A Review of the Prevalence and Diagnostic Points of Cryptosporidium Species in Immunocompromised and Healthy Human Samples in Iran

Cryptosporidium is a protozoan parasite of the phylum Apicomplexa that infects the margins of the gastrointestinal tract epithelial microvilli in a wide range of vertebrate hosts including humans (1). Cryptosporidium infection (cryptosporidiosis) is recognized as acute and selflimiting gastroenteritis in people with healthy immune systems, while it is regarded as a persistent and lifethreatening infection in people with defective immune systems (2). It is estimated that millions of cases occur annually in developing and underdeveloped countries. This parasite is a critical factor in endemic infections, traveler’s diarrhea, and epidemics (3). Frequent patterns of the disease are described based on age, season, geographical location, and routes of transmission (4). Cryptosporidiosis is more dominant among young children in developing countries that causes severe diarrhea similar to cholera (5, 6). Different species of Cryptosporidium can infect humans and a wide range of animals. In addition, the widespread occurrence of parasitic oocysts in the environment facilitates the acquisition of human infection in different ways (6). Person-to-person transmission may play a major role in the spread of Cryptosporidium infection in children and elderly populations, especially in kindergartens and nursing homes (7). Cryptosporidium is transmitted feco-orally via ingestion of water or food contaminated by parasite oocysts (8) A Review of the Prevalence and Diagnostic Points of Cryptosporidium Species in Immunocompromised and Healthy Human Samples in Iran

Dis Diagn. Vol 10, No 4, 2021 170 http://ddj.hums.ac.ir http which are physically and chemically resistant, can survive in the environment for a long time, and are insensitive to water chlorination (9)(10).

Cryptosporidium Species Isolated From Humans Cryptosporidium parvum
Cryptosporidium parvum is one of the most frequently reported species among mammals and was initially identified in mice. The oocyst of this species is smaller than that of Cryptosporidium muris and, its habitat is usually the small intestine. More than 150 mammalian species have been identified as hosts of C. parvum and other similar parasites. Molecular characterization of the parasite has shown the occurrence of various genotypes with different base sequences and infectivity. DNA of some of the genotypes is now known as a distinct species such as C. hominis and C. parvum as human class II and I genotypes, respectively (11).
Other genotypes of the parasite are found in mice, pigs, marsupials, monkeys, mice, and mink. Currently, most researchers suggest that a species isolated from mammals is called C. parvum (11).

Cryptosporidium hominis
Although Cryptosporidium species that infect humans have previously been referred to as C. parvum, a human genotype (genotype 1) or genotype H has recently been identified as well. Thus, in recent years, studies conducted on biological and molecular differences, have categorized the human Cryptosporidium under a separate species called C. hominis (12,13). These studies have not only demonstrated wide biological genetic differences between C. hominis and C. parvum (bovine genotype or genotype II) but also evidenced that the human species are antigenically more stable than the bovine species (13).

Cryptosporidium meleagridis
Stages of parasite growth were observed on the epithelial cells of the distal third of the small intestine of turkeys (14). A child with diarrhea in Mazandaran province of Iran was identified to be infected by C. meleagridis. In this study, it was found that the last two species (i.e., C. parvum and C. meleagridis) potentially had a shared transmission between humans and animals (15).
C. meleagridis is spherical and measures 4.6 by 5.2 µm with sporozoites, but it is not easily detectable inside the oocyst. The disease is associated with diarrhea, but the mortality rate is low. The parasite completes its entire life cycle on the surface of the intestinal epithelium and does not seem to invade host tissues (16).
Further studies have indicated that, in addition to turkeys, other birds such as chickens and parrots are susceptible to infection induced by C. meleagridis. Likewise, molecular studies have shown that C. meleagridis is a species distinct from other species and is the third most common Cryptosporidium parasite found in humans (17,18).

Important Predisposing Factors for Cryptosporidiosis Age
In tropical and temperate countries, Cryptosporidium is an important agent causing diarrhea in children. The effect of age may be related to the immune status of the young host and the infectivity of the Cryptosporidium species. Epidemiological studies have shown that cryptosporidiosis reaches the peak at two extremes of age. The first is under five years old and the second one occurs in adulthood (twenty to forty years old), which can be due to their occupational contacts (19).

Individual Immune System
As previously stated, cryptosporidiosis is a self-limiting gastrointestinal disorder in people with healthy immunity, while it causes persistent and life-threatening diarrhea in people with immunodeficiency (primary or secondary). Suppression of the immune system for any reason will increase the likelihood of cryptosporidiosis (20).
Other predisposing factors associated with cryptosporidiosis include weather, season, travel, occupation, contact with the animals, nutritional status, and personal health status (21).

Importance of Diagnostic Points
Similar to most intestinal parasites, Cryptosporidium is detected by microscopic examination of the stool. Stool samples are usually stored in 10% formalin. Fresh samples are also used for direct examination, but bear an increased risk of infecting the examiner (22).
Care should be taken when using stool preservatives, for example, polyvinyl alcohol interferes with staining techniques and is usually not recommended. Frozen stool samples and 2.5% potassium dichromate can be used for enzyme-linked immunosorbent assay (ELISA) and for preserving this parasite in stool samples, respectively (23).
Direct diagnosis of this parasite using a microscope is extremely challenging due to its wide spectrum of hosts and discrepancies in its oocyst size compared to other parasites transmitted feco-orally (24).
Cryptosporidium can be found elsewhere in the body that is suitable for parasite development, such as respiratory epithelium. However, the main site of the parasite is the surface of the small intestinal epithelium, causing recurrent and chronic diarrhea as well as diarrhea interspersed with constipation. Therefore, stool and sputum examination are the more preferred ways to diagnose the parasite. However, factors such as small size, transparency, and similarity of the parasite oocyst with other similar parasites make it impossible for direct examination to be considered as a gold standard test (25,26).

Direct Stool Test Method
In this method, the parasite oocyst, which has been obtained by reliable methods of concentration (e.g., Shitter), is detected by a phase-contrast microscope or bright background (27).

Floatation
This method includes the Shitter method or flotation by sugar-water and the use of various salts (i.e., chloride, ZnSo4, and NaCl), which are less sensitive and specific than the Shitter method. Further, they prevent the oocyst wall from rapid crystallization during the direct detection and diagnosis of parasites (28).

Sedimentation
Some references consider the formalin-ether deposition method to be a better option than the shitter flotation method (29). Nevertheless, Pal et al assume that the use of formalin-ethylacetate can reduce the number of oocysts, and the sediment obtained in this method due to having additional materials must be painted by appropriate painting techniques (30).

Smear Staining Methods
Two points are important to be considered in this method: First, the type of sample used (direct or concentrated), and second, the type of smear staining. In the case of the samples, direct, concentrated, or deposited samples can be used depending on the type of work.

Histopathological Method
This was the first test to detect the Cryptosporidium parasite in intestinal tissues. The cell forms of the parasite turn into purple with hematoxylin-eosin staining. Tissues become available only after invasive procedures, and organisms are not always detected on biopsies (32).

Serological Method
This method employs immunofluorescent tests based on monoclonal antibodies (copro-antigen-copro antibodies) that are specific to Cryptosporidium oocysts. The sensitivity of this method is 10 times greater than that of acid-fast dyeing methods. The direct immunofluorescence method using monoclonal antibodies is currently regarded as the gold standard test for fecal samples. The rate of parasite antigen detection using commercial kits in the form of ELISA and immunochromatographic tests is increasing. The ELISA tests are designed with high sensitivity and specificity )25).

Cryptosporidium Culture
Technically, the cultivation of Cryptosporidium faced some challenges in the past, but significant progress has been made in recent years. Today, the asexual stages of the parasite can be produced in antimicrobial systems (33).

Molecular Methods
The inability to identify and differentiate Cryptosporidium at the species and subspecies level delimits our knowledge of natural history, epidemiology, and the risk of zoonosis. Assessing the risks of water and food contamination by oocysts for public health is considered compounded (31). Given the new findings from the Cryptosporidium strains and their apparent similarities, this point becomes more evident and even more important when discussing Cryptosporidium. It is required to identify this parasite in terms of species, genotype, and its relationship with the age of the host via molecular pathways (34). Furthermore, the small size of oocysts in different species of Cryptosporidium with an extremely thin line demarcating their characteristics makes us realize the importance of using molecular methods (34,35).
Currently, the use of such molecular methods as polymerase chain reaction (PCR), PCR-Restriction fragment length polymorphism (RFLP), nested-PCR, and DNA hybridization to detect Cryptosporidium parasites is increasing. These methods have higher sensitivity and specificity than the microscopic methods (36,37).
The application of these methods is especially important in diagnosing clinical and environmental samples with low parasite numbers, detecting species differences and the genetic relationship between Cryptosporidium parasites, and examining the relationship between Cryptosporidium and other Apicomplexa. These methods are also used in epidemiological studies and geographical diversity of parasites (38), The details of which are provided in Table 1 (39-60).

Discussion
The aim of this study was to examine the prevalence and diagnostic points of Cryptosporidium species in immunocompromised and healthy human samples  in Iran, and studies conducted in the last decade were reviewed accordingly. The existence of different methods of transmission and the intricate zoonotic relationship of this parasite makes the epidemiology of cryptosporidiosis a complex entity. Investigating the prevalence of cryptosporidiosis through molecular tools helps in better understanding the risk factors, sources of the infection, epidemiology, transmission, and determination of specific host-species relationships (61).
In most of the above-mentioned studies, stool samples were first examined by the formalin-ether concentration method, and then, after microscopic examination, the modified acid-fast staining would be performed if the test was likely to be positive. Preparation of direct smears from stool or use of formalin-ether precipitate to prepare modified Ziehl-Neelsen staining smear is a common protocol for the microscopic detection of Cryptosporidium oocytes. However, this method requires a skilled technician to distinguish Cryptosporidium oocytes from yeasts and other cyclospora. Further, this method requires a considerable number of oocytes in the stool for better sensitivity. Therefore, there is a need to use a combination of the modified Ziehl-Neelsen staining method and molecular methods to get the best results (49).
In some studies that used molecular methods, after confirmation by staining, the parasite DNA was extracted from the stool samples using a kit for molecular tests. Most molecular studies performed in Iran used PCR, nested-PCR-RFLP, and sequences analysis methods. These studies applied ribosomal 18SsRNA genes, GP60, and TRAP-C2. Most of these studies used the nested-PCR method and the ribosomal 18SsRNA gene sequence methods, which can detect low-count parasites in samples. These methods are recommended to be used in cryptosporidiosis epidemiological studies, environmental studies, and water resources testing (43).
Sharbatkhori et al used the gp60 gene to determine the significant genetic heterogeneity between C. parvum and C. hominis in this gene and found different families in both species (52).
Some of the studies in Iran used ELISA and direct fluorescent-antibody serology methods and evidenced that molecular methods are more sensitive and specific compared to staining and serology methods. Moreover, one study in Iran used the uracil DNA glycosylasesupplemented loop-mediated isothermal amplification technique based on the target gene S-adenosyl methionine synthetase from Cryptosporidium gene to detect Cryptosporidium species in human immunodeficiency virus (HIV)-positive patients. This study tried to eliminate false-positive results following contamination (57).
The obtained results from various studies indicated that C. parvum and C. hominis accounted for 90% of human infections in most areas, and some studies reported C. meleagridis (62).
The prevalence of Cryptosporidium species varies around the world, and the distribution of the species may also vary within a country. Studies on human samples demonstrated the predominance of C. parvum, C. hominis, and C. meleagridis, suggesting that their cycle frequency and routes of disease transmission are higher than that of other species. Therefore, it is possible to target appropriate routes for the transmission and spread of the parasite and control and prevent the spread of this parasite using appropriate strategies specifically aimed at sources of infection (63).
In most Iranian studies, children and immunocompromised adults were most susceptible to the parasites which can be mainly attributed to poor hygiene and weakened immune systems. Subsequently, the growing size of these sub-populations indicated an increased risk of infection in the community. Therefore, it is recommended that safety points, as well as healthcare and hygiene principles be well observed in dealing with sources of infection, especially in children. Further, the lack of effective treatment for this parasite necessitates focusing attention on the principles of parasite prevention and control as well as sources of the infection. Some important points highlighted by most of these studies include the health status of the community and sources of infection (e.g., consumable water) as factors affecting the prevalence (64).
No definitive treatment and proper treatment protocol have yet been proven for the disease. Thus, prevention seems to be the main factor to control this disease, especially in immunocompromised individuals since the development and clinical manifestations of cryptosporidiosis are affected by the individual's level of immunity (65).
Cryptosporidiosis appears with severe and, sometimes, fatal symptoms in children and immunocompromised individuals (66). In some reports, immunodeficiency is the most common risk factor for cryptosporidiosis in humans (39). However, the ability of many protozoan parasites to invade and cause disease in children and immunocompromised individuals, as well as the difficulty to distinguish Cryptosporidium parasites from other pathogenic protozoans via direct microscopic examination are the factors that pronounce the importance of molecular methods, especially parasite genotyping, in parasite identification (67).
Molecular research-based epidemiological studies allow researchers to gain a better understanding of the parasite transmissibility and pathogenicity pathogenic in humans, animals, and environmental samples (68).
Moreover, the use of molecular methods is highly useful in evaluating the common potential of humans and animals for infection by different species of Cryptosporidium (63). More than 35 Cryptosporidium genotypes have so far been identified, some of which have recently been recognized as new species. Human infection by several species of Cryptosporidium has also been reported. Different species of Cryptosporidium are morphologically indistinguishable from each other while being genomically heterogeneous. Therefore, using molecular methods, especially genotyping, is essential to find out the origin of human or animal infections (69).
The results of this study indicated that due to the importance of cryptosporidiosis in people, especially in HIV patients, the need for conducting more studies has arisen in this field. In addition, it is important to identify and select other high-risk individuals who have defective immune systems.

Conclusion
In this review article data were collected regarding the prevalence of cryptosporidiosis in high-risk individuals such as children and immunocompromised individuals. The results revealed that the higher prevalence of C. parvum in Iranian studies in the last 10 years may indicate the transmission of infection from animal sources.