مطالعه اپیدمیولوژی کریپتوسپوریدیوز در کودکان مبتلا به اسهال در استان چهارمحال و بختیاری

Introduction:

Cryptosporidiosis is caused by the species Cryptosporidium, a parasite that has been classified as a pathogen by the Centers for Disease Control and Prevention (CDC). The organism infects the gastrointestinal epithelium and causes diarrhea, which is a self-limiting infection in healthy people but is life-threatening in people with immunodeficiency such as children, and people with acquired immunodeficiency syndrome (AIDS). Infection with this parasite accounts for up to 6% of all diarrheal diseases in people with weakened immune systems. The infection is also present in 24% of people with AIDS and diarrhea worldwide (1, 2). Cryptosporidium is an intracellular parasite in the Apicomplexa group. Ten species are now known based on differences in host characteristics and oocyte morphology. Cryptosporidium parvum is the most common species in humans. C. felis, C. muris, and C. meleagridis have also been identified in immunocompromised individuals. Cryptosporidium is can complete all stages of its development (asexual and sexual) in one host.  Humans become infected when they ingest cryptosporidium oocytes, oocytes rupture in the gastrointestinal tract, releasing infectious sporozoites. The sporozoite attaches to the apical membrane of the host epithelial cell. Such a transplant reorganizes the host cell actin cytoskeleton and protrudes the host cell membrane around the sporozoite to form a vacuole in which the organism remains intracellular but outside the cytoplasm (3, 4, 5). The sexual form of the parasite repeats the life cycle after fertilization in the gastrointestinal tract, producing thin-walled oocysts that release sporozoites upon re-sporulation. This condition can lead to spontaneous infection and severe and persistent infections with widespread oocyst shedding in the feces of an infected patient (6, 7, 8). Cryptosporidium became one of the most common intestinal pathogens in immunocompromised individuals worldwide in 1976, when it was first reported as human infection. The total proportion of egg-laying populations in developed countries is 1 to 3% and in developing countries 10%. Cryptosporidiosis is an important clinical problem in patients without access to antiretroviral therapy and in malnourished children, as well as in people undergoing transplantation or chemotherapy. Infection is often transmitted from person to person, by animals, and indirectly through the environment (especially water). Transmission from cattle and sheep to humans is well known and these animals are currently the most important reservoirs of human disease. Cryptosporidium oocysts can be found in all types of water, including untreated surface water, swimming pool water, and even chlorinated or disinfected drinking water, or food (9, 10, 11). The widespread prevalence of contaminated water and food has affected almost the entire world. Although its pathogenesis is weak, there is currently no fully effective treatment for this infection. Currently, there are limited and scattered reports of the frequency of this protozoan in Iran, but the results showed that the rate of cryptosporidium infection in Iran has a wide range between 0.83 to 24% (12).

Due to the health importance of this protozoan, especially in children with diarrhea, the present study was designed and performed to investigate the prevalence of cryptosporidiosis in children with diarrhea in Chaharmahal and Bakhtiari province.

Materials and Methods

Sampling: The present cross-sectional study was performed in Shahrekord. Stool samples of children with diarrhea during autumn, winter, spring, and summer of 1399 (2020) were collected from the pediatric ward of Ayatollah Kashani Hospital and Shahrekord Central Laboratory. In addition to urban children, rural children were also referred to this hospital. Sampling was performed in the age group under 12 years which was divided into two age groups of 1-6 and 7-12 years. In each season, 100 samples and a total of 400 samples were taken from patients with diarrhea for one year. To find out the factors affecting cryptosporidia infection, as well as a comparison between the factors considered in this study, a questionnaire containing information such as sex and age of the infected person, referral season, contact with the animal, place of residence was completed by patients.

Identification of Cryptosporidia: The Modified Ziehl Neelsen staining was used to detect Cryptosporidium in feces. Briefly, one gram of feces was first mixed well with 11 ml of 1.5% saline and then filtered and transferred to centrifuge tubes. One millimeter of ether was then added to each tube and centrifuged at 35 rpm for 11 minutes. A smear of the precipitate was prepared on the slide, and after drying, a few drops of ethyl alcohol were added and placed in an oven at 50 ° C for 2 minutes to dry completely. The smears were then immersed in 96% methanol for 3 minutes to be completely fixed, heated over a flame for a few seconds, and immersed in Karbel Fuchsin (0.34% Fuchsin and 4% Phenol) for 20 minutes. In the next step, they were placed in 5% sulfuric acid, and finally, 5% malachite green was used. Stained smears were evaluated by light microscopy for the presence of cryptosporidium parasite oocysts. In the microscopic evaluation for each sample, five microscopic fields were studied, and based on the average number of oocysts counted, the severity of infection was + (1-4 oocysts), ++ (5-25 oocysts) and +++ (more than 25 oocysts) were determined in each microscopic field (13).

DNA Extraction: The QIAamp mini stool test kit was used according to the manufacturer's instructions with minor modifications. Briefly, 200 mg of each stool sample was poured into an Eppendorf tube and stored frozen at minus 20 ° C. Before thawing, completely dissolved in 1.4 ml of ASL buffer contained in the extraction kit. The samples were then boiled for 5 minutes and then subjected to three cycles of freezing in liquid nitrogen for 1 minute and boiling for 2 minutes to destroy the cyst wall and release DNA. It was then centrifuged at 13,000 rpm for 1 minute and 1.2 ml of supernatant was pipetted into a new tube, and an inhibitory tablet was added to remove possible PCR inhibitors. After incubation at room temperature for 1 minute and two centrifuges at 13000 rpm for 3 minutes, 200 mg of supernatant was mixed with proteinase K (final concentration 1 mg/ml) and 200 μl of AE buffer. After vortexing the samples, incubation was performed at 70 ° C for 10 minutes and 200 μl of 96% ethanol was added, and the samples were vortexed again. They were then centrifuged at 13,000 rpm for 1 minute. After washing with buffers and centrifugation twice, DNA was removed from the silica column for 1 minute using 200 μl AE buffer and centrifugation at 13,000 rpm.

To control the mechanical disturbance, the negative stools were divided into several sections of 200 mg and used for DNA extraction according to the above protocol in parallel with the samples without three freezing and boiling cycles (14).

Nested PCR: PCR reaction with a volume of 50 μl including 10 mM Tris-HCl (pH 8.3), 50 mM KCl solution, 2.5 mM MgCl2 solution, 0.5 µM of each external primer (Table 1), 1.5 U of Taq polymerase enzyme, and µM 100 nucleotides were performed. For the second step, the solutions were the same as mentioned, except that 1μl of the first PCR product and 0.5 µM of each of the internal primers (Table 1) were added. Reaction mixtures with external primer set once at 94 ° C for 5 minutes, then 35 times at 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C, respectively. The steps were performed for 1 minute and then the final extension step was performed at 72 ° C for 5 minutes. For Nested PCR products, the thermal cycle was performed as follows: once at 94 ° C for 5 minutes, 30 times at 94 ° C for 30 seconds, and at 72° C for 1 minute, then Once at 72 ° C for 5 minutes (14, 15).

Table 1- Primer Sets used in the Identification of C. parvum 18S rDNA

Data Analysis: The data are presented by descriptive statistics and related to factors such as age, sex, location, and contact with animals using the Chi-square test and Fisher's exact test by Sigma stat 4 statistical software. P <0.05 is considered a significant difference.

Results.

The results obtained from the modified Ziehl Neelsen staining showed that out of 400 samples, 14 samples (3.5%) contained Cryptosporidium protozoa (Fig. 1).

Based on the results, the rate of infection was 3.7% and 3.3% in males and females, respectively. The results of the statistical test showed no statistically significant relationship between sex and the rate of cryptosporidium infection (P> 0.05). (Table 2).

Among those who tested positive for cryptosporidium, 12 (5.5%) were under 6 and 2 (1.1%) were over 6 years old. There was a statistically significant difference between the infection in the ages under 6 and more than 6 years old (P <0.05) (Table 3).

Out of 400 samples, 208 samples were related to rural cases and 192 samples were related to urban cases. Among the positive cases of Cryptosporidium, 4 samples (2.1%) were related to urban individuals, and 10 samples (4.9%) were related to rural communities. The results of the statistical test showed a significant relationship between habitat and cryptosporidiosis (P <0.05) (Table 4).

The results showed that the highest prevalence of cryptosporidiosis was in summer (6%) and the lowest was in winter (1%) (Table 5).

Fig. 1- Cryptosporidium oocysts Staining by the Modified Ziehl Neelsen Method

Table 2- Frequency of Cryptosporidium Infection between the Sexes in Children with Diarrhea

Similar letters in each column indicate no statistically significant relation (P> 0.05)

Table 3- Frequency of Cryptosporidium Infection at Different Ages in Children with Diarrhea

Different letters in each column indicate a statistically significant relation (P< 0.05)

Table 4- Prevalence of Cryptosporidium Infection in Urban and Rural Children with Diarrhea

Different letters in each column indicate a statistically significant relation (P< 0.05)

Table 5- Frequency of Cryptosporidium Infection in Children with Diarrhea in Different Seasons

Different letters in each column indicate a statistically significant relation (P< 0.05)

Data analysis showed that 6.7% of infected children had been in contact with the animal in the previous month, and 1.9% had no contact with the animal. The results of the statistical test showed that there was a significant relationship between the contact with the animal and infection with Cryptosporidium (P <0.05) (Table 6).

In the PCR test, the bands of the 676 bp and 285 bp region represent the nucleotide chain selected to confirm the presence of Cryptosporidium parvum contamination in the samples. The results of individuals with positive results for the presence of Cryptosporidium oocysts in their feces showed that all individuals were infected with Cryptosporidium parvum (Fig 2).

Table 6- Frequency of Cryptosporidium Infection in Children with Diarrhea and its Relationship with Animal Contact

Different letters in each column indicate a statistically significant relation (P< 0.05)

Fig. 2- The Nested PCR Test Results to Identify Cryptosporidium Parvum. M marker, CN negative control, CP positive control, 1, 2, and 3 positive samples. 676 bp band belongs to outer primer and 285 bp band belongs to inner primer.

Discussion

Cryptosporidiosis is caused by an obligatory intracellular parasite, which was first identified in 1907 as an opportunistic pathogen. Cryptosporidium infection raises public health concerns in developed and developing countries. Globally, it appears to be more prevalent in the United States, Canada, Australia, and Europe, especially the United Kingdom, Germany, and Ireland. Cryptosporidiosis was first reported in a rural child and an immunocompromised individual and is now reported in more than 90 countries on all continents (16). Studies show that Cryptosporidium parvum and Cryptosporidium hominis are present in children and immunocompromised ones (17). Based on the results of the present study, the prevalence of cryptosporidiosis in children with diarrhea was 3.5%. The infection rate was 3.7% in males and 3.3% in females. Other studies in Iran and other countries also show that the prevalence of this parasite varies depending on the region. In a review study in 2017, information on the prevalence of Cryptosporidium in Iran was comprehensively reviewed and summarized. The prevalence of cryptosporidiosis in children was 3.65% and in other people with diarrhea was 2.94%. Also, the infection rate in healthy individuals and immunocompromised patients was 1.29 and 54.4%, respectively (12). In another study conducted in Tehran, the rate of this infection was 2.57% in patients with gastroenteritis (18). In Urmia, during a study, the prevalence of this parasite in people admitted to the ward of kidney patients was reported between 3.88 to 11.5% (19). In other countries, the prevalence of this parasite has been reported differently, a study in different parts of India in children with diarrhea showed that the prevalence of this parasite is from 2.55% to 18.5% (20). Also in another report from China, the cases of this infection ranged from 8% to 16.44% (21).

The results of the present study showed that the infection rate of Cryptosporidium parasite in children under 6 years is 5.5% and in children over 6 years is 1.1%. Other studies in Iran have also confirmed the relationship between children's age and infection with this parasite. In 2017, Reza Brahmat et al., in a review study reported the prevalence of cryptosporidiosis in children and healthy individuals as 3.65 and 1.29%, respectively (12). In other countries such as India and China, children are more infected with the cryptosporidium parasite than adults (20, 21).  According to the results of the present study, the infection rate of Cryptosporidium parasite in children living in rural areas was 4.9% and in children living in urban areas was 2.1%. The reasons for the higher level of infection in rural areas can be due to two reasons. First, the level of hygiene and compliance with health standards in rural areas is lower than in urban areas, this is evident even in different countries that have different levels of health. Studies show that African, South, and South American countries, Asia, and other parts of the Pacific and Caribbean have the highest prevalence of the infection, while North America and Europe have the lowest prevalence of cryptosporidiosis. Also, in rural areas due to low levels of health, lifestyle, lack of facilities, etc., the prevalence of cryptosporidium is higher (22).

Also, in the present study, it was found that there is a relationship between contact with animals and the rate of infection so among infected children, 6.7% had contact with animals and 1.9% had no history of contact with animals. Other studies have shown an association between contact with animals and infection with Cryptosporidium (23). Cacciò et al.(2006) stated that the most important factor for the transmission of Cryptosporidium parvum is contact with animals (24). The existence of this relationship can also justify the higher level of infection in rural areas than in urban areas because due to the lifestyle and employment of villagers in agriculture and animal husbandry, the possibility of contact with animals is much higher than in urban areas. Studies also indicate the presence of this infection in livestock, especially in calves, sheep, and goats in Iran (18). However, keeping pets such as dogs and cats may also contribute to the spread of cryptosporidiosis in urban areas.

In the present study, it was shown that there is a relationship between the rate of Cryptosporidium infection and the season, so that in hot seasons, especially summer the prevalence was higher (6%) than in cold seasons, such as winter (1%). The other studies summarized the effect of season on the prevalence of cryptosporidiosis (21, 22, 25).

Based on the results of the PCR test, all positive samples were Cryptosporidium parvum. Other studies have shown that parvum species are more prevalent than other species of this parasite. Studies have shown that the most well-known risk factor for Cryptosporidium parvum infection is contact with animals (21, 23).

In conclusion, the results of the present study showed that Cryptosporidium parvum is the predominant species in Chaharmahal and Bakhtiari province, which is more prevalent in children under 6 years old, in warm seasons and rural areas, and in people who have more contact with animals.

Acknowledgments

The authors of this study thank the Vice Chancellor for Research of Shahrekord University for supporting this study.

Conflict of interests

There is no conflict of interest between the authors of this article.