Identification and determination of the viability of Giardia lamblia cysts and Cryptosporidium parvum and Cryptosporidium hominis oocysts in human fecal and water supply samples by fluorescent in situ hybridization (FISH) and monoclonal antibodies.
In the present study, fluorescent in situ hybridization (FISH) and monoclonal antibodies (MAbs) were evaluated for species-specific detection and viability determination of Giardia lamblia, Cryptosporidium parvum, and Cryptosporidium hominis in human fecal and water supply samples. A total of 50 fecal human samples positive for G. lamblia cysts, 38 positive for C. parvum, and 23 positive for C. hominis were studied. Also, 18 water supply samples positive for Giardia spp. and Cryptosporidium spp. by the United States Environmental Protection Agency (USEPA) Method 1623 were studied by FISH and fluorescein isothiocyanate (FITC)-conjugated MAbs. Eighteen percent of the fecal samples parasitologically positive for G. lamblia presented viable and nonviable cysts, and 5% of those positive for Cryptosporidium spp. presented viable and nonviable oocysts. Of the 18 water supply samples analyzed, 6 (33%) presented Giardia spp. viable and nonviable cysts and 2 (11%) presented viable and nonviable Cryptosporidium spp. oocysts. G. lamblia identification was confirmed by polymerase chain reaction (PCR) and sequencing of the beta-giardin gene in the fecal and water samples found positive by FISH and FITC-conjugated MAbs. C. parvum and Cryptosporidium muris were identified, by PCR and sequencing of the small subunit of ribosomal RNA gene, in seven and one water samples, respectively. Our results confirm that this technique enables simultaneous visualization, species-specific identification, and viability determination of the organisms present in human fecal and water supply samples.
Lemos
Vanessa
V
Unidade de Protozoários Oportunistas/VIH e Outras Protozooses, Unidade de Parasitologia e Microbiologia Médicas (UPMM), Instituto de Higiene e Medicina Tropical, Rua da Junqueira, 96, 1349-008 Lisbon, Portugal.
Graczyk
Thaddeus K
TK
Alves
Margarida
M
Lobo
Maria Luísa
ML
Sousa
Maria C
MC
Antunes
Francisco
F
Matos
Olga
O
D000818:Animals; D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan / Q000032:analysis* / Q000276:immunology; D003457:Cryptosporidiosis / Q000469:parasitology; D003458:Cryptosporidium / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D003598:Cytoskeletal Proteins / Q000235:genetics; D016054:DNA, Protozoan / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D005243:Feces / Q000469:parasitology*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D016829:Giardia lamblia / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D005873:Giardiasis / Q000469:parasitology; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D013194:Staining and Labeling; D014867:Water / Q000469:parasitology*; D014881:Water Supply
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D003598:Cytoskeletal Proteins; D016054:DNA, Protozoan; D015800:Protozoan Proteins; C038207:giardin protein, Giardia lamblia; D014867:Water; D016650:Fluorescein-5-isothiocyanate