Cystoisospora belli is an intracellular parasite associated with gastrointestinal disease in immunocompromised hosts. Although infection has been classically associated with intestinal disease, studies have identified Cystoisospora in the gallbladder of immunocompetent patients based on hematoxylin-eosin morphology. Recently, the identity of this histologic finding as Cystoisospora has been questioned based on negative results of nucleic acid studies.
To determine the prevalence of this histologic feature in pediatric patients, we retrospectively reviewed all cholecystectomy specimens from a pediatric hospital during a 24-month period.
In 180 cholecystectomy specimens, we identified 11 cases (6.1%) with classical histologic features previously described to represent Cystoisospora organisms. To further investigate these structures, we retrieved tissue from paraffin-embedded blocks and performed electron microscopy.
Ultrastructural examination identified ovoid perinuclear cytoplasmic structures composed of dense fibrillar aggregates rather than organisms. Patients with positive cases were similar in age to controls (positive cases: mean patient age 13.4 years [range, 2–23 years]; negative cases: mean patient age 14.7 years [range, 12 weeks–31 years]; P = .35). There was no significant association of this finding with cholelithiasis (54.5% versus 65.1%, P = .52), cholesterolosis (0% versus 22.5%, P = .12), acute cholecystitis (9.1% versus 10.1%, P > .99), or chronic cholecystitis (45.5% versus 66.3%, P = .20).
To our knowledge, this is the first positive identification of these structures as cytoplasmic fibrillar aggregates rather than parasitic inclusions by ultrastructural examination, and the first study of this histologic finding in pediatric cholecystectomies.
Cystoisospora belli (formerly known as Isospora belli) is a coccidian parasite that may cause gastrointestinal disease in humans. It is found in a global distribution, but is most prevalent in tropical and subtropical regions. Infection occurs secondary to ingestion of food or water contaminated with mature oocysts.1 These ingested oocysts release sporozoites, which invade human gastrointestinal epithelial cells. The obligate intracellular parasitic organism subsequently undergoes a merogonic and sporogonic stage. When the infected epithelial cells die and are shed, oocysts are released and excreted in stool, propagating the cycle.2
Immunocompetent individuals with intestinal cystoisosporiasis typically have transient to mild disease, with fever, nausea, vomiting, and watery diarrhea. Life-threatening and fatal disease is rare in the immunocompetent host.1,3 Severe disease with extraintestinal manifestations is more commonly seen in immunocompromised individuals, classically in the setting of human immunodeficiency virus.1,4 In the immunocompromised host, Cystoisospora infections are frequently chronic and recurrent. This may be associated with its propensity to infect extraintestinal locations and form treatment-resistant unizoic cysts.5
Cystoisospora in the gallbladder and biliary tract has been reported in both immunocompetent and immunosuppressed individuals.6–12 Recently, the identity of these structures in the gallbladder has been questioned based on negative nucleic acid studies using primers directed against C belli.13 To understand the prevalence of this histologic finding, we examined cholecystectomy specimens from a pediatric institution and performed electron microscopy from paraffin-embedded specimens to investigate the ultrastructure of the eosinophilic structures that have been attributed to Cystoisospora, with the hypothesis that parasitic inclusion would be demonstrable by electron microscopy, similar to previous examples of Cystoisospora.6
MATERIALS AND METHODS
After institutional review board approval was obtained, the pathology records from Nationwide Children's Hospital (Columbus, Ohio) were searched for cholecystectomy specimens obtained during a 24-month interval (August 2015–August 2017), and 193 cases were identified. After exclusion of 13 cases without well-preserved gallbladder epithelium, the study population consisted of 180 gallbladders.
Hematoxylin-eosin sections of the specimens were reviewed by at least 1 of 6 participating pathologists. The presence of acute cholecystitis, chronic cholecystitis, cholesterolosis, and cholelithiasis was recorded. Each case was assessed for the presence of eosinophilic perinuclear cytoplasmic structures, and a second pathologist confirmed all cases flagged as positive. Difficult or indeterminate cases were evaluated with a periodic acid–Schiff stain with diastase to highlight the cytoplasmic structures as reported previously in cholecystectomy specimens.9 The density of the cytoplasmic structures was classified as rare (up to 1 per high-power field), few (2–5 per high-power field), or many (>5 per high-power field). A more detailed histologic evaluation of gallbladders with eosinophilic cytoplasmic structures and an equal number of negative control cases was subsequently performed. Features that were assessed and compared between groups were epithelial vacuolation evident at low power, increased intraepithelial lymphocytosis, lymphoid aggregates, prominent mucosal eosinophils, prominent mucosal plasma cells, and epithelial atrophy (defined as a decreased height to width ratio of the mucosal epithelial cells).
Electron microscopy was performed on paraffin-embedded tissue from 2 cases that were selected based on the presence of numerous cytoplasmic structures. Following removal of tissue from the paraffin block and deparaffinization in xylene, tissues were processed and embedded in Epon resin. Ultrathin sections were stained with osmium tetroxide and examined using a Hitachi H7650 electron microscope.
Categorical variables were compared between the positive and negative gallbladders using a 2-tailed Fisher exact test. Numerical variables were compared using a 2-tailed t test. A P value of less than .05 was considered statistically significant.
Overall, 11 of 180 cases (6.1%) showed eosinophilic perinuclear cytoplasmic structures. These eosinophilic structures were oval-, wedge-, or banana-shaped (Figure 1, A through D). The structures were found in subnuclear and/or supranuclear areas of cytoplasmic clearing, and were usually located more basally within the epithelial cells. Similar to previous reports, periodic acid–Schiff staining with diastase (Figure 2, A) and trichrome staining (Figure 2, B) highlighted the structures. Only mucosal epithelial cells contained the eosinophilic structures. Nine cases (81.8%) had foci with more than 5 structures per high-power field, and 2 cases (18.2%) had 2 to 5 structures per high-power field. Only 2 of the 11 positive cases were identified prospectively, highlighting the challenge of identifying these structures. Of note, both cases identified prospectively were collected after tweets about Cystoisospora in the gallbladder appeared on Twitter,14,15 suggesting that awareness of this finding was raised by social media.
The demographics of patients with eosinophilic perinuclear cytoplasmic structures were similar to those of patients without eosinophilic cytoplasmic structures (Tables 1 and 2). Positive cases showed a male-skewed male to female ratio (1:1.2 versus 1:3.1); however, this did not reach statistical significance (P = .15). No diagnoses showed statistically significant correlation with the presence of eosinophilic cytoplasmic structures. This included cholelithiasis, cholesterolosis, and acute or chronic cholecystitis. Cholesterolosis was not seen in 11 cases with eosinophilic cytoplasmic structures, compared with 38 of the 169 negative cases; however, this was not statistically significant (P = .12) (Table 1).
We did not identify any histologic features associated with the presence of eosinophilic cytoplasmic structures. In a case-controlled comparison of the 11 positive and 11 negative controls, we found no differences in the prevalence of epithelial vacuolation, intraepithelial lymphocytosis lymphoid aggregates, mucosal eosinophilia, prominent mucosal plasma cells, or epithelial atrophy (Table 3).
Of the 11 cholecystectomy specimens that contained eosinophilic cytoplasmic structures, 1 specimen (Figure 3, A) was collected concurrently with biopsies of the upper gastrointestinal tract (esophagus, stomach, and duodenum). No eosinophilic cytoplasmic structures were identified in the concurrent biopsies of the duodenum (Figure 3, B and C).
To determine the identity of these structures, we processed previously paraffin-embedded tissue for electron microscopy. Ultrastructural examination identified cytoplasmic structures that were identical in size and location within the cytoplasm as compared with the eosinophilic structure seen in hematoxylin-eosin sections (compare Figure 4, A and B). The structures in the cytoplasm were composed of aggregates of fine fibrils and/or tubular structures, without evidence of a surrounding membrane or internal structures characteristic of a protozoan (Figure 4, C and D).
There have been multiple publications9–12,16 reporting gallbladder cystoisosporiasis, most notably a study by Lai et al9 that identified Cystoisospora in cholecystectomy specimens from 18 immunocompetent individuals. In each study, organisms were identified in the gallbladder epithelium based on hematoxylin-eosin and special stain features with no further confirmation by molecular or ultrastructural methods.
A recent study by Swanson et al13 reported negative nucleic acids studies and suggested the cytoplasmic structures used previously to identify patients with gallbladder cystoisosporiasis are artifacts. Our study supports the conclusions of Swanson et al13 by failing to identify organisms and positively identifying these eosinophilic cytoplasm structures as fibrillar aggregates by ultrastructural examination. In addition to negative molecular and ultrastructural support for the presence of Cystoisospora in affected gallbladders, we note that neither our review nor any of the previously published studies that showed a high prevalence of infection in the gallbladder of immunocompetent hosts have demonstrated inclusions that would represent the entire life cycle of C belli. This is in sharp contrast to classical presentation of C belli, in which the organisms are seen in various life cycle stages.6
The precise composition of the fibrillar eosinophilic aggregates identified in our ultrastructural studies remains unclear. However, the formation of fibrillar aggregates suggests that these intracytoplasmic structures may be misfolded cytoplasmic proteins. We demonstrate that fibrillar aggregates in the gallbladder epithelium are present in 6% of specimens with evaluable epithelium. The lack of associated clinical or pathologic features suggests that these aggregates are incidental features of cholecystectomy specimens and not definitively diagnostic for Cystoisospora infection as previously suggested. Awareness of this diagnostic pitfall is important to avoid misdiagnoses as C belli in the immunocompetent host.
The authors have no relevant financial interest in the products or companies described in this article.
Portions of this work were presented at the United States and Canadian Academy of Pathology Annual Meeting; March 20, 2018; Vancouver, British Columbia, Canada.