الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Blastocystis spp. are the most common eukaryotes reported to colonize humans, yet,
remain an enigma on many levels. Despite having been described more than 100 years ago,
the question of whether Blastocystis causes disease or is a commensal of the human gut
still has no definitive answer. Moreover, its genetic diversity, host specificity and zoonotic
potential remain very incomplete.
The prevalence of Blastocystis has been reported in many parasite surveys performed
across the world. Published infection rates fall anywhere between 0.5 and 62%. High
prevalence of the parasite was observed in Egypt (33%). The diagnostic technique used is a
potentially significant variable that can influence the reported prevalence as microscopy is
generally thought to be less sensitive than culture.
The zoonotic possibility of Blastocystis transmission is enhanced by its higher
prevalence among animal handlers, zoo-keepers and abattoir workers, indicating that
animals may pose a significant zoonotic source of Blastocystis for humans.
For Blastocystis genotyping, SSU-rDNA barcoding is highly applicable, easily
detectable, sensitive, well conserved within species but shows variations between subtypes
and it was recommended as the best method for Blastocystis subtyping and phylogenetic
analysis
Genus Blastocystis was found to comprise at least 17 subtypes based on SSU rDNA
analysis. Of the nine definite STs detected in humans, only four are common – ST1, ST2,
ST3, and ST4; making up around 90% of all human Blastocystis. Other STs (5–9), were
most frequently found in non-human hosts and may be zoonotically transmitted.
Summary and Conclusion
74
The present work aimed to detect Blastocystis spp. in stool samples of humans and
cattle using direct wet mount smears and in vitro culture technique with molecular
characterization of the cultured isolates.
One hundred thirty-six individuals and 190 cattle from a rural area in Kafr El-Sheikh
Governorate, Egypt were recruited for this study after making informed consent and
obtaining ethical approval. Personal information as name, age, gender and clinical history
were recorded. Also, data on animal origin whether household or cattle farms were collected.
Stool samples were collected from humans and cattle. At the Parasitology Department of the
MRI; part of each stool sample was immediately processed by direct wet mount technique;
another part was directly cultured in Jones’ medium for detection of Blastocystis..
DNA was extracted from each Blastocystis positive culture using ZR Fecal DNA
MiniPrep™kit (Zymo Research Corp.) according to manufacturer’s instructions. The
extracted DNA was stored at -20ºC until use. Extracted DNA samples were subjected to
PCR amplification using BhRDr and RD5 primers followed by visualization by
electrophoresis on agarose gel stained with ethidium bromide. The amplicons with a length
of approximately 600 bp (SSU-rDNA barcode region) were excised from the gel and DNA
was purified using the QIAamp Gel Extraction Kit (Qiagen, Hilden, Germany) according
to the manufacturer’s recommendations
Cycle sequencing was performed with BhRDr and RD5 primers using BigDye
Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Darmstadt, Germany)
according to the recommendations of the manufacturer. Sequencing products were
analyzed with a Genetic Analyzer ABI PRISM 3130 (Applied Biosystems). Based upon
sequence data, a split network tree was constructed with cluster tree neighbour-joining
analysis using the bioinformatics tools of Geneious R10.2.3 analysis.
Summary and Conclusion
75
The data were entered, verified and analyzed using SPSS software using appropriate
statistical tests. Difference and association were considered statistically significant at P< 0.05.
1) Findings related to Blastocystis infection in human
B. hominis was detected by direct wet mount and/or culture technique in 39% of
participants; the majority (88.6% ) were singly infected with B.hominis, while only 11.4%
were coinfected with other parasites. There was a good agreement between direct wet
mount and culture techniques in the diagnosis of B.hominis.
Individuals aged 30 years or over had higher rate of Blastocystis infection compared
to the younger age groups, but the difference was not statistically significant. Moreover,
being male or female is not considered a risk factor for acquiring the infection.
A lower B. hominis infection rate (33.8%) was observed among individuals who were in
contact with animals compared to those who had no animal contact (44. %). Yet, the difference
between the two groups was not statistically significant.
Among Blastocystis infected subjects, the majority (66 %) were asymptomatic while
34% complained of diarrhea and/or abdominal pain with non-statistically significant
difference between the different age groups regarding the presence or absence of symptoms.
2) Findings related to Blastocystis infection in cattle
To the best of our knowledge, the current study is considered the first report to detect
Blastocystis among cattle in Egypt. Blastocystis spp. were detected in 37 cattle with an
overall infection rate of 19.4%.Significant difference was demonstrated between farm
cattle and household cattle with infection rates of14.2% and 27%, respectively.
Summary and Conclusion
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There was a good agreement between direct wet mount and culture in the diagnosis
of Blastocystis infection in animals.
3) Findings related to molecular detection and genotyping of human and animal Blastocystis STs
Blastocystis SSU rDNA was successfully amplified by PCR in 55 out of 71 culture
positive samples. Among these samples, 24 samples could be sequenced. The SSU rDNA
barcode region gave good sequence results in 13 samples (six human and seven animal
isolates).
Regarding human samples, STs 1-3 were detected.ST1was identified in two samples,
one from an asymptomatic female and the other belonged to a male complaining of
abdominal pain. ST2was detected in three participants, two of them (one male and one
female) were asymptomatic and one male had abdominal pain. ST3 was found in a male
patient complaining of diarrhea.
Regarding the seven sequenced animal samples, three different subtypes were found,
namely STs 4, 10 and 14. ST 14 was the most common, being detected in five samples, all
belonged to household cattle. ST 4 and ST10 were less frequent; each was detected in one
farm cattle sample.
Phylogenetic analysis revealed that the two detected human ST1 in the present study
were related to the isolated human and dog Malaysian ST1. One of the human ST2 isolated in
the present study was identical to ST2 isolated from a dog in France. The human isolated ST3
was genetically related to the animals isolated ST3 from China. Moreover, it shared the main
clade with ST4 and ST10. Genetic similarity was also observed between cattle ST4 of the
present study and human isolated ST4 in Japan, United Kingdom and France. Further analysis
of the tree confirmed the restricted reporting of ST10 and ST14 to animals.
Summary and Conclusion
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In conclusion, Blastocystis is a common organism identified in human and animal
stool samples. Animal contact does not increase the risk of acquiring the infection.
Moreover, humans and animals harbor different Blastocystis STs. Although, these findings
may exclude the probability of zoonotic transmission of Blastocystis, the close
phylogenetic relation between human and animal isolates sheds light on the potential for
zoonotic transmission of STs 1- 4.