High-throughput genotyping assay for the large-scale genetic characterization of Cryptosporidium parasites from human and bovine samples.
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.
Departamento de Anatomía Patolóxica e Ciencias Forenses, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
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Abal-Fabeiro
Departamento de Anatomía Patolóxica e Ciencias Forenses, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
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Maside
Xenómica Comparada de Parásitos Humanos, IDIS, Santiago de Compostela, Spain.
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Llovo
Departamento de Anatomía Patolóxica e Ciencias Forenses, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
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Bello
Centro Nacional de Genotipado, Universidade de Santiago de Compostela, Spain.
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Torres
Servicio de Microbioloxía e Parasitoloxía, Complexo Hospitalario Universitario de Santiago de Compostela, 15706 Santiago de Compostela, Spain.
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Treviño
Servicio de Microbioloxía e Parasitoloxía, Complexo Hospitalario Universitario de Santiago de Compostela, 15706 Santiago de Compostela, Spain.
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Moldes
Departamento de Microbioloxía e Parasitoloxía, Facultade de Bioloxía, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
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Muñoz
Departamento de Anatomía Patolóxica e Ciencias Forenses, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
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Carracedo
Departamento de Anatomía Patolóxica e Ciencias Forenses, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
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Bartolomé
D000483:Alleles; D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000453:epidemiology* / Q000469:parasitology; D003457:Cryptosporidiosis / Q000453:epidemiology* / Q000469:parasitology; D003458:Cryptosporidium / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D016054:DNA, Protozoan; D005819:Genetic Markers; D015800:Protozoan Proteins