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High-throughput genotyping assay for the large-scale genetic characterization of Cryptosporidium parasites from human and bovine samples

Published online by Cambridge University Press:  15 November 2013

J. L. ABAL-FABEIRO ,
X. MASIDE ,
J. LLOVO ,
X. BELLO ,
M. TORRES ,
M. TREVIÑO ,
L. MOLDES ,
A. MUÑOZ ,
A. CARRACEDO  and
C. BARTOLOMÉ
J. L. ABAL-FABEIRO
Affiliation:
Departamento de Anatomía Patolóxica e Ciencias Forenses, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Medicina Xenómica, CIMUS, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Xenómica Comparada de Parásitos Humanos, IDIS, Santiago de Compostela, Spain
X. MASIDE
Affiliation:
Departamento de Anatomía Patolóxica e Ciencias Forenses, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Medicina Xenómica, CIMUS, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Xenómica Comparada de Parásitos Humanos, IDIS, Santiago de Compostela, Spain
J. LLOVO
Affiliation:
Xenómica Comparada de Parásitos Humanos, IDIS, Santiago de Compostela, Spain Servicio de Microbioloxía e Parasitoloxía, Complexo Hospitalario Universitario de Santiago de Compostela, 15706 Santiago de Compostela, Spain
X. BELLO
Affiliation:
Departamento de Anatomía Patolóxica e Ciencias Forenses, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Medicina Xenómica, CIMUS, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Xenómica Comparada de Parásitos Humanos, IDIS, Santiago de Compostela, Spain
M. TORRES
Affiliation:
Centro Nacional de Genotipado, Universidade de Santiago de Compostela, Spain
M. TREVIÑO
Affiliation:
Servicio de Microbioloxía e Parasitoloxía, Complexo Hospitalario Universitario de Santiago de Compostela, 15706 Santiago de Compostela, Spain
L. MOLDES
Affiliation:
Servicio de Microbioloxía e Parasitoloxía, Complexo Hospitalario Universitario de Santiago de Compostela, 15706 Santiago de Compostela, Spain
A. MUÑOZ
Affiliation:
Departamento de Microbioloxía e Parasitoloxía, Facultade de Bioloxía, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain
A. CARRACEDO
Affiliation:
Departamento de Anatomía Patolóxica e Ciencias Forenses, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Fundación Pública Galega de Medicina Xenómica (SERGAS)-CIBERER (USC), Complexo Hospitalario Universitario de Santiago de Compostela, 15706 Santiago de Compostela, Spain Center of Excellence in Genomic Medicine Research, King Abdulaziz University, 21589 Jeddah, Kingdom of Saudi Arabia
C. BARTOLOMÉ*
Affiliation:
Departamento de Anatomía Patolóxica e Ciencias Forenses, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Medicina Xenómica, CIMUS, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Xenómica Comparada de Parásitos Humanos, IDIS, Santiago de Compostela, Spain
*
*Corresponding author: CIMUS, Despacho P2D2, Avda. Barcelona s/n, 15782 Santiago de Compostela, Galicia, Spain. E-mail: carolina.bartolome@usc.es
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Summary

The epidemiological study of human cryptosporidiosis requires the characterization of species and subtypes involved in human disease in large sample collections. Molecular genotyping is costly and time-consuming, making the implementation of low-cost, highly efficient technologies increasingly necessary. Here, we designed a protocol based on MALDI-TOF mass spectrometry for the high-throughput genotyping of a panel of 55 single nucleotide variants (SNVs) selected as markers for the identification of common gp60 subtypes of four Cryptosporidium species that infect humans. The method was applied to a panel of 608 human and 63 bovine isolates and the results were compared with control samples typed by Sanger sequencing. The method allowed the identification of species in 610 specimens (90·9%) and gp60 subtype in 605 (90·2%). It displayed excellent performance, with sensitivity and specificity values of 87·3 and 98·0%, respectively. Up to nine genotypes from four different Cryptosporidium species (C. hominis, C. parvum, C. meleagridis and C. felis) were detected in humans; the most common ones were C. hominis subtype Ib, and C. parvum IIa (61·3 and 28·3%, respectively). 96·5% of the bovine samples were typed as IIa. The method performs as well as the widely used Sanger sequencing and is more cost-effective and less time consuming.

Keywords

Cryptosporidium epidemiologyhigh-throughput SNV genotypingMALDI-TOF mass spectrometry
Type
Research Article
Information
Parasitology , Volume 141 , Issue 4 , April 2014 , pp. 491 - 500
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Copyright
Copyright © Cambridge University Press 2013 

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