Ten Minute Introduction to Blastocystis
for Physicians and Scientists

"The great enemy of the truth is very often not the lie--deliberate, contrived and dishonest--but the myth--persistent, persuasive and unrealistic." 
- John Fitzgerald Kennedy

Q: When I was in medical school, they taught me that Blastocystis hominis was harmless.  How can it be pathogenic?

Did you attend medical school in the United States before 1989?  Then the organism that your professor showed you probably was harmless.  But things have changed since then.  Some parasites only infect a single host, which is called "host specificity".  So microbiologists often name a parasite after the host from which it is identified.  Blastocystis shows host specificity if you are a rodent, python, lizard, or bird.  But there is something different about humans.  Humans can be infected with any one of seven subtypes of Blastocystis.  This color-coded phylogenetic chart of Blastocystis shows which sub-species of Blastocystis have been found in various hosts:

KEY (see below for recent studies)
RED=Identified as pathogenic in humans by all recent studies
YELLOW=Identified as pathogenic in humans by most recent studies
GREEN=Identified as commensal in humans [Kaneda, 2001]

The chart is redrawn from a 2005 study performed at the Pasteur Institute in France, and the Woods Hole Oceanographic Institute, two of the world's leading Eukaryotic genetics research centers [1].  Note that some subtypes are specific to certain groups of animals. Subtype 2 and 4 are the Avian Subtypes.  Subtype 7 is the Rodent Subtype.

But a closer look shows that humans can be infected by all seven variants.  Human infection with Subtype 6 was reported last year [2]  The pathogenic subtypes are more common in Less Developed Countries [3].  However, over the last 20 years, this may have changed, as returning vacationers contracted symptomatic infections overseas and brought them back to the United States.

Q: Why has it taken so long to figure this out?

A: Understanding the complexity of Blastocystis requires special equipment to perform genetic investigation.  The infection has low mortality so it is not a focus of research.  The result is a large number of case reports, but little scientific research.  There has been no clinical study of Blastocystis infection in United States patients published since 1993.  A search of NIH's CRISP database for Blastocystis shows that the last funding for research occurred in 1993.

There are a number of other complexities which are beyond the scope of this introduction, but involve the proper way to identify and subtype the infection.  One important finding has been that the mainstay of gastrointestinal diagnosis - direct microscopy - fails to detect most infections.

Q: Can't biologists tell the types apart with a microscope?

Unfortunately, genetic techniques or advanced culture techniques are needed to distinguish types reliably.  A good analogy may be the emergence of Enterotoxigenic E. coli (ETEC) and now Enteroaggregative E. Coli (EAEC).  Both of these are variations of the harmless E. Coli found in humans.  EAEC is common in Less Developed Countries, and ETEC can't be distinguished with microscopy.

In the case of Blastocystis, though, the subtypes are not serotypes - they can be considered individual species [1].  There is sufficient genetic distance between them, and they have different numbers of chromosomes [4].   Within each subtype, there is substantial variation as well -- about 2% based on ribosomal DNA sequencing [5]. 

Q: I heard that Koch's Postulate has not been proven with Blastocystis.

A: Koch's postulates, which are widely viewed as sufficient proof of an organism's pathogenicity [6],  have been fulfilled in three studies [7-9].  For a list of animal studies, please see the References Section.

Q: So which subtype is the original harmless Blastocystis hominis?

A: BRF believes what was originally called "Blastocystis hominis" in the United States is Subtype #5 in the chart above.  This subtype is the only one which is unique to primates.  This subtype was shown to be non-pathogenic in 91% (21/23) of human hosts from whom it was isolated in Japan [10].  It constituted 45% (29/64) of the Blastocystis infections in a Japanese survey [10]. Until recently, this may have been the dominant type in the United States.

Q: Which subtypes make patients ill?

A: Researchers have consistently identified three Blastocystis subtypes as pathogenic or being associated with symptoms in patients: Subtype 1 [10,11,14], Subtype 3 [10,13,14], and Subtype 7 [13,15].  The non-human hosts of these subtypes are Farm Animals, Cattle, Pigs, and Rodents. Note that the ability of a subtype to infect cattle and rodents seems to be a good predictor of whether it will be pathogenic in humans.

BRF's research partners are currently repeating the experiment performed by Stensvold.  They are sequencing ribosomal DNA from samples taken from symptomatic patients.  We anticipate submitting a paper shortly.

Q: What is different about the subtypes which make patients ill?

Two of the subtypes come from the "Right Hand" branch of the Blastocystis tree.  Isolates from this side of the tree tend to have more chromosomes (13-16) and exhibit amoeboid morphological forms.  The amoeboid form may be an adaptation needed by the organism to infect rodents, cattle, and reptiles.  The amoeboid form has also been reported in Subtype 1 isolates from symptomatic individuals [14].

Here is a photomicrograph comparison showing a special culture of Blastocystis taken from an asymptomatic individual (left hand side) to that from a symptomatic individual (right hand side). [16]

Q: How do we know that it is Blastocystis making patients sick, and not some as-yet-undiscovered organism?

A: An NIH study showed that patients diagnosed with symptomatic Blastocystis infection exhibit an elevated immune response to the Blastocystis antigen that is not present in asymptomatic carriers [17].  A similar study performed with Egyptian patients produced equivalent results [18].

When cultured in the presence of intestinal bacteria, Blastocystis isolates from symptomatic patients produce large amoeboid forms which are not present in Blastocystis isolates from asymptomatic carrier [16].  When the isolates from this study were genetically analyzed, they were shown to belong to a separate phylogenetic branch of Blastocystis [19]. 

Q: If it is a pathogen, where are the patients?

In the early 1990's, it is likely that few symptomatic cases existed in the US.  The dominant form of Blastocystis was most likely Subtype 5.  Most symptomatic cases were due to travelers returning from less developed countries [3].

According to CDC and private laboratory data, the prevalence of Blastocystis infection saw a major increase between 1987 and 2000, from 2.6% of samples examined to 23% of samples examined.  In the same period, the incidence of Giardia actually decreased.  Many infections may be diagnosed as irritable bowel syndrome, as the symptoms are similar.

(c) 2007 BRF

Figure 3.  Comparison of the prevalence of four common gastrointestinal protozoa in stool samples submitted to diagnostic labs in 1987 and 2000 nationally in the United States.  The prevalence of Giardia dropped significantly, while the prevalence of Cryptosporidium increased.  The largest increase was in Blastocystis, which was previously present in 2.6% of samples, but was present in 23% of samples submitted to this lab. 1987 Data taken from [Kappus, 1991 - view abstract on PUBMED  or contact me for paper] 2000 Data taken from [Amin, 2002 - view paper]

Q: Why doesn't the infection respond to short courses of oral antibiotics

The pathogenic variant of Blastocystis has been shown to produce large non-motile adhesive amoeboid forms [16].  In fungal infections, this type of behavior leads to aggregations of infectious material which can be difficult to treat.  Treatment efficacy should be evaluated with direct PCR testing of stool samples, as direct microscopy is particularly insensitive in the detection of infections [see references page], and may lead to the confusing phenomenon of patients testing negative for the parasite while continuing to show symptoms.

Q: What is the relationship to IBS (or Leaky Bowel Syndrome or Multiple Food Allergies)

Research from the Middle East has found that patients with IBS exhibit an elevated response to the Blastocystis antigen [20].  However, the researcher was unable to identify Blastocystis with direct microscopy in stool samples in these patients.  He did not have data on the patient's treatment history, so he assumed that the infection had been eradicated, and the immune response remained due to the persistence of the cellular material of Blastocystis.

When this research was performed, the genetic diversity of Blastocystis was not understood.  We now know that infection with some of the zoonotic variants can be difficult to detect with direct microscopy and even with stool culture [2] [13]. The most reliable method for detecting infection is direct PCR analysis of stool specimens [2] [13].

BRF is currently working with several research partners to examine Blastocystis subtypes in individuals diagnosed with symptomatic Blastocystis infection.  Because of the difficulty in detecting Blastocystis infection [2] [13] [21-23], we are also investigating infection in patients with "nebulous" gastrointestinal diseases (Leaky Gut Syndrome, Multiple Food Allergies and Irritable Bowel Syndrome).   Our research partners anticipate submitting a publication this summer.

Q: How can the diarrhea and abdominal pain be explained in the absence of endoscopic findings?  How can diffuse symptoms such as headaches, fatigue, and depression be explained?

Pathogens can cause diarrhea in two ways.  They can attack the host causing damage, or they can secrete a substance that alters gastrointestinal permeability.  For example, the bacteria that causes Cholera does not destroy tissue - it causes diarrhea by secreting a substance.

Both symptomatic Blastocystis infection and Entamoeba histolytica infection are unusual in that they can produce a sustained secretory diarrhea alternating with constipation.  An investigation into the secretory properties of Entamoeba histolytica showed that it manufactures serotonin, a neurohormonal substance [24].  A further study showed that patients who have contracted Entamoeba histolytica infection exhibit elevated serum serotonin levels [25].  Most of the symptoms described by Blastocystis patients are serotonin mediated: headaches, depression, fatigue, sleep disturbances.  An investigation of a similar pathogenesis may be productive.  

References

1.  Noel C, Dufernez F, Gerbod D, Edgcomb VP, Delgado-Viscogliosi P, Ho LC, Singh M, Wintjens R, Sogin ML, Capron M, Pierce R, Zenner L, Viscogliosi E.  Molecular phylogenies of Blastocystis isolates from different hosts: implications for genetic diversity, identification of species, and zoonosis. J Clin Microbiol. 2005 Jan;43(1):348-55.  PMID: 15634993 [Download paper]

2. Parkar U, Traub RJ, Kumar S, Mungthin M, Vitali S, Leelayoova S, Morris K, Thompson RC. Direct characterization of Blastocystis from faeces by PCR and evidence of zoonotic potential. Parasitology. 2007 Mar;134(Pt 3):359-67. Epub 2006 Oct 19. PMID: 17052374 [View abstract]

3. Sheehan DJ, Raucher BG, McKitrick JC. Association of Blastocystis hominis with signs and symptoms of human disease. J Clin Microbiol. 1986 Oct;24(4):548-50. PMID: 3771743
[Download paper].

4. Carbajal JA, del Castillo L, Lanuza MD, Villar J, Borras R. Karyotypic diversity among Blastocystis hominis isolates. Int J Parasitol. 1997 Aug;27(8):941-5. PMID: 9292310  [View abstract]

5. Stensvold CR, Suresh GK, Tan KS, Thompson RC, Traub RJ, Viscogliosi E, Yoshikawa H, Clark CG. Terminology for Blastocystis subtypes--a consensus. Trends Parasitol. 2007 Mar;23(3):93-6.PMID: 17241816 [View abstract]

6. Evans AS. Causation and disease: the Henle-Koch postulates revisited. Yale J Biol Med 49:175–195.  1976

7. Moe KT, Singh M, Howe J, Ho LC, Tan SW, Chen XQ, Ng GC, Yap EH. Experimental Blastocystis hominis infection in laboratory mice. Parasitol Res. 1997;83(4):319-25.  PMID: 9134552 [View abstract]

8. Yao FR, Qiao JY, Zhao Y, Zhang X, Yang JH, Li XQ. Experimental infection of mice with Blastocystis hominis.   Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2005 Dec 30;23(6):444-8. 
PMID: 16566218 [View abstract]

9. Zhang HW, Li W, Yan QY, He LJ, Su YP.  Impact of blastocystis hominis infection on ultrastructure of intestinal mucosa in mice.  Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2006 Jun;24(3):187-91. PMID: 17094618 [View abstract]

10. Kaneda Y, Horiki N, Cheng XJ, Fujita Y, Maruyama M, Tachibana H.  Ribodemes of Blastocystis hominis isolated in Japan. Am J Trop Med Hyg. 2001 Oct;65(4):393-6. PMID: 11693890 [Download paper] Note that Kaneda uses Ariuse subtypes - you can use the table below to translate between subtypes from different papers

11. Yan Y, Su S, Lai R, Liao H, Ye J, Li X, Luo X, Chen G. Genetic variability of Blastocystis hominis isolates in China. Parasitol Res. 2006 Oct;99(5):597-601. PMID: 16688468  [View Abstract]

12. Omitted

13. Stensvold R, Brillowska-Dabrowska A, Nielsen HV, Arendrup MC. Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reaction. J Parasitol. 2006 Oct;92(5):1081-7. PMID: 17152954 [View abstract]

14. Personal communication from Professor Suresh Kumar, University of Malaysia, 10/2006.  Dr. Kumar's laboratory published the two papers below comparing isolates from symptomatic and asymptomatic patients [16] [19].  He indicated that all isolates from symptomatic patients in these studies belonged to (Yoshikawa) Subtype 3, which was consistent with his other patients.  He indicated that some Subtype 1 isolates had been found in symptomatic individuals, but only in cancer patients.

15.  Puthia MK, Sio SW, Lu J, Tan KS. Blastocystis ratti induces contact-independent apoptosis, F-actin rearrangement, and barrier function disruption in IEC-6 cells. Infect Immun. 2006 Jul;74(7):4114-23. PMID: 16790785 [Abstract]  [Download paper]

16.  Tan TC, Suresh KG. Predominance of amoeboid forms of Blastocystis hominis in isolates from symptomatic patients. Parasitol Res. 2006 Feb;98(3):189-93.  PMID: 16323025  [View Abstract]

17.Zierdt CH, Nagy B. Antibody response to Blastocystis hominis infections. Ann Intern Med. 1993 Jun 15;118(12):985-6. PMID: 8489119  [Download paper].

18. Mahmoud MS, Saleh WA. Secretory and humoral antibody responses to Blastocystis hominis in symptomatic and asymptomatic human infections. J Egypt Soc Parasitol. 2003 Apr;33(1):13-30. PMID: 12739797  [View Abstract]

19. Tan TC, Suresh KG, Thong KL, Smith HV.  PCR fingerprinting of Blastocystis isolated from symptomatic and asymptomatic human hosts. Parasitol Res. 2006 Sep;99(4):459-65.
PMID: 16628457 [View Abstract]

20. Hussain R, Jaferi W, Zuberi S, Baqai R, Abrar N, Ahmed A, Zaman V. Significantly increased IgG2 subclass antibody levels to Blastocystis hominis in patients with irritable bowel syndrome. Am J Trop Med Hyg. 1997 Mar;56(3):301-6.  PMID: 9129532  [View Abstract]

21.Termmathurapoj S, Leelayoova S, Aimpun P, Thathaisong U, Nimmanon T, Taamasri P, Mungthin M. The Usefulness of Short-Term In-Vitro Cultivation for the Detection and Molecular Study of Blastocystis hominis in Stool Specimens. Parasitology Research. 2004 Aug;93(6):445-7. Epub 2004 Jul 9. {Pubmed ID 15243800}  [View Abstract]

22. Suresh K, Smith H. Comparison of Methods for Detecting Blastocystis minis. European Journal of Clinical Microbiology and Infectious Diseases. 2004 Jun;23(6):509-11. Epub 2004 May 28. {Pubmed 15168139} [View Abstract]

23.  Leelayoova S, Taamasri P, Rangsin R, Naaglor T, Thathaisong U, Mungthin M. In-vitro Cultivation: A Sensitive Method for Detecting Blastocystis hominis. Annals of Tropical Medicine and Parasitology. 2002 Dec;96(8):803-7. {Pubmed 12625935}  [View Abstract]

24. McGowan K, Kane A, Asarkof N, Wicks J, Guerina V, Kellum J, Baron S, Gintzler AR, Donowitz M.
Entamoeba histolytica causes intestinal secretion: role of serotonin. Science. 1983 Aug 19;221(4612):762-4.PMID: 6308760  [View Abstract]

25. Banu, Naheed et al. Neurohumoral Alterations and their role in Amoebiasis. Indian Journal of Clinical Biochemistry, 2005. 20 (2) 142-145. [Download Paper]