2 research outputs found
Investigation of the Presence of Blastocystis spp. in Stool Samples with Microscopic, Culture and Molecular Methods
Blastocystis species are enteric protozoa frequently detected in human
and animals. Seventeen subtypes (STs) have now been identified, nine of
them isolating from humans. The pleomorphic structure and genetic
diversity of Blastocystis spp. and the absence of standardized
diagnostic methods complicate the evaluation of current data.
Microscopic methods such as native-lugol and trichrome staining are most
frequently used methods in routine diagnosis, while culture and
molecular methods are preferred for research purposes. The aims of this
study were to investigate the presence of Blastocystis spp. in the stool
samples of patients with gastrointestinal complaints by microscopic and
culture methods, and to detect the subtypes of isolates by polymerase
chain reaction (PCR). A total of 350 stool samples collected from
patients with diarrhea (n= 157) and without diarrhea (n= 193) were
included in the study. Presence of Blastocystis spp. in the samples were
investigated by native-lugol examination, trichrome staining and direct
fluorescent antibody (DFA) methods. Ringer's solution containing 10\%
horse serum and 0.05\% asparagine was used for cultivation. The cultures
were evaluated after 3-4 days of incubation at 37 degrees C by
microscopic examination. The subtypes of Blastocystis spp. strains
isolated from the cultures have been identified by PCR using
sequence-tagged site primers. A total of 66 (19\%) stool samples, of
them 26 (16.6\%) were from diarrheal and 40 (21\%) from non-diarrheal
cases, yielded Blastocystis sp. growth in culture. Among the evaluated
samples, 12\% (42/350) were found positive with native-lugol
examination, 17\% (58/350) with trichrome staining, and 19\% (66/350)
with DFA method. The agreement of culture and native-lugol method was
estimated as strong (kappa= 0.752), while it was very strong between
culture with trichrome staining and DFA methods (kappa= 0.922 and kappa=
1.00, respectively). When the culture was accepted as reference method,
the sensitivity and specificity rates of native-lugol method were 65\%
and 100\%, trichrome staining method were 88\% and 100\%, and DFA method
were 100\% and 100\%, respectively. Forty-three (65\%) of Blastocystis
spp. positive samples were subtyped by PCR, while 23 isolates could not
be subtyped. The most frequent detected subtype was ST3 (12/43; 28\%),
followed by ST1 (6/43; 13.9\%), ST4 (5/43; 11.6\%) and ST7 (5/43;
11.6\%), ST2 (3/43; 7\%) and ST6 (1/43; 2.3\%). ST5 was not detected in
this study and 11(25.6\%) samples have been identified to have mixed
subtypes. The differences of Blastocystis spp. positivity rates and the
distribution of the subtypes between the patients with or without
diarrhea were not found statistically significant (p> 0.05). In our
study, ST3 was the most frequently identified Blastocystis spp. subtype,
similar to the previous national studies, however ST6 and ST7 have been
identified for the first time. In conclusion, as the sensitivity of
native-lugol examination is low, culture is time-consuming and laborious
and PCR methods are costly and non-standardized, rapid, practical and
high sensitive DFA is considered as the favourable method in the
diagnosis of Blastocystis spp. in routine laboratories