Molecular Characterization of Cryptosporidium spp. in Children from Mexico

Cryptosporidiosis is a parasitic disease caused by Cryptosporidium spp. In immunocompetent individuals, it usually causes an acute and self-limited diarrhea; in infants, infection with Cryptosporidium spp. can cause malnutrition and growth retardation, and declined cognitive ability. In this study, we described for the first time the distribution of C. parvum and C. hominis subtypes in 12 children in Mexico by sequence characterization of the 60-kDa glycoprotein (GP60) gene of Cryptosporidium. Altogether, 7 subtypes belonging to 4 subtype families of C. hominis (Ia, Ib, Id and Ie) and 1 subtype family of C. parvum (IIa) were detected, including IaA14R3, IaA15R3, IbA10G2, IdA17, IeA11G3T3, IIaA15G2R1 and IIaA16G1R1. The frequency of the subtype families and subtypes in the samples analyzed in this study differed from what was observed in other countries.


Introduction
Cryptosporidiosis is a parasitic disease caused by Cryptosporidium spp. These parasites belong to the phylum Apicomplexa and are intracellular protozoa that infect mammals, birds, reptiles and amphibians. They are cosmopolitan, mostly affecting people with immunodeficiency and, in some cases, can be deadly. In immunocompetent individuals, Cryptosporidium causes acute diarrhea, usually self-limited, nausea, vomiting, loss of appetite, weight loss and fever [1][2][3][4][5]. In infants, infection with Cryptosporidium spp. may cause malnutrition and permanently affect growth, resulting in a functional decline in physical fitness and cognitive ability [6][7][8][9][10]. Parasite transmission occurs via the fecal-oral route through the ingestion of contaminated water or food, person-person contact, or animal contact [11][12][13].
In Mexico, there have been very few studies of human cryptosporidiosis [41][42][43], and none of these determined Cryptosporidium species and subtypes. The objective of this work was to characterize Cryptosporidium spp. identified in stool samples of children in Mexico.

Ethics Statement
The protocol of this project was approved by the Ethics Committee of Hospital Infantil del Estado de Sonora. Informed consent was obtained from each Cryptosporidium-infected patient who voluntarily participated after a clear explanation of the research objectives. Parents or guardians signed consent on behalf of the children enrolled in this study. Samples obtained from Hospital Infantil Federico Gómez in Mexico City were originally submitted for parasitic analysis, positive samples to Cryptosporidium included in this study were kindly provided by Dr. Rosa Maria Bernal. The stool samples of infants were carriers of Cryptosporidium spp., diagnosed on microscopic observation of oocysts [44]. The inclusion criteria of participation was: Cryptosporidium spp. infected patients regardless of age, gender, with or without clinical symptoms and patients who consented to the study, whereas the exclusion criteria were those who were not Cryptosporidium spp infected and who did not give their consent to participate in the study. Clinical data were obtained from patient's medical record with patient's consent and permission from health authorities. Fecal samples were stored at 4uC for further analysis.

Stool Samples
Stool samples were analyzed from 12 children (2 girls and 10 boys) from 7 months to 14 years of age who were carriers of Cryptosporidium spp. as diagnosed on microscopic observation of oocysts [44]. Four samples were obtained from the Hospital Infantil del Estado de Sonora, and 8 were obtained from the Hospital Infantil Federico Gómez in Mexico City (Table 1). They were diagnosed as Cryptosporidium-positive by modified Kinyoun method described by Henriksen [44]. The cases included in this study occurred from October 2010 to July 2013 (Table 1).

Oocyst Concentration
1 to 2 g of feces was homogenized with 10 ml of physiological saline solution (PSS). The suspension was filtered through cheesecloth into a 15-ml conical tube and centrifuged at 2,000 rpm for 1 minute. The supernatant was decanted, and the pellet was resuspended with 15 ml of PSS. The process was repeated 2 to 3 times until the supernatant was clear. Then, 10 ml of 5% formalin was added to the supernatant, mixed and allowed to stand 10 minutes. Next, 5 ml of ethyl acetate was added, and then the tube was capped and shaken vigorously for 30 seconds, uncovered carefully and centrifuged at 1,500 rpm for 2 minutes. A wooden applicator was inserted into the tube to release the border of the layers. Carefully, the layers were decanted without disturbing the sediment. The sediment was resuspended in 3 ml of 0.2 N NaOH. The mixture was incubated at 37uC for 30 minutes, washed twice with PSS and centrifuged at 2,000 rpm. The PSS was removed with a pipette and, the number of oocysts in the sediment was determined by staining of a smear of the sediment using the Kinyoun method described above.

DNA Extraction
DNA extraction was performed directly from ,200 ml of stool or ,200 ml of the oocyst concentrate using the QIAamp DNA Stoll Mini Kit (QIAGEN Inc, Valencia, CA) following the recommendation of the supplier after 5 cycles of freezing and thawing (270uC to boiling) of the oocysts. The extracted DNA obtained was stored at 220uC until further processing. As positive controls, we used DNA preparations (one each) from C. parvum and C. hominis from HIV patients in Peru, previously identified by PCR-RFLP analysis of the SSU rRNA gene and DNA sequencing of the gp60 gene [45].

Molecular Characterization of Cryptosporidium
The molecular characterization of Cryptosporidium spp. was conducted by nested PCR analyses of 3 molecular markers: the small subunit rRNA (SSU rRNA) [21], COWP gene and GP60 [38], generating products that were of 826 to 864, 540 and 350 bp, respectively. To determine the species of Cryptosporidium, the nested PCR product of the 18S rRNA gene was digested using the VspI restriction enzyme (Promega, USA) [21]. To identify subtype families and subtypes of Cryptosporidium, PCR products of the GP60 gene were sequenced and subtype families were named as proposed by Strong et al., [35]. Each species of Cryptosporidium identified was assigned a Roman numeral; C. hominis was assigned I and C. parvum II. After indicating the species, the subtype family was identified with a lower case letter. For C. hominis the subtype families included Ia, Ib, Id, Ie, If and Ig; for C. parvum the subtype families included IIa, IIb, IIc, IId, IIe, IIf, lih, IIi, IIj, IIk. Subtypes within each subtype family were named according to the nomenclature proposed by Sulaiman [34], depending on the trinucleotide (TCA, TCG, and TCT) encoding the amino acid serine. Each time these sequences were repeated, they were assigned the capital letters A, G, and T, respectively. For C. parvum subtype family IIa, Sulaiman [46]

Age and Gender of Children with Cryptosporidiosis
Of the 12 children included in the study, 83% were boys (10/ 12) and 17% were girls (2/12). Of the children infected with C. hominis, 75% (9/12) were 0 to 4 years of age. The only 2 cases of C. parvum were boys, ages 5 and 9 years old, who were also diagnosed with HIV ( Table 1).

Species of Cryptosporidium Identified
After determining the presence of Cryptosporidium using the Kinyoun method, the SSU rRNA gene was amplified and nested PCR products were digested with the restriction enzyme Vspl, producing the characteristic bands of C. hominis (102/104 bp and 561 bp) in 10 children (2 girls and 8 boys) and C. parvum (102/ 104 bp and 628 bp) in 2 boys (Figure 1), this result was confirmed by sequence analysis of the nested PCR products. The COWP gene was amplified and nested PCR products was confirmed by sequence analysis (Figure 2).

Discussion
In this work, we determined the species, subtype families, and subtypes of Cryptosporidium in stool samples in the state of Sonora and Mexico City. Of the 12 cases of cryptosporidiosis included in this study, we identified C. hominis in 10 cases and C. parvum in 2 cases. Our results are consistent with findings in developing countries where C. hominis is considered the predominant species in humans [47][48][49]. Despite the small number of Cryptosporidiumpositive specimens in this study, we identified all 4 common subtype families of GP60 in C. hominis, including Ia, Ib, Id and Ie. Previously, Ib was the most frequently identified C. hominis subtype family [36], although in this study Ia and Ie were identified in 8 of 10 C. hominis samples. In this study, subtype IbA10G2 was only identified in one 9-month-old child, although it is the most common Ib subtype (88.5% within subtype Ib). The IbA10G2 subtype is considered the most common cause of outbreaks of waterborne cryptosporidiosis [16,36,50,51], and has been identified in humans in Africa [39], Asia [34], Australia [50,52], and in cattle in South American [16,36,50,53].
The Id subtype, which was only identified in a 3-year-old child, was IdA17. There have been a few reports of this subtype isolated in humans in Australia (Western Region), Netherlands and Kenya (Nairobi) [54,55]. In contrast, the IeA11G3T3 is the most  prevalent Ie subtype [36]. In this study, IeA11G3T3 was one of the most prevalent and was identified in 3 patients in Sonora. These results agreed with the data by other researchers in India (Kolkata) [56], United Kingdom [57], US [58], Australia [52], Kuwait [34], Ecuador, Pakistan and Uganda [37,56] and no reports in animals. In this study, 2 subtypes of family subtype Ia were identified: IaA15R3 (in 2 patients) and IaA14R3 (in 3 patients).
Most of the subtypes found in this study have been previously reported in humans in various countries, except IaA15R3 subtype, which has only been reported once by Hadfield in 2011 in a patient in the United Kingdom (GenBank HQ149032) [57].
In this study, 3 children participated in the study were diagnosed with HIV; unfortunately one of the children died. Two of the three children were infected with C. parvum; the child who died had IIaA15G2R1, and another child had the IIaA16G1R1 ( Table 1). The two subtypes have been identified in farm animals [59][60][61][62] [62][63][64][65]. In addition, IIaA15G2R1 e has been reported as the most common subtype in HIV+ patients in Malaysia [66], and is commonly seen in humans in other countries such as Australia [50], Egypt [67] and the Netherlands [63]. The IIaA16G1R1 subtype has also been identified in humans in Slovenia [65] and the US [68,69]. The subtype family IIa is the most common C. parvum (57.8%) and is the second most frequently reported subtype family in humans (25.5%), with a global distribution (26 countries) [36].
In conclusion, in this work, C. hominis was the predominant species in 12 Cryptosporidium-positive children analyzed in Mexico. The frequency of the C. hominis subtypes identified in this study appear to be different from what was reported in other areas of the world. However, additional studies with a larger sample size in multiple states are needed to determine the subtypes of C. hominis and C. parvum in the country, better understand the transmission of cryptosporidiosis in humans, and assess the role of zoonotic transmission in cryptosporidiosis epidemiology.