The influence of plant extracts on viability of ST3 and ST7 subtypes of Blastocystis sp.

Blastocystis sp. is one of the most frequently detected protozoa during stool specimen examination. In the last decade, the studies about the pathogenic potential of Blastocystis sp. have intensified. Additionally, treatment approaches against this parasite are still disputable. The study aimed to investigate the in vitro activity of the substances of natural origin against two subtypes (ST) of Blastocystis sp.-ST3 and ST7. Garlic and turmeric extracts exhibited the highest inhibitory effect in relation to the ST3 viability. While horseradish and turmeric were found to be the most effective extracts to the ST7 viability. The study showed that ginger, garlic, horseradish, and turmeric extracts have potent antimicrobial activity against Blastocystis ST3 and ST7, with the half-maximal inhibitory concentration (IC50) ranging from 3.8 to 4.8 µg/ml and from 3.3 to 72.0 µg/ml, respectively, and thus may be useful in the prevention and control of Blastocystis infections. Additionally, this research confirmed that Blastocystis ST7 is more resistant to the selected plant extracts treatment than Blastocystis ST3 which in consequence may bring some difficulties in its eradication.

Blastocystis spp. and Other Intestinal Parasites in Polish Soldiers Deployed to Lebanon and Iraq.

Intestinal parasitic infections are one of the most common infectious diseases worldwide, particularly in developing countries. A distinct group at increased risk of infection is military personnel deployed overseas for extended periods, typically six months at a time. The aim of this study was to determine the prevalence of Blastocystis spp. and other intestinal parasites in Polish military personnel returning from deployments to Lebanon (n = 206) and Iraq (n = 220). In this group of subjects, we found Blastocystis spp. (13.6%), Dientamoeba fragilis (3.3%), Entamoeba coli (0.9%), and Endolimax nana (0.5%). Entamoeba histolytica sensu lato and Chilomastix mesnili infections were detected only in one soldier returning from Lebanon and Iraq, respectively. Blastocystis subtype (ST) 3 was predominant in soldiers returning from Lebanon, followed by ST2 and ST1. ST1 infection was predominant in soldiers returning from Iraq, followed by ST3 and ST2. Our study affirms that, deployment abroad is of no influence of the prevalence of parasitic protozoa. However, it would be worth to monitor parasite infection in military personnel returning from tropical zone even if they have no actual symptoms. In addition, it is very important to determine the subtypes of Blastocystis-this may help to clearly define their pathogenicity, especially considering the scarcity of studies on Blastocystis genotypes in Iraqi and Lebanese residents.

Identification of Blastocystis spp. in Urban Rodents of Different Districts in Southwestern Iran: Subtype Distribution and Possible Zoonotic Potential.

PURPOSE: Rodents are one of the most abundant and diverse species of mammals and have recently been identified as carriers of numerous human pathogens. The current study was conducted to assess the prevalence, subtype (STs) distribution, and zoonotic potential of Blastocystis spp. in various species of rodents in Shiraz, southwestern Iran. METHODS: For this aim, a total of 120 fresh fecal samples were collected from Mus musculus (n = 40), Rattus norvegicus (n = 40), and Rattus rattus (n = 40) in various municipality districts of Shiraz (6 out of 10 districts) between February and November 2020. Upon detecting parasites using light microscopy, a DNA fragment of the Blastocystis SSU rDNA gene was amplified using conventional PCR. RESULTS: By employing direct wet mount examination, 8 out of 120 fecal samples (6.7%; 2 from house mice, 3 from black rats, and 3 from brown rats) tested positive. Similarly, 5% (2/40) of house mice, 7.5% (3/40) of black rats, and 7.5% (3/40) of brown rats tested positive using the molecular method. Phylogenetic analysis revealed that the Blastocystis infecting different rodent species in Shiraz belonged to two potentially zoonotic STs (ST1 and ST4). Accordingly, rodents should not be overlooked as potential reservoirs of zoonotic Blastocystis infections. Different sampled urban districts and their statistical association with reported prevalence rates were analyzed separately. CONCLUSION:  Overall, the issue of the frequency and ST distribution of Blastocystis in urban rodents of Iran is still open to question and for a proper understanding, wider and more comprehensive studies are needed.

The Significance of Opportunistic Parasitosis and Blastocystosis in Patients with Gastric Cancer: a Study with Control Group.

Objective: The aim of this study was to determine the prevalence of opportunistic parasites and Blastocystis spp. in patients with gastric cancer (CA) and to determine the significance of these parasite. Methods: The patient group and the control group were composed of 100 people each. The stool samples were examined under the microscope for intestinal parasites with the native-Lugol method. Then, samples were multiplied by formol-ethyl acetate method and stained with modified acid-fast method. Results: Intestinal parasite positivity was indicated in 14% of the gastric CA, and 2% of the healthy individuals (p=0.001). Blastocystis spp. (p=0.009) was identified in 11%, Cryptosporidium spp. was identified in 4%, G. intestinalis was identified in 2%, and C. cayetanensis was identified in 1% of the patient group. There were significant differences between the intestinal parasite positivity (p=0.012), abundant Blastocystis spp. positivity (p=0.041) and all Blastocystis spp. positivity (p=0.037) in patient and control groups. Most of the patients who were positive for parasites had diarrhea. Conclusion: Based findings, it was concluded that it would be beneficial to evaluate gastric CA patients, especially those with diarrhea, for intestinal parasites.

Occurrence and Genotypic Identification of Blastocystis spp., Enterocytozoon bieneusi, and Giardia duodenalis in Leizhou Black Goats in Zhanjiang City, Guangdong Province, China.

Blastocystis spp., Enterocytozoon bieneusi, and Giardia duodenalis are three common zoonotic intestinal parasites that cause severe diarrhea and enteric diseases. Leizhou black goats are characterized by a high reproductive rate, fast growth, and good meat quality, making them one of the pre-eminent goat breeds in China. Goats are reportedly common reservoirs of these three intestinal pathogens, but no information on their prevalence or genotypic distributions in black goats in Guangdong Province, China, is available. A total of 226 fecal samples were collected from goats in Zhanjiang city and genomic DNA was extracted from them. The presence of the three pathogens was detected using nested PCR targeting the sequences encoding SSU rRNA (Blastocystis spp.), the internal transcribed spacer of rRNA (ITS; E. bieneusi), as well as beta-giardin, glutamate dehydrogenase, and triosephosphate isomerase (G. duodenalis). All PCR products were sequenced to determine the species and genotypes of the organisms. The total prevalence rates of Blastocystis spp., E. bieneusi, and G. duodenalis were 33.63% (76/226), 17.70% (40/226), and 24.78% (56/226), respectively. Four subtypes of Blastocystis spp. were detected: ST5 (n = 6), ST10 (n = 50), ST14 (n = 14), and ST21 (n = 6). Among them, ST10 was the dominant genotype, accounting for 65.79% of strains, followed by the genotypes ST14 (18.42%), zoonotic ST5 (7.89%), and ST21 (7.89%). Four genotypes of E. bieneusi were detected: CHG3 (n = 32), CM21 (n = 4), CHG1 (n = 2), and ET-L2 (n = 2). Among these, CHG3 was the dominant genotype. Assemblage E (n = 54) and concurrent assemblages A and E (n = 2) were identified in the G. duodenalis-positive goats using multilocus genotyping. Blastocystis spp., E. bieneusi, and G. duodenalis infections were common in Leizhou black goats, all of which have zoonotic genotypes, indicating the potential risk of zoonotic transmission. Our results provide basic data for the prevention and control of these three intestinal pathogens. Further studies are required to better understand their genetic characteristics and zoonotic potential in Guangdong Province.

Parasitic Contamination of Fresh Leafy Green Vegetables Sold in Northern Lebanon.

Contaminated, raw or undercooked vegetables can transmit parasitic infections. Here, we investigated parasitic contamination of leafy green vegetables sold in local markets in the Tripoli district, Lebanon, during two consecutive autumn seasons (2020-2021). The study involved the microscopic examination of 300 samples of five different types of vegetables (60 samples per type) and used standardized qualitative parasitological techniques for some protozoa and helminths. The results showed that 16.7% (95% interval for p: 12.6%, 21.4%) (50/300) of the vegetable samples were contaminated with at least one parasite. The most frequently detected parasite was Blastocystis spp. (8.7%; 26/300); this was followed in frequency by Ascaris spp. (3.7%; 11/300). Among the different vegetable types, lettuce (23.3%; 14/60) was the most contaminated, while arugula was the least contaminated (11.7%; 7/60). The statistical analysis did not reveal any significant association between the prevalence of parasitic contamination and the investigated risk factors, which included collection date, vegetable type, market storage status, and wetness of vegetables at the time of purchase (p > 0.05). The high prevalence of parasitic contamination also suggested the potential presence of other microbial pathogens. These findings are important because leafy green vegetables are preferentially and heavily consumed raw in Lebanon. Thus, implementing effective measures that target the farm-to-fork continuum is recommended in order to reduce the spread of intestinal pathogens.

Systematic Review and Meta-Analysis: Epidemiology of Human Blastocystis spp. Infection in Malaysia.

Blastocystis spp. is a unicellular enteric protozoan parasite in humans with a controversial role in disease etiology. It is common in developing countries among immunocompromised patients and people who have close contact with animals. In this study, we have systematically reviewed previous studies on the distribution and genotypes of human Blastocystis infection in Peninsular Malaysia. Studies examining the prevalence of Blastocystis in diverse demographics, including rural, urban, comorbid conditions, and high-risk populations, were taken into consideration. The infection has been reported in nine states; the total percentage of infection was 17.8% (1671/9397), with the most cases in Pahang (27.3%) and the least in Johor (3.4%). Molecular studies revealed the presence of six subtypes: ST1, ST2, ST3, ST4, ST5, and ST6. ST3 was reported as the predominant subtype in all the states, with a prevalence of 54.7% (338/618). The findings provide greater clarity on the epidemiology of Blastocystis in Malaysia, which will help in policy making towards planning and strategizing control measures against the parasite.

Investigation of intestinal parasites by conventional and molecular methods in children with gastrointestinal system complaints.

Intestinal parasitic infections are a global health problem that causes morbidity and mortality, especially in children living in rural areas. In this study, stool samples of pediatric patients with gastrointestinal complaints were examined by conventional and molecular methods to determine the prevalence of intestinal parasites. A total of 100 pediatric patients with gastrointestinal complaints and 50 healthy children were included in the study. Stool samples were collected from each child and examined by direct microscopic examination (native-Lugol method), formol-ethyl acetate concentration technique, Kinyoun's acid-fast staining, and Wheatley trichrome staining methods. Real-time PCR was used for the detection of Blastocystis spp. and D. fragilis in the stool samples. Sanger sequencing was used to identify Blastocystis spp. subtypes. One or more intestinal parasites were found in 12% (n = 100) of the patient group and 1% (n = 50) of the control group using conventional techniques. By using real-time PCR, Blastocystis spp. was discovered in 14% (14/100) of the patient group and 8% (4/50) of the control group. There was no significant difference in the frequency of Blastocystis spp. between the two groups. The most prevalent Blastocystis subtype was ST1 and the most frequent allele was a2 among the samples successfully amplified and sequenced. D. fragilis was detected in 17% (17/100) of the patient group and 8% (4/50) of the control group by real-time PCR. The prevalence of D. fragilis was not significantly different between the patient and control groups, as well. Blastocystis spp. and D. fragilis were found in high prevalence in pediatric patients with gastrointestinal complaints in this study. Although the role of these protists as a pathogen in humans is still controversial, it is supposed to the presence of the parasites are associated with gastrointestinal disorders such as diarrhea, abdominal pain, nausea, and vomiting. More case-control studies are needed to understand the pathogenic or commensal role of these parasites on the intestinal microbiota, especially in both patients with gastrointestinal disorders and healthy individuals.

Cryptosporidium spp. and Blastocystis spp. coinfection as a reason of an acute diarrhea in a young healthy veterinary Polish student - Case report.

OBJECTIVES: Cryptosporidium belongs to enteric parasites responsible for prolonged symptoms in the gastrointestinal tract, both in immunocompetent and immunocompromised individuals. One of the risk factors of infection is contact with an infected person or animals (cattle). The case is described of a young man admitted to the Department of Tropical and Parasitic Diseases of the Medical University in Poznan, Poland, because of watery diarrhea with high fever and in whom symptomatic treatment did not produce any improvement. MATERIAL AND METHODS: A 21-year -old male was examined and his epidemiologic history obtained. Primary blood test, anti- Toxocara IgG (ELISA) and anti-Toxoplasma gondii IgG and IgM (ELISA) were performed. PCR detected 16 enteropathogens in a stool sample. Microscopic parasitic stool examination was also performed based on Ziehl-Neelsen method, which allowed the assessment of the presence of cryptosporidium life stages. RESULTS: Epidemiology data provided information that the patient was a veterinary student who therefore had many contacts with domestic animals. Multiplex PCR detected a genetic material of Cryptosporidium. The result was confirmed with repeated positive direct stool examinations which gave the evidence of Cryptosporidium spp. oocysts and vacuolar forms of Blastocystis spp.. CONCLUSIONS: 1) Cryptosporidium is responsible for watery diarrhea in healthy individuals. 2) Contact with animals (cattle) is a potential risk factor for infection. 3) Protozoan co-infection should be treated to shorten the symptomatic period and to avoid post-infection complications. 4) Different diagnostic methods increase the possibility to establish appropriate diagnosis.

Detection of Blastocystis spp., Cryptosporidium spp. and Encephalitozoon spp. among wild animals from Eastern Slovakia.

The aim of this study was to draw attention to the risk of transmission of Encephalitozoon, Cryptosporidium and Blastocystis infection due to high animal migration and to point out that even wild animals can be a source of many zoonotic diseases. Encephalitozoon cuniculi, Cryptosporidium spp. and Blastocystis spp. are frequent microscopic organisms that parasitise humans, domestic and wild animals. Two hundred and fifty-five faecal specimens were collected from wild boars, badgers, wolves, bears, foxes and deer from 15 locations in Slovakia. Sequencing of positive PCR products and subsequent sequence comparison with GenBank sequences identified Blastocystis spp. in five wild boars. The ST 5 (n = 4) and ST 10 (n = 1) subtypes were determined by genotyping. We identified Encephalitozoon cuniculi in five wild boars, and genotype II (n = 5) was determined on the basis of ITS repeat sequences. Cryptosporidium scrofarum was sequenced in wolves (n = 4) and wild boars (n = 1), while Cryptosporidium suis only in wild boars (n = 2). None of the wild boars had a mixed infection.

Relationship between Blastocystis spp. with Accompanying Food Hypersensitivity and Chronic Spontaneous Urticaria.

INTRODUCTION: The relationship between Blastocystis spp. and chronic spontaneous urticaria (CSU) is unclear. The aim of this study was to evaluate the role of this parasitic infection on CSU and to search for risky groups in CSU patients with this parasite. METHODS: Seventy adult CSU patients with Blastocystis spp. in their stool samples forming Group A and 70 CSU patients without any parasite as Group B were prospectively compared regarding urticaria activity score-7 (UAS7), medication scores (MS), and laboratory parameters. All patients received CSU treatment, and additionally, those in group A received an antiparasitic antibiotic. Eight months later, the same parameters were compared between the ones in remission (group A1) and those still having CSU symptoms (group A2) in group A. RESULTS: UAS7 and MS were lower in group A than in group B (p: 0.007, p < 0.001) 8 months later, while the initial scores were similar. The presence of food hypersensitivity reactions (FHRs) was higher in group A than in group B (p < 0.001) and was detected as a significant risk factor in the presence of Blastocystis spp. infection (p: 0.002, OR [CI] = 0.151 [0.045-0.502]). In group A, UAS7, MS, serum total IgE levels, and blood eosinophil counts decreased 8 months later (p < 0.001, p < 0.001, p: 0.003, p: 0.004, respectively). Additionally, total IgE levels and eosinophil counts decreased in group A1 (p: 0.033, p: 0.002) while they did not change in group A2. DISCUSSION/CONCLUSION: The eradication of Blastocystis spp. can improve the disease activity in CSU and the presence of FHRs seems to be risky in CSU patients with Blastocystis spp.

Dinámica de la evolución de las formas de Blastocystis spp. en un medio de cultivo simple / Dynamics of the evolution of the forms of Blastocystis spp. in a simple culture medium

Resumen Blastocystis spp. es un parásito muy frecuente en materia fecal humana, pero la naturaleza polimórfica y el número de Blastocystis en la muestra pueden complicar su detección por microscopía. El objetivo del trabajo fue describir la dinámica de los morfotipos de Blastocystis a corto plazo en un medio de cultivo simple y determinar su aplicabilidad para utilizarlo como complemento del análisis coproparasitológico y para estudios morfológicos, bioquímicos y moleculares del parásito. Se sembraron 10 muestras de materia fecal con Blastocystis en un medio Pavlova adaptado, se examinaron diariamente por examen microscópico durante 6 días y se registraron las formas y el recuento. El desarrollo fue regular y abundante y las formas fueron de tamaños variables y claramente identificables. El cultivo ensayado puede ser útil para la detección de Blastocystis cuando existan dudas diagnósticas por microscopía, para estudios de sensibilidad y especificidad diagnóstica o cuando se requiera aumentar la carga para realizar otros estudios.

Abstract Blastocystis spp. is a very frequent parasite in human fecal matter, but the polymorphic nature and the number of Blastocystis in a sample can complicate its detection by microscopy. The objective of the present work was to describe the dynamics of Blastocystis morphotypes in the short term in a simple culture medium and to determine its applicability to use it as a complement to coproparasitological analysis and for morphological, biochemical and molecular studies of the parasite. Ten stool samples with Blastocystis were cultured in an adapted Pavlova medium and examined during 6 days by microscopy to record the forms and the count. The development was regular and abundant and the shapes were of variable sizes and clearly identifiable. The tested culture could be used for the detection of Blastocystis when microscopic diagnosis is dubious, for studies of diagnostic sensitivity and specificity or when it is necessary to increase the load to perform other studies.

Resumo Blastocystis spp. é um parasita muito frequente nas fezes humanas, mas a natureza polimórfica e o número de Blastocystis na amostra podem complicar a sua detecção através do microscópio. O objetivo do trabalho foi descrever a dinâmica dos morfotipos de Blastocystis no curto prazo em um meio de cultura simples e determinar sua aplicabilidade para ser utilizado como complemento da análise coproparasitológica e para estudos morfológicos, bioquímicos e moleculares do parasita. Foram semeadas dez amostras de fezes com Blastocystis em um meio Pavlova adaptado e examinadas diariamente através de exame microscópico durante 6 dias, registrando as formas e fazendo recontagem. O desenvolvimento foi regular e abundante e as formas foram de tamanhos variáveis e claramente identificáveis. A cultura testada pode ser útil para a detecção de Blastocystis quando houver dúvidas diagnósticas por microscopia; para estudos de sensibilidade e especificidade diagnóstica ou quando for necessário aumentar a carga para a realização de outros estudos.

First Report of Blastocystis spp. Infection in Pet Birds in Henan Province, Central China.

Blastocystis spp. are common intestinal parasites found in humans and many kinds of animals. Blastocystis spp. infection is associated with a variety of symptoms, including diarrhea, abdominal pain, and chronic urticaria, among which asymptomatic infection is the most common. Among the 11 potentially zoonotic subtypes of Blastocystis spp., 9 subtypes have been reported in bird species. The purpose of this study was to detect the infection rate and gene subtype distribution of Blastocystis spp. in pet birds in Henan Province, Central China, to provide a foundation for preventing and controlling Blastocystis spp. in pet birds. Fecal DNA was extracted from 382 fresh fecal samples of pet birds collected from five areas in Henan Province, Central China. Twenty-three species of pet birds from four orders, from local pet trading markets, parks, and individuals, were sampled. All DNA samples were investigated by PCR, and positive samples were sequenced to analyze the gene subtypes based on the small ribosomal subunit (SSU rRNA) gene. Blastocystis spp. was detected in 0.8% of the samples. Further DNA sequencing and phylogenetic analyses resulted in the identification of two known zoonotic subtypes, ST1 (n = 2) and ST7 (n = 1). As far as we know, this is the first time that ST1 subtype has been reported in Chinese birds. It is found that pet birds may be the hosts of zoonotic Blastocystis spp. subtypes, and the role of birds in transmitting Blastocystis spp. to humans needs to be further studied.

Protozoan Intestinal Parasitic Infection in Patients with Hematological Malignancies.

The aim of this study was to evaluate the frequency of gastrointestinal protozoan infection in patients with hematological malignancies (HMs) undergoing intensive hemato-oncological treatment and to determine the influence of certain biological factors on the incidence of intestinal parasite infection. Stool samples were collected from hematological malignancy patients (n = 50) hospitalized at the Department of Hematology and Transplantology of the Pomeranian Medical University in Szczecin. The control group consisted of 50 healthy participants. We used a direct smear examination and a commercial immunoenzymatic test. Intestinal protozoans were detected in 16% of patients with hematological malignancies and in 6% of individuals in the control group. In stool samples from patients with HM, cysts of Giardia intestinalis (2%), oocysts of Cryptosporidium spp. (10%), vacuolar forms of potentially pathogenic Blastocystis spp. (2%), and cysts of nonpathogenic Entamoeba coli (2%) were found. Cryptosporidium spp. and Giardia intestinalis coproantigens were detected in 5 (10%) and 1 (2%) patients with HM, respectively. In three participants from the control group, vacuolar forms of Blastocystis spp. were found. In the patients with HM, a significantly higher prevalence of intestinal parasite infection was found in individuals working in the garden without protective gloves and those in contact with animals. In patients with hematological malignancies, intestinal parasites should be excluded, even during intensive chemotherapy treatment.

Standardization of molecular techniques for the detection and characterization of intestinal protozoa and other pathogens in humans.

Background: The intrinsic sensitivity limitations of basic parasitological methods, along with the particular biological characteristics of parasites, make these methods ineffective to differentiate morphologically indistinguishable species. Molecular detection and characterization techniques could be used to overcome these problems. The purpose of this work was to standardize molecular polymerase chain reaction (PCR) techniques, described in the literature, for the detection and molecular characterization of intestinal protozoa and other pathogens in humans. Methods: DNA was extracted from human or animal feces, previously washed or cultured in Boeck Drbohlav's Modified Medium. DNA extraction was performed with Machery-Nagel extraction kits. The standardization of the PCR, nested-PCR or RFLP techniques was carried out according to the literature. For each molecular technique performed, the sensitivity of the test was determined based on the minimun quantity required of DNA (sensitivity A) and the minimum quantity of life forms that the test detected (sensitivity B). Results: Sensitivity A was 10 fg for G. duodenalis, 12.5 pg for Entamoeba histolytica or Entamoeba dispar, 50 fg for Cryptosporidium spp., 225 pg for Cyclospora spp. and 800 fg or 8 fg for Blastocystis spp. after performing a 1780 bp PCR or 310 bp nested PCR, respectively. The sensitivity B was 100 cysts for G. duodenalis, 500 cysts for E. histolytica or E. dispar, 1000 oocysts for Cyclospora spp. and 3600 or four vegetatives forms for PCR or nested PCR of Blastocystis spp., respectively. Conclusions: The molecular detection of protozoa and chromist was achieved and the molecular characterization allowed the genotyping of some of the parasites such as Giardia duodenalis, Cryptosporidium spp., and Blastocystis spp. This study summarizes the molecular techniques for epidemiological studies in humans and animals, and helps in the investigation of their transmission sources in countries where intestinal parasites are a public health problem.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) technique for rapid, accurate, and specific detection of Blastocystis spp. in AIDS patients.

BACKGROUND: Blastocystis spp. is one of the most prevalent intestinal parasites with worldwide distribution. Various diagnostic methods with different sensitivities and specificities have been used to detect Blastocystis in clinical samples. The present study aims to develop and evaluate a LAMP assay to detect Blastocystis spp. in AIDS patients for the first time. METHODS: In this cross-sectional study, 98 AIDS patients with an average CD4 + T lymphocyte count lower than 150 cells/mm3 participated in the study. The presence of Blastocystis spp. in the stool samples of AIDS patients was examined by parasitology (direct wet mount and concentration assays) and molecular (PCR and LAMP) methods. The 18 SSU rRNA genomic target was used to design the specific primers for the PCR and LAMP assays. The specificity of designed primers for the LAMP assay was evaluated using the sequencing of a conventional PCR product by the external LAMP primers. The data were analyzed using the SPSS software and chi-square test and Fischer's exact tests were used and Cohen's Kappa calculates the agreement of the molecular tests. Associations were tested using odd ratios (OR) and 95% confidence intervals (CI) after adjustments. RESULTS: Out of 98 stool samples from patients with AIDS, 9 (9.18%), 13 (13.26%), and 15 (15.30%) samples were detected positive for Blastocystis spp. by parasitology, PCR, and LAMP techniques, respectively. PCR amplification and subsequent sequencing of the product sequences revealed that the obtained partial sequences were identical to the corresponding 18 SSU rRNA sequences reported in GenBank. The higher positivity rate for Blastocystis spp. among studied AIDS patients by LAMP technique compared to other diagnostic methods showed the higher potential and effectiveness of this relatively new described molecular assay for the detection of Blastocystis spp. in AIDS patients. CONCLUSION: The accurate and rapid detection of emerging intestinal protozoa such as Blastocystis is of clinical importance for better prevention and timely treatment of the disease, especially in immunocompromised patients. The results obtained for the first time showed that the sensitivity and accuracy of the LAMP technique in the diagnosis of Blastocystis spp. in AIDS patients is very high.

First report of Blastocystis spp. subtypes in ZOO animals in Slovakia, Central Europe.

Blastocystis spp. has been reported in wildlife, domestic animals and animals housed in ZOO. To-date, 17 genetically diverse lines have been reported in mammals and birds (designated ST) based on differences in the SSU rRNA. In this study, faeces samples were collected from 24 ZOO animals with clinical signs suggestive of gastrointestinal disease in Kosice ZOO, Slovakia. After DNA isolation, PCR was conducted to amplify the SSU region of DNA of Blastocystis species. Forward primer- Blast F and reverse primer- Blast R were used in the reaction. From 25 faeces samples, Blastocystis spp. was detected in 5 animals (3 mammals, 2 birds), with a prevalence of 20%. Subsequent molecular analyses identified the ST 5 (n = 3), ST 7 (n = 1), and ST 12 (n = 1) subtypes, where the ST 5 subtype was identified in the mammalian group and birds, and the ST 7 and ST 12 subtypes were identified only in mammals. Based on these findings, focusing on ZOO animals as a potential source of infection for humans is highly recommended.

Standardization of molecular techniques for the detection and characterization of intestinal protozoa and other pathogens in humans

Background: The intrinsic sensitivity limitations of basic parasitological methods, along with the particular biological characteristics of parasites, make these methods ineffective to differentiate morphologically indistinguishable species. Molecular detection and characterization techniques could be used to overcome these problems. The purpose of this work was to standardize molecular polymerase chain reaction (PCR) techniques, described in the literature, for the detection and molecular characterization of intestinal protozoa and other pathogens in humans. Methods: DNA was extracted from human or animal feces, previously washed or cultured in Boeck Drbohlav's Modified Medium. DNA extraction was performed with Machery-Nagel extraction kits. The standardization of the PCR, nested-PCR or RFLP techniques was carried out according to the literature. For each molecular technique performed, the sensitivity of the test was determined based on the minimun quantity required of DNA (sensitivity A) and the minimum quantity of life forms that the test detected (sensitivity B). Results: Sensitivity A was 10 fg for G. duodenalis, 12.5 pg for Entamoeba histolytica or Entamoeba dispar, 50 fg for Cryptosporidium spp., 225 pg for Cyclospora spp. and 800 fg or 8 fg for Blastocystis spp. after performing a 1780 bp PCR or 310 bp nested PCR, respectively. The sensitivity B was 100 cysts for G. duodenalis, 500 cysts for E. histolytica or E. dispar, 1000 oocysts for Cyclospora spp. and 3600 or four vegetatives forms for PCR or nested PCR of Blastocystis spp., respectively. Conclusions: The molecular detection of protozoa and chromist was achieved and the molecular characterization allowed the genotyping of some of the parasites such as Giardia duodenalis, Cryptosporidium spp., and Blastocystis spp. This study summarizes the molecular techniques for epidemiological studies in humans and animals, and helps in the investigation of their transmission sources in countries where intestinal parasites are a public health problem.(AU)

Blastocystis spp. infection in cases of diarrhea: A pilot study from a tertiary care teaching hospital in Rishikesh, Uttarakhand, with a brief review of literature.

CONTEXT: Intestinal parasitic infections (IPI) are among the most common infections throughout the world. Blastocystis spp. is a mysterious parasite which is commonly encountered in tropical countries. Its pathogenic status is unknown and there is a paucity of literature about this organism from the state of Uttarakhand, India. AIMS: The aim was to estimate the prevalence of Blastocystis spp. in diarrheal stools. SETTINGS AND DESIGN: This was a cross-sectional study conducted from January 2018 to July 2019. SUBJECTS AND METHODS: Nonrepetitive stool samples of 187 consecutive patients of diarrhea attending the inpatient department and outpatient department of a tertiary care teaching hospital located in Rishikesh, Uttarakhand, were collected after obtaining informed written consent. These samples were subjected to wet mount microscopy and permanent staining. STATISTICAL ANALYSIS USED: Fisher's exact test and Kappa coefficient were used in this study. RESULTS: The mean age ± standard deviation of the patients was 36.04 ± 11.31 years with a male-to-female ratio of 1.49:1. The prevalence of IPI was 36.09%. Giardia intestinalis was the most common parasite. Blastocystis spp. was observed in 6.42% of the stool samples, majority of which were obtained from cases of chronic diarrhea. Moderate agreement (0.48) was observed between wet mount microscopy and permanent staining in the identification of Blastocystis spp. CONCLUSIONS: This is the first study to assess the burden and role of different epidemiological and clinical profiles of Blastocystis spp. in Uttarakhand. More studies are required to know its pathogenesis and its role as opportunistic pathogen.

Prevalence of intestinal parasites and molecular characterization of Giardia intestinalis, Blastocystis spp. and Entamoeba histolytica in the village of Fortín Mbororé (Puerto Iguazú, Misiones, Argentina).

BACKGROUND: Intestinal parasites (IPs) are widely distributed worldwide and are one of the major contributors to gastrointestinal disease. Their prevalence is associated with poor access to water, sanitation and hygiene (WASH). The objective of this study was to identify the prevalence of IPs, including soil-transmitted helminths (STH), and their relation to socioeconomic characteristics, as well as a first approach to molecularly characterize the types of Giardia intestinalis, Blastocystis spp. and Entamoeba histolytica present in an indigenous community from Puerto Iguazú, Misiones, Argentina. METHODS: A cross-sectional study was conducted in the rural settlement of Fortin Mbororé between January and March 2018. Socioeconomic variables, household characteristics, and stool and blood samples were collected. Standard coprological techniques were used to analyze stool samples, and a complete hemogram was performed on the blood samples. Giardia intestinalis microscopy-positive samples were genetically typed by the ß-giardin (bg) gene. Molecular identification of Blastocystis spp. subtypes and E. histolytica were carried out by amplification and sequencing of a partial fragment of the small subunit ribosomal RNA gene (SSU rDNA). RESULTS: The overall prevalence of IPs was 92.7%, with 72.0% specifically for hookworm. IPs were significantly more prevalent in preschool- and school-age children (P < 0.05). No formal education (P = 0.035), the presence of unimproved floors (P = 0.001) and overcrowding (P = 0.005) were significantly associated with IP infection. Hookworm was associated with anemia (P = 0.019). Molecular characterization revealed the presence of E. histolytica sub-assemblages AII (12.5%), AIII (87.5%) and BIV (100%); one case of sub-assemblage D for G. intestinalis; and the presence of subtypes ST1 (14.8%), ST2 (14.8%) and ST3 (70.4%) of Blastocystis spp. CONCLUSIONS: Protozoans detected in this study are transmitted mainly through water contaminated with fecal matter, evidencing the need to improve the quality of water and sanitation for the inhabitants of Fortín Mbororé. Molecular characterization showed that domestic animals can be implicated in the zoonotic transmission of G. intestinalis and Blastocystis spp. to humans. A hyperendemic area for STH was found, with hookworm prevalence greater than 50%. Therefore, improvements in WASH as well as mass deworming programs need to be implemented in this area to control and decrease the prevalence of IPs in general and STH in particular.