Cryptosporidium ubiquitum in Venezuela: First report in a paediatric patient with acute diarrhoea

The enteric protozoa parasite Cryptosporidium is a significant contributor to the global burden of childhood diarrhoea worldwide.¹ In Venezuela Cryptosporidium infections have been previously documented in community and adult HIV patient populations,^2,3 whereas the prevalence of cryptosporidiosis has been reported in the range of 7–89% in children under five years old.^4,5 In the only molecular study conducted to date, three Cryptosporidium species (C. hominis, C. parvum, and C. canis) were found infecting HIV-positive patients.⁶

A 19-month-old male child was admitted in January 2014 complaining of gastrointestinal symptoms compatible with cryptosporidiosis, including acute, non-bloody watery diarrhoea, abdominal pain, and weight loss. The patient lived in a peri-urban dwelling endowed with public drinking water supply and sewage system. He maintained frequent contact with the poultry rose in the family smallholding, but no other potential risk factors for Cryptosporidium infection could be identified.

Search of enteric protozoan and helminth parasites was performed by microscopy on wet mounts (saline and 1% Lugol's iodine solutions, Giemsa staining) of fresh faecal samples concentrated by the formol–ether sedimentation method of Ritchie. The modified Ziehl–Neelsen stain was also used for the specific detection of coccidian, including Cryptosporidium.

Total DNA was extracted from concentrated faecal material and subjected to downstream molecular methods (PCR and sequence analyses of the obtained amplification products). The identification of the Cryptosporidium species was carried out by a nested-PCR protocol targeting a partial fragment of the small-subunit ribosomal RNA (SSU rRNA) gene of the parasite.⁷ Molecular characterization at the subtype level was conducted by PCR amplification of the near complete sequence of the gene codifying for the 60-kDa glycoprotein (GP60) of C. ubiquitum.⁸

Microscopic examination of modified Ziehl–Neelsen-stained faecal smears showed 60–80 Cryptosporidium oocysts per field examined. No other enteric parasite species could be detected, neither in fresh preparations with 1% Lugol's iodine solution nor in Giemsa-stained faecal smears. The SSU rRNA PCR generated the expected amplicon of 470–491 bp. Sequence analysis of the corresponding PCR product confirmed the presence of C. ubiquitum. We also managed to successfully amplify the GP60 gen of C. ubiquitum, obtaining the expected product of ∼948 bp. Subsequent sequence analyses allowed the assignment of this isolate to the XIId sub-type family of C. ubiquitum. The nucleotide sequence data obtained in this study at the SSU rRNA and the GP60 loci were deposited in GenBank under accession numbers KP899827 and KP899828, respectively.

This case represents the first report of cryptosporidiosis by C. ubiquitum in Venezuela, and the second in the whole South America region after the identification of this Cryptosporidium species in a human isolate from Peru.⁹C. ubiquitum, previously known as the Cryptosporidium cervine genotype, the W4 genotype or genotype 3, was formally described and proposed as a taxonomically valid species in 2010.⁹ Because of its wide distribution and broad host range, C. ubiquitum has been proposed as a zoonotic pathogen emerging in humans, with cases described in Canada, New Zealand, Peru, Slovenia, Spain, Turkey, UK, and USA.^8–10

Molecular discrimination at the sub-type level of C. ubiquitum isolates of animal, human, or environmental origin has been based on the intrinsic genetic heterogeneity of the GP60 gen, allowing the identification of six sub-type families (XIIa-XIIf).⁸ In this recent study, sequence analyses of 170 C. ubiquitum isolates from animal and human samples revealed host adaptation at this locus. For instance, all field specimens (n = 70) from domestic and wild ruminants in both the Old and New World belonged to the XIIa sub-type family, whereas human infections (n = 41) were predominantly caused by the sub-types XIIa, XIIb, and XIId.⁸ Our molecular data are in agreement with those preliminary findings, conclusively demonstrating that the paediatric case reported here was infected by the sub-type XIId of C. ubiquitum. This isolate showed 100% sequence identity to that found in a human patient from USA (GenBank accession number JX412922). To date, the sub-type XIId has been only found in 14 human samples worldwide, including 12 cases in USA⁸ and an additional 2 in UK¹⁰. Based on the epidemiological and environmental information currently available, it remains unclear whether the C. ubiquitum infection of this paediatric patient was attributable to zoonotic or anthroponotic transmission.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This study was funded by the Carlos III Institute of Health, Ministry of Economy and Competitiveness under projects CP12/03081 and PI13/01103. The authors are grateful to Eyes Araque and Begoña Bailo for technical assistance.