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Mei X, Wei L, Su C, Yang Z, Tian X, Zhang Z, Wang S. Advances in the axenic isolation methods of Blastocystis sp. and their applications. Parasitology 2024; 151:125-134. [PMID: 38087868 PMCID: PMC10941048 DOI: 10.1017/s0031182023001300] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2023] [Revised: 11/24/2023] [Accepted: 12/05/2023] [Indexed: 01/06/2024]
Abstract
Blastocystis sp. is a prevalent protistan parasite found globally in the gastrointestinal tract of humans and various animals. This review aims to elucidate the advancements in research on axenic isolation techniques for Blastocystis sp. and their diverse applications. Axenic isolation, involving the culture and isolation of Blastocystis sp. free from any other organisms, necessitates the application of specific media and a series of axenic treatment methods. These methods encompass antibiotic treatment, monoclonal culture, differential centrifugation, density gradient separation, micromanipulation and the combined use of culture media. Critical factors influencing axenic isolation effectiveness include medium composition, culture temperature, medium characteristics, antibiotic type and dosage and the subtype (ST) of Blastocystis sp. Applications of axenic isolation encompass exploring pathogenicity, karyotype and ST analysis, immunoassay, characterization of surface chemical structure and lipid composition and understanding drug treatment effects. This review serves as a valuable reference for clinicians and scientists in selecting appropriate axenic isolation methods.
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Affiliation(s)
- Xuefang Mei
- Xinxiang Key Laboratory of Pathogenic Biology, Department of Pathogenic Biology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Lai Wei
- Xinxiang Key Laboratory of Pathogenic Biology, Department of Pathogenic Biology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Changwei Su
- Xinxiang Key Laboratory of Pathogenic Biology, Department of Pathogenic Biology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Zhenke Yang
- Xinxiang Key Laboratory of Pathogenic Biology, Department of Pathogenic Biology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Xiaowei Tian
- Xinxiang Key Laboratory of Pathogenic Biology, Department of Pathogenic Biology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Zhenchao Zhang
- Xinxiang Key Laboratory of Pathogenic Biology, Department of Pathogenic Biology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan, PR China
| | - Shuai Wang
- Xinxiang Key Laboratory of Pathogenic Biology, Department of Pathogenic Biology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan, PR China
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Defaye M, Nourrisson C, Baudu E, Warwzyniak I, Bonnin V, Bonnet M, Barnich N, Ardid D, Delbac F, Carvalho FA, Poirier P. Efficient and reproducible experimental infections of rats with Blastocystis spp. PLoS One 2018; 13:e0207669. [PMID: 30452467 PMCID: PMC6242359 DOI: 10.1371/journal.pone.0207669] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2018] [Accepted: 11/05/2018] [Indexed: 12/15/2022] Open
Abstract
Although Blastocystis spp. infect probably more than 1 billion people worldwide, their clinical significance is still controversial and their pathophysiology remains poorly understood. In this study, we describe a protocol for an efficient and reproducible model of chronic infection in rats, laying the groundwork for future work to evaluate the pathogenic potential of this parasite. In our experimental conditions, we were unable to infect rats using vacuolar forms of an axenically cultivated ST4 isolate, but we successfully established chronic infections of 4 week-old rats after oral administration of both ST3 and ST4 purified cysts isolated from human stool samples. The infection protocol was also applied to 4 week-old C57BL/9, BALB/C and C3H mice, but any mouse was found to be infected by Blastocystis. Minimal cyst inoculum required for rat infection was higher with ST3 (105) than with ST4 (102). These results were confirmed by co-housing experiments highlighting a higher contagious potential of ST4 in rats compared to ST3. Finally, experiments mimicking fecal microbiota transfer from infected to healthy animals showed that Blastocystis spp. could easily infect a new host, even though its intestinal microbiota is not disturbed. In conclusion, our results provide a well-documented and robust rat model of Blastocystis chronic infection, reproducing "natural" infection. This model will be of great interest to study host parasite interactions and to better evaluate clinical significance of Blastocystis.
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Affiliation(s)
- Manon Defaye
- Université Clermont Auvergne, 3iHP, CNRS, Laboratoire Microorganismes: Génome et Environnement, Clermont-Ferrand, France
- Université Clermont Auvergne, 3iHP, Inserm U1107, NeuroDol, Clermont-Ferrand, France
| | - Céline Nourrisson
- Université Clermont Auvergne, 3iHP, CNRS, Laboratoire Microorganismes: Génome et Environnement, Clermont-Ferrand, France
- Université Clermont Auvergne, CHU, 3iHP, CNRS, Laboratoire Microorganismes: Génome et Environnement, Clermont-Ferrand, France
| | - Elodie Baudu
- Université Clermont Auvergne, 3iHP, Inserm U1107, NeuroDol, Clermont-Ferrand, France
- Université Clermont Auvergne, 3iHP, Inserm U1071, USC INRA 2018, Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte, Clermont-Ferrand, France
| | - Ivan Warwzyniak
- Université Clermont Auvergne, 3iHP, CNRS, Laboratoire Microorganismes: Génome et Environnement, Clermont-Ferrand, France
| | - Virginie Bonnin
- Université Clermont Auvergne, 3iHP, Inserm U1071, USC INRA 2018, Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte, Clermont-Ferrand, France
| | - Mathilde Bonnet
- Université Clermont Auvergne, 3iHP, Inserm U1071, USC INRA 2018, Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte, Clermont-Ferrand, France
| | - Nicolas Barnich
- Université Clermont Auvergne, 3iHP, Inserm U1071, USC INRA 2018, Microbes, Intestin, Inflammation et Susceptibilité de l'Hôte, Clermont-Ferrand, France
| | - Denis Ardid
- Université Clermont Auvergne, 3iHP, Inserm U1107, NeuroDol, Clermont-Ferrand, France
| | - Frédéric Delbac
- Université Clermont Auvergne, 3iHP, CNRS, Laboratoire Microorganismes: Génome et Environnement, Clermont-Ferrand, France
| | | | - Philippe Poirier
- Université Clermont Auvergne, 3iHP, CNRS, Laboratoire Microorganismes: Génome et Environnement, Clermont-Ferrand, France
- Université Clermont Auvergne, CHU, 3iHP, CNRS, Laboratoire Microorganismes: Génome et Environnement, Clermont-Ferrand, France
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Ajjampur SSR, Tan KSW. Pathogenic mechanisms in Blastocystis spp. - Interpreting results from in vitro and in vivo studies. Parasitol Int 2016; 65:772-779. [PMID: 27181702 DOI: 10.1016/j.parint.2016.05.007] [Citation(s) in RCA: 101] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2015] [Revised: 05/04/2016] [Accepted: 05/11/2016] [Indexed: 01/20/2023]
Abstract
Blastocystis spp. are commonly reported intestinal protists but whose clinical significance remains controversial. Infections have ranged from asymptomatic carriage to non-specific gastrointestinal symptoms and have also been linked to irritable bowel syndrome and urticaria in some patient populations. In vitro studies showed that both parasite and parasite lysates have damaging effects on intestinal epithelial cells causing apoptosis and degradation of tight junction proteins occludin and ZO1, resulting in increased intestinal permeability. Adhesion of trophic forms to the intestinal epithelium and release of cysteine proteases appear to be the major triggers leading to pathogenesis. Two putative virulence factors identified are cysteine proteases legumain and cathepsin B. Blastocystis spp. also have immuno-modulatory effects including degradation of IgA, inhibition of iNOS and upregulation of proinflammatory cytokines, IL8 and GM-CSF in intestinal epithelial cells and IL1β, IL6 and TNFα in murine macrophages. Blastocystis spp. have also been reported to dampen response to LPS in intestinal epithelial cells and monocytes. Studies in rodent models and naturally infected pigs have shown that the parasite localizes to the lumen and mucosal surface of the large intestine mostly in the caecum and colon. The parasite has been found to cause mucosal sloughing, increase in goblet cell mucin, increased intestinal permeability and to induce a pro-inflammatory cytokine response with upregulation of TNFα, IFNγ and IL12. In this review, we summarize findings from in vitro and in vivo studies that demonstrate pathogenic potential but also show considerable inter and intra subtype variation, which provides a plausible explanation on the conflicting reports on clinical significance.
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Affiliation(s)
- Sitara S R Ajjampur
- Laboratory of Molecular and Cellular Parasitology, Department of Microbiology, National University of Singapore, Singapore
| | - Kevin S W Tan
- Laboratory of Molecular and Cellular Parasitology, Department of Microbiology, National University of Singapore, Singapore.
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Evaluation of cellular retinoic acid binding protein 2 gene expression through the retinoic acid pathway by co-incubation of Blastocystis ST-1 with HT29 cells in vitro. Parasitol Res 2016; 115:1965-75. [PMID: 26911149 DOI: 10.1007/s00436-016-4939-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2015] [Accepted: 01/20/2016] [Indexed: 10/22/2022]
Abstract
Blastocystis is a parasitic protist with a worldwide distribution that is commonly found in patients with colon and gastrointestinal pathological symptoms. Blastocystis infection has also commonly been reported in colorectal cancer and HIV/AIDS patients with gastrointestinal symptoms. To understand the pathway networks of gene regulation and the probable mechanisms influencing functions of HT-29 host cells in response to parasite infection, we examined the expression of 163 human oncogenes and kinases in human colon adenocarcinoma HT-29 cells co-incubated with Blastocystis by in-house cDNA microarray and PCR analysis. At least 10 genes were shown to be modified following Blastocystis co-incubation, including those with immunological, tumorigenesis, and antitumorigenesis functions. The expression of genes encoding cellular retinoic acid binding protein 2 (CRABP2) and proliferating cell nuclear antigen (PCNA) was markedly upregulated and downregulated, respectively. Reverse transcriptase-PCR validated the modified transcript expression of CRABP2 and other associated genes such as retinoic acid (RA)-related nuclear-receptor (RARα). Together, our data indicate that CRABP2, RARα, and PCNA expressions are involved in RA signaling regulatory networks that affect the growth, proliferation, and inflammation of HT-29 cells.
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Encystation—survival of Blastocystis hominis in immunocompetent mice abdomen cavity. Parasitol Res 2010; 106:1315-20. [DOI: 10.1007/s00436-010-1801-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2010] [Accepted: 02/09/2010] [Indexed: 11/26/2022]
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Tan KSW. New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin Microbiol Rev 2008; 21:639-65. [PMID: 18854485 PMCID: PMC2570156 DOI: 10.1128/cmr.00022-08] [Citation(s) in RCA: 459] [Impact Index Per Article: 27.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
SUMMARY Blastocystis is an unusual enteric protozoan parasite of humans and many animals. It has a worldwide distribution and is often the most commonly isolated organism in parasitological surveys. The parasite has been described since the early 1900s, but only in the last decade or so have there been significant advances in our understanding of Blastocystis biology. However, the pleomorphic nature of the parasite and the lack of standardization in techniques have led to confusion and, in some cases, misinterpretation of data. This has hindered laboratory diagnosis and efforts to understand its mode of reproduction, life cycle, prevalence, and pathogenesis. Accumulating epidemiological, in vivo, and in vitro data strongly suggest that Blastocystis is a pathogen. Many genotypes exist in nature, and recent observations indicate that humans are, in reality, hosts to numerous zoonotic genotypes. Such genetic diversity has led to a suggestion that previously conflicting observations on the pathogenesis of Blastocystis are due to pathogenic and nonpathogenic genotypes. Recent epidemiological, animal infection, and in vitro host-Blastocystis interaction studies suggest that this may indeed be the case. This review focuses on such recent advances and also provides updates on laboratory and clinical aspects of Blastocystis spp.
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Affiliation(s)
- Kevin S W Tan
- Department of Microbiology, Laboratory of Molecular and Cellular Parasitology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore.
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Sohail MR, Fischer PR. Blastocystis hominis and travelers. Travel Med Infect Dis 2005; 3:33-8. [PMID: 17292002 DOI: 10.1016/j.tmaid.2004.06.001] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2004] [Revised: 06/25/2004] [Accepted: 06/27/2004] [Indexed: 11/22/2022]
Abstract
B. hominis is a unicellular protozoan commonly identified in stool specimens of travelers who have returned from tropical countries. It has a world-wide distribution, and infection is more common in developing countries compared to industrialized nations. Clinical features of illness which have been attributed to Blastocystis include nausea, anorexia, abdominal pain, flatulence and acute or chronic diarrhea. The preferred method of diagnosis is a permanently stained smear of an unconcentrated stool specimen. The presence of B. hominis in stool specimens of symptomatic travelers should prompt clinicians to search for other unrecognized co-pathogens. Due to controversy regarding the pathogenicity of B. hominis in humans, clinicians are often faced with the dilemma of whether or not they should offer treatment for B. hominis infection in returned travelers. The most commonly used drugs for treatment include metronidazole and trimethoprim-sulfamethoxazole (TMP-SMX), when treatment is deemed necessary. Prevention in travelers should focus on food and water precautions as the organism is transmitted by the fecal-oral route.
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Affiliation(s)
- Muhammad R Sohail
- Division of Infectious Diseases, Department of Medicine, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA
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Requena I, Hernández Y, Ramsay M, Salazar C, Devera R. [Prevalence of Blastocystis hominis among food handlers from Caroni municipality, Bolivar State, Venezuela]. CAD SAUDE PUBLICA 2004; 19:1721-7. [PMID: 14999338 DOI: 10.1590/s0102-311x2003000600016] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
A cross-sectional survey was conducted to determine the prevalence of Blastocystis hominis infection in a random sample of apparently healthy food handlers. A total of 415 individuals attending the Manoa Urban Outpatient Clinic (Caroní Municipality, Bolívar State, Venezuela) in the Adult Hygiene Program and who requested health certification to work as food handlers were studied. Stool samples obtained by spontaneous evacuation were examined by direct microscopy and the Willis concentration method. A total of 150 individuals were infected (36.14%), 107 (25.78%) of whom with B. hominis. There was no difference between males and females (p > 0.05), but there was a significant difference between ages (chi(2) = 12.17; g.l. = 4), with infection more frequent between 18 and 27 years. In 71.02% of the cases, B. hominis was the only parasite. Giardia lamblia was the parasite most frequently associated with B. hominis (2.41%). In the majority (85%) of infected individuals, less than five microorganisms per microscopic field were observed. We conclude that B. hominis is a frequent intestinal parasite among food handlers in Caroní Municipality, Bolivar State, Venezuela.
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Affiliation(s)
- Ixora Requena
- Departamento de Parasitología y Microbiología, Escuela de Medicina, Universidad de Oriente, Ciudad Bolívar, Estado Bolívar, Venezuela
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Wang KX, Li CP, Wang J, Cui YB. Epidemiological survey of Blastocystis hominis in Huainan City, Anhui Province, China. World J Gastroenterol 2002; 8:928-32. [PMID: 12378644 PMCID: PMC4656589 DOI: 10.3748/wjg.v8.i5.928] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To provide scientific evidence for prevention and controlling of blastocystosis, the infection of Blastocystis homonis and to study its clinical significance in Huainan City, Anhui Province, China.
METHODS: Blastocystis homonis in fresh stools taken from 100 infants, 100 pupils, 100 middle school students and 403 patients with diarrhea was smeared and detected with method of iodine staining and hematoxylin staining. After preliminary direct microscopy, the shape and size of Blastocystis homonis were observed with high power lens. The cellular immune function of the patients with blastocystosis was detected with biotin-streptavidin (BSA).
RESULTS: The positive rates of Blastocystis homonis in fresh stools taken from the infants, pupils, middle school students and the patients with diarrhea, were 1.0% (1/100), 1.0% (1/100), 0% (0/100) and 5.96% (24/403) respectively. Furthermore, the positive rates of Blastocystis homonis in the stool samples taken from the patients with mild diarrhea, intermediate diarrhea, severe diarrhea and obstinate diarrhea were 6.03% (14/232), 2.25% (2/89), 0% (0/17) and 12.31% (8/65) respectively. The positive rates of Blastocystis homonis in fresh stools of male and female patients with diarrhea were 7.52% (17/226) and 3.95% (7/177) respectively, and those of patients in urban and rural areas were 4.56% (11/241) and 8.02% (13/162) respectively. There was no significant difference between them (P > 0.05). The positive rates of CD3+, CD4+, CD8+ in serum of Blastocystis homonis-positive and-negative individuals were 0.64 ± 0.06, 0.44 ± 0.06, 0.28 ± 0.04 and 0.60 ± 0.05, 0.40 ± 0.05 and 0.30 ± 0.05 respectively, and the ratio of CD4+/CD8+ of the two groups were 1.53 ± 0.34 and 1.27 ± 0.22. There was significant difference between the two groups (P < 0.05, P < 0.01).
CONCLUSION: The prevalence of Blastocystis hominis as an enteric pathogen in human seems not to be associated with gender and living environment, and that Blastocystis hominis is more common in stool samples of the patients with diarrhea, especially with chronic diarrhea or obstinate diarrhea. When patients with diarrhea infected by Blastocystis hominis, their cellular immune function decreases, which make it more difficult to be cured.
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Affiliation(s)
- Ke-Xia Wang
- Department of Etiology and Immunology, School of Medicine, Anhui University of Science Technology, Huainan 232001, Anhui Province, China
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Tan KSW, Singh M, Yap EH. Recent advances in Blastocystis hominis research: hot spots in terra incognita. Int J Parasitol 2002; 32:789-804. [PMID: 12062550 DOI: 10.1016/s0020-7519(02)00005-x] [Citation(s) in RCA: 90] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Despite being discovered more than 80 years ago, progress in Blastocystis research has been gradual and challenging, due to the small number of laboratories currently working on this protozoan parasite. To date, the morphology of Blastocystis hominis has been extensively studied by light and electron microscopy but all other aspects of its biology remain little explored areas. However, the availability of numerous and varied molecular tools and their application to the study of Blastocystis has brought us closer to understanding its biology. The purpose of this review is to describe and discuss recent advances in B. hominis research, with particular focus on new, and sometimes controversial, information that has shed light on its genetic heterogeneity, taxonomic links, mode of transmission, in vitro culture and pathogenesis. We also discuss recent observations that B. hominis has the capacity to undergo programmed cell death; a phenomenon similarly reported for many other unicellular organisms. There are still many gaps in our knowledge of this parasite. Although there is a growing body of evidence suggesting that B. hominis can be pathogenic under specific conditions, there are also other studies that indicated otherwise. Indeed, more studies are warranted before this controversial issue can be resolved. There is an urgent need for the identification and/or development of an animal model so that questions on its pathogenesis can be better answered. Another area that requires attention is the development of methods for the transfection of foreign/altered genes into B. hominis in order to facilitate genetic experiments.
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Affiliation(s)
- Kevin S W Tan
- Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597.
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