Presentation of Quiz #57

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A 5-year old girl was first seen in the clinic because of failure to thrive and vague symptoms like poor appetite and failure to gain weight. There was one other child in the family, a 7-year old sister who was in good health. The child was apparently fine until about two months prior to becoming symptomatic. She began to have diarrhea, however, this condition was not always present. She complained of lack of appetite, and general malaise. Although stool examinations were performed, it was unclear exactly what diagnostic methods were used and the initial examinations were reported as negative.

Additional parasitology examinations were performed on stool specimens that had been collected in the two-vial system (one of 10% formalin and one in PVA – zinc-based) (O&P exam: concentration, permanent stained slide). Note that the direct wet mount was not performed since the specimen was submitted in preservative – this is the correct approach. The direct wet mount is not performed unless fresh stool is submitted, and the specimen is liquid or very soft.

Laboratory results revealed the following on the second and third stool examinations from the trichrome stained smear; the first specimen was reported as "No Parasites Seen":

  

This is from a trichrome-stained permanent stained smear; the organism was seen using the 100X oil immersion objective of the microscope. Although a specific organism was not suspected from the history, organism identification could not be confirmed without examination of the permanent stained slide, and this was the only protozoan type seen in the entire examination; the first specimen had been reported as negative.

QUESTIONS:

1.            From this image seen on the permanent stained smear, can you identify the organism to the genus and species levels?

2.            How should this organism be reported? Could this organism be responsible for the patient’s symptoms?

 

 

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ANSWER AND DISCUSSION OF DIAGNOSTIC QUIZ #57

This image contains several Blastocystis spp. (Blastocystis hominis) central body forms; these organisms measured approximately 10 to 25 microns. The organisms were reported as: "moderate Blastocystis spp. " seen.

ORGANISM COMMENTS:

Blastocystis spp. is a common intestinal parasite, with a prevalence in developing countries of up to 50% or higher. Infection has been reported as asymptomatic, acute symptomatic, and chronic symptomatic. This wide range of responses to infection could be related to the genetic diversity of morphologically indistinguishable specimens obtained from infected hosts. The former name Blastocystis hominis is now reported as Blastocystis spp. because of its genetic diversity. Blastocystis hominis is recognized as a complex of nine different subtypes (half of which are pathogenic and half are nonpathogenic) that have not been fully characterized as independent species.  Molecular studies have successfully classified subtypes or subgroups among B. subtypes and also revealed the presence of zoonotic strains.  In light of the genetic similarities and differences observed among human and nonhuman Blastocystis isolates, it is reasonable to speculate that the transmission of this organism occurs either by human-to-human or animal-to-human routes.  Also, results support the hypothesis that asymptomatic and symptomatic Blastocystis isolates may represent different subtypes/species with varying pathogenic potential.  However, all the organisms isolated from patients have similar morphologies; thus the appearance does not indicate pathogen vs. nonpathogen.

 

Blastocystis is capable of pseudopod extension and retraction, reproduces by binary fission or sporulation, and has a membrane bound central body (previously called a vacuole) that takes up 90% of the cell and functions in sexual and asexual reproduction. Other structures with unknown function are not yet defined. The organism is strictly anaerobic, normally requires bacteria for growth, and is capable of ingesting bacteria and other debris.

The classic form that is usually seen in the human stool specimen varies tremendously in size, from 6 to 40 µm, and is characterized by a large central body, which may be involved with carbohydrate and lipid storage (visually like a large vacuole). The more amebic form is occasionally seen in diarrheal fluid but may be extremely difficult to recognize. Generally, Blastocystis will be identified on the basis of the more typical round form with the central body (“Central Body form”).

Both thin and thick walled cysts have been confirmed. It is suggested that the thin walled cysts are autoinfectious, leading to multiplication of the organism in the intestinal tract. The thick walled cysts are probably responsible for external transmission via the fecal oral route. This type of life cycle might explain the high percentage of positive carriers in many studies, showing that the percentage of patients with Blastocystis is often much higher than those who are infected with other intestinal protozoa.

CLINICAL DISEASE:

When Blastocystis is present in the absence of other pathogenic parasites, bacteria, or viruses, it may be the cause of diarrhea, cramps, nausea, fever, vomiting, and abdominal pain and may require therapy. In one recent study of patients with irritable bowel syndrome, there was a set of patients in whom the presence of Blastocystis did not appear to be incidental. Possible relationships between Blastocystis and intestinal obstruction and perhaps even infective arthritis have been suggested. In patients with other underlying conditions, the symptoms may be more pronounced. The incidence of this organism appears to be higher than suspected in stools submitted for parasite examination. In symptomatic patients in whom no other etiologic agent has been identified, Blastocystis should certainly be considered the possible pathogen.

Other studies suggest that when a symptomatic Blastocystis infection responds to therapy, the improvement probably represents elimination of some other undetected pathogenic organism (E. histolytica, G. lamblia, D. fragilis). Data from other geographic areas indicate that Blastocystis is thought to be a mix of pathogenic and nonpathogenic subtypes, all of which have the same morphology. Although for a number of years the true role of this organism in terms of colonization or disease was still somewhat controversial, it is now generally considered a causative agent of intestinal disease. It has also been suggested that ribodemes I, III, and VI may be responsible for gastrointestinal symptoms.

DIAGNOSIS:

Routine stool examinations are very effective in recovering and identifying Blastocystis, although the permanent stained smear is the procedure of choice since the examination of wet preparations may not easily reveal the organism. If the fresh stool is rinsed in water before fixation (for the concentration method), Blastocystis organisms, other than the cysts, will be destroyed, thus possibly yielding a false negative report. The organisms should be quantitated on the report form, i.e., as rare, few, moderate, or many. It is also important to remember that other possible pathogens should be adequately ruled out before a patient is treated for Blastocystis.

Remember, there is still controversy regarding quantitation of these organisms on the laboratory report form. Although there was thought to be correlation between numbers of organisms present and patient symptoms, some physicians have seen symptomatic patients where even small numbers of Blastocystis are present and there appear to be no other causative agents present in the stool specimen(s). For this reason, quantitation of the organisms is still recommended. However, it is important to communicate with your physician clients so that they know how you are reporting and/or quantitating this organism. The approach must be determined by each laboratory after discussions with physician clients.

Both enzyme linked immunosorbent assay (ELISA) and fluorescent antibody tests have been developed for detection of serum antibody to Blastocystis infections. By using the ELISA and a threshold dilution of 1/50, 27 of 30 serum samples (28 patients with no protozoan parasites other than Blastocystis) were positive at 1/50 or higher; 42 control blood bank serum samples were all negative at 1/50 (61). A strong antibody response is consistent with the ability of this organism to cause symptoms. Also, demonstration of serum antibody production both during and after Blastocystis symptomatic disease is immunological evidence for the pathogenic role for this protozoan, although it may take 2 years or more with chronic infections to develop a serologic response.

 

EPIDEMIOLOGY AND CONTROL:

From present information, it appears that Blastocystis is transmitted via the fecal oral route through contaminated food or water. Although other possible modes of transmission are not defined, the incidence and apparent worldwide distribution indicate the traditional route of infection. Recent studies suggest the existence of numerous zoonotic isolates with frequent animal-to-human and human-to-animal transmissions and of a large potential reservoir in animals for infections in humans. Prevention would probably involve improved personal hygiene and sanitary conditions.

 

TREATMENT:

Although there is not a great deal of clinical evidence, there have been studies on in vitro susceptibility of Blastocystis to numerous drugs. At present, metronidazole (Flagyl) appears to be the most appropriate drug. Diiodohydroxyquin (Yodoxin) has also been effective, and dosage schedules for these two drugs are as recommended for other intestinal protozoa. The development of a new drug sensitivity assay may improve our ability to scientifically evaluate the activities of various drugs against this organism.

 

Two images of Blastocystis from positive stool specimens (from left to right: central body form seen in wet preparation from fecal concentration; central body form undergoing division in a wet preparation from concentration sediment. Note the nuclei around the edges of the central body area are more difficult to see (wet preparation, high dry examination), while the morphology is more clearly seen on the trichrome stain, oil immersion examination).

 

REFERENCES:

Boreham, P. F. L., J. A. Upcroft, and L. A. Dunn. 1992. Protein and DNA evidence for 2 demes of Blastocystis from humans. Int. J. Parasitol. 22:49-53.

Garcia, LS, 2016. Diagnostic Medical Parasitology, 6th Ed., ASM Press, Washington, DC.

Garcia, L.S. 2009. Practical Guide to Diagnostic Parasitology, 2nd Ed., ASM Press, Washington, D.C.

Noel, C., F. Dufernez, D. Gerbod, V. P. Edgcomb, P. Delgado-Viscongliosi, L. Ho, M. Singh, R. Wintjens, M. L. Sogin, M. Capron, R. Pierce, L. Zenner, and E. Viscogliosi. 2005. Molecular phylogenies of Blastocystis isolates from different hosts: Implications for genetic diversity, identification of species, and zoonosis. J. Clin. Microbiol. 43:348-355.