Diagnosis of Enterocytozoon bieneusi (microsporidia) infections by polymerase chain reaction in stool samples using primers based on the region coding for small-subunit ribosomal RNA.
OBJECTIVE
Enterocytozoon bieneusi is the most prevalent microsporidian causing chronic diarrhea in patients with acquired immunodeficiency syndrome. The current methods used for routine diagnosis of infections caused by microsporidia are based on microscopic detection of the microorganism spores in stained smears. We evaluated the usefulness of the polymerase chain reaction (PCR) technique as a tool to diagnose Enterocytozoon bieneusi infections, using the species-specific diagnostic primer pair EBIEF1/EBIER1 on stool samples that were also analyzed by optical microscopy.


METHODS
To perform PCR in such samples, we developed a novel protocol to obtain DNA free of PCR inhibitors. This protocol was based on disruption of spores using glass beads and overnight digestion with proteinase K; final purification was accomplished with the RapidPrep Micro Genomic DNA isolation Kit for Cells and Tissues (Pharmacia Biotech Inc, Piscataway, NJ). We also evaluated this approach on aliquots of a sample fixed in formalin from 1 to 10 days. PATIENTS AND SAMPLES: We evaluated the PCR technique on 64 stool samples obtained from patients with acquired immunodeficiency syndrome who had persistent chronic diarrhea. Patients were from Spain, Brazil, Germany, and the United States.


RESULTS
Using this approach, we could confirm the presence of E bieneusi in all 17 positive samples; no false-positive results were observed. We could also amplify E bieneusi DNA in 10 aliquots of one sample fixed up to 10 days in 10% formalin.


CONCLUSIONS
We conclude that PCR technology is very suitable for species identification of microsporidia in stool samples and may have a potential application in prospective studies in formalin-fixed samples.
da Silva
A J
AJ
Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341-3724, USA.
Bornay-Llinares
F J
FJ
del Aguila de la Puente
C del A
Cdel A
Moura
H
H
Peralta
J M
JM
Sobottka
I
I
Schwartz
D A
DA
Visvesvara
G S
GS
Slemenda
S B
SB
Pieniazek
N J
NJ
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry*; D016054:DNA, Protozoan / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005243:Feces / Q000469:parasitology*; D005404:Fixatives; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D007411:Intestinal Diseases, Parasitic / Q000175:diagnosis* / Q000469:parasitology / Q000473:pathology; D016816:Microsporida / Q000235:genetics / Q000302:isolation & purification*; D016881:Microsporidiosis / Q000175:diagnosis* / Q000469:parasitology / Q000473:pathology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000032:analysis*
D017931:DNA Primers; D016054:DNA, Protozoan; D005404:Fixatives; D012335:RNA, Ribosomal; D005557:Formaldehyde