DOI: https://doi.org/10.21203/rs.3.rs-2706210/v1
Background: Cystoisospora belli inhabits the epithelium of the upper human small intestine, producing resistant oocysts that are infrequently detected in stool specimens but can spread and cause infection.
Objectives: To determine the rate of C. belli infection among children and correlate the rate of infection with the patient's variables.
Patients and Methods: In this cross-sectional study, 156 fresh stool samples were collected from children suffering from diarrhoea or abdominal discomfort in Dr Jamal Ahmed Rashid Pediatric Teaching Hospital, Sulaimaniyah, Iraq, from July to August 2021. Slides for direct wet mount and fixed smears stained with modified Ziehl-Neelson stain were prepared for each sample. Additionally, the correlation between the bacterial infection rate and patients' variables (age, gender, residency, duration of infection, and type of drinking water/stool) was determined.
Results: None examined stool samples were positive for C. belli oocyst by direct wet mount technique, while the modified ZN staining method revealed 42 (26.92%) positive samples. The highest rate of C. belli infection (15.38%) was found among children aged 4-6 years (p=0.000), male patients (15.38%), patients who lived in an urban area (23.08%) (p≥0.05), had infection for 1-3 days (15.38%) (p=0.038), took bottle water (15.38%) (p=0.02), and patients had liquid/semiliquid stool type (11.54%) (p=0.485). On the other hand, modified ZN stain showed coinfections with other protozoa, including Cryptosporidium spp., Microsporidia spp., Cyclospora spp. and Blastocyst hominins at rates of 10.26%, 5.13%, 3.85% and 1.28%, respectively.
Conclusion: There was a relatively high rate of C. belli infection among children in Sulaimaniyah, Iraq, despite other types of protozoa.
Parasites, specifically those inhabiting the gastrointestinal tract of humans, account for about 3.5 billion infections worldwide. Coccidian parasites are among the causes of intestinal protozoan infection in humans and animals. Primary symptoms include diarrhoea, which may become bloody in severe cases. Most cases infected with coccidia are asymptomatic, but young or immunocompromised patients may suffer from severe symptoms and death [1].
Cystoisospora belli, previously known as Isospora belli, is a coccidian, unicellular protozoa belonging to the phylum Apicomplexa that parasitizes the upper small intestine epithelium of humans and causes diarrhoea [2]. Infection with this parasite is cosmopolitan in distribution but more common in tropical/subtropical climates, the Middle East, and areas with poor sanitation [3]. It is associated with outbreaks of diarrhoea in mental wards and daycare centres [4].
As with another coccidian, C. belli has a sexual and asexual life cycle. Humans are the only known host for this parasite, and the infection is acquired through the ingestion of sporulated oocysts through contaminated water and food [5]. After the invasion of the small intestinal epithelium and completes its life cycle within the cytoplasm of the enterocytes, Oocysts (diagnostic stage) are excreted with the faeces and develop outside the host into mature cysts, each with two sporocysts which contain four sporozoites each (infective stage) [6].
Most infections are asymptomatic, and symptomatic cystoisosporiasis is characterized by watery diarrhoea, colicky abdominal pain, vomiting, dehydration, malabsorption, weight loss, and peripheral eosinophilia. Infection is typically self-limited in immunocompetent hosts, but chronic and severe illnesses in infants, young children, and healthy adults have been reported [2].
The diagnosis of C. belli relies on direct or concentrated wet smears of fresh or preserved stool specimens that must be repeated because of the intermittent shedding of its cysts in stools. In wet smears, oocysts are pale transparent, oval, measures 20–33 µm × 10–19 µm, and generally contain only one or two immature sporoblasts. The acid-fast stain is another detection method by which the oocyst is stained red [7].
C. belli considers itself an opportunistic pathogen; however, it causes severe clinical conditions under immunosuppression [8]. Thus, this study aimed to determine the infection rate of C. belli among children with diarrhoea and abdominal discomfort.
This cross-sectional study was conducted on fresh stool samples of 156 children with diarrhoea or abdominal discomfort at Dr Jamal Ahmed Rashid Pediatric Teaching Hospital, Sulaimani, Iraq, in July-August 2021.
A well-prepared questionnaire was used to collect patient data (including age, gender, residency, duration of infection, source of drinking water, and type of stool) from their mothers through direct interviews.
Children aged > 1 year old with diarrhoea and abdominal discomfort, regardless of gender and nationality.
Children with chronic diseases (such as diabetes mellitus) and debilitated diseases (such as cancer) were not enrolled.
The collected stool specimens were placed in a clean, sterile, and well-sealed plastic container with a cover, then labelled with the date and name of the patient and transported directly into the laboratory without fixative in a cooled icebox. Next, each stool sample was examined macroscopically for consistency, colour, and presence of pus, mucus or blood. At the same time, microscopic examinations were done based on two preparations for each stool sample.
A drop of normal saline was placed on the centre of a clean slide. Then, with an applicator stick, a small portion of stool specimen was picked up, mixed with normal saline, covered with a cover slip, and examined under a microscope using the objective lens of 10×, then 40× magnification power.
A small amount of stool sample was picked up with a clean applicator stick and spread by rolling the stick over the middle part of the slide. Then, the smear was dried, fixed with 2–3 drops of absolute methanol, and let to dry. Finally, smears were stained with Modified Ziehl-Neelson (ZN) acid-fast stain to detect oocysts of C. belli and other coccidian parasites.
Statistical analysis was done using SPSS, Chicago, USA (Version 21). Frequency and percentage were calculated for all variables, and the correlations between the variables were determined using Chi–square test. A p-value of 0.05 or less was considered significant.
In a total of 156 examined stool specimens, 42 (26.92%) samples were positive, and 114 (73.08%) were negative for the oocysts of C. belli using modified Z-N staining (Fig. 1). At the same time, direct wet mount examination didn’t reveal any oocyst of C. belli.
Furthermore, microscopical examination showed the presence of other parasites, including Entamoeba histolytica cyst (9.0%), monilia (5.1%), cysts and trophozoites of Giardia lamblia (0.6%), and Entrobius vermicularis ova (0.6%) (Fig. 2).
The highest rate of infection (15.38%) was found among children aged 4–6 years, followed by the age group > 9 years (7.69%), and then 1–3 years (3.85%) with no infection among children aged 7–9 years. There was a significant difference between the rate of C. belli infection and the age groups (p = 0.000). Regarding gender, a higher infection rate was recorded in males (15.38%) than in females (11.54%). Also, urban citizens reported a higher infection rate (23.08%) than rural citizens. However, there was no significant association between the rate of C. belli infection with both gender and residency (p ≥ 0.05). The highest rate of C. belli was reported among those infected within 1–3 days (15.38%), followed by 4–6 days (7.69%), then 7–9 days (3.85%) with no detected protozoa in patients > 9 days being ill. Significant differences existed between the infection rate and illness duration (p = 0.038). The current study found a higher rate (15.38%) of disease among children who consume bottled water, followed by tap water (11.54%), and no infection among those who drank tap/bottled water. Also, significant differences between the disease rate and the drinking water type were observed (p = 0.02). Furthermore, the same infection rate (11.54%) was observed in both liquid and semiliquid stools. The formed stool was reported at 3.85%, with no significant association between the infection rate and patient chair types (Table 1).
| Variable | No. of sample | C. belli detection using ZN test | P-value | ||
| Negative | Positive | ||||
| Number, % | |||||
| Age (Year) | 1–3 | 62 (39.7) | 56 (35.9) | 6 (3.85) | 0.000** |
| 4–6 | 64 (41.0) | 40 (25.64) | 24 (15.38) | ||
| 7–9 | 10 (6.4) | 10 (6.41) | 0 (0.00) | ||
| > 9 | 20 (12.8) | 8 (5.13) | 12 (7.69) | ||
| Gender | Male | 76 (48.7) | 52 (33.33) | 24 (15.38) | 0.201 |
| Female | 80 (51.3) | 62 (39.740 | 18 (11.54) | ||
| Residency | Urban | 139 (89.1) | 103 (66.03) | 36 (23.08) | 0.41 |
| Rural | 17 (10.9) | 11 (7.05) | 6 (3.85) | ||
| Duration of Infection (Day) | 1–3 | 107(68.6) | 83 (53.21) | 24 (15.38) | 0.038* |
| 4–6 | 27 (17.3) | 15 (9.62) | 12 (7.69) | ||
| 7–9 | 16 (10.3) | 10 (6.41) | 6 (3.85) | ||
| > 9 | 6 (3.8) | 6 (3.85) | 0.0 (0.0) | ||
| Type of Drinking water | Tap water | 88 (56.4) | 70 (44.87) | 18 (11.54) | 0.02* |
| Bottle water | 57 (36.5) | 33 (21.15) | 24 (15.38) | ||
| Tap & bottle | 11 (7.05) | 11 (7.05) | 0 (0.0) | ||
| Type of stool | Formed | 18 (11.5) | 12 (7.69) | 6 (3.85) | 0.485 |
| Semiliquid | 79 (50.6) | 61 (39.10) | 18 (11.54) | ||
| Liquid | 59 (37.8) | 41 (26.28) | 18 (11.54) | ||
| *: Significant difference, **: Highly significant difference using Chi-square test, ZN: Ziehl-Neelson acid-fast stain | |||||
Moreover, modified ZN stain revealed the presence of other protozoa, including Cryptosporidium spp., Microsporidia, Cyclospora spp., and Blastocyst hominins accounting for 10.26%, 5.13%, 3.85%, 1.28%, respectively (Fig. 3).
Cystoisospora belli is an intestinal protozoon distributed worldwide; however, it is a less common cause of protozoal diarrhoea than Toxoplasma and Cryptosporidium [9]. Studies showed that many parasitic infections are prevalent and endemic in Iraq, including Sulaimaniyah; however, research on C. belli infections is limited in Iraq, with no reported data in Sulaimaniyah.
The direct wet mount technique was used in this study. Still, it didn’t reveal any oocyst of C. belli in the examined stool samples of the children, which might be related to the transparent appearance of the oocysts that could be overlooked in direct faecal smears. Diagnosis can be challenging owing to intermittent and low-grade shedding of oocysts that may not be found despite actual infection [10]. However, a modified ZN stain detected a high burden of Cystoisosporiasis (26.92%) in children. This outcome is inconsistent with that found by Abdel-Hafeez et al., 2012 in Egypt (9.7%) [11], Vouking et al., 2014 in Cameron (10.08%), Barcelos et al., 2018 in Brazil (3.8%), Al-Saeed et al., 2019 in Erbil, Iraq (3.8%) [12], Kumar et al., 2017 in India (2%) [13], and Mbae et al., 2013 in Kenya (1.2%) [14]. However, it is in line with that found by Casmo et al., 2018 in Mozambique (25%), but it is lower than that observed by Swathirajan et al., 2017 in India (88.8%) [15]. These variations were referred to the differences in the study area and designs, methods of diagnosis, the season of studies and geographic location and patient populations.
Additionally, microscopical examination in this study showed the presence of other microbial agents rather than C. belli, such as Entamoeba histolytica, monilia, Giardia lamblia, and Entrobius vermicularis. Similarly, other studies found various microbial agents, including Giardia lamblia [12], Ascaris lumbricoides, Entamoeba histolytica, Trichuris trichiura, Giardia intestinalis, Hymenolepis nana, and Schistosoma mansoni [16]. At the same time, the most common protozoan infection in immunosuppressed children was Cryptosporidium parvum (60.2%), Blastocystis hominins (12.1%), Cyclospora caytenensis (7.8%), Entamoeba histolytica (24.6%), and Giardia lamblia (17.6%) [11].
In the current study, age (4–6 years), duration of infection (1–3 days), and type of drinking water (bottled water) (p < 0.05) were considered as the main predisposing and risk factors for development and progression of cystoisosporiasis in diarrheal children and on the other hand, gender, residency, and type of the school had no impact on the development and progression of the disease in children with diarrhoea. Thus, other risk factors for developing intestinal parasites were reported in other studies, such as high waste disposal habits, open field defecation, drinking water sources, and hand washing habit before meals [17].
Cryptosporidium spp., Cyclospora spp., Toxoplasma spp., and C. belli are related taxonomically [3]. Thus, the modified ZN stain revealed the presence of other protozoa, including Cryptosporidium spp., Microsporidia, Cyclospora spp., and Blastocyst hominins. In this regard, Cryptosporidium spp was detected in 34.3% and C. belli in 1.5% of patients with HIV/AIDS in South Ethiopia [18]. In comparison, another study reported Cryptosporidium parvum (43.6%), C. belli (15.5%) and Blastocystis hominis (10.5%) in HIV/AIDS patients in North West Ethiopia [19]. Moreover, Adamu et al., 2006 in Addis Ababa found emerging opportunistic parasites in diarrhoeal children, including Cryptosporidium parvum (8.1%), C. belli (2.3%), Enterocytozoon bieneusi/Encephalitozoon intestinalis (0.5%), Ascaris lumbricoides (0.5%), Trichuris trichiura (0.9%), Giardia lamblia (6.3%), Entamoeba histolytica/ E. dispar (1.4%), Blastocystis hominis (5.9%) and Hymnolepis nana (0.5%) [20].
This study concludes that there was a relatively high rate of C. belli infection among children in our locality. At the same time, other microbial agents, including protozoa, parasites, bacteria, and fungi, were also detected. In addition, age, type of drinking water, and duration of infection significantly correlated with the protozoal rate. However, limitations of the study include lack of funding and using traditional non-quantitative techniques for C. belli detection. Thus, it is recommended to conduct more investigations using more advanced diagnostic methods to detect C. belli oocysts from stool samples.
Ethical approval and consent to participate
The protocol of this study was revised and approved by the Scientific and Ethical Committee of Sulaimani Technical Institute, Sulaimaniyah, Iraq (No. 7993/12/07/2021). The patients and their parents or/and legal guardian filled out a written consent form and were informed about the purpose of the study. Also, they were allowed free to quit at any time without giving a reasonable declaration.
Consent for publication
Not applicable.
Availability of data and materials
The data used to support the findings of this study are included within the article.
Competing interest
The author declares there is no conflict of interest in this study.
Funding
This study has not received any grant or fund from any national or international company, University, or organization and is entirely self-funded.
Acknowledgements
The author would like to express her heartfelt thanks to all the healthcare and physician of Dr Jamal Ahmed Rashid Pediatric Teaching Hospital, Sulaimaniyah, Iraq.
Author contribution
FMA: Conceptualization, study registration, study administration, methodology, data analysis, resources, validity, visualization, wrote the original manuscript text.