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Journal of Clinical Microbiology, November 2006, p. 4246-4249, Vol. 44, No. 11
0095-1137/06/$08.00+0     doi:10.1128/JCM.00091-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Cryptosporidium spp. from Children in Kolkata, India

Pradeep Das,^1* Seuli Saha Roy,² Kakali MitraDhar,² Phalguni Dutta,² Mihir K. Bhattacharya,² Abhik Sen,² Sandipan Ganguly,² Sujit K. Bhattacharya,² Altaf A. Lal,³ and Lihua Xiao³

Rajendra Memorial Research Institute of Medical Sciences, Agam Kuan, Patna 800 007, India,¹ National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme-XM, Beliaghata, Kolkata 700 010, India,² National Center for Infectious Diseases, Centers for Disease Control and Prevention, Building 22, Mail Stop F-12, 4770 Buford Highway, Atlanta, Georgia 30334³

Received 16 January 2006/ Returned for modification 24 March 2006/ Accepted 30 August 2006

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The intracellular parasite Cryptosporidium is responsible for severe diarrhea in immunocompromised persons in developing countries. Few studies on the characterization of the parasite in India are available. In this study, molecular characterization of the parasite from diarrheic children was carried out by PCR-restriction fragment length polymorphism analysis. At least three genotypes were identified. Out of 40 positive samples, 35 were positive for C. hominis, 4 were positive for C. parvum, and 1 was positive for C. felis. This study clearly suggests that cryptosporidiosis in this region is caused largely by anthroponotic transmission.

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Cryptosporidium, an intracellular (extracytoplasmic) protozoon once thought to be rare and host specific, is ubiquitous and has multiple hosts such as calves, lambs, foals, piglets, humans, and other vertebrates including fish, birds, and reptiles (4, 7). It is responsible for acute self-limiting diarrhea in immunocompetent persons and life-threatening diarrhea in immunocompromised hosts, particularly in persons receiving immunosuppressive drugs and AIDS patients (3, 5). Among the animal hosts, calves are the most important from a public health point of view, as they may serve as a source of human infection (8, 10). Because of the durability of its oocyst, Cryptosporidium is a significant water- and food-borne pathogen of humans as well as animals.

Because of the lack of clear diagnostic features that allow the differentiation of Cryptosporidium species, we do not have a full understanding of infection sources in humans, the burden of disease attributable to different species or strains/genotypes, and the role of species and strains/genotypes in virulence or transmission in humans (10).

Molecular characterization using isozyme profiles (1), random amplified polymorphic DNA analysis (14), nucleotide sequence characterizations, and PCR-restriction fragment length polymorphism (RFLP) analysis of several genes (2, 20, 21) indicated that this genus is complex and contains at least 16 different species (22). Cryptosporidium hominis has been implicated as the major cause of cryptosporidiosis in humans in most areas (15). Other species that have been found in humans include C. parvum, C. meleagridis, C. felis, C. canis, C. muris, C. suis, and the Cryptosporidium cervine genotype (11, 13, 17).

In India, information on the prevalence of Cryptosporidium species in humans and their role in human disease is meager. An attempt has been made in the present study to genotype Cryptosporidium specimens from children with diarrhea admitted to the B. C. Roy Children's Hospital in Kolkata.

Stool specimens submitted for routine diagnosis of Cryptosporidium and other enteric pathogens from children who attended the diarrhea treatment and training unit of the B. C. Roy Memorial Children's Hospital, Kolkata, in 2003 and 2004 were used in the study. Age and clinical history accompanying the laboratory diagnosis request form were recorded. A total of 1,338 stool samples from diarrheic and nondiarrheic patients up to 5 years of age were collected. Stool samples were first examined under a microscope, and aliquots of positive samples were kept at 4°C in 2.5% potassium dichromate for molecular characterization.

For microscopic examinations, four slides were prepared for each sample, two for routine examination of all enteric parasites and the other two for coccidian protozoan parasites. For Cryptosporidium, air-dried fecal smears were first fixed in methanol and stained by Kinyoun modified acid-fast staining (9) and counterstained by methylene blue. Slides were observed under a microscope at a magnification of x1,000. Cryptosporidium oocysts (size, 4 to 5 µm) are pink against a light blue background.

The Immunofluorescence Easy stain (BTF Pvt. Ltd., Australia) was used to confirm some positive stool samples on a microscopic slide. These slides were examined using a confocal microscope (LSM 510; Zeiss, Germany) at an excitation wavelength of 488 nm and an emission wavelength of 518 nm. The oocysts have an apple green fluorescent color.

For DNA isolation, DNA was extracted from all 40 microscopically positive stool samples by using a QIAGEN (Valencia, Calif.) DNA stool mini kit according to the manufacturer's suggested protocol. The eluated DNA was stored at –20°C for further use.

For species identification, an 830-bp fragment of the small-subunit (SSU) rRNA gene was amplified by nested PCR as described previously (23). To identify the species and genotype of Cryptosporidium isolates present in the sample, the nested PCR products were digested with SspI and VspI endonuclease enzymes. Five microliters of the secondary PCR products was digested in a 25-µl reaction mixture containing 5 U of the enzyme SspI or VspI (Fermentas, Canada) and 2.5 µl of 10x restriction buffer at 37°C for 2 h in a water bath. The digested products were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. Cryptosporidium species and genotypes were diagnosed by comparing banding patterns with those published previously (23).

To confirm the RFLP results, all secondary PCR products that were positive for Cryptosporidium species were sequenced in both directions using an ABI PRISM 3100 genetic analyzer (Applied Biosystems) with forward and reverse primers. Sequences obtained were analyzed and assembled using SEQUENCHER software (Gene Codes Corp.). The obtained nucleotide sequences were used to search the GenBank nucleotide sequence database for sequence similarities using BLAST software (NCBI, Bethesda, MD). Multiple alignments of these sequences were made using the BioEdit program.

Out of 1,338 human stool samples examined from diarrheic and nondiarrheic cases, Cryptosporidium was detected by microscopy in 40 (2.98%) samples, with a prevalence of 4.6% in diarrheic cases and 1.2% in nondiarrhea cases. Among the positive cases with diarrhea, dehydration was observed in 70% of the cases, watery diarrhea was observed in 100% of the cases, fever was observed in 20% of the cases, and vomiting was observed in 30% of cases. Age-specific distribution of cryptosporidiosis showed the highest prevalence in the 0- to 24-month age group compared to other age groups (Fig. 1). The occurrence of cases was high (6.3%) between the months of June and October, when the rainfall was high in both the study years. In contrast, the prevalence in the dry winter season was negligible and low (2.7%) in the summer months (between April and June) (Fig. 2). In diarrheic cases, 8.6% and 5.2% of samples were positive for Cryptosporidium in the rainy season and summer, respectively; in nondiarrheic cases, 3.7% showed cryptosporidiosis during the rainy season. Positivity was related to rainfall irrespective of cases with or without diarrhea.

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FIG. 1. Age-associated distribution of Cryptosporidium isolates in children. Age is represented in months.

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FIG. 2. Seasonal prevalence of Cryptosporidium in children in Kolkata, India. (A) Seasonal prevalence of Cryptosporidium hominis. (B) Seasonal prevalence of Cryptosporidium species other than Cryptosporidium hominis. Rainfall data were obtained from the Kolkata Meteorological Office.

All 40 microscopically positive samples showed amplification in the secondary PCR. RFLP analysis using SspI digestion of nested PCR products yielded three bands of about 444 bp, 247 bp, and 106 bp for all samples except one, which showed bands at the 426-bp and 390-bp regions. The latter banding pattern suggested that Cryptosporidium felis was present (Fig. 3). The digestion of nested PCR products with VspI yielded two distinct bands of 556 bp and 104 bp for most samples, thereby suggesting that Cryptosporidium hominis was present in the sample. In the RFLP analysis of the nested PCR products of four Cryptosporidium samples, VspI digestion showed bands of 628 bp and 104 bp, suggesting the presence of Cryptosporidium parvum. However, VspI digestion of the sample that was potentially positive for C. felis showed bands of 476 bp and 182 bp (Fig. 3), confirming the identification of C. felis by SspI RFLP analysis.

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FIG. 3. RFLP analysis of PCR products. The VspI-and SspI-digested products of the SSU rRNA gene were fractionated on a 2% agarose gel and visualized by ethidium bromide staining. Lanes 1, 2, 5, 6, 8, 9, 10, 11, 12, 13, and 15 are C. hominis; lane 3 is C. felis; and lane 7 is C. parvum. The upper panel is VspI RFLP products, and the lower panel is SspI RFLP products. M, molecular weight marker.

SSU rRNA sequences were obtained from all 40 Cryptosporidium-positive samples. DNA sequencing and nucleotide homology analysis showed that the sequence of C. felis (GenBank accession number DQ899783) present in one sample was identical to the C. felis sequence reported in GenBank under accession number AF159113. Similarly, the sequences of four human stool samples (accession number DQ899782) were identical to that of C. parvum reported under accession number AJ493544, and the sequences from 35 samples (accession number DQ899781) were identical to that of C. hominis reported under accession number AJ493079. There was full agreement in genotyping results between RFLP analysis and DNA sequencing.

Previous studies showed that human cryptosporidiosis is common in Indian children. Our present study from India showed similar observations. The prevalence rates of 4.6% and 1.2% in diarrheic and nondiarrheic cases, respectively, and the peak occurrence of infection in the 0- to 12-month age group are consistent with previous observations of Pal et al. (16) and Das et al. (6) from the same hospital. This finding is also similar to observations from other developing countries (19) but does not agree with observations described previously by Mathan et al. (12), who reported Cryptosporidium infection rates as high as 9.8% in nondiarrheic persons in South India. The reason for the observed difference in results is unclear at this point. The seasonal transmission of Cryptosporidium infection and its association with rainfall have been previously reported (18).

The results described here clearly indicate the potential use of molecular tools for studying the transmission of Cryptosporidium species in India. PCR-RFLP analysis demonstrated the existence of at least three species of Cryptosporidium in humans in India: C. hominis, C. parvum, and C. felis. Of these three species, C. hominis is almost exclusively a human parasite (15, 20, 23, 24). In contrast, C. parvum and C. felis (20, 23, 24) are also responsible for cryptosporidiosis in ruminants (cattle, sheep, and goat) and cats, respectively. The majority of the children in this study came from the urban slums of Kolkata, where they live in overcrowded rooms and belong to low socioeconomic classes with poor hygiene but do not have direct contact with those animals. Thus, direct person-to-person transmission probably played an important role in cryptosporidiosis epidemiology in the children in this study. In addition, most of the infected children consumed either municipality-supplied water or water from wells. A common observation was that the drinking water was stored in wide-mouth vessels. Family members and children usually dipped their hands into the vessels to collect water, which might contaminate the drinking water. Urban slum areas of Kolkata are well known for the presence of all types of enteropathogens including bacteria, viruses, and parasites. These findings suggest that zoonotic transmission of Cryptosporidium is probably occurring infrequently in the Indian population studied.

This is the first report on the molecular typing of Indian Cryptosporidium samples. The findings clearly suggest the existence of three genotypes, including two of potential animal origins. Further genotyping studies with large sample sizes and extensive collection of epidemiological data are needed for a better characterization and understanding of cryptosporidiosis transmission in India.

Nucleotide sequence accession numbers. The sequences obtained were submitted to GenBank, and the following accession numbers were obtained: DQ899781 for C. hominis, DQ899782 for C. parvum, and DQ899783 for C. felis.

 
* Corresponding author. Mailing address: Rajendra Memorial Research Institute of Medical Sciences, Agam Kuan, Patna 800 007, India. Phone: 91-612-2631565. Fax: 91-612-2634379. E-mail: dasp{at}cal2.vsnl.net.in.

Published ahead of print on 13 September 2006.

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Journal of Clinical Microbiology, November 2006, p. 4246-4249, Vol. 44, No. 11
0095-1137/06/$08.00+0     doi:10.1128/JCM.00091-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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• Ajjampur, S. S. R., Gladstone, B. P., Selvapandian, D., Muliyil, J. P., Ward, H., Kang, G. (2007). Molecular and Spatial Epidemiology of Cryptosporidiosis in Children in a Semiurban Community in South India. J. Clin. Microbiol. 45: 915-920 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Das, P. Articles by Xiao, L. Search for Related Content PubMed PubMed Citation Articles by Das, P. Articles by Xiao, L.