Infectious Disease Pathology

Transmural Granulomatous Inflammation with Perforation of Small Intestine Due to Disseminated Infection with Histoplasma capsulatum

Stephen D. Allen
Indiana University School of Medicine
Indianapolis, IN


Clinical Summary:
A 43-year-old man was transferred from an outside hospital for further evaluation and management of abdominal pain. The patient had abdominal pain for approximately 10 days, and the pain had been worsening. He noted difficulty in swallowing and had a 30 pound unintentional weight loss. He also stated he had gotten surgery for hemorrhoids and "intestine hanging out of my anus" approximately three weeks ago. The patient noted tenesmus with significant straining to produce a bowel movement. Although he also gave a history of diarrhea in recent weeks, his last bowel movement was normal and occurred three days ago. He had occasional nausea, but no emesis. He denied any chills but had been having high grade fevers. Otherwise, he denied any shortness of breath or chest pain. His abdominal pain was cramping and worse in his left lower quadrant and suprapubic regions. His past medical history was significant for AIDS with his last CD4 count two weeks ago of 10/µL. In addition, the patient had been incarcerated in northern Indiana and was a recovering narcotic and alcohol addict. He had immigrated to the US from Puerto Rico in 1998. The patient denied a history of opportunistic infection and tuberculosis. He denied HAART, but outside records indicated he was admitted on a regimen of emtricitabine, kaletra, lamivudine, levoquin, and bactrim. On physical examination, his initial temperature in the emergency room was 105.5° F. He was a cachectic Latino man with a mildly toxic appearance. HEENT examination was unremarkable. Chest examination revealed rales bilaterally. His abdomen was soft, mildly distended, and moderately tender diffusely, greatest in the left lower quadrant and suprapubic regions. There was a 2 cm, mobile mass in his scrotum. Laboratory data was notable for a white blood count of 1900/µL with 13% bandemia, hemoglobin 9.6, platelets 85, and AST 223 (H). Blood and urine cultures were pending. A chest x-ray demonstrated a large amount of free intraperitoneal air and a suspected small left pleural effusion. A head CT was notable for mild generalized cerebral and cerebellar volume loss with a few scattered hypodense foci within the central pons. A CT of the abdomen and pelvis, with oral and intravenous contrast, demonstrated free intraperitoneal air over the upper abdomen with periportal, retroperitoneal, and mesenteric lymphadenopathy as well as pleural effusion with bibasilar atelectasis, left greater than right. The patient underwent urgent exploratory laparotomy for suspected small bowel perforation.

Images Provided:
One scanned glass slide from the suspected perforation site in the jejunum (hematoxylin and eosin [H&E]) and images (H&E and Gomori methenamine silver [GMS] stains as part of the handout [Figures 1-9].


Slide 1
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Figure 1
Shows a low power view of the inflammatory infiltrate within the mucosa and lamina propria of the small intestine specimen (H&E).
Figure 2
Is a low power view adjacent to that shown in Figure 1 revealing transmural inflammation (H&E). Adjacent to this area was a prominent ulcer which extended from the mucosa to the serosal surface.
Figure 3
Shows diffuse granulomatous inflammation in the submucosa (H&E).

Figure 4
Shows nonnecrotizing granulomas within the muscle layers (H&E).
Figure 5
Demonstrates a prominent necrotizing granuloma (H&E).
Figure 6
High magnification reveals clusters of small yeasts within macrophages (H&E x500).

Figure 7
Higher magnification reveals clusters of small yeasts within macrophages. Note the retraction of the fungus cytoplasm from the cell wall ("halo") giving the false impression of a capsule (H&E x1000). The halo effect is not seen using Gomori methenamine silver (GMS) stain as demonstrated in Figures 8 and 9..
Figure 8
GMS stain (x500) revealing myriads and aggregates of dark stained intracellular and extracellular budding yeasts.
Figure 9
GMS stain (x1000) high power view revealing details of small yeasts, 2-to-4 µm in diameter, having single buds with narrow necks.


Pathologic Findings:
Histopathologic examination revealed a combination of microscopic findings. These included a prominent ulcer, diffuse lymphohistiocytic infiltrates, non-necrotizing granulomas, and necrotizing granulomas. The inflammatory process extended from the ulcerated mucosal surface to the serosal surface. In addition to lymphocytes and macrophages, there were many granulocytes and plasma cells. Multinucleated giant cells were present but not in large number. H&E stained sections revealed abundant small yeasts clustered within macrophages. Extracellular yeasts were also present in large number. GMS stain confirmed the presence of myriads of small yeasts, 2-to-4 µm in diameter, having single buds with narrow necks.

Diagnosis:
Transmural Granulomatous Inflammation with Perforation of Small Intestine Due to Disseminated Infection with Histoplasma capsulatum This 43-year-old Spanish-speaking-only male underwent a small bowel resection on 11/4/08. This was for an acute abdomen, secondary to perforation approximately 150 cm distal to the ligament of Treitz (thought to be in the mid-jejunum). Three diagnostic results became available within one day of each other. First, the small bowel specimen revealed transmural granulomatous inflammation with perforation due to Histoplasma capsulatum. Second, a test for urinary Histoplasma antigen (MiraVista Diagnostics, Indianapolis, IN) was positive (> 39 units). Third, a fungal gel immunodiffusion test for antibody against Histoplasma antigen was M band positive (H band not detected).

After the diagnosis was made, the patient received amphotericin B (Abelcet) intravenously for 13/14 days; he did not tolerate it well. He subsequently was to be treated with itraconazole (200 mg po BID) for the remainder of his life. Following the surgery, however, the hospital course was complicated by a bowel obstruction. This involved the duodenum due to lymphadenopathy (extrinsic). It was elected to place a gastrostomy-jejunostomy (GJ) tube for enteral nutrition, and the patient was discharged back to prison. At the time, he refused to take his HIV medication due to nausea. He returned to surgery clinic on 12/9/08. He had continued to lose weight, despite being on approximately 1800 calories of tube feeds through his J-tube. On exam, the GJ tube was in good position, his incision was well-healed, and he had no abdominal tenderness. The patient subsequently removed his own gastric tube and had since noted pain and drainage from his external abdominal wall. Radiographic studies (upper GI) during his last appointment (1/23/09) revealed marked improvement with no evidence of duodenal or other obstruction.

Discussion
More than a century ago, Samuel T Darling, a U.S. Army pathologist assigned to Panama, first described a disease entity now recognized as disseminated histoplasmosis [5, 20]. Although the gross lesions resembled disseminated tuberculosis, microscopic examination showed intracellular organisms Darling first thought were Leishmania; however, they lacked kinetoplasts. Darling observed the organisms in histiocytes, thought they resembled plasmodium-like protozoans and that the clear spaces around the organisms were capsules. Thus, he proposed the name Histoplasma capsulata [23, 28] . Although further investigation showed this was not a protozoan, it took many years to demonstrate that H. capsulatum is a thermal dimorphic fungus that occurs as a filamentous mold in soil or on an agar medium incubated at 25 °-30 ° C; that soil, especially soil fertilized by bird droppings or bat guano, is the environmental reservoir; and that histoplasmosis is primarily a pulmonary infection acquired through inhalation of microconidia [7, 8, 18]. The yeast form is found in tissues from infected warm-blooded animals and on enriched media (e.g., brain heart infusion agar) incubated at 35 ° -37° C in the laboratory.

In humans, cases of histoplasmosis caused by H. capsulatum var. capsulatum have been documented in ³ 60 countries on most continents (but not Antarctica) [20]. The endemic areas with highest prevalence are in the midwestern and south central United States (e.g., the Ohio, and Mississippi River valleys) and much of Latin America [20]. H. capsulatum var. duboisii, which causes "African histoplasmosis", will not be addressed in this presentation. The remainder of the discussion is confined to H. capsulatum var capsulatum, or simply H. capsulatum and histoplasmosis caused by this organism.

Gastrointestinal histoplasmosis.
Interestingly, ileal ulcerations were noted at autopsy in one of Darling's first three patients [6, 16]. Confirmation of enteritis and colitis in disseminated histoplasmosis was reported many years later [14]. At autopsy, H. capsulatum can be demonstrated in the GI tracts of 70-90% of patients who have disseminated histoplasmosis [9, 10, 13]. During life, however, GI disease due to H. capsulatum (GIH) is recognized in only 3-12% of patients, thus raising concern that GIH may be underdiagnosed [16, 29, 35].

Microconidia of H. capsulatum may be inhaled when dispersed in the air during various activities that disrupt soil, dirt, chicken coops, other dusts where birds have roosted, or bat guano (e.g., in caves or attics). Once inhaled, the conidia attract neutrophils and macrophages, causing pneumonitis. When ingested by macrophages, the conidia transform into yeasts, which multiply unchecked in the non-immune individual. Before cellular immunity to H. capsulatum develops, infection occurs within the lungs, and the organism disseminates hematogenously throughout the reticuloendothelial system. About a month after infection, specific T cell immunity develops, progression of the infection stops, and the organisms are eliminated or contained in > 99% of individuals [12]. After low inoculum exposure, most individuals remain asymptomatic. Only a few develop symptoms (usually a flu-like respiratory illness). However, heavy exposure may lead to diffuse lung involvement with more severe pulmonary illness proportional to the level of exposure. In addition, while previously infected individuals may have milder illnesses because of pre-existing immunity, more severe illness occurs in individuals who are immunosuppressed. Progressive dissemination of H. capsulatum with various clinical consequences occurs in immunocompromised individuals [16].

Reactivation of quiescent infection with progressive dissemination has been assumed to occur in some individuals when they become immunosuppressed [16, 32], though evidence for this is considered weak (Wheat, LJ, Histoplasmosis; see recent review at www.miravistalabs.com). Accordingly, an argument against reactivation as a common mode of pathogenesis is that the frequency of histoplasmosis in immunosuppressed patients is very low [30]. As reviewed elsewhere by Wheat, the frequency of histoplasmosis is " ~ 1% of patients with AIDS [34], < 0.5% following transplantation [30], and < 0.1% after anti-TNF treatment [31, 38]. " When the T cell response works, sterilizing immunity presumably leads to macrophage killing of intracellular Histoplasma [4, 19]. Data are lacking to support the concept of "latent" infection in histoplasmosis. As Wheat also has pointed out, cultures of old calcified granulomas show no growth, even though yeasts may be seen microscopically. In the presence of old calcified granulomas, reinfection [11], rather than reactivation, is more likely to occur. Thus, at least in endemic areas, it is postulated that progressive disseminated histoplasmosis in immunosuppressed individuals probably is due to exogenous infection (Wheat, LJ, Histoplasmosis; see recent review at www.miravistalabs.com).

GIH may be the result of mediastinal histoplasmosis through impingement on the esophagus by enlarged, inflamed lymph nodes, or involvement of the esophagus in patients with fibrosing mediastinitis [16]. Factors predisposing individuals to progressive dissemination of H. capsulatum include the following: AIDS, immunosuppressive medications, extremes of age, idiopathic CD4 lymphocytopenia, Job's syndrome, common variable immunodeficiency, other immune disorders, and biologic agents that interfere with the action of tumor necrosis factor µ (e.g., infliximab and etanercept) [1, 2, 16, 17, 22, 38].

Pathology.
The pathologic spectrum of GIH was described by Lamps and colleagues in an excellent review of 52 patients with disseminated disease from endemic and nonendemic areas [21]. Both gastrointestinal and hepatic lesions were present in 60% of their cases and about half were immunocompromised. Ulcers (0.2 to 4.0 cm in diameter), the most common gross lesions in the GI tract, were most frequently observed in the ileum but also found in the jejunum, colon, esophagus, and stomach. Most of the ulcers were multiple and annular with raised borders and were associated with hyperemia, hemorrhage, and a necrotic base. Microscopically, diffuse lymphohistiocytic infiltrates, ulcers, lymphohystiocytic nodules involving the mucosa and submucosa, or minimal inflammatory reactions were seen. The inflammatory cell infiltrates contained relatively large numbers of lymphocytes, plasma cells, and macrophages as well as many neutrophils and eosinophils. Yeasts were usually seen within the cytoplasm of macrophages but also extracellularly in severe GI infections. Well-formed granulomas were rare.

A more recent review by Kahi and associates provides additional descriptions of GIH pathology and clinical findings [16]. In H&E stained sections, H. capsulatum is likely to be seen when the yeasts are abundant (Figures 6 & 7). As in the present case, they are small, 2-4 µm, uniform, ovoid-to-spherical, uninucleate yeasts with narrow-based buds. The yeasts are often present in clusters within the cytoplasm of macrophages. The cytoplasm of H. capsulatum retracts from its cell wall, leaving a "halo" or clear zone around the organism (Fig. 7). The Gomori (or Grocott) methenamine silver stain is especially useful for demonstrating H. capsulatum in tissue (Figures 8 & 9), even when only small numbers of yeasts are present. In our experience, a periodic acid-Schiff (PAS) stain is not as reliable for visualizing this organism.

For GIH, the differential diagnosis includes Crohn's disease, ulcerative colitis (or idiopathic inflammatory bowel disease) and sarcoidosis [16, 21]. Other microorganisms that could be confused with H. capsulatum include the following: Candida species, particularly C. glabrata, Cryptococcus neoformans, Blastomyces dernatitidis, Pneumocystis jirovecii, Leishmania spp., and Trypanosoma cruzi.

Laboratory diagnosis of histoplasmosis.
In addition to histopathology, a variety of other methods can be used to diagnose histoplasmosis. Histoplasma antigen can be detected in the urine of over 90% of patients who have disseminated histoplasmosis [16]. Antigen testing can also aid in monitoring treatment. However, antigen testing has not been well studied specifically in patients with GIH. In patients with localized pulmonary histoplasmosis, antigen is detected in urine in only about 25% of cases. Serologic tests for antibodies to H. capsulatum are recommended for all cases of suspected histoplasmosis. Complement fixation tests are positive (titers ³ 1:8) in 90% of patients who have pulmonary histoplasmosis. Immunodiffusion tests are positive for M bands in 76% and positive for H bands in only 23% of such patients [27, 33, 37]. Serologic tests tend to be less useful in patients with disseminated disease [33, 35, 37]. Direct microscopic examination of fresh clinical specimens (e.g., frozen sections and/or touch preparations of lymph nodes, liver biopsies, or GI biopsies) stained with Diff-Quik, Giemsa, GMS, and calcofluor white, and peripheral blood buffy coat smears stained with Wright –Giemsa, aid in rapid diagnosis of patients who have disseminated infection. Direct microscopic exams are not as sensitive as culture. Cultures are positive in about 85% of cases with disseminated histoplasmosis, though multiple cultures are recommended to improve the yield (16). H. capsulatum tends to grow slowly; cultures must be held 4-6 weeks before they are reported as showing no growth. If disseminated histoplasmosis is suspected, at least three sets of fungal blood cultures (i.e., using lysis-centrifugation method) should be collected. Molecular diagnostic methods, though not commercially available, are being developed and used in some reference/referral laboratories.

Treatment guidelines from the Infectious Diseases Society of America for treatment of histoplasmosis were published in 2007 [36]. Without treatment, mortality of disseminated histoplasmosis is 80%; with amphotericin B treatment, mortality can be reduced to 25% [24, 25, 26]. Liposomal amphotericin B (AmBisome) was shown to be more effective than standard amphotericin B [15, 16]. Itraconazole is the treatment of choice for patients with less severe histoplasmosis or following initial treatment with amphotericin B [15, 16, 36].

Acknowledgements
I thank Dr. Xiaoyan Wang who originally consulted with me on the case and provided further clinical details and follow-up information. I also appreciate the clinical perspective and additional information on the patient provided by Dr. Virginia Caine.

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