Guest Commentary
Laboratory diagnosis of Blastocystis spp Blastocystis is one of at least two stramenopiles organisms parasitic to humans. A strict anaerobe, it is a parasite of the colon, transmitted by feces, and colonizing humans and a large variety of nonhuman hosts, ranging from nonhuman primates, over birds, to insects, including cockroaches.[1] Colonizing probably more than 1 billion people globally, its clinical and public health significance remains obscure, primarily because it is common in both healthy individuals and patients suffering from intestinal symptoms, including common manifestations such as diarrhea and irritable bowel syndrome; [2,3] hence, it is currently not possible to distinguish between colonization and infection. Drugs or drug combinations reported to enable the eradication of Blastocystis fail to do so consistently, impeding studies aiming to explore the effect of treatment.[4,5] Blastocystis exhibits extreme genetic diversity, currently comprising seventeen subtypes (ST1–ST17), nine of which have been identified in humans, and one of the current working hypotheses is that differences in clinical outcome may rely on differences in subtypes.
not particularly informative. Indeed, this predicament may have significantly hampered our ability to develop a more detailed understanding of the role of Blastocystis in health and disease.[6] The insensitivity of microscopy of fecal concentrates likely stems from disintegration of Blastocystis during technical processing, but it should also be kept in mind that there is no consensus regarding Blastocystis morphology. Many stages have been accounted for, although it appears that there are no benchmark criteria for the identification of the vacuolar, granular, avacuolar, mutlivacuolar, amoeboid, and cyst stages, and it is possible that some of the stages are artifacts arising from, e.g., exposure to oxygen. To many, the morphology of Blastocystis may be seen as a continuum of these different “stages,” and the morphological classification of Blastocystis remains a bit of a mystery. Surprisingly, the cyst stage, which is probably responsible for transmission, is very rarely reported of and may require special procedures for accurate detection.[7]
Accurate diagnosis is critical to clinicoepidemiological studies addressing the role of Blastocystis in health and disease. Several methods have been used to diagnose the parasite. Surveys reporting on intestinal parasites and their prevalence often use classical parasitological techniques, including microscopy of fecal concentrates. These methods are applicable to a wide variety of intestinal parasites and typically known as the “Ova and Cysts” (or–misleadingly–“Ova and Parasites”) methods. Blastocystis is often seen listed in data collections arising from studies employing these methods, but consensus is emerging that such methods are impaired by significant lack of sensitivity, which is why data on Blastocystis obtained by the Ova and Cysts method is
In their article, Elghareeb et al.[17] compare different methods for detecting Blastocystis in 1200 fecal samples from diarrheic patients in Qualyobia, Egypt, demonstrating a vast range in diagnostic sensitivity. Hence, 22.8% (n = 274) of the samples were positive for Blastocystis by xenic in vitro culture (XIVC), while 12.3%, 10.0%, 6.0%, and 3.5% were positive by trichrome staining of fixed fecal smears, formol ether concentration, iodine‑stained smear, and direct smear, respectively. Interestingly, XIVC was more sensitive than any of the traditional parasitological techniques. Intriguingly, the authors include a table of the different morphological stages detected by the five methods, and here it appears that culture was the only method enabling detection of all four morphological stages reported of in the study, including the cyst stage. It is interesting that the authors report on cysts in culture since it might be expected that cysts would excyst when incubated in culture under anaerobic conditions at 37°C. Approaches to differentiating morphological stages by diagnostic methods as seen here should be encouraged, but clear definitions on the criteria used for morphological classification are warranted.
Access this article online Quick Response Code:
Website: www.tropicalparasitology.org DOI: 10.4103/2229-5070.149885
Tropical Parasitology
3
Jan 2015 | Volume 5 | Issue 1 |
Stensvold: Blastocystis and diarrhoea
People who have worked with Blastocystis cultures know how easy Blastocystis organisms are to pick up by microscopy of a few microliters of positive culture, and hence specificities lower than 100% are unexpected. Surprisingly, Younis et al. indicates the specificity of culture relative to trichrome staining as only 88%. Contrary to evaluating cultures for the presence of Blastocystis, examining permanently stained preparations of fecal smears requires skill and experience. Parasites such as Blastocystis and Dientamoeba may be difficult to pick up in giemsa‑stained fecal smears even by highly experienced parasitologists; for instance, they may be confused with leukocytes or even epithelial cells. Compared with giemsa, trichrome staining is more commonly used for Blastocystis; yet, in the absence of molecular confirmation, the diagnostic performances of the various methods evaluated in the study by Younis may be difficult to appreciate.
showed a Blastocystis prevalence of 100% in a larger group of children in Senegal.[16] Having DNAs available for further analysis, they were able to map the distribution of subtypes. For surveys, it might be interesting to know the proportion of individuals colonized, but it might be even more important to know what subtypes are present, and so a positive result should preferably be expanded by subtype analysis. In cases where posttreatment tests are carried out to test whether Blastocystis has been eradicated or not, obtaining a qualitative result is obviously useful, but also here, adding subtype–even allele–information would be valuable to learn whether any strain detected post-treatment is identical to the one detected prior to treatment. These thoughts should also accompany decisions on whether or not to implement other, non‑DNA based, qualitative methods such as antigen detecting ELISA systems. Despite rapid advances in the field of sequencing, the use of DNA‑based diagnostic methods are still relative laborious and expensive, requiring quite advanced technological infrastructures. Subtle, easy‑to‑use, and low‑cost subtype‑differentiating methods should be developed to enable screening of large batches of fecal samples along the lines of fecal dipstick tests or tests exploiting synthetic biology technology.
Meanwhile, state‑of‑the‑art diagnosis is real‑time polymerase chain reaction (PCR), such as the assays proposed by Poirier et al. and Stensvold et al.,[8,9] not only due to high sensitivity, but also since DNAs positive for Blastocystis by real‑time PCR can be used for molecular characterization using barcoding, [10,11] which has recently been facilitated and standardized thanks to the “Blastocystis Subtype (18S) and Sequence Typing (MLST) Databases” available at http://www. pubmlst.org/blastocystis.[12] It was recently shown that the sensitivity of culture compared with DNA‑based methods is in the range of 52–79%.[8,9]
Christen Rune Stensvold Department of Microbiology and Infection Control, Laboratory of Parasitology, Statens Serum Institut, Artillerivej 5, DK‑2300 Copenhagen S, Denmark. E‑mail: [email protected]
Little is known about factors potentially influencing the sensitivity of culture as a diagnostic method, including the time lag from sampling to culture, culture medium used, whether cysts are excysting during XIVC, and whether culture amplifies all subtypes equally well. Jones’ medium has been used by many, probably because it is easy and inexpensive to manufacture, but many different types of media would work with Blastocystis.[13] Svensson[14] pointed out that Blastocystis is likely to grow avidly in any starch‑containing medium, provided that the starch is split (typically by starch splitting bacteria). Jones’ medium does not contain starch, and so using a starch‑containing medium may enable a higher sensitivity of culture. Of note, long‑term culture may favor one subtype over another in cases of mixed subtype infections,[15] but experience is limited.
REFERENCES 1. 2.
3.
4. 5.
Although it is still unclear whether intensity of colonization is linked to existence of symptoms in carriers of Blastocystis, one aspect of Blastocystis diagnosis that is very rarely touched upon, if ever, is the clinical and epidemiological value of a qualitative result, that is, a result simply indicating whether a sample is positive or negative. Using real‑time PCR, El Safadi et al. recently Jan 2015 | Volume 5 | Issue 1 |
6. 7.
4
Clark CG, van der Giezen M, Alfellani MA, Stensvold CR. Recent developments in Blastocystis research. Adv Parasitol 2013;82:1‑32. Scanlan PD, Stensvold CR, Rajilić‑Stojanović M, Heilig HG, De Vos WM, O’Toole PW, et al. The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota. FEMS Microbiol Ecol 2014;90:326‑30. Krogsgaard LR, Engsbro AL, Stensvold CR, Nielsen HV, Bytzer P. The prevalence of intestinal parasites is not greater among individuals with irritable bowel syndrome: A Population‑based Case‑control Study. Clin Gastroenterol Hepatol 2014;[Epub ahead of print]. Stensvold CR, Smith HV, Nagel R, Olsen KE, Traub RJ. Eradication of Blastocystis carriage with antimicrobials: Reality or delusion? J Clin Gastroenterol 2010;44:85‑90. Nagel R, Bielefeldt‑Ohmann H, Traub R. Clinical pilot study: Efficacy of triple antibiotic therapy in Blastocystis positive irritable bowel syndrome patients. Gut Pathog 2014;6:34. Stensvold CR. Blastocystis: Genetic diversity and molecular methods for diagnosis and epidemiology. Trop Parasitol 2013;3:26‑34. Rene BA, Stensvold CR, Badsberg JH, Nielsen HV. Subtype analysis of Blastocystis isolates from Blastocystis cyst excreting patients. Am J Trop Med Hyg 2009;80:588‑92. Tropical Parasitology
Stensvold: Blastocystis and diarrhoea
8. Stensvold CR, Ahmed UN, Andersen LO, Nielsen HV. Development and evaluation of a genus‑specific, probe‑based, internal‑process‑controlled real‑time PCR assay for sensitive and specific detection of Blastocystis spp. J Clin Microbiol 2012;50:1847‑51. 9. Poirier P, Wawrzyniak I, Albert A, El Alaoui H, Delbac F, Livrelli V. Development and evaluation of a real‑time PCR assay for detection and quantification of Blastocystis parasites in human stool samples: Prospective study of patients with hematological malignancies. J Clin Microbiol 2011;49:975‑83. 10. Scicluna SM, Tawari B, Clark CG. DNA barcoding of Blastocystis. Protist 2006;157:77‑85. 11. Stensvold CR. Comparison of sequencing (barcode region) and sequence‑tagged‑site PCR for Blastocystis subtyping. J Clin Microbiol 2013;51:190‑4. 12. Stensvold CR, Alfellani M, Clark CG. Levels of genetic diversity vary dramatically between Blastocystis subtypes. Infect Genet Evol 2012;12:263‑73. 13. Clark CG, Diamond LS. Methods for cultivation of luminal parasitic protists of clinical importance. Clin Microbiol Rev 2002;15:329‑41.
14. Svensson R. Studies on human intestinal protozoa, especially with regard to their demonstrability and the connection between their distribution and hygienic conditions. Acta Med Scand 1935;Suppl 70:1‑115. 15. Parkar U, Traub RJ, Kumar S, Mungthin M, Vitali S, Leelayoova S, et al. Direct characterization of Blastocystis from faeces by PCR and evidence of zoonotic potential. Parasitology 2007;134:359‑67. 16. El Safadi D, Gaayeb L, Meloni D, Cian A, Poirier P, Wawrzyniak I, et al. Children of Senegal river basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infect Dis 2014;14:164. 17. Elghareeb AS, Younis MS, El Fakahany AF, Nagaty IM, Nagib MM. Laboratory diagnosis of Blastocystis spp. in diarrheic patients. Trop Parasitol 2015;5:24-29. How to cite this article: Stensvold CR. Laboratory diagnosis of Blastocystis spp. Trop Parasitol 2015;5:3-5. Source of Support: Nil. Conflict of Interest: None declared. DOA: 21-12-2014, DOP: 22-01-2016
“Quick Response Code” link for full text articles The journal issue has a unique new feature for reaching to the journal’s website without typing a single letter. Each article on its first page has a “Quick Response Code”. Using any mobile or other hand-held device with camera and GPRS/other internet source, one can reach to the full text of that particular article on the journal’s website. Start a QR-code reading software (see list of free applications from http://tinyurl.com/ yzlh2tc) and point the camera to the QR-code printed in the journal. It will automatically take you to the HTML full text of that article. One can also use a desktop or laptop with web camera for similar functionality. See http://tinyurl.com/2bw7fn3 or http://tinyurl.com/3ysr3me for the free applications.
Tropical Parasitology
5
Jan 2015 | Volume 5 | Issue 1 |
Copyright of Tropical Parasitology is the property of Medknow Publications & Media Pvt. Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Laboratory diagnosis of Blastocystis spp Blastocystis is one of at least two stramenopiles organisms parasitic to humans. A strict anaerobe, it is a parasite of the colon, transmitted by feces, and colonizing humans and a large variety of nonhuman hosts, ranging from nonhuman primates, over birds, to insects, including cockroaches.[1] Colonizing probably more than 1 billion people globally, its clinical and public health significance remains obscure, primarily because it is common in both healthy individuals and patients suffering from intestinal symptoms, including common manifestations such as diarrhea and irritable bowel syndrome; [2,3] hence, it is currently not possible to distinguish between colonization and infection. Drugs or drug combinations reported to enable the eradication of Blastocystis fail to do so consistently, impeding studies aiming to explore the effect of treatment.[4,5] Blastocystis exhibits extreme genetic diversity, currently comprising seventeen subtypes (ST1–ST17), nine of which have been identified in humans, and one of the current working hypotheses is that differences in clinical outcome may rely on differences in subtypes.
not particularly informative. Indeed, this predicament may have significantly hampered our ability to develop a more detailed understanding of the role of Blastocystis in health and disease.[6] The insensitivity of microscopy of fecal concentrates likely stems from disintegration of Blastocystis during technical processing, but it should also be kept in mind that there is no consensus regarding Blastocystis morphology. Many stages have been accounted for, although it appears that there are no benchmark criteria for the identification of the vacuolar, granular, avacuolar, mutlivacuolar, amoeboid, and cyst stages, and it is possible that some of the stages are artifacts arising from, e.g., exposure to oxygen. To many, the morphology of Blastocystis may be seen as a continuum of these different “stages,” and the morphological classification of Blastocystis remains a bit of a mystery. Surprisingly, the cyst stage, which is probably responsible for transmission, is very rarely reported of and may require special procedures for accurate detection.[7]
Accurate diagnosis is critical to clinicoepidemiological studies addressing the role of Blastocystis in health and disease. Several methods have been used to diagnose the parasite. Surveys reporting on intestinal parasites and their prevalence often use classical parasitological techniques, including microscopy of fecal concentrates. These methods are applicable to a wide variety of intestinal parasites and typically known as the “Ova and Cysts” (or–misleadingly–“Ova and Parasites”) methods. Blastocystis is often seen listed in data collections arising from studies employing these methods, but consensus is emerging that such methods are impaired by significant lack of sensitivity, which is why data on Blastocystis obtained by the Ova and Cysts method is
In their article, Elghareeb et al.[17] compare different methods for detecting Blastocystis in 1200 fecal samples from diarrheic patients in Qualyobia, Egypt, demonstrating a vast range in diagnostic sensitivity. Hence, 22.8% (n = 274) of the samples were positive for Blastocystis by xenic in vitro culture (XIVC), while 12.3%, 10.0%, 6.0%, and 3.5% were positive by trichrome staining of fixed fecal smears, formol ether concentration, iodine‑stained smear, and direct smear, respectively. Interestingly, XIVC was more sensitive than any of the traditional parasitological techniques. Intriguingly, the authors include a table of the different morphological stages detected by the five methods, and here it appears that culture was the only method enabling detection of all four morphological stages reported of in the study, including the cyst stage. It is interesting that the authors report on cysts in culture since it might be expected that cysts would excyst when incubated in culture under anaerobic conditions at 37°C. Approaches to differentiating morphological stages by diagnostic methods as seen here should be encouraged, but clear definitions on the criteria used for morphological classification are warranted.
Access this article online Quick Response Code:
Website: www.tropicalparasitology.org DOI: 10.4103/2229-5070.149885
Tropical Parasitology
3
Jan 2015 | Volume 5 | Issue 1 |
Stensvold: Blastocystis and diarrhoea
People who have worked with Blastocystis cultures know how easy Blastocystis organisms are to pick up by microscopy of a few microliters of positive culture, and hence specificities lower than 100% are unexpected. Surprisingly, Younis et al. indicates the specificity of culture relative to trichrome staining as only 88%. Contrary to evaluating cultures for the presence of Blastocystis, examining permanently stained preparations of fecal smears requires skill and experience. Parasites such as Blastocystis and Dientamoeba may be difficult to pick up in giemsa‑stained fecal smears even by highly experienced parasitologists; for instance, they may be confused with leukocytes or even epithelial cells. Compared with giemsa, trichrome staining is more commonly used for Blastocystis; yet, in the absence of molecular confirmation, the diagnostic performances of the various methods evaluated in the study by Younis may be difficult to appreciate.
showed a Blastocystis prevalence of 100% in a larger group of children in Senegal.[16] Having DNAs available for further analysis, they were able to map the distribution of subtypes. For surveys, it might be interesting to know the proportion of individuals colonized, but it might be even more important to know what subtypes are present, and so a positive result should preferably be expanded by subtype analysis. In cases where posttreatment tests are carried out to test whether Blastocystis has been eradicated or not, obtaining a qualitative result is obviously useful, but also here, adding subtype–even allele–information would be valuable to learn whether any strain detected post-treatment is identical to the one detected prior to treatment. These thoughts should also accompany decisions on whether or not to implement other, non‑DNA based, qualitative methods such as antigen detecting ELISA systems. Despite rapid advances in the field of sequencing, the use of DNA‑based diagnostic methods are still relative laborious and expensive, requiring quite advanced technological infrastructures. Subtle, easy‑to‑use, and low‑cost subtype‑differentiating methods should be developed to enable screening of large batches of fecal samples along the lines of fecal dipstick tests or tests exploiting synthetic biology technology.
Meanwhile, state‑of‑the‑art diagnosis is real‑time polymerase chain reaction (PCR), such as the assays proposed by Poirier et al. and Stensvold et al.,[8,9] not only due to high sensitivity, but also since DNAs positive for Blastocystis by real‑time PCR can be used for molecular characterization using barcoding, [10,11] which has recently been facilitated and standardized thanks to the “Blastocystis Subtype (18S) and Sequence Typing (MLST) Databases” available at http://www. pubmlst.org/blastocystis.[12] It was recently shown that the sensitivity of culture compared with DNA‑based methods is in the range of 52–79%.[8,9]
Christen Rune Stensvold Department of Microbiology and Infection Control, Laboratory of Parasitology, Statens Serum Institut, Artillerivej 5, DK‑2300 Copenhagen S, Denmark. E‑mail: [email protected]
Little is known about factors potentially influencing the sensitivity of culture as a diagnostic method, including the time lag from sampling to culture, culture medium used, whether cysts are excysting during XIVC, and whether culture amplifies all subtypes equally well. Jones’ medium has been used by many, probably because it is easy and inexpensive to manufacture, but many different types of media would work with Blastocystis.[13] Svensson[14] pointed out that Blastocystis is likely to grow avidly in any starch‑containing medium, provided that the starch is split (typically by starch splitting bacteria). Jones’ medium does not contain starch, and so using a starch‑containing medium may enable a higher sensitivity of culture. Of note, long‑term culture may favor one subtype over another in cases of mixed subtype infections,[15] but experience is limited.
REFERENCES 1. 2.
3.
4. 5.
Although it is still unclear whether intensity of colonization is linked to existence of symptoms in carriers of Blastocystis, one aspect of Blastocystis diagnosis that is very rarely touched upon, if ever, is the clinical and epidemiological value of a qualitative result, that is, a result simply indicating whether a sample is positive or negative. Using real‑time PCR, El Safadi et al. recently Jan 2015 | Volume 5 | Issue 1 |
6. 7.
4
Clark CG, van der Giezen M, Alfellani MA, Stensvold CR. Recent developments in Blastocystis research. Adv Parasitol 2013;82:1‑32. Scanlan PD, Stensvold CR, Rajilić‑Stojanović M, Heilig HG, De Vos WM, O’Toole PW, et al. The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota. FEMS Microbiol Ecol 2014;90:326‑30. Krogsgaard LR, Engsbro AL, Stensvold CR, Nielsen HV, Bytzer P. The prevalence of intestinal parasites is not greater among individuals with irritable bowel syndrome: A Population‑based Case‑control Study. Clin Gastroenterol Hepatol 2014;[Epub ahead of print]. Stensvold CR, Smith HV, Nagel R, Olsen KE, Traub RJ. Eradication of Blastocystis carriage with antimicrobials: Reality or delusion? J Clin Gastroenterol 2010;44:85‑90. Nagel R, Bielefeldt‑Ohmann H, Traub R. Clinical pilot study: Efficacy of triple antibiotic therapy in Blastocystis positive irritable bowel syndrome patients. Gut Pathog 2014;6:34. Stensvold CR. Blastocystis: Genetic diversity and molecular methods for diagnosis and epidemiology. Trop Parasitol 2013;3:26‑34. Rene BA, Stensvold CR, Badsberg JH, Nielsen HV. Subtype analysis of Blastocystis isolates from Blastocystis cyst excreting patients. Am J Trop Med Hyg 2009;80:588‑92. Tropical Parasitology
Stensvold: Blastocystis and diarrhoea
8. Stensvold CR, Ahmed UN, Andersen LO, Nielsen HV. Development and evaluation of a genus‑specific, probe‑based, internal‑process‑controlled real‑time PCR assay for sensitive and specific detection of Blastocystis spp. J Clin Microbiol 2012;50:1847‑51. 9. Poirier P, Wawrzyniak I, Albert A, El Alaoui H, Delbac F, Livrelli V. Development and evaluation of a real‑time PCR assay for detection and quantification of Blastocystis parasites in human stool samples: Prospective study of patients with hematological malignancies. J Clin Microbiol 2011;49:975‑83. 10. Scicluna SM, Tawari B, Clark CG. DNA barcoding of Blastocystis. Protist 2006;157:77‑85. 11. Stensvold CR. Comparison of sequencing (barcode region) and sequence‑tagged‑site PCR for Blastocystis subtyping. J Clin Microbiol 2013;51:190‑4. 12. Stensvold CR, Alfellani M, Clark CG. Levels of genetic diversity vary dramatically between Blastocystis subtypes. Infect Genet Evol 2012;12:263‑73. 13. Clark CG, Diamond LS. Methods for cultivation of luminal parasitic protists of clinical importance. Clin Microbiol Rev 2002;15:329‑41.
14. Svensson R. Studies on human intestinal protozoa, especially with regard to their demonstrability and the connection between their distribution and hygienic conditions. Acta Med Scand 1935;Suppl 70:1‑115. 15. Parkar U, Traub RJ, Kumar S, Mungthin M, Vitali S, Leelayoova S, et al. Direct characterization of Blastocystis from faeces by PCR and evidence of zoonotic potential. Parasitology 2007;134:359‑67. 16. El Safadi D, Gaayeb L, Meloni D, Cian A, Poirier P, Wawrzyniak I, et al. Children of Senegal river basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infect Dis 2014;14:164. 17. Elghareeb AS, Younis MS, El Fakahany AF, Nagaty IM, Nagib MM. Laboratory diagnosis of Blastocystis spp. in diarrheic patients. Trop Parasitol 2015;5:24-29. How to cite this article: Stensvold CR. Laboratory diagnosis of Blastocystis spp. Trop Parasitol 2015;5:3-5. Source of Support: Nil. Conflict of Interest: None declared. DOA: 21-12-2014, DOP: 22-01-2016
“Quick Response Code” link for full text articles The journal issue has a unique new feature for reaching to the journal’s website without typing a single letter. Each article on its first page has a “Quick Response Code”. Using any mobile or other hand-held device with camera and GPRS/other internet source, one can reach to the full text of that particular article on the journal’s website. Start a QR-code reading software (see list of free applications from http://tinyurl.com/ yzlh2tc) and point the camera to the QR-code printed in the journal. It will automatically take you to the HTML full text of that article. One can also use a desktop or laptop with web camera for similar functionality. See http://tinyurl.com/2bw7fn3 or http://tinyurl.com/3ysr3me for the free applications.
Tropical Parasitology
5
Jan 2015 | Volume 5 | Issue 1 |
Copyright of Tropical Parasitology is the property of Medknow Publications & Media Pvt. Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
