Detection rate of Blastocystis sp. by microscopy and culture methods in symptomatic and asymptomatic persons in Kharkiv city, Ukraine

Abstract

Introduction. Blastocystis sp. - is the most prevalent anaerobic intestinal protozoan parasite in humans and many animals; from 1 to 2 billion people in the world are colonized by this pathogen. Blastocystis sp. is found both in faecal samples (FS) of healthy people (asymptomatic persons) and - patients (symptomatic persons) with nonspecific symptoms of gastrointestinal tract, skin, joints and other organs lesions. The prevalence of people affected by Blastocystis sp. of both cohorts in the world vary widely (from 0.08% to about 90%) depending on the degree of the country's economic development, sanitary and hygienic conditions, cultural values, etc. Currently, microscopic, cultural, immunological and molecular genetic methods are used for Blastocystis sp. detection in stool samples. Each group of methods of Blastocystis sp. detection/identification in FS has its advantages and disadvantages. The goal of this study was to determine the prevalence of Blastocystis sp. in faecal samples in different cohorts of people (clinically healthy and symptomatic people with symptoms of gastrointestinal lesions) in Kharkiv by microscopic and cultural methods. Materials and Methods. Cohort of surveyed residents of Kharkiv (n=169) included 72 clinically healthy individuals and 97 symptomatic individuals with gastrointestinal tract diseases.   All 169 FSs (their precipitates) were subjected to microscopic examination after the formalin-phosphate-salt buffer (FPBSCS) enrichment (concentration) procedure (pH=7.4) at 500 g for 10 minutes.  Blastocystis sp. identification was carried out by means of microscopy of the faecal smears, which were stained by Wheatley's modification trichrome stain (mWTS) and by Heidenhain's iron-hematoxylin stain (HIHS). The inoculated material was a filtered suspension of native FS (200 μl) which was inoculated in 5 ml of liquid media RPMI/IMDMEM (mixture of equal volumes of RPMI and IMDMEM media) with antibiotics and serum. Blastocystis sp. culture growth was carried out under anaerobic conditions at 37 oC for 5 days. The blastocysts final identification was carried out by means of light microscopy of suspensions smears stably stained with mWTS HIHS. Results & Discussion. It was carried out a comparative evaluation of the effectiveness Blastocystis sp. detection methods as microscopy (smears of enriched faecal material stained with mWTS or HIHS) and cultivation (on RPMI/IMDMEM medium) based on the results of parallel studies of 169 FS from different groups of people by both methods. An insignificant increase (4.1%) of the Blastocystis sp. frequency detection/identification by means of cultural method in comparison with the frequency of microscopic parasites detection in all FS was determined: in FS from asymptomatic individuals (n = 72) only by 2.7%, and in FS from symptomatic individuals (n = 97) - by 5.2% (p> 0.05). From all FS in which Blastocystis sp. was detected microscopically, the growth of these parasite primary cultures was obtained. Among the total results (negative + positive) Blastocystis sp. detection / identification by microscopic and cultural methods in all FS from humans r_ф reaches +0.92, and for groups FS from asymptomatic and symptomatic individuals - r_ф=+0.94 and r_ф=+0.90, respectively. In the sample of only positive results  detection / identification of Blastocystis sp. by microscopic and cultural methods, the value of r_ф is: + 0.59 for all studied FS from humans, + 0.20 - for FS from asymptomatic individuals and + 0.66 - for FS from symptomatic individuals.  Conclusion. According to the results of a parallel study of microscopic and cultural methods of 169 FS from different groups of people it was  found that the cultural method dominates over microscopic in sensitivity of Blastocystis sp. detection in FS (20.6%) and is characterized by a much higher level of specificity (accuracy of parasite identification), which reaches 100%. The method of in vitro diagnostics helps to increase the efficiency of parasites detection in human FS, can be used for epidemiological studies to establish the population prevalence of protozoa, to determine the sensitivity of Blastocystis sp. cultures to drugs, control of the etiotropic blastocystosis therapy effectiveness, obtaining parasites antigens, study the disease pathogenesis and the virulence potential of pathogen strains of different origin, etc.

Keywords: Blastocystis sp., diagnostics, in vitro. microscopic examination.

DOI: 10.5281/zenodo.6634866