Development of a real-time PCR assay for quantitative detection of Encephalitozoon intestinalis DNA.
A new real-time PCR assay for quantitation of Encephalitozoon intestinalis DNA was developed which used a TaqMan fluorescent probe for specific detection. Serial dilutions of E. intestinalis spore suspensions obtained from tissue culture were used as external standards. The detection limit of the technique was 20 spores per ml, with a good interassay reproducibility (coefficient of variation of 7.1% for the suspension containing 20 spores/ml, 5.0% for the suspension containing 75 spores/ml and below 3.5% for higher concentrations). Quantitative detection of E. intestinalis DNA was similar whether the serial dilutions of spores were made in distilled water or in a stool suspension, allowing the use of the assay for stool specimens. The assay was then applied to 14 clinical specimens from 8 immunocompromised patients with proven E. intestinalis infection. The quantitation of the parasitic burden was achieved in stools, blood, urine, tissue biopsies, and bronchopulmonary specimens. The highest parasitic burdens were noted in stools, urine, and bronchopulmonary specimens, reaching 10(5) to 10(6) spores/g or ml. Dissemination of the infection was also evidenced in some patients by demonstration of E. intestinalis DNA in blood and serum. We conclude that real-time PCR is a valuable tool for quantitation of E. intestinalis burden in clinical specimens.
Menotti
Jean
J
Laboratory of Parasitology-Mycology, Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, and Faculté de Médecine Lariboisière-Saint-Louis, Université Paris VII, Paris, France. jean.menotti@sls.ap-hop-paris.fr
Cassinat
Bruno
B
Sarfati
Claudine
C
Liguory
Olivier
O
Derouin
Francis
F
Molina
Jean-Michel
JM
D017088:AIDS-Related Opportunistic Infections / Q000175:diagnosis / Q000469:parasitology; D000818:Animals; D016054:DNA, Protozoan / Q000032:analysis*; D016819:Encephalitozoon / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D016890:Encephalitozoonosis / Q000175:diagnosis* / Q000469:parasitology; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007411:Intestinal Diseases, Parasitic / Q000175:diagnosis* / Q000469:parasitology; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D033761:Spores, Protozoan / Q000302:isolation & purification; D019914:Taq Polymerase
D016054:DNA, Protozoan; D005456:Fluorescent Dyes; D019914:Taq Polymerase