parasites or after chemotherapy, activation of proteolytic enzymes produced by the egg, and allergic manifestation [34,35]. However, the value of neutrophiles, lymphocytes, basophils, and monocytes were not significantly different between S. mansoni infected patients and apparently healthy controls (P>0.05). This finding agreed with a report of experimental studies conducted in Alexandria Egypt, and Gabon [36,37].

The median RBC count, Hgb level, and the value of RBC indices such as RDW, MCV, MCHC, and MCH were significantly lower among S. mansoni infected patients compared to healthy controls (P<0.001). This finding was in line with reports from Northwest Ethiopia [23] and Western Burkina Faso [15]. In this study, there was no significant difference in HCT values between S. mansoni infected adults and healthy controls (P<0.062).

The median platelet count was lower in S. mansoni infected patients (244 ×103) compared to the healthy controls (277×103). However, this difference was not statistically significant (P>0.05). This finding disagrees with a study done in Brazil, where the difference was statistically significant [16]. In this study, 26.7% of S. mansoni infected patients were thrombocytopenic. The mechanism of thrombocytopenia in S. mansoni infected patients is reported to be multifactorial. According to the finding of different studies, the most common cause of thrombocytopenia among S. mansoni infected patients are gastrointestinal bleeding, massive adhesion of platelets to the S. mansoni egg shell in the vascular endothelium, and S. mansoni infected patients producing anti-schistosome antibodies that cross-react with platelet and this leads to clearance of the platelet and cause thrombocytopenia [38,39]. In addition, splenomegaly caused by congestion due to obstruction, hyperplasia of reticulo-endothelial cells, fibrosis due to immunological stimulation, and hypersplenism were reported to cause thrombocytopenia [12,39,40]. Patients with chronic hepatosplenic schistosomiasis are more risky to develop complex hemostatic abnormality, that leads to risk of life threatening bleeding from ruptured esophageal varices [41].

Erythrocytic sedimentation rate (ESR) was significantly higher in S. mansoni infected patients than healthy controls. This finding was agreed with reports from Nigeria [32]. The elevation of ESR in S. mansoni patients might be due to blood loss as a result of bleeding during migration of worms in the intestine and consumption of blood by the worm.

In the present study, the overall prevalence of anemia among S. mansoni infected patients was 23.3%. This finding was in line with a study done in Kenya that reported 25.5% (13.1–38.0) prevalence of anemia among patients with heavy intensity of S. mansoni infection [42]. However, the finding was lower than a study conducted in Brazil [40]. The difference might be due to variation in immune status of the study participants, difference in strains of the parasite, and the intensity of infection. The prevalence of anemia among males and females was 18.8% and 28.6%, respectively. Regarding the severity, about 12.5% and 6.25% of males had mild and moderate anemia, respectively. Of the anemic female participants, 21.4% of them were mild anemic and 7.14% were moderately anemic. Anemia is one of the hematological abnormalities in S. mansoni infected patients that contribute to schistosomiasis associated morbidity and mortality [43]. The possible mechanisms of anemia in S. mansoni infected patients are iron deficiency due to extra-corporeal blood loss from egg movement through the intestinal wall, red blood cells, sequestration due to splenomegaly, anemia of inflammation, autoimmune hemolysis, gastrointestinal bleeding due to movement of laterally spined S. mansoni egg and anemia of inflammation induced by proinflamatory cytokines [44,45]. Schistosomiasis associated anemia is also contribute to fatigue, weakness, reduced cognitive function and impose socioeconomic burden [45].

Schistosoma mansoni infected patients were found to have elevated levels of ALT, AST, DBIL, and ALP. These parameters are important marker of liver injury in patients with schistosomiasis. This finding was in line with a study conducted in Northwest Ethiopia that reported a significantly elevated amounts of AST, and ALT among S. mansoni infected patients compared to apparently healthy controls [23] and other studies reported significantly higher AST, ALT, ALP and bilirubin among patients with hepatosplenic schistosomiasis than healthy controls [17-19,46]. The elevated value of liver function tests in S. mansoni infected patients might be due to the impairment of liver function due to S. mansoni egg deposition in the liver that later induces early granuloma formation and later on portal fibrosis and enlarged fibrotic portal tract [11]. The total protein and albumin levels were lower in 3.3% and 10% of the S. mansoni infected patients, respectively. This finding was supported by a study in Brazil that reported significantly lower levels of albumin in S. mansoni infected patients compared to the healthy controls [17]. In addition, the level of TC and TG was significantly lower in S. mansoni infected adults than the noninfected one. This finding was supported by the finding of a study conducted in Brazil [47]. The low level of cholesterol in schistosomiasis patients is reported to be associated with the parasite need but cannot synthesize cholesterol. So, the explanation is the adult worm internalize the host cholesterol through their tegument, shedding of Glycosyl-phosphatidylinositol sequestered with the host lipoprotein, and lipoprotein removal by neutrophil endocytosis [48,49].

The non-parametric Mann-Whitney U tests showed a significant difference in ALT, AST, DBIL, and ALP between S. mansoni infected patients and healthy controls (P-value <0.04). This was supported by studies conducted in Brazil and Northwest Ethiopia [23,50].

The findings of this study showed 55.6%, 28.9%, and 15.6% light, moderate, and heavy intensity of S. mansoni infection among the study participants, `respectively. This finding was in line with a report from Southwest Ethiopia that reported 57% light, 26.7% moderate, and 16.3% heavy intensity of infection [51]. However, the finding was quite different from reports in Sanja town, Northwest Ethiopia, that reported 69.1% light, 28.2% moderate, and 2.7% heavy intensity of infection [23] and Sanja Primary hospital Northwest Ethiopia that reported 40.0% light, 24.0% moderate and 36.0% heavy intensity of infection respectively [52]. According to the Kruskal Wallis H test, the median WBC count, RBC count, MCV, RDW, MCHC, MCH, MPV, PDW, and ALB in patients with moderate and heavy intensity of infection was significantly lower than in patients with light intensity of infection and healthy controls (P<0.05). on the other hand, the values of AST, ALT, DBIL, and ALP were significantly elevated among patients with Moderate and heavy intensity of infection compared to those with light intensity of infection and healthy controls (P<0.05).

This study was conducted with some limitations. Imaging techniques such as magnetic resonance angiography (MRA) and magnetic resonance imaging (MRI) was not done to assess to the morphological alteration of the liver and spleen due to schistosomiasis. The disease clinical severity parameters like size of the spleen, size of liver sign of fibrosis, and thickness of portal vein at entrance and secondary branch and its association with the various hematological and biochemical parameters were not evaluated. In addition, chronicity of the disease, and the markers of fibrosis and granulomatous inflammatory cytokine were not determined. Further study addressing these limitations are recommended to be conducted. As anemia is the leading hematological abnormalities, further studies that can investigate the mechanism and type of anemia among patients with schistosomiasis is also recommended.

Conclusion

The findings of this study showed significantly altered hematological and biochemical parameters among S. mansoni infected patients compared to apparently healthy controls. Considering hematological and biochemical abnormalities in schistosomiasis patients, and screening of patients with hematological and/or biochemical abnormalities for S. mansoni infection in endemic areas is an important measure to reduce schistosomiasis associated morbidity and mortality as well as to improve the health of the society. In addition, anemia and thrombocytopenia are found to be a major health problem among S. mansoni infected patients. As most of the hematological parameters declined with increased intensity of S. mansoni infection, treatment of the infection and the underlying hematological and biochemical abnormalities is recommended. Considering the range of infection intensity and associated clinical morbidity is also vital for patient management. In addition, the finding of this study indicates the necessity of considering schistosomiasis as a cause of the abnormality or as a co-morbidity while interpreting hematological and biochemical profiles in schistosomiasis endemic areas.

Questionnaire for assessing the Hematological and Biochemical changes in Schistosoma mansoni infected patients at Haik Primary Hospital, North-East Ethiopia: A comparative cross-sectional study.

(DOCX)

Click here for additional data file.

18 Apr 2022

Dear Mr. Bisetegn,

Thank you very much for submitting your manuscript "Hematological and Biochemical changes in Schistosoma mansoni infected patients at Haik Primary Hospital, North-East Ethiopia: A comparative cross-sectional study" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers.

In light of the reviews (below this email and in attachment), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments. Amongst others, reviewers found that details regarding the sampling procedure and a careful discussion of the limitations and implications of the study are needed.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Alberto Novaes Ramos Jr

Associate Editor

PLOS Neglected Tropical Diseases

Simone Haeberlein

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Sampling

1. “Systematic random sampling technique was employed to recruit the study participants at Haik Primary Hospital based on the inclusion criteria.” - how was the systematic random sampling conducted? Please elaborate.

2. “According to the rule of thumb recommended by van Voorhis and Morgan, 30 participants per group are required to detect real differences which could lead to about 80% power [21].” - We cannot rely on “rule of thumb”. Please describe appropriate sampling procedure. If random sampling from an adequate sample size was not done, then this should be described in limitations. This would not also be probability sampling

Methods/data collection

1. Both direct fecal smear and KK were used, but only KK was only used in quantification of ova. KK has higher sensitivity than DFS so please explain why KK was not used in determining if a sample is positive.

2. “A blood sample was collected from each study participant and healthy controls following standard operating procedures (SOPs) by trained laboratory technologists” – What are these SOPs? Please describe or at least put a reference or supplementary information.

3. Pictures of the processes are recommended

4. Patient-level (anonymised) were not provided

5. Please include the tools (e.g., questionnaires) used.

Ethical considerations

1. Please provide documentation of ethics clearance - include clearance number and attach clearance

Financial disclosure

1. The study involved fieldwork and specimen processing, so it is strange that the authors declared not receiving funding. Please elaborate how the project expenses (whether in cash or in kind) were funded.

Reviewer #2: The study objectives are well articulated and the study design is appropriate. The study population and the control groups are matching. The sample size is sufficient to draw conclusion and appropriate statistical method is used. Ethical considerations are clearly followed.

Reviewer #3: Yes, the proposed objectives are in accordance with the suggested hypothesis;

Yes, the study design is in line with the objectives;

The population is adequate, however, it lacks a better description of the region of origin of this population in relation to schistosomiasis related to endemicity;

Yes, the sample size is sufficient, however, it needs to better describe the control group;

Yes, the statistical analysis are corrects;

It's okay with ethical requirements.

Reviewer #4: Study Design and Methods:

Study design or procedure: The description of the stages of the study was not shown. For instance, biological samples ( stool and blood) were collected. Did the individuals undergo sample collection at any time during patient assistance at the primary hospital or only at the admission of the study? No details were informed.

Ethical statement was not included in ” Methods”.

“Thus, a total of 180 (90 S. mansoni infected adults and 90 apparently healthy controls) were enrolled in the study”. There was no definition of “apparently healthy controls” described at “Eligibility criteria”. Did any of the controls have a previous history of treated schistosomiasis? If yes, how long ago did they get the medication and proved treated before they were enrolled in the study?

Since other parasitic infections associated with anaemia may be a confounder, did the author excluded them before enrolling in the study? Is malaria an important issue in the study area? If yes, were both S.mansoni infected and controls investigated for malaria?

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: 1. Please describe how the data sets meet the requirements/assumptions for Kruskall-Wallis

2. Please include CIs in all estimates.

Reviewer #2: The presented result matches the analysis plan. and completely presented. comments for consideration are indicated in the last section.

Reviewer #3: Results are well presented, tables and graphs are clear, and statistical tests are well used.

I suggest adding the standard deviation to the graphs.

Reviewer #4: The data were presented mostly in tables that should be reviewed.

Tables and Figures:

No footnotes were added to the respective Tables. Abbreviations were available only at the end of the text. It should be included in the footnotes under each table.

In Table 2 and 3, "normal", "Low" and "High" were not defined ( laboratory reference values should be displayed in the footnote).

Figure 1 showed a title without legend. Also, there is no identification of the ordinate ( does it represent percentage?). It is advisable to properly review it.

Results:

In "Intensity of infection, biochemical and hematological values": .....The WBC and RBC count was significantly lower in patients with moderate and heavy intensity of infection compared to patients with light intensity of infection and apparently healthy controls". The results described were not shown.( Table? statistical analysis?)

Discussion:

An extensive literature review could improve the discussion of some issues. Example:

"Erythrocytic sedimentation rate (ESR) was significantly higher in S. mansoni infected patients than

healthy controls. This finding was agreed with reports from Nigeria [23]. The elevation of ESR in

S. mansoni patients might be due to blood loose as a result of bleeding during migration of worms

in the intestine and consumption of blood by the worm". Should ESR be used to diagnosis and monitor inflammatory/fibrotic

response? Was ESR elevated equaly in men and women? May other comorbidities be confounders?

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: 1. The implications of the study are not clear. Please elaborate what will happen if we now know that schistosomiasis may result to biochemical changes

2. Please justify how we the conclusions have global relevance given the limited sample size.

Reviewer #2: Conclusion and recommendation supports the data presented. Authors had discussed the data is helpful and showed public health relevance.

Reviewer #3: The results support the conclusions, however limitations of the study were not described. Although the data are useful for a better understanding of the subject, there was no approach of this importance in relation to public health.

Reviewer #4: The use of hematological and biochemical markers in schistosomiasis management should be done in parallel wirh other tests ( Image). For instance, intensity and extension of fibrotic liver response correlates with progression to chronic and advanced schistosomiasis. Although, authors propose the use of hematological and biochemical markers for screening the individuals infected with Schistosoma, the limitations of the this strategy were not fully discussed. The conclusions are weakly supported by the results. Also, added value of the study and implications of the results in the field were described.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Formatting

1. Include site map

2. Use indentation

3. Please use line numbers

Technical writing

1. Please elaborate what “Institution-based comparative cross-sectional study” means. Not a term I often encounter.

2. “regular chat chewer” - What is chat? Please describe.

3. Please improve English writing and proofread

3.1. “S. mansoni infected adults (≥18 years old) attending Haik Primary Hospital” - people do not attend hospitals

3.2. It’s median, not “media”

4. “Three species of the genus Schistosoma (Schistosoma mansoni, Schistosoma hematobium, and Schistosoma japonicum) are responsible for the majority of Schistosomiasis in the World [2].” – I am not sure if there other genus which causes schistosomiasis?

Reviewer #2: General: all scientific names should be italicized

Abstract

Conclusion section “Therefore, screening of S. mansoni infected patients for various hematological and biochemical parameters and providing treatment to the underlying abnormalities is very crucial to avoid schistosomiasis associated morbidity and mortality of S. mansoni infected patients.” Should read “Therefore, screening of S. mansoni infected patients for various hematological and biochemical parameters and providing treatment to the underlying abnormalities is very crucial to avoid schistosomiasis associated morbidity and mortality.”

Introduction: Paragraph 3- remove “appendix”

Method section: since the sociodemographic characters collected are few, delete “Structured” in the questionnaire.

Statistical analysis: “Variables were expressed as median and interquartile range is” repeated

Result:

Table 2- show the value for RBC

Under the “Prevalence and severity of anemia among S. mansoni infected patients” section correct “Moreover, 21.4% and 7.14% of females had mild and moderate anemic (Figure 1)” to “…….of females had mild and moderate anemia”

Figure 1 needs revision to show the value for females study subjects and correction of “Non-amenic” to Non-anemic

Discussion: ESR section instead of “blood loose” it should read “blood loss”

Confidence interval for intensity of infection should not be repeated in the discussion section.

Reviewer #3: No editorial suggestions

Reviewer #4: The data presentation should be completely reviewed ( Tables and Figure).

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The article touches on a topic seldom studied. However, unless the authors could justify the global implications of the study, the article may be better published in a regional journal.

Reviewer #2: The work is important to show healthcare points to be considered in schistosomiasis patients. The manuscript needs minor language editorial.

Reviewer #3: File in attachment.

Reviewer #4: This is a cross-sectional study that addresses both hematological and biochemical Schistosoma mansoni-induced alterations in infected individuals living in endemic área and attending medical assistance at a primary hospital in Haik town ( North-East Ethiopia). The study population includes adult individuals with schistosomiasis and a group of “apparently” healthy individuals as controls. The findings showed anemia and thrombocytopenia as the major laboratory-based disturbances in addition to abnormal biochemical results in Schistosoma infected individuals. Despite the lack of information about this particular geographical area, the results are not exactly new. Hematological and biochemical profiles of Schistosoma infected individuals were recently described in Amhara region in Ethiopia [Dessie et al, 2020]. Dessie et col. describe the hematological and biochemical profile of Schistosoma mansoni – infected individuals living in a study area with similar altitude, population density and schistosomiasis prevalence as the present study [ Dessie N et al. J Trop Med.2020: 4083252. doi: 10.1155/2020/4083252; Bisetegn H et al. Trop Dis Travel Med Vaccines. 2021 ;7(1):30. doi: 10.1186/s40794-021-00156-0]. The importance of the studies on the subject is undeniable. To stablish the profile of Schistosoma-induced disease by using less expensive laboratory markers may help at least in two ways: to clarify aspects of Schistosoma infection pathological responses and improve disease management. The last one is an important achievement in high-moderate endemic areas in low-income countries. However, the present manuscript brings a mild discussion on the main subject of the study wich should be greatly improved.

--------------------

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Submitted filename: PLOS NTD-2022.pdf

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2 Jun 2022

Submitted filename: Point by point response to the editors and reviewers.docx

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2 Jul 2022

Dear Mr. Bisetegn,

Thank you very much for submitting a revised version of your manuscript "Hematological and Biochemical changes in Schistosoma mansoni infected patients at Haik Primary Hospital, North-East Ethiopia: A comparative cross-sectional study" for consideration at PLOS Neglected Tropical Diseases. Based on the new reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations. Please pay attention to the reviewer comments below.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Alberto Novaes Ramos Jr

Associate Editor

PLOS Neglected Tropical Diseases

Simone Haeberlein

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Can we properly calculate the sample size based on your research question and study design? I am not sure the article in Tutorials in Quantitative Methods for Psychology is an authoritative reference for sample size estimation.

"The quality of the blood sample was maintained by collecting and processing it according to the229

standard operating procedures (SOPs) - the authors said that they had already provided a reference, but there is still none.

Reviewer #2: The objectives of the study are clearly stated and the study design is appropriate to address it. the sample size is appropriate according to WHO standard. The statistical analysis is is appropriate to address the objectives.

Reviewer #3: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: The results are clear and completely presented with tables.

Reviewer #3: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: the conclusion stems from the study findings . There is no limitation of analysis described.. The study finding is helpful to advance the understanding of the topic under study and has public health importance.

Reviewer #3: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: Accept it.

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Great work on the revisions. I just have minior comments. To ensure transparency, please provide the anonymised/coded data you used in the analysis. Note that policy of PLOS - "The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, except in cases where the data are legally or ethically restricted (for example, participant privacy is an appropriate restriction)" The ethical restriction should not be an issue if the each participant is coded.

Reviewer #2: None

Reviewer #3:

My main suggestions and recommendations I sent in an attached file. From what I observed in the answers of the authors, they did not access this file. As for the other suggestions made in the general questionnaire, I fully agree.

I am attaching here again the PDF file mentioned above.

PLOS NTD--CONSIDERATIONS-2022.pdfHematological and Biochemical changes in Schistosoma mansoni infected patients at

Haik Primary Hospital, North-East Ethiopia: A comparative cross-sectional study

GENERAL CONSIDERATIONS AND SUGGESTIONS

Methodology

It is necessary to clarify the origin of the control group because it is a little strange to have a control group chosen within a hospital. The criterion used to determine the participants in this group has been based only on clinical information as mentioned in the item Socio-demographic characteristics and clinical data? Stool tests has been done? If yes, which method has been used?

The authors only mention (in the introduction) that schistosomiasis mansoni is endemic in Haik town, not revealing this endemicity index, an important factor in the analysis and determination of the groups.

A more detailed description of the area from which these patients originated was lacking. It is important to know if the region has S. haematobium, for example, and if this was also used as an exclusion criterion.

In the Item “Stool sample collection and processing”, the last period about blood collection must be removed and added in the following item – “Blood sample collection processing”

Patients with splenomegaly were excluded and those with hepatomegaly, too? If these conditions are present in patients with severe forms of schistosomiasis, wouldn't it be interesting to keep them in the study group? An association could be made between the presence of these clinical forms and the hematological and biochemical indices analyzed.

Results.

The results are clear and well-presented between tables and graphs, I suggest, however, that you add the standard deviation in the graphs.

Discussion

The discussion in relation to hematological and biochemical indices is well founded, however I missed the approach on clinical and socio-demographic data.

There was a lack of an approach to public health using indicators from the region and also, the limitations found.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

18 Jul 2022

Submitted filename: point by point response PLOS NTD.docx

Click here for additional data file.

10 Aug 2022

Dear Mr. Bisetegn,

We are pleased to inform you that your manuscript 'Hematological and Biochemical changes in Schistosoma mansoni infected patients at Haik Primary Hospital, North-East Ethiopia: A comparative cross-sectional study' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests. Please also pay attention to the requested Editorial and Data Presentation Modifications raised by reviewer 2, shown below this email.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Sincerely,

Alberto Novaes Ramos Jr

Academic Editor

PLOS Neglected Tropical Diseases

Simone Haeberlein

Section Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: The objectives of the study are clearly presented. The study design and the sample size is appropriate to evaluate the hypothesis. Correct statistical methods are used to analyze the data to generate conclusion. The work is done following ethical considerations.

Reviewer #3: The objective, study design, sample size and statistical analysis with corrections to the suggestions made are in accordance with the purpose of the manuscript.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: The analysis presented match with the analysis plan. All results are presented according to the objectives of the study. Figures and Tables are of good quality.

Reviewer #3: The results are well presented and better visualized with the present figures. The tables are well made.

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: The conclusion is supported by the data obtained from the study. Limitations are also addressed. Moreover, it had shown the public health importance.

Reviewer #3: The conclusions support the data presented. The authors report the limitations of the work, however I still think there is still a need for a better discussion of the impact of these results in the region using the local public health indicators as parameters.

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: The MS PDF is presented in double copies.

Author summary first sentence needs rewriting. Introduction line 80 83 should be rewritten clearly through avoiding long sentence. In the method section the geographical location should be put in correct description. Line 136 needs rewriting. Line number 290 add 'for' after values. Line334 "health" should read 'healthy". The description of Line 340-347 is not supported by the current study outcome. Hence, needs revision.

Reviewer #3: No suggestion.

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Authors should submit a tracked changes version of manuscript or a version where only the changes are highlighted

Patient-level data (anonymised) should be provided. I think it is a requirement that data used in coming up with the results should be available

Responses to the comments should have been incorporated in the text. For instance, the following response should be incorporated in the manuscript.

Response: Institution-based mean the study is conducted in health facilities/institution/ and it is

not community based/survey. While comparative crossectional study is a a form of cross-sectional

study where data on the two groups (in our case S. mansoni infected patients and healthy control)

are collected at a point on time

Response: The variables are continuous variables. The distributions are not normally distributed

as checked by Kolmogorov-Smirnov and Shapiro Wilk normality tests and the test should be non-

parametric. It is four group (patient with light intensity, moderate intensity, heavy intensity of

infection and healthy controls). If the number of groups to be compared are three and more, the

Kruskal-Wallis H test is used to compare the variable.

Reviewer #2: The work has significant value as a public health concern. It needs minor grammatical revision and italicizing scientific names.

Reviewer #3: I understand that with the modifications made by the reviewers, the article is more robust and that the editor's discretion can follow for publication.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

25 Aug 2022

Dear Mr. Bisetegn,

We are delighted to inform you that your manuscript, "Hematological and Biochemical changes in Schistosoma mansoni infected patients at Haik Primary Hospital, North-East Ethiopia: A comparative cross-sectional study," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases