Epidemiology of free-living amoebae in the Philippines: a review and update.

Free-living amoebae (FLA) are considered environmental pathogens and thus pose a public health threat. Their ubiquity in natural sources may magnify the potential severity of health outcomes in the future. However, less attention was given despite several probable public health risks that arise from the presence of pathogenic strains in the environment. Here, we provide epidemiological data based on investigations involving the distribution and occurrence of free-living amoebae in the Republic of the Philippines. This aims to connect data of fragmented studies of these organisms and provide potential roadmaps in FLA research in the country. The majority of the reviewed articles (n = 19) focused on characterization studies (36.8%; 7/19) while environmental isolation and isolation from biological samples had an equal frequency of 31.6% (6/19) each. There is a great disparity between the established ubiquity in environmental sources and the number of cases of FLA infections in the country. FLA-related research in the Philippines is still in its inceptive stage with several gaps to fill, which can be used to formulate policy briefs in the future regarding its isolation, identification, diagnosis, therapeutic management, and control of FLA infections in the country.

Amoebal coculture and enrichment methods as a proposal for water quality control in Brazil.

Free living amoebae (FLA) can be found in different environments, where they feed on diverse microorganisms. Some bacteria preyed by FLA are called amoeba-resistant bacteria (ARB), as they can resist to lysosomal fusion and are capable of multiplying and evading FLA after internalization, propagating in the environment. Despite the health risks due to the existence of pathogenic and opportunistic species that are ARB and the pathogenicity of some FLA species, there are no water quality protocols to analyze the presence of ARB or FLA. In this sense, our study aimed to isolate FLA through amoebal enrichment and to identify ARB using amoebal coculture in water samples from a public park and two hospitals in southern Brazil. As a result, 9 different microorganisms genera have been identified through amoebal coculture, including fastidious Legionella spp. and Bosea vestrisii. From the positive samples for FLA, by amoebal enrichment, Acanthamoeba spp., Vermamoeba vermiformis and Naegleria spp. were identified in 14 amoebic isolates. The methodologies used in this work proved to be effective as simple and low-cost methods to be used in the implementation in water quality control of anthropogenic environments.

Usefulness of a 3D-printing air sampler for capturing live airborne bacteria and exploring the environmental factors that can influence bacterial dynamics.

We created a handmade 3D-printed air sampler to effectively collect live airborne bacteria, and determined which environmental factors influenced the bacteria. Bacterial colony forming units (CFUs) in the air samples (n = 37) were monitored by recording the environmental changes occurring over time, then determining the presence/absence of correlations among such changes. The bacterial CFUs changed sharply and were significantly correlated with the DNA concentrations, indicating that the captured bacteria made up most of the airborne bacteria. Spearman's rank correlation analysis revealed significant correlations between the bacterial CFU values and some environmental factors (humidity, wind speed, insolation, and 24-h rainfall). Similarly the significant associations of CFU with humidity and wind speed were also found by multiple regression analysis with box-cox transformation. Among our panel of airborne bacteria (952 strains), 70 strains were identified as soil-derived Bacillus via the production of Escherichia coli- and Staphylococcus aureus-growth inhibiting antibiotics and by 16S rDNA typing. Soil-derived protozoa were also isolated from the air samples. We conclude that the airborne bacteria mainly derived from soil can alter in number according to environmental changes. Our sampler, which was created by easy-to-customize 3D printing, is a useful device for understanding the dynamics of live airborne bacteria.

Detection of free-living amoebae in domestic cats with and without naturally-acquired keratitis.

Pathogenic free-living amoebae, most notably Acanthamoeba spp., are important pathogens of the human cornea. The importance of infection with free-living amoebae in cats with keratitis is currently unclear. The aim of this study was to determine the frequency of amoeba detection in corneas of cats with naturally-acquired keratitis and in the ocular surface microflora of cats without ocular disease. Clinical ophthalmic and in vivo corneal confocal microscopic examinations were performed on 60 cats with keratitis. Corneal scrapings were analyzed by amoeba culture; cytological evaluation; and Acanthamoeba, Hartmannella, and Vahlkampfia PCR assays. Following ophthalmic examination, conjunctival specimens collected from 60 cats without clinically apparent ocular disease were analyzed similarly. In one cat with ulcerative keratitis, amoeba cysts and trophozoites were detected by in vivo corneal confocal microscopy; an Acanthamoeba sp. was isolated from corneal specimens and detected by Acanthamoeba PCR assay; and suppurative corneal inflammation was present cytologically. An Acanthamoeba sp. was isolated from conjunctival specimens from one cat without clinically apparent ocular disease, but with suppurative inflammation demonstrated cytologically. Both Acanthamoeba isolates belonged to the T4 genotype. Naegleria-like amoebae were isolated in samples from two cats with keratitis and seven cats without clinical ocular disease, but amoebae were not detected by the other assays in these samples. Amoeba detection by culture was significantly (P = 0.01) associated with cytologically diagnosed corneoconjunctival inflammation. This study identified naturally-acquired Acanthamoeba keratitis in cats. Detection of Naegleria-like amoebae in samples from cats with and without keratitis is of uncertain pathological significance.

Evaluation of the occurrence of pathogenic free-living amoeba and bacteria in 20 public indoor swimming pool facilities.

Recently, indoor swimming pool activities have increased to promote health-enhancing physical activities, which require establishing suitable protocols for disinfection and water quality control. Normally, the assessment of the microbial quality of the water in the pools only considers the presence of different bacteria. However, other less frequent but more resistant pathogens, such as free-living amoebas (FLA), are not contemplated in both existing recommendation and research activities. FLA represent a relevant human health risk, not only due to their pathogenicity but also due to the ability to act as vehicles of other pathogens, such as bacteria. Therefore, this work aimed to study the physicochemical characteristics and the occurrence of potentially pathogenic FLA and bacteria in water samples from 20 public indoor swimming facilities in Northern Portugal. Our results showed that some swimming pools presented levels of pH, free chlorine, and conductivity out of the recommended limits. Pathogenic FLA species were detected in two of the facilities under study, where we also report the presence of both, FLA and pathogenic bacteria. Our findings evidence the need to assess the occurrence of FLA and their existence in the same environmental niche as pathogenic bacteria in swimming pool facilities worldwide and to establish recommendations to safeguard the health of the users.

Isolation and identification of Legionella spp. from hot spring water in Algeria by culture and molecular methods.

AIMS: Due to infectious risk associated with the presence of Legionella in warm water, we determined the prevalence of living Legionella spp. in hot spring water in Algeria. METHODS AND RESULTS: Detection of Legionella by culture was done by using two methods, direct culture on agar plates and co-culture with amoeba. Fifty samples were taken from different hot springs in northern Algeria, including swimming pools, showers and thermal sources. Legionella pneumophila serotypes were predominant, accounting for 60% of positive samples. Direct method allowed the isolation of 13 L. pneumophila only of 50 samples (26%), whereas co-culture using a panel of three free living amoeba allowed the isolation of 119 Legionella species from the same samples (80%) CONCLUSIONS: Amoeba co-culture allowed the isolation of several Legionella sp., while direct culture allowed the isolation of L. pneumophila only. Remarkably, Legionella longbeachae, usually isolated from soil and compost, was isolated for the first time in thermal water in three samples using Vermamoeba vermiformis co-culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Legionella in the water of hot springs in Algeria, which are mainly frequented by individuals at risk of Legionellosis, requires urgent control measures.

Identification of free-living amoebas and amoeba-resistant bacteria accumulated in Dreissena polymorpha.

To identify the free-living amoeba (FLA) and amoeba-resistant bacteria (ARB) accumulated in zebra mussels and in the water in which they are found, mussels were collected at two locations in the Ebro river basin (North East Spain). FLAs and bacteria were isolated from mussel extracts and from natural water. PCR techniques were used to identify the FLAs and endosymbiont bacteria (Legionella, Mycobacterium, Pseudomonas and cyanobacteria), and to detect Giardia and Cryptosporidium. The most frequently found FLAs were Naegleria spp. The presence of Legionella, Mycobacterium and Pseudomonas inside the FLA was demonstrated, and in some cases both Legionella and Pseudomonas were found together. Differences between FLAs and ARB identified inside the mussels and in the water were detected. In addition, Escherichia coli, Clostridium perfringens, Salmonella spp. and Enterococcus spp. were accumulated in mussels in concentrations unconnected with those found in water. The results show the ability of the zebra mussel to act as a reservoir of potentially pathogenic FLAs, which are associated with potentially pathogenic ARB, although the lack of association between microorganisms inside the mussels and in the water suggests that they are not useful for monitoring microbiological contamination at a specific time.

Identification of Free-Living Amoebas in Tap Water of Buildings with Storage Tanks in Korea.

Free-living amoebas (FLAs) can cause severe disease in humans and animals when they become infected. However, there are no accurate survey reports on the prevalence of FLAs in Korea. In this study, we collected 163 tap water samples from buildings, apartments, and restrooms of highway service areas in 7 Korean provinces with high population density. All these buildings and facilities have water storage tanks in common. The survey was separated into categories of buildings, apartments, and highway service areas. Five hundred milliliters of tap water from each building was collected and filtered with 0.2 µm pore filter paper. The filters were incubated in agar plates with heated E. coli at 25°C. After axenization, genomic DNA was collected from each FLA, and species classification was performed using partial 18S-rDNA PCR-sequencing analysis. We found that 12.9% of tap water from buildings with storage tanks in Korea was contaminated with FLAs. The highway service areas had the highest contamination rate at 33.3%. All of the FLAs, except one, were genetically similar to Vermamoeba vermiformis (Hartmannella vermiformis). The remaining FLA (KFA21) was very similar to Acanthamoeba lugdunensis (KA/E26). Although cases of human infection by V. vermiformis are very rare, we must pay attention to the fact that one-third of tap water supplies in highway service areas have been contaminated.

Genome-wide association mapping and accuracy of predictions for amoebic gill disease in Atlantic salmon (Salmo salar).

Amoebic gill disease (AGD) is a parasitic disease caused by the amoeba Paramoeba perurans, which colonizes the gill tissues and causes distress for the host. AGD can cause high morbidity and mortalities in salmonid and non-salmonid fish species. To understand the genetic basis of AGD and improve health status of farmed A. salmon, a population of ~ 6,100 individuals belonging to 150 full-sib families was monitored for development of AGD in the sea of Ireland. The population was followed for two rounds of AGD infections, and fish were gill scored to identify severity of disease in first (N = 3,663) and the second (N = 3,511) infection with freshwater treatment after the first gill-scoring. A subset of this gill-scored population (N = 1,141) from 119 full-sib families were genotyped with 57,184 SNPs using custom-made Affymetrix SNP-chip. GWAS analyses were performed which resulted in five significantly associated SNP variants distributed over chromosome 1, 2 and 5. Three candidate genes; c4, tnxb and slc44a4 were found within QTL region of chromosome 2. The tnxb and c4 genes are known to be a part of innate immune system, and may play a role in resistance to AGD. The gain in prediction accuracy obtained by involving genomic information was 9-17% higher than using traditional pedigree information.

Molecular detection of free-living amoebae from Namhangang (southern Han River) in Korea.

The free-living amoebae Naegleria spp. and Acanthamoeba spp. exist in the natural environment and are sometimes causal agents of lethal primary amoebic meningoencephalitis (PAM), amoebic keratitis (AK) and granulomatous amebic encephalitis (GAE) in humans, respectively. To ascertain the existence of free-living amoebae in Korea, water samples were collected from the Korean hydrosphere, Namhangang (southern Han River), an active location for water skiing and recreation. Samples underwent two-step filtration and were cultured on non-nutrient agar medium with inactivated E. coli. The remaining samples were subjected to PCR for primarily the 18S small ribosomal RNA gene and gene sequencing. Similarities in 18S rDNA sequences, in comparison with various reference amoebae in GenBank, showed 86~99% homology with N. gruberi, N. philippinensis, N. clarki, A. polyphaga, A. castellannii, and Hartmannella (Vermamoeba) vermiformis. Therefore, this study will be useful for seasonal detection of free-living amoebae from various Korean hydrospheres in future studies.

Isolation and Phylogenetic Analysis of Free-Living Amoebae (Acanthamoeba, Naegleria, and Vermamoeba) in the Farmland Soils and Recreational Places in Iran.

PURPOSE: Free-living amoeba (FLA) including Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria are among the soil-born parasites. There are reports of FLA-related keratitis with a history of contact with soil and dust sources, particularly among the farmers. Due to lack of the previous studies on the farmland soils and a limited number of researches conducted on recreational soils in Iran, the present study was conducted. METHODS: A total of 93 soil samples including farming lands and recreational places were tested for the presence of Acanthamoeba spp. Balamuthia mandrillaris, Naegleria, and Vermamoeba using morphological key and sequencing-based tools. Pathogenicity of Acanthamoeba positive strains was also evaluated. To verify genetic associations and taxonomic status of isolated amoeba, a phylogenetic tree was built by MEGA 5.05 software inferred by the 18S rRNA gene based on maximum likelihood algorithm. RESULTS: Overall, 28 samples (30%) were contaminated with potentially pathogenic FLA, and according to the sequencing data, 17 strains were successfully sequenced. The isolated Acanthamoeba belonged to T2, T4, T5, mixed T4 and T5 contaminations, and T11. ITS sequencing revealed the occurrence of one strain of Naegleria canariensis. Four strains of Vermamoeba vermiformis were also confirmed. Morphological survey and PCR assay failed to show any positive results for Balamuthia mandrillaris. Pathogenic potential of the Acanthamoeba strains showed that T2, T4, and T11 genotypes were highly pathogenic, whereas T5 genotypes demonstrated lower pathogenic potential. CONCLUSION: The results indicate that soil could be a serious hazard to human health, and therefore, further studies are expected to investigate the source of infection in patients developing FLA-related diseases. The present study is the first to investigate FLA in the farmland soils in Iran and the first to report the presence of N. canariensis in the country.

Investigation of the Physical, Chemical Characteristics and Microbial Contamination of the Indoor Swimming Pools

Objective: The aim of this study was to investigate the physical, chemical and microbiological contamination of indoor swimming pools. Methods: Pool water specimens were collected using a plastic polypropylene sterilized bottle. The physical and chemical qualities of the waters were analyzed in terms of temperature, turbidity, pH, and free residual chlorine, with the standard methods for the examination of water. Bacteriological (routine methods) and parasitological (molecular methods) tests were carried out on pools water. Results: The mean temperature, pH, and residual chlorine of the indoor pools were 31.2 °C, 7.6 and 1.5 mg/L, respectively. Turbidity was not observed in any of the pools. The pH and temperature values were in standard ranges in 92.3% and 15.4% of the waters of swimming pools, respectively. The prevalence rates of bacterial and amoebic contaminations of the water in the swimming pools were 53.8% and 46.2%, respectively. One pool (7.7%) was contaminated with both bacteria and amoeba. Streptococcus viridans, Staphylococcus epidermidis, Pseudomonas stutzeri, Cryptosporidium and Bacillus spp. were isolated from the pool waters. Conclusion: In this study, some microorganisms were identified from the water pools. Effective management of swimming pools and proper control of the physical, chemical and microbiological property of water pools can produce the healthy recreational activity.

Long-term persistence of infectious Legionella with free-living amoebae in drinking water biofilms.

Prolific growth of pathogenic Legionella pneumophila within engineered water systems and premise plumbing, and human exposure to aerosols containing this bacterium results in the leading health burden of any water-related pathogen in developed regions. Ecologically, free-living amoebae (FLA) are an important group of the microbial community that influence biofilm bacterial diversity in the piped-water environment. Using fluorescent microscopy, we studied in-situ the colonization of L. pneumophila in the presence of two water-related FLA species, Willaertia magna and Acanthamoeba polyphaga in drinking water biofilms. During water flow as well as after periods of long-stagnation, the attachment and colonization of L. pneumophila to predeveloped water-biofilm was limited. Furthermore, W. magna and A. polyphaga showed no immediate interactions with L. pneumophila when introduced to the same natural biofilm environment. A. polyphaga encysted within 5-7 d after introduction to the tap-water biofilms and mostly persisted in cysts till the end of the study period (850 d). W. magna trophozoites, however, exhibited a time delay in feeding on Legionella and were observed with internalized L. pneumophila cells after 3 weeks from their introduction to the end of the study period and supported putative (yet limited) intracellular growth. The culturable L.pneumophila in the bulk water was reduced by 2-log over 2 years at room temperature but increased (without a change in mip gene copies by qPCR) when the temperature was elevated to 40 °C within the same closed-loop tap-water system without the addition of nutrients or fresh water. The overall results suggest that L. pneumophila maintains an ecological balance with FLA within the biofilm environment, and higher temperature improve the viability of L. pneumophila cells, and intracellular growth of Legionella is possibly cell-concentration dependent. Observing the preferential feeding behavior, we hypothesize that an initial increase of FLA numbers through feeding on a range of other available bacteria could lead to an enrichment of L. pneumophila, and later force predation of Legionella by the amoeba trophozoites results in rapid intracellular replication, leading to problematic concentration of L. pneumophila in water. In order to find sustainable control options for legionellae and various other saprozoic, amoeba-resisting bacterial pathogens, this work emphasizes the need for better understanding of the FLA feeding behavior and the range of ecological interactions impacting microbial population dynamics within engineered water systems.

Novel data from Italian Vermamoeba vermiformis isolates from multiple sources add to genetic diversity within the genus.

Vermamoeba vermiformis represents one of the most common free-living amoebae identified in worldwide environmental surveys. We analyzed 56 water samples with varying characteristics, including temperature and the particular settings in which humans may be exposed to water, plus one corneal scraping from a keratitis patient, with the following aims: (i) to investigate the presence of V. vermiformis; (ii) to identify the isolate subtypes; (iii) to place the Italian isolates in the broader picture of the genetic diversity within V. vermiformis. Twenty-two isolates were identified upon culturing and sequencing of > 600 bp in the 18S ribosomal RNA (rRNA) gene sequence, bringing to 27 the number of sequences recovered from Italian sources. By adding deposited sequences, we assembled a dataset of 74 isolates. Three of our isolates were characterized by allelic code 7-5-1-1, never reported before, and two showed 100% identity with an uncultured eukaryote and carried the 719T>C variant. We show that the variable segments E5, E3, F, and G convey most of the information on diversity, enabling the clustering of the isolates in a replicable fashion. The presence of different strains in natural thermal waters and in distribution systems indicated heterogeneity of the amoebic populations. Also, ours and the only other sequence from human infection were mapped in different clades. Overall, we enlarged the repertoire of single nucleotide and indel variants and the list of allelic codes, proceeding one step further in the description of the diversity within the genus.

Isolation and molecular identification of free-living amoebae from dishcloths in Tenerife, Canary Islands, Spain.

In this work, the presence of free-living amoebae (FLA) in dishcloths collected from human activity related places was evaluated. Once in the laboratory, 6 cm2 pieces of each dishcloth were cut and washed with Page's Amoeba Solution (PAS) in sterile tubes. After washing, the dishcloth pieces were removed, and the tubes were centrifuged (1500 rpm for 10 min). The obtained pellets were seeded onto 2% non-nutrient agar (NNA) plates, incubated at room temperature and were monitored daily an inverted microscope. Once clonal cultures were obtained (only one type of FLA observed), molecular analyses were carried out in order to characterize the isolated FLA strains at the genus/genotype level. From the 31 dishcloths which were processed, FLA strains were isolated in NNA plates in 13 the samples (13/31, 42%). However, and due to bacterial overgrowth, only six strains were characterized at the molecular level (PCR and sequencing). Among the PCR positive strains, 83.33% (5/6) of the PCR positive samples belonged to Acanthamoeba genus (80% (4/5) to genotype T4 and 20% (1/5) to genotype T11). Furthermore, one strain was identified as a member of Allovahlkampfia genus using both morphological and molecular approaches. To the best of our knowledge, this is the first report on the isolation of Allovahlkampfia genus from dishcloths and in the Spanish territory. The presence of FLA in dishcloths should raise awareness to improve hygienic strategies in food- and domestic-related environments, in order to prevent contamination with these protozoa, which are able to be pathogenic and even to act as vehicles of other pathogenic agents.

One-year survey of opportunistic premise plumbing pathogens and free-living amoebae in the tap-water of one northern city of China.

In this study, qPCR was used to quantify opportunistic premise plumbing pathogens (OPPPs) and free-living amoebae in 11 tap water samples collected over four seasons from a city in northern China. Results demonstrated that the average numbers of gene copies of Legionella spp. and Mycobacterium spp. were significantly higher than those of Aeromonas spp. (p < 0.05). Legionella spp. and Mycobacterium spp. were 100% (44/44) positively detected while P. aeruginosa and Aeromonas spp. were 79.54% (35/44) and 77.27% (34/44) positively detected. Legionella pneumophila was only detected in 4 samples (4/44), demonstrating its occasional occurrence. No Mycobacterium avium or Naegleria fowleri was detected in any of the samples. The average gene copy numbers of target OPPPs were the highest in summer, suggesting seasonal prevalence of OPPPs. Average gene copy numbers of OPPPs in the taps of low-use-frequency were higher than in taps of high-use-frequency, but the difference was not significant for some OPPPs (p > 0.05). Moderate negative correlations between the chlorine concentration and the gene copy numbers of OPPPs were observed by Spearman analysis (rs ranged from -0.311 to -0.710, p < 0.05). However, no significant correlations existed between OPPPs and AOC, BDOC, or turbidity. Moderate positive correlations were observed between the target microorganisms, especially for Acanthamoeba spp., through Spearman analysis (p < 0.05). Based on our studies, it is proposed that disinfectant concentration, season, taps with different-use frequency, OPPP species, and potential microbial correlations should be considered for control of OPPPs in tap water.

Potentially pathogenic genera of free-living amoebae coexisting in a thermal spring.

Little is known about the prevalence of Balamuthia mandrillaris within the environment due to its difficult isolation, but once an axenic culture is established, it is relatively easy to maintain. As most of the time researchers are interested mainly in isolating B. mandrillaris from environmental samples, the flora that accompanies it becomes second in importance. Therefore, this study aimed to determine which potentially pathogenic free-living amoebae, in addition to B. mandrillaris, could be found co-inhabiting a source of natural thermal water called "Agua Caliente" (Mexico), where this amoeba has previously been detected twice by molecular methods. A third sampling from this same source was carried out to try to isolate B. mandrillaris and other free-living amoebae using 37 and 45 °C as isolation temperatures. For PCR techniques, specific primers were used for B. mandrillaris, Naegleria fowleri, and Acanthamoeba species, plus a universal primer set for the eukaryotic 18S SSU rRNA gene for other isolated amoebae. PCR products were sequenced for final identification. 42 strains of the primary isolate were obtained, but only 34 could be kept in culture. Of them, 23 strains were identified as Naegleria lovaniensis, eight strains as Acanthamoeba jacobsi, two strains as Stenamoeba sp. and only one was identified as Vermamoeba vermiformis. The isolation of B. mandrillaris was once again not successful, but the presence of potentially pathogenic and nonpathogenic free-living amoebae is reported for the first time in this type of water in Mexico thanks to molecular methodology.

Identification of free-living amoebae isolated from tap water in Istanbul, Turkey.

Free-living amoebae (FLA) are widely spread in the environment and also known to cause rare but often serious infections. The present work focuses on a local survey on FLA. It is essential to know the prevalence and distribution of these microorganisms in order to get infections caused by them under control. In this study, FLA isolated from domestic tap water samples from homes of contact lens wearers were identified by morphology and by 18S rRNA gene sequence analysis. Morphological analysis and partial sequencing of the 18S rDNA revealed the presence of Acanthamoeba genotype T4 and Vermamoeba vermiformis in the investigated tap water samples. Naegleria fowleri, Balamuthia mandrillaris, and Sappinia spp. were not detected during this study. It was shown that species of FLA known to cause eye infections in humans are widely distributed in tap water in Istanbul, Turkey. Contact lens wearers should be aware of the risk of contamination from tap water and strictly apply stringent contact lens hygiene. With this study, we established Acanthamoeba genotype T4 and Vermamoeba vermiformis as contaminants of tap water in Istanbul.

Intestinal helminths and protozoan infections in patients with colorectal cancer: prevalence and possible association with cancer pathogenesis.

The purpose of the present study was to determine the prevalence of intestinal helminths and protozoa in colorectal cancer (CRC) patients and to evaluate the possible association between the prevalence and CRC pathogenesis. A total of 200 CRC patients and 200 residents of Tashkent, who had no complaints related to the gastrointestinal tract, were examined by triple coproscopy using a concentration method and estimations of protozoan infection intensity. Of the CRC patients tested, 144 were classified as T1-4N0M0 (without metastases) and 56 were classified as T1-4N1-2M0-1 (with metastases). Parasitological examination was performed during CRC diagnosis before and after surgery and chemotherapy. A significantly higher prevalence of Blastocystis sp., Chilomastix mesnili, Jodamoeba butschlii, and Endolimax nana was found in CRC patients than in the control population (p < 0.0001), amounting to 80, 20, 22.5, and 11.5%, respectively. The high prevalence of Blastocystis sp., as well as the patterns of infection intensity, was stable at all stages of examination. The ratio of the number of CRC patients with and without Blastocystis sp. in the T1-4N0M0 and T1-4N1-2M0-1 groups amounted to 3.3 and 7.0, respectively. The ratios for C. mesnili, E. coli, J. butschlii, and E. nana in both groups were 0.2 and 0.2, 0.07 and 0.07, 0.3 and 0.16, and 0.18 and 0.01, respectively. The prevalence of helminths and Giardia lamblia in CRC patients and the control population was not significantly different. Taken together, these data indicate a possible role for Blastocystis sp. in CRC pathogenesis. Diagnosis, treatment, and further observation of patients with Blastocystis sp. are necessary at all stages of CRC, including during diagnosis and before and after surgery and chemotherapy.