INTRODUCTION
Schistosomiasis is a water-based parasitic disease that affects over 200 million people worldwide, in addition to 800 million at risk.[1,2] It is a disease of poverty that shakes communities of the lowest socioeconomic status.[3]Schistosomamansoni infection with egg deposition in tissues gives rise to inflammation and granuloma formation leading to subsequent pathology.[4] Common symptoms and pathologic changes range from anemia, abdominal pain, and diarrhea with or without blood, to pseudopolyps and microulcerations.[5,6]
In areas where schistosomiasis is endemic, ongoing efforts over the past few decades have been embraced by the WHO to control and eliminate schistosomiasis. The declared objective by the WHO plan was to refocus on long-term and sustainable morbidity control through the scale-up of praziquantel (PZQ) mass drug administration.[7,8] Yet, the repertoire of simple, inexpensive, and noninvasive methods to measure the intestinal morbidity associated with schistosomiasis is rather limited, and new assays are urgently needed to estimate the power of control programs through the assessment of morbidity in treatment situations.[9]
Novel attempts to identify markers of intestinal morbidity were described, including fecal inflammatory markers. They comprise a heterogeneous group of substances that are generated by inflamed intestinal mucosa.[10] Lactoferrin, S100 A12 protein, fecal calprotectin (FC), and fecal occult blood (FOB), concurrently with questionnaire-based methods, are useful markers for assessing intestinal morbidity.[11,12]
FC is a calcium and zinc binding heterocomplex protein, mainly exhibited in and released from the cytoplasm of neutrophils, and is also found in monocytes and early-stage macrophages. In response to local inflammation, degranulation of an increased number of white blood cells in the bowel leads to a rise in FC. It remains stable against bacterial degradation during the intestinal passage, and hence can be quantified readily in feces, using an immunoassay. This method provides a simple and possible measurement of FC that can be used in an outpatient setting.[13,14]
FOB is an evident general marker of intestinal morbidity. It can be indicative of intestinal pathologic changes. There are various causes of positive FOB, including infection with some intestinal parasites.[11,15] Even though S. mansoni eggs perforate the intestinal mucosa and cause a small release of blood into the bowel, few studies have recommended that FOB can be used as a direct marker of schistosomiasis morbidity. The used tests showed differing sensitivities and detection limits.[16,17]
The aim of the present study was to investigate FC and FOB as morbidity markers for S. mansoni before and after PZQ treatment and, accordingly, their prospective use as indicators of intestinal morbidity.
MATERIALS AND METHODS
Ethics approval
The study was approved by the Ethics Committee of the Medical Research Institute, Alexandria University. Consents were obtained from the adults and parents and/or guardians on behalf of all children enrolled in the study. All cases positive for S. mansoni and/or other parasites diagnosed after Kato-Katz were treated with conventional drugs.
Study setting and population
Based on a field survey that was carried out earlier in 2017 in Motobus village, it was found that this area is highly endemic for schistosomiasis. The prevalence of S. mansoni was 50.9% by Kato-Katz.[18] Accordingly, Motobus village in Kafr El-Sheikh governorate, located 100 km away from Alexandria, was chosen as a study area for this work [Figure 1]. The study was conducted on 205 school children and adults symbolizing early and late stages of morbidity and was categorized as follows:
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Figure 1: A map of Kafr El-Sheikh governorate showing Motobus village
Group 1:117 school children from primary/preparatory schools.
Group 2: 88 adult individuals were randomly selected from the entire population of Motobus village.
Exclusions
Individuals with other parasitic infections, those who reported intake of intestinal antiseptics or antimicrobials within the previous month, and those diagnosed with having inflammatory bowel diseases or any chronic diseases, including chronic kidney or liver disease, and family history of malignancy or diabetes mellitus were excluded from the study.
Questionnaire
All participants were interviewed about symptoms of S. mansoni infection and treatment, history of blood in stool, diarrhea, and abdominal pain. Results were used to generate binary variables (negative or positive responses).
Stool samples collection
The school children and randomly chosen families were visited on two consecutive days every week. On the 1st day, tightly closed plastic containers were distributed, labeled with the participant’s identification number and name, for stool collection. On the next day, the containers were collected from all participants and returned to the parasitology laboratory in the Medical Research Institute for processing and microscopic examination.
Stool microscopy, parasitological detection, and praziquantel treatment
All stool samples were examined microscopically after the Kato-Katz technique (duplicate slides of 41.7 mg/slide/sample).[19] All parasites were identified, S. mansoni eggs, when found, were counted and recorded [Figure 2]. Cases positive for S. mansoni were treated with PZQ at a single oral dose of 40 mg/kg body weight, and stools were recollected 1 month later. Part of each fecal specimen before and after treatment was stored at 2212 − 20°C for studying FC levels. FOB assay was performed on fresh stool samples. For the exclusion of protozoan infections and newly emerging protozoa, formalin ethyl acetate technique and modified Ziehl–Neelsen-stained smears were performed. Only cases positive for S. mansoni after Kato-Katz, and negative for protozoa and helminths, were subjected to studying FC and FOB before and after PZQ treatment.
Figure 2: A photo of Schistosoma mansoni ova (×400)
Biomarker assays
Fecal calprotectin
FC was measured by a quantitative enzyme immunoassay (The EDI™ Epitope Diagnostics, Inc., USA) as per the manufacturer’s instructions, without knowledge of patients’ data. Concentrations were expressed as micrograms per gram of stool. FC in patients with gut inflammation were those with values more than 43.2 μg/g, which is the calculated cutoff read directly from the generated standard curve of a given set of standards.
Fecal occult blood
A qualitative, lateral flow immunoassay was employed for FOB detection (ABON™, One Step FOB Test Device, P.R. China), following the manufacturer’s instructions. Briefly a small amount of feces was homogenized in a liquid buffer after collection. Two drops of stool suspension were applied to a test cassette, and results were visually read after 5 min.
Statistics
Data collected were coded, tabulated, and analyzed using the Statistical Package for the Social Science Software version 20.0. (SPSS, Armonk, NY: IBM Corp). Data were expressed as a number, percentage, arithmetic mean ± standard deviation and geometric mean counts (GMEC) and 95% confidence interval (CI) were used for normally distributed quantitative data. Chi-square test was applied if qualitative variables were compared. Fisher’s exact and Monte Carlo tests were used if more than 20% of the expected cell counts were <5. Paired t-test was used to compare values before and after treatment among the same individual. Student’s t-test was used for comparing values before and after treatment among different individuals. All tests were applied two-sided with a level of significance of P < 0.05.
RESULTS
Among the 205 participants, 117 children aged from 8 to 14 years, of which 63 were males, while the 88 adults aged from 18 to 60, 37 were males. Prevalence rates of S. mansoni were 20.5% and 11.36% among children and adults, respectively, with an overall prevalence of 16.58%.
As for infection intensity, at 95% CI, 62.5% and 80% of children and adults had light infections with GMEC of 32.28 and 27.53 eggs, respectively, and few had moderate and high infection intensity [Table 1].
Table 1: Distribution of Schistosoma mansoni cases among children and adults by the intensity of infection
Referring to the questionnaire, among S. mansoni-positive cases, diarrhea was the most frequent complaint reported by 50% of positive children, followed by blood in stool and abdominal pain (33.3% each). On the other hand, abdominal pain was the dominant symptom in adults (90%), followed by diarrhea (70%) and blood in stool (30%) [Table 2].
Table 2: Self-reported symptoms among the two studied groups
Calprotectin and occult blood in stool of Schistosoma mansoni cases before and after praziquantel treatment
Fecal calprotectin
The cut-off value for FC concentration by the enzyme-linked immunosorbent assay kit was 43.2 μg/g stool, above which cases were considered positive. In the present work, among children, the mean FC level was 43.825 μg/g before treatment and became 38.06 μg/g after treatment with borderline statistical significance (P = 0.0546). One case of high infection intensity gave a shooting positive response of 468.2 μg/g and turned negative after treatment. As for adults, the mean FC was 41.15 μg/g before treatment versus 25.05 μg/g after treatment. No statistically significant difference regarding the mean levels of FC before and after treatment was observed (P > 0.05) [Table 3].
Table 3: Comparison between fecal calprotectin levels before and after treatment among children and adults
By referring to individual results, six positive diarrheic children, three with moderate and three with heavy infection intensity [Table 1], showed FC levels higher than the cut-off value before treatment and became negative after treatment with statistically significant differences. All adults showed negative results before and after treatment.
Fecal occult blood
By comparing FOB before and 30 days after treatment among the studied groups, four diarrheic children (of moderate and heavy intensity) out of 17 (23.5%) had occult blood in their stool before treatment, and they turned negative after treatment. Regarding adults, all cases were negative for FOB before and after treatment [Table 4].
Table 4: Comparison between fecal occult blood before and 30 days after treatment among school children and adults
DISCUSSION
Morbidity control program indicators for intestinal schistosomiasis originated from the concept that “risk of morbidity is due to heavy infection.” This was when PZQ was not widely available and morbidity was defined as severe fibrotic manifestations of chronic infection mostly in the liver and the intestinal wall.[20]
PZQ has a high safety margin and is very well tolerated, with no long-lasting crucial adverse effects on humans to date.[2] It is quickly and almost completely absorbed into the bloodstream yet with no or slight effect on juvenile worms and eggs.[21] Release of tissue-dwelling eggs in feces continues for a number of weeks following treatment.[22] Hence, follow-up visits are preferably conducted 4–6 weeks post infection after a single dose of 40 mg/kg body weight, where 70%–100% of cases cease to excrete eggs.
Nowadays, there is an opportunity to update these concepts, given the recent understanding of morbidity and more sensitive diagnostic techniques. Hence, morbidity control would depend on the identification of a level of infection below which little to no morbidity occurs that rest on a marker readily measurable in the field or laboratory.[23]
The present studied FC and FOB as appropriate measures for possible monitoring of intestinal schistosomiasis morbidity in pre- and post-treatment situations. Based on Kato-Katz, S. mansoni infection rate was 20.5% among children and 11.3% among adults with GMEG <100 eggs/g, pointing to the low prevalence and infection intensity due to the sustainable application of schistosomiasis control measures by the Egyptian governorate.
A questionnaire was designed to investigate the past or current experience of relevant symptoms. The results of the present study showed that diarrhea was the most frequent complaint among children, followed by blood in stool and abdominal pain. On the other hand, abdominal pain was the most dominant self-reported symptom in adults, followed by diarrhea and blood in stool. Betson et al. found that a self-reported history of blood in stool or diarrhea showed a positive association in S. mansoni-infected children but not in adults.[10] They concluded that questionnaire data were not reliable as morbidity indicators when studying small numbers of children. Symptoms such as blood in stool, abdominal pain, and diarrhea are not specific for intestinal schistosomiasis. They can be caused by a number of different infections, and the recall of symptoms may be imperfect.
FC, a stable neutrophil-specific marker, was proposed as a morbidity marker and was studied among positive schistosomiasis cases. In the present study, 6/17 diarrheic children of moderate and heavy intensity showed positive FC levels before treatment with a borderline significant difference pre- and post-PZQ. All adult participants gave negative results. This finding accords with those of Yang et al., who reported that inflammatory processes in the intestine play an important role in schistosomiasis progression.[24] Chronic inflammation associated with S. mansoni eggs appears to involve the recruitment of eosinophils rather than neutrophils, which may explain the absence of a detectable increase in levels of FC among some infected individuals.[25] In Uganda, Betson et al. reported no association between schistosomiasis infection and FC in children, although a positive inverse relationship was reported in mothers.[10] On the contrary, Bustinduy et al. concluded that FC and FOB correlated strongly and positively with moderate and heavy egg patent intensities of S. mansoni infection.[11] They reported a positive improvement response after PZQ treatment, indicative of a short-term reversion of morbidity. The absence of significant association in the present study may be due to the dominance of low infection intensity with low-grade inflammation.
FOB, cryptic blood in feces, the utility for assessment of morbidity associated with intestinal schistosomiasis has been previously investigated with tests of differing sensitivities. A FOB test was used to assess occult intestinal bleeding as a morbidity marker due to S. mansoni infection before and after treatment. In the present study, 23% were positive for FOB, particularly evident for S. mansoni-infected children and not for adults. All positive cases had moderate and heavy infection intensities. On follow-up, all infected cases gave negative results for FOB with no significant association before and after treatment. This finding could be explained by the fact that S. mansoni infected children may have intestinal bleeding more than long-standing infections in adults. In addition, negative FOB after treatment may be due to absence and/or reduction in the number of eggs produced by adult worms alongside to chemotherapy.[25]
Similar to the present study, a positive correlation between the intensity of S. mansoni infection and FOB was reported in Brazil and Zimbabwe.[26,27] On the same line, high-intensity S. japonicum-infected cases revealed a threefold positive FOB level than those with light and moderate intensities.[28] Betson et al. reported a chronological positive correlation in young children (<5 years of age) between FOB and S. mansoni egg patency at baseline, 6 months and 12 months after PZQ treatment.[10] In 2012, they found that FOB tests were highly specific for S. mansoni infection but not sensitive, likely in cases with low infection intensities.[16] Studies in China and Saudi Arabia reported no relation between FOB and intestinal schistosomiasis.[29]
Several possible explanations for the strong association between morbidity markers and intestinal schistosomiasis were reported comprising a complex interplay between host and parasite; genotypic partitioning in the S. mansoni parasite in different regions. This may be associated with differences in the morbidity induced by the parasite, and tribal differences, which may lead to differences in immune responses to Schistosoma eggs and other infections, which could lead to FOB masking the relationship with schistosomiasis.[25,30]
Conclusively, positive results both for FC and FOB were few to drive any solid decisions. However, the absence of significant evidence that intestinal schistosomiasis was associated with either positive FC and/or FOB before or after treatment among adults may be due to low intensity of infection. Yet, the presence of FC and FOB among children with moderate and high infection intensity might point to the possible use of such markers for S. mansoni morbidity at an early age particularly they reverted to negative after treatment. Further studies in various endemic areas with different age and infection intensity are needed to reach more assured results.
Study limitation
The small number of infected cases is considered a limitation of this study.
Ethical clearance
The study was approved by the Research Ethics Committee of the Medical Research Institute, Alexandria University, Egypt (IORG0008812).
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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