الفهرس 
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Abstract
Microsporidia are widespread obligatory intracellular eukaryotic parasites,
infecting a diversity of hosts including invertebrates and vertebrates. Intestinal
microsporidiosis is the most common cause of chronic diarrhea in
immunocompromised patients.
Laboratory diagnosis of microsporidiosis was initially based on the
detection of microsporidial spores in stool specimens by light microscopy
using special stains. Electron microscopy, wherever possible, was also used
for detection of micro sporidia. Molecular technique such as polymerase chain
reaction (PCR) became available later for confirmation of micropsoridia
Additionally, species specific PCR can be used to identify different species of
micro sporidia.
However, little is known about the sensitivity or limits of detection for
molecular methods in Egypt, so the present study was undertaken to find out
sensitivity, specificity and accuracy of electron microscopy and modified
trichrome staining for detection of microsporidia in human stool samples
compared to multiplex real-time PCR as a golden standard for detection and to
test the ability of multiplex real-time PCR, electron microscopy and modified
trichrome staining to differentiate between Enterocytozoon bieneusi and
Encephalitozoon intestinalis in human fecal specimens.
The current study included 78 immune compromised patients, suffering
from diarrhea, had attended the internal medicine, pediatrics, oncology and
renal dialysis units in Suez Canal University Hospital, Ismailia city from from
March to November 2011.
Study population was interviewed using the structured questionnaire. Stool
samples were collected from all patients in plastic clean containers and
divided into two parts. The first part was preserved in 10% formalin solution,
stained by Modified trichome and were subjected to light microscopy and
electron microscopy. Second part of stool samples was subjected to DNA
extraction and the extracted DNA was stored at -20°C until used for real-time
PCR.
The me.an age of the studied population was found to be 61.6 years,
ranging from 37 to 77 years old. There were 45 females and 33 males. 53.8%
of the subjects were living in rural areas, while 46.2% were living in urban
areas and all patients had gastrointestinal manifestations.
Diarrhea was the most common presenting complain (98.7%) followed by
abdominal pain (91%), while flatulence was the least common presenting
complain (6.4%). However, among the studied population, 38 patients had had
cancer; bladder cancer and breast cancer were the most common types of
cancer found (10% each), while lymphoma was the least common type of
cancer found among the studied population. Meanwhile, forty subjects had had
associated medical conditions; renal failure and diabetes mellitus were the
most common medical conditions presented in the studied subjects (17.9% and
14.1 %) followed by corticosteroid intake (11.5%) and splenectomy (7.7%).
On the other hand, out of the 78 diarrheal stool samples, twenty cases
(25.6%) were positive for intestinal microsporidial infection by PCR. Light
microscopy with Modified trichome stain showed microsporidial spores in
twenty-two (28.2%) samples whereas; only twelve (15.3%) cases were
detected by electron microscopy. Of these twenty samples positive by PCR,
seven (35%) were identified as Enterocytozoon bieneusi, eight (40%) were
On using of light microscopy with modified trichome staIn VclSUS rcai-tillie
PCR (as a golden standard) for detection of micro sporidia revealed 75%
sensitivity and 87.9% specificity whereas, on using electron microscopy
versus real-time PCR as a gold standard for the diagnosis of microsporidia
infection showed that electron microscopy had 60% sensitivity for detection
of micro sporidia with 1 OO%specificity.
Among the twenty positive samples by real time PCR, the mean threshold
cycles (C, value) for micro sporidia was 26.9±6.5, in 9(45%) of them, C, value
was 20-25. There was a highly statistically significant relationship between C,
value detected by real time PCR and number of spores detected using
modified trichrome staining (p <0.001). In addition, there was a strong inverse
correlation between the two values (r=-0.9), which was highly significant
(p<O.OOl).
from our study, we concluded that Multiplex real-time PCR is the best
diagnostic method for detection of micro sporidia and differentiation between
E. bieneusi and E. intestinalis which was proved by the lower sensitivity and
specificity of electron microscopy and modified trichrome stain when
compared with real-time PCR, also it proved to be the most accurate and time
saving method.