الفهرس 
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Abstract
D. fragilis is an enteric protozoan parasite that remains obscure and neglected. Studies utilizing electron microscopy and molecular phylogenetics defined it as a Trichomonad which lacks flagella. It is a pathogenic protozoan parasite which inhabits the gut and causes gastrointestinal disease in human. Because in most studies only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with variants that display similar morphologies but different pathogenicities. There are two major D. fragilis genotypes, genotype 1 and genotype 2 with genotype I being the most common subtype.
A great deal of controversy exists on the mode of transmission of D. fragilis. It has been postulated that E. vermicularis may serve as a vector for D. fragilis. Recently, D. fragilis DNA has been detected in E. vermicularis eggs. Moreover, they found a higher incidence of coinfection with D. fragilis and E. vermicularis than D. fragilis infections alone which further strengthening the hypothesis of D. fragilis transmission via E. vermicularis ova. A recent report described the discovery of a new cyst stage in its life cycle. It was found that fecal-oral transmission of cysts may play a role in transmission of D. fragilis yet many of the specific details remain unidentified.
Globally, the prevalence rates of D. fragilis infection vary depending on the identification tool used. Using the light microscopy, the rates of infections oscillate between 0.4% and 52%. Previous studies done in Egypt reported prevalence rates between 1 and 29.8% using light microscopy and culture techniques. As regards the age distribution, numerous studies reported a higher incidence rate in children less than 18 years old.
It is difficult to identify the trophozoites of D. fragilis morphologically. So, for accurate diagnosis prompt fixation of the clinical specimen is essential as D. fragilis trophozoites degrade rapidly in the environment. Different fixatives and stains have been used over the years to detect D. fragilis in clinical specimens. The fixatives that are currently used in the laboratories are polyvinyl alcohol (PVA) and sodium acetate acetic acid formalin (SAF) along with trichrome and iron Hematoxylin stains, respectively.
There are many in vitro culture media for D. fragilis such as MBD medium; Robinson’s medium; TYGM-9 medium and Loeffler’s medium. Loeffler’s medium, a biphasic medium that is currently used for in vitro cultivation of D. fragilis, has been proven to give higher trophozoite numbers twice that obtained in other media.
The current gold standard for diagnosis is to detect the small subunit rRNA gene of D. fragilis using fresh or frozen infected stool samples by polymerase chain reaction (PCR), which is more rapid, sensitive and specific than microscopy. Few Egyptian studies were done for molecular diagnosis of D. fragilis using conventional and qPCR techniques However, all these studies were done on adults and in a localized area of Suez Canal zone.
The current study aimed to detect D. fragilis as a neglected pathogen in children aged 6-12 years old, complaining of gastrointestinal disturbances by nPCR and compare the results of nPCR with those of light microscopy and culture. One hundred fresh stool samples subjected to the following diagnostic methods, light microscopic examination of iron hematoxylin stained stool smears, culture using a loeffler’s culture medium and n PCR.
Out of the 100 patients that participated in the study, 55 % were aged ≤ 8 years, 45 % were aged >8 years. As regards to the gender, 48 % were from females and 52 % were from males. Out of 100 patients presenting with gastrointestinal manifestations, 41 (41%) presented with abdominal pain, 38(38%) with diarrhea, 16 (16%) with flatulence and 5 (5%) with loss of appetite. All stool samples were examined macroscopically. As regards to consistency (38%) had formed stool, (44%) had loose stool and (18%) had semi- formed stool.
Examination of the 100 stool samples by direct smear, formalin-ethyl acetate concentration technique, iron hematoxylin stain and modified Ziehl Neelsen stain(mZN). Out of one hundred stool samples, 69(69%) were parasite-free and 31 (31%) revealed parasitic infections other than D. fragilis when examined by direct smear (saline and iodine wet mount). Of these, 8 had G. lamblia, 2 H. nana, 6 E. coli, 9 B. hominis”, 3 cases had mixed infections “B. hominis with E. coli and 3 cases had mixed infections G. lamblia with B. hominis.
After formalin-ethyl acetate concentration technique, out of 100 stool samples cases 52 (52%) were parasite-free and 48 (48%) revealed other parasitic infections, i.e. 17 of the cases who gave negative results by direct smear method exhibited parasitic infections when subjected to concentration technique, staining with iron- hematoxylin stain and mZN stain. Of these 17 cases, 2 cases (B. hominis), 2 cases of mixed infection (B. hominis and E. coli), one case (Iodamoeba butschlii),4 cases (Endolimax nana), one case (mixed infection G. lamblia and Endolimax nana) and 8 cases (Microsporidia).
Out of 100 iron- hematoxylin stained stool smears ,7samples (7%) were positive for D. fragilis. Out of 100 fecal samples subjected to be cultured in Loeffler’s culture medium, 2 (2%) were positive for D. fragilis while out of 100 DNA extracted stool samples,4 samples (4%) were successfully amplified but 96 samples gave negative results. To independently compare all three methods for the detection of D. fragilis, an extended reference standard was used for true positive stool samples. We considered true positive samples as those samples positive for D. fragilis by two or more diagnostic methods. A true-negative result was defined as a specimen with at least two negative results among three available tests.
Based on the obtained results, the frequency of D. fragilis among the study population was 4% (4/100) by nPCR. nPCR showed the highest sensitivity and specificity 100%. Culture exhibited 50% sensitivity, 100% specificity while light microscopy showed 50% sensitivity, 95% specificity. The correlation between the results of iron hematoxylin stain, culture and nPCR used for detecting D. fragilis in stool samples revealed that there was a moderate agreement between nPCR and both culture and microscopy (k=0.41). The statistical difference between nPCR and both culture and microscopy for the detection of D. fragilis infection was significant (p-value <0.05).
There was no statistically significant association in regards to gender, age and D. fragilis infection (p-value >0.05). As regards to clinical manifestations, there was no statistically significant association between D. fragilis infection and clinical manifestations (p-value >0.05). As regards to stool consistency, there was no statistically significant association between D. fragilis infection and stool consistency (p-value >0.05).
In conclusion, the present study has demonstrated that n PCR assay has the highest performance compared to microscopy and culture in the diagnosis of D. fragilis infection. Culture cannot be applied for routine diagnosis, because the methods for in vitro culture are often complex, their quality control is difficult, need repeated subcultures for better yield. Also, the results of culture need to be confirmed by staining. NPCR is recommended as the diagnostic method of choice for detection of D. fragilis in stool samples. If the PCR technique is not available in the diagnostic laboratory, it is recommended that at least two different diagnostic techniques should be used for detection of D. fragilis infection. These results showed the need to be cautious when interpreting prevalence reports, especially for studies that rely solely on microscopy. The detection rate for D. fragilis in 100 symptomatic children resident in different rural areas belonged to Naser center, Beni-Suef governorate, Egypt was 4%.