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| ORIGINAL ARTICLE |
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| Year : 2022 | Volume
: 12
| Issue : 2 | Page : 87-93 |
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Evaluation of microscopy and PCR for detection of Dientamoeba fragilis
Mona Mohamed Tolba1, Amal Farahat Allam1, Safia Saleh Khalil1, Wagdy Makram Elshouki2, Amel Youssef Shehab1
1 Department of Parasitology, Medical Research Institute, University of Alexandria, Alexandria, Egypt
2 Department of Zoology, Faculty of Science, University of Alexandria, Alexandria, Egypt
| Date of Submission | 17-Nov-2021 |
| Date of Decision | 09-Feb-2022 |
| Date of Acceptance | 03-Mar-2022 |
| Date of Web Publication | 24-Nov-2022 |
Correspondence Address:
Amel Youssef Shehab
Department of Parasitology, Medical Research Institute, 165 El Horreya Avenue, El Hadara, Alexandria
Egypt
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/tp.tp_93_21
 Abstract | | |
Introduction: Dientamoeba fragilis (D. fragilis) diagnosis is an intestinal protozoan parasite globally found in rural and urban areas and is attracting a growing interest. Its prevalence in stool varies from 0.2% to more than 19% depending upon the population studied.
Materials and Methods: This study was based on the examination of 100 stool samples of randomly referred cases in a rural area in Motobus district, Kafr El-Sheikh governorate, Egypt. Our aim was to investigate the presence of D. fragilis in stool of the examined individuals using conventional polymerase chain reaction (PCR) compared to wet mount and trichrome stain with confirmation of infection by transmission electron microscopy.
Results: D. fragilis was detected in 13/100 of the stool samples examined using wet mount smears, while trichrome stain detected 17/100. Conventional PCR diagnosed 41 cases of D. fragilis in the studied group. A very good agreement was found between wet mount and trichrome stain for diagnosing D. fragilis, while there was fair agreement between conventional PCR and both microscopy methods. Transmission electron microscope was performed on pooled positive samples that revealed the internal structures of D. fragilis trophozoite with its characteristic nucleus.
Conclusions: PCR technique was superior to microscopy for the detection of D. fragilis. Trichrome stain remains vital for microscopic diagnosis.
Keywords: Conventional polymerase chain reaction, diagnosis, Dientamoeba fragilis, trichrome stain, wet mount
How to cite this article:
Tolba MM, Allam AF, Khalil SS, Elshouki WM, Shehab AY. Evaluation of microscopy and PCR for detection of Dientamoeba fragilis. Trop Parasitol 2022;12:87-93 |
| Introduction | |  |
Dientamoeba fragilis (D. fragilis) is a single-celled trichomonad flagellated protozoan parasite with a worldwide distribution, commonly inhabiting the human bowel. The pathogenicity of this organism has remained controversial, however, in recent years, numerous clinical studies have substantiated D. fragilis as a significant enteropathogen with a wide range of symptoms.[1] Some patients may be asymptomatic and others may present with symptoms including nausea, anorexia, malaise, weight loss and the most common are intermittent diarrhea, abdominal pain, and fatigue.[2],[3] Children were reported more susceptible to infection with D. fragilis, and presented with clinical symptoms at higher rates than adults.[4],[5]
The diagnostic tools available for D. fragilis are limited, however, definitive diagnosis is based primarily on microscopy.[6] In wet preparations, D. fragilis appears as a nonspecific rounded mass and the nuclear structure cannot be seen in either saline or iodine preparations. As trophozoites degenerate rapidly, prompt fixation of the specimen is necessary.[7] Successful microscopic diagnosis of D. fragilis is closely associated with utilizing permanent stains of fixed fecal smears. A wide variety of permanent stains have been used, the most common being iron hematoxylin and trichrome stains.[8],[9] However, microscopy usually lacks sensitivity and specificity, moreover, the accuracy for detecting D. fragilis differs from one laboratory to another, as it relies often on the competency of the microscopist as well as on sample preparation.[10]
Novel molecular biology techniques such as polymerase chain reaction (PCR) offer a highly sensitive and specific alternative to traditional microscopy diagnostic approaches.[11] However, these assays are not employed routinely by most diagnostic laboratories. Recent studies have reported the development of a conventional PCR by amplification of the small subunit rRNA gene to detect D. fragilis with remarkable sensitivity and specificity.[12],[13]
To date, only a few studies have described the ultrastructure of D. fragilis trophozoites by transmission electron microscopy (TEM).[7],[14]
The aim of the study was to investigate the presence of D. fragilis in stool of individuals from Motobus district, Kafr El-Sheikh governorate, Egypt, using conventional PCR compared to wet mount and trichrome stain. Transmission electron microscope was used to confirm the obtained results.
| Materials and Methods | | |
The study was carried out on 100 randomly referred individuals from private laboratories in Borg Meghizel village, Motobus district, Kafr El-Sheikh governorate. Sample size was calculated using the OpenEpi free open-source software for epidemiologic statistics using average prevalence for D. fragilis stated in the meta-analysis done by Barratt et al., 2011.[15] Fecal specimens were collected in dry and clean plastic containers. Each sample was divided into two parts: one part for microscopic examination in the Borg Meghizel laboratory health unit by the Parasitology Department technicians from the Medical Research Institute. The remaining part was transferred to the Parasitology Department at the Medical Research Institute and stored at − 20°C for Dientamoeba DNA detection by PCR.
Parasitological study
For each participant, a total of three slides/specimens were examined microscopically for D. fragilis detection at ×100 and ×400 magnifications using wet mount and trichrome stain of fresh stools instantly after sample arrival at the laboratory.[14]
Molecular study
Genomic DNA was extracted from 150 mg of each fecal sample using the DNA Stool Mini Kit (Bioline, UK) following the manufacturer's instructions. The extracted DNA was stored at − 20°C. The fecal-recovered DNA was amplified using a set of D. fragilis-specific primers targeting a 662-bp amplicon size of the 18S SSU rRNA gene – DF1 primer (forward): (5'-CTCATAATCTACTTGGAACCAATT-3', positions 100–123) and DF4 primer (reverse): (5'-CCCCGATTATTCTCTTTGATATT-3', positions 739–761). The PCR reaction included 1 μl of template DNA, 10 pmoles of each primer, 12.5 μl of 2X MyTaq™ red mix (Bioline, UK), and water-free RNase in a reaction volume of 25 μl. The mixture was then transferred into a thermal cycler (Applied Biosystems, USA). The cycling conditions were initial denaturation at 95°C for 5 min, followed by annealing for 1.5 min at 55°C for 1.5 min and final extension at 72°C for 5 min after 35 cycles.[16] Amplified fragments were run on 1% agarose gel with ethidium bromide staining (0.5 μg/ml) and visualized by ultraviolet light. A 100-bp DNA ladder (Bioron, Germany) was included as a size marker. Dientamoeba-positive DNA specimens and distilled water were included in each run of PCR as positive and negative controls, respectively, to validate results.
Transmission electron microscopy
Positive stool samples for D. fragilis were pooled and prepared for TEM according to Banik et al.[7] In brief, heavily infected D. fragilis-positive stool samples were selected and pooled. They were centrifuged at 10,000 × g for 1 min and prepared for TEM. The supernatant was decanted and the pellet was fixed in 5% (v/v) glutaraldehyde in phosphate-buffered saline (0.1 M, pH 7.2) at room temperature overnight. The solution was then centrifuged at 5000 g for 1 minute, the supernatant was decanted and fresh buffer was added to the pellet and washed three times, 5 minutes each. The pellet was put in 1% agarose. The agar blocks were cut, fixed, dehydrated, cut by microtome, stained, and processed as described by Banik et al.[7] Different ultrastructural organelles were revealed and described using TEM (JEOL, JSM-2100 Plus, Japan).
Statistical tests
Statistical analyses were carried out using SPSS software (version 20.0, SPSS Inc., Chicago, IL). P < 0.05 was considered statistically significant. Differences between proportions were evaluated by the Chi-square test associated with Cohen's kappa agreement to test the strength of association: values of 0–0.20 as poor, 0.21–0.40 as fair, 0.41-0.60 as moderate, 0.61–0.80 as good, and 0.81–1 as very good.[17]
| Results | | |
One hundred participants were enrolled in the present study, 42 males and 58 females. The mean age was 10.5 years, ranging from 4 to 57 years. The overall percentage of intestinal parasites as diagnosed by direct wet mount was 71%. Using trichrome stain, a lower infection rate of 64% was found [Table 1]. Cryptosporidium, Cyclospora, Isospora, and Microsporidia were not detected. Blastocystis hominis (B. hominis) showed the highest percentage of infection. D. fragilis trophozoites came second by both techniques; it was detected in 13% and 17% of the patients examined after wet mount and trichrome stain, respectively. Trophozoites had 1 or 2 nuclei. The nuclear chromatin appeared fragmented, and no peripheral chromatin on the nuclear membrane could be seen. The cytoplasm appeared vacuolated containing ingested debris and uniform granules. No D. fragilis cyst stage was detected. Giardia lamblia (G. lamblia) had an equal infection rate (13%). Eggs of A. lumbricoides, A. duodenale, and H. nana were not detected by the trichrome stain method as they are normally distorted during the fixation steps [Figure 1] and [Figure 2]. | Table 1: Intestinal parasitic infections among the 100 studied participants diagnosed by direct wet mount and trichrome stain
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 | Figure 1: Dientamoeba fragilis trophozoite using wet mount (magnification, ×400)
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 | Figure 2: Dientamoeba fragilis trophozoite with trichrome stain (oil immersion, magnification, ×1000)
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Regarding co-infections, D. fragilis was detected solely in two individuals only; one of them was diarrheic. D. fragilis and B. hominis were detected together in nine cases; two of them were symptomatic. D. fragilis was detected together with B. hominis and G. lamblia in four cases; three of them were symptomatic. One case had D. fragilis together with B. hominis and E. coli and complained of bloating. A. lumbricoides was detected in one case in association with D. fragilis and B. hominis complaining of abdominal pain [Table 2]. | Table 2: Intestinal parasitic co-infections with Dientamoeba fragilis among the 100 studied participants diagnosed by direct wet mount and/or trichrome stain
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Molecular detection
PCR analysis outperformed both microscopic examination techniques; it revealed 28 and 24 additional positive stool samples that failed to be detected by wet mount and trichrome stain, respectively. Trichrome stain came next with 17% infection rate, and the lowest infection rate was 13% as diagnosed by wet mount. A total of 44 D. fragilis-infected cases were diagnosed by all three techniques [Figure 3]. | Figure 3: Gel electrophoresis of Dientamoeba fragilis PCR with primers DF1 and DF4. Lanes 1, 2, 3, 4: 662 bp bands specific for Dientamoeba fragilis SSu rRNA. Lane N: Negative control, Lane M: DNA marker
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Distribution of Dientamoeba fragilis infection by age and gender as diagnosed by the three techniques
[Table 3] shows the frequencies of D. fragilis infection in relation to the age of participants. All age groups were infected, and the highest infection rate of dientamoebiasis (50%) was among those aged 11–<21. All other age groups were almost equally infected. Statistically, there was no significant difference among the various age groups. Regarding gender, a higher infection rate in females (51.7%) than males (33.3%) was found, yet the difference was not significant.
Agreement studies
The agreement between both microscopic techniques in detecting D. fragilis infection was very good (k = 0.844). On the other hand, fair agreement between the two microscopic techniques and the conventional PCR method in diagnosing D. fragilis infection was found (k = 0.216 and 0.319) for wet mount and trichrome stain, respectively [Table 4]. | Table 4: Agreements between wet mount, trichrome stain, and polymerase chain reaction for the detection of Dientamoeba fragilis
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Transmission electron microscopy
D. fragilis has a spherical or oval shape with a granular, vacuolated cytoplasm and a nuclear matrix (N). The nuclear matrix contains electron-dense material. Digestive or food vacuoles were commonly found throughout the cytoplasm. Phagosomes were formed which allow digestion of the food particles. Lysosomes were also seen near digestive vacuoles and other cytoplasmic organelles [Figure 4]. | Figure 4: TEM showing mononucleated Dientamoeba fragilis trophozoite. N: nucleus. Dv: digestive vacuole, L: lysosome, Ph: phagosome
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| Discussion | | |
Intestinal parasitic infections caused by protozoa and helminths are common worldwide; Egypt is no exception. The diagnosis of D. fragilis and other protozoa in stool samples needs specific techniques besides expertise.[8]
In the present study, two microscopic stool examination modalities, wet mount and trichrome stain, were used to estimate the total parasitic infection rate among the examined individuals. A low prevalence of helminths was diagnosed by wet mount which highlights efficient control measures, while trichrome stain failed to detect any helminth ova. A. lumbricoides, A. duodenale, and H. nana gave the lowest infection rates (5% and 1%).
As to protozoa, D. fragilis was the second most frequently detected protozoan parasite after B. hominis, with an equal infection rate to that of G. lamblia. The prevalence rates of D. fragilis were 13%, 17%, and 41% as detected by wet mount, trichrome, and PCR techniques, respectively, with no correlation to either age or gender. Such infection rates point to the fact that D. fragilis is a common enteric protozoan parasite that should be considered in any differential diagnosis of gastrointestinal disease. Nearly half of the patients were symptomatic from just gastrointestinal discomfort to diarrhea and the rest were asymptomatic. This was in concordance with Barratt et al., 2011, who stated that in symptomatic patients harboring D. fragilis and no other pathogens, it should be considered the etiological agent and treated as such.[15] Reported prevalence rates of D. fragilis in Egypt varied between 21% and 34% as diagnosed by wet mount method.[18] In agreement with most of the literature, no cyst stage was detected among the examined samples.[19],[20],[21] However, Munasinghe et al. reported that cysts were observed in mice and Stark et al. reported the detection of cyst forms of D. fragilis in human fecal samples.[1],[3],[22],[23]
The results of the present study agreed with those of Pietilä et al. who reported the presence of Dientamoeba in formalin-fixed trichrome-stained samples.[14] On the contrary, Dientamoeba was not found by direct smear or formalin-ether concentration methods as stated by Hamidi et al.[24] Previous studies reported that identification and differentiation of Dientamoeba by direct smear and formalin-ether concentration techniques lack accuracy and are laborious and time-consuming compared to molecular assays.[25],[26]
Molecular PCR techniques offer a highly sensitive and specific alternative to the traditional microscopic approach. By studying the agreement between the three techniques, a very good agreement between wet mount technique and trichrome permanent stain was found, yet trichrome stain is more sensitive compared to wet mount. On the same line, as reported previously in many studies, trichrome stain remains essential for D. fragilis detection.[19],[27]
The agreement between wet mount and conventional PCR was fair; three cases were missed by PCR, while wet mount stool examination missed a total of 31 cases. Again, a fair agreement between trichrome stain and PCR was found; 27 cases diagnosed positive by PCR were missed by trichrome stain. Although three cases were missed by PCR, it showed a higher sensitivity (91%) compared to wet mount and trichrome stain (29% and 31%, respectively).
A possible explanation for the considerable number of patients missed by microscopy is that the majority of patients provided a single stool specimen for examination; if multiple samples were examined, the infection rate may increase. The delay between the time of sample collection and preparation of wet mount and/or permanent staining might play a role in the false-negative results. Similarly, Garcia reported that D. fragilis trophozoites have a survival period of 24–48 h and its morphological features will be missed if the specimen is not examined or maintained in an appropriate fixative as soon as possible after defecation. He also recommended that better diagnostic results could be obtained by using trichrome or iron-hematoxylin-stained fecal smears and examined with an oil immersion objective (×1000). Furthermore, microscopy is influenced by the variable and intermittent shedding of D. fragilis trophozoites, while PCR needs only the DNA of the studied organism not depending on the presence of the whole organism with its characteristic morphology. The few cases missed by PCR might be considered false negatives due to the inhibitors to DNA amplification that are probably expected to exist in stool samples.[27]
Nevertheless, it was reported that even under ideal conditions, with prompt preservation of stool and evaluation by a skilled technologist, permanent stained smears are only 34% sensitive compared to molecular methods.[28] Likewise, many authors confirmed that molecular methods are more sensitive compared to microscopy, Ögren et al. reported that all samples positive by microscopy for D. fragilis were also positive by PCR. Moreover, PCR showed 20% of an increased parasite recovery compared to microscopy.[29],[30]
To confirm the obtained results, TEM was performed on pooled positive samples of D. fragilis to verify the internal structure of the organism. The ultrastructural characterization of D. fragilis trophozoites was comparable to those of Banik et al. and Garcia.[7],[27]
| Conclusion | | |
This study reported a high prevalence of D. fragilis by PCR, highlighting its importance in diagnosis. However, and despite the fact that conventional PCR technique was superior to microscopy in the diagnosis of D. fragilis, trichrome stain remains indispensable for D. fragilis diagnosis in areas of limited resources.
Ethical clearance
The study protocol was approved by the Research Ethics Committee of the Medical Research Institute, Alexandria University, Egypt. The outline of the study was explained to the participants and their consent to submit stool samples were obtained.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]
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