Polymerase chain reaction-based diagnosis of infection with Cryptosporidium in children with primary immunodeficiencies.
BACKGROUND
Patients with deficient cell-mediated immunity are prone to chronic biliary tract infection with Cryptosporidium, which can lead to the development of sclerosing cholangitis and acute cryptosporidiosis after bone marrow transplantation (BMT). The organism is very difficult to detect during asymptomatic periods.


METHODS
PCR techniques were compared with standard microscopy for detecting the organism in such patients. Amplification targets were two fragments of the 18S ribosomal RNA gene (unnested) and part of the Cryptosporidium oocyst wall protein gene (nested and unnested). Twenty eight-patients with primary immunodeficiencies were studied including: CD40 ligand deficiency (13); undefined combined immunodeficiency (10); major histocompatibility complex II deficiency (2); and other defects (3). Samples analyzed included stool, bile and liver tissue.


RESULTS
Of 25 patients tested prospectively, Cryptosporidium could be detected by PCR but not by microscopy in 12, only 3 of whom had a known history of infection. Five of this group had sclerosing cholangitis. Nine of the PCR-positive patients subsequently underwent BMT and 5 developed acute posttransplant diarrhea and cholangiopathy associated with Cryptosporidium excretion. Of the 13 PCR-negative patients, 3 had cholangiopathy (sclerosing cholangitis in 1 and minor changes in 2). Four of these underwent BMT and none developed cryptosporidiosis. In 3 patients, studied only after developing post-BMT cholangiopathy and diarrhea, Cryptosporidium was detected by PCR but not by microscopy. Genotyping and sequencing showed multiple types of Cryptosporidium in approximately one-third of positive cases.


CONCLUSIONS
These results indicate that PCR-based procedures are more sensitive than microscopy for detecting Cryptosporidium in patients with immunodeficiencies.
McLauchlin
James
J
Food Safety Microbiology Laboratory, Public Health Laboratory Service, Central Public Health Laboratory, Great Ormond Street Hospital for Children, London WC1N 3JH, UK.
Amar
Corinne F L
CF
Pedraza-Díaz
Susana
S
Mieli-Vergani
Giorgina
G
Hadzic
Nedim
N
Davies
E Graham
EG
D000293:Adolescent; D000818:Animals; D016026:Bone Marrow Transplantation / Q000276:immunology; D002648:Child; D002675:Child, Preschool; D003457:Cryptosporidiosis / Q000175:diagnosis* / Q000276:immunology*; D003458:Cryptosporidium / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000032:analysis; D005260:Female; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000276:immunology*; D008297:Male; D009894:Opportunistic Infections / Q000175:diagnosis* / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012494:Sampling Studies; D012680:Sensitivity and Specificity; D014181:Transplantation Immunology
D016054:DNA, Protozoan