—  SPECIALTY CONFERENCE  —

Renal Pathology

Case 1 - Acute granulomatous interstitial nephritis due to Encephalitozoon cuniculi

Sherry L. Werner, University of Texas Health Sciences Center, San Antonio, TX





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Clinical History
A 56 year-old man underwent left lung transplantation for idiopathic pulmonary fibrosis. Initial immunosuppression included anti-thymocyte globulin followed by tacrolimus, mycophenolate mofetil and prednisone. Early post-op course was complicated by cellular rejection with a humoral component which was treated with steroids and rituximab. The patient had a history of hypertension and worked as a cattle rancher with herding dogs. Five to six months after transplantation, he was admitted to the hospital twice with episodic fever, weight loss, persistent cough, fluctuating altered mental status, diarrhea and renal insufficiency. Serum creatinine increased from a baseline of 0.8 mg/dl to 1.2 mg/dl. Chest CT scans showed small ground glass infiltrates in the upper lobe of the transplanted lung. The patient underwent an extensive evaluation for fever including two transbronchial biopsies that showed no significant histologic changes. MRI of the brain was unrevealing. Colonoscopy and stool cultures were negative. Blood, urine, sputum and CSF cultures were negative for bacteria and fungi. Despite an extensive evaluation for fever over a four week period and empiric therapy with multiple antimicrobial agents including Vancomycin and Cefepine as well as Acyclovir, the fever persisted and the renal function worsened. Tacrolimus blood levels were high (11- 23 ng/ml) and renal function transiently improved with decreased tacrolimus doses. However, the patient developed acute renal failure with a peak serum creatinine of 4.3 mg/dl requiring dialysis. Urinalysis demonstrated sterile pyuria, pleomorphic rod-shaped organisms and tubular epithelial cells with inclusions. A renal biopsy established the diagnosis on electron microscopic studies and the patient was immediately begun on the appropriate therapy.

Pertinent Laboratory Data:
Other labs: PCR tests for HIV, CMV, EBV, HSV, BK and JC virus DNA were negative. Serum studies were negative for: Hepatitis B and C, coccidiomycosis/histoplasma antibody, cryptococcal antigen, C. difficile fecal toxin, ANA and ANCA.


Case 1 - Figure 1
Unstained urine sediment shows rod-shaped organisms and tubular epithelial cells with intracytoplasmic inclusions.
Case 1 - Figure 2
Low-power view showing granulomatous interstitial nephritis with poorly formed granulomas, moderate inflammatory cell infiltrate, severe fibrosis and tubular atrophy.(H&E stain)
Case 1 - Figure 3
High-power view showing inflammatory cell infiltrate extending into tubules and resulting in complete tubular necrosis. (H&E stain)
Case 1 - Figure 4
Low-power view showing granulomatous inflammation surrounding a vessel that also has fibrinoid necrosis of the wall. Glomeruli were unremarkable. (H&E stain)

Case 1 - Figure 5
At high-power, granulomas showed small organisms within epithelioid histiocytes. (H&E stain)
Case 1 - Figure 6
Under oil immersion, numerous small purplish pleomorphic rod-shaped organisms were identified in some tubular lumens and in tubular epithelial cells. (H&E stain)
Case 1 - Figure 7
Oil immersion of kidney section shows irregularly stained gram-positive pleomorphic rod-shaped organisms with a transverse "belt-like stripe." (Gram stain)
Case 1 - Figure 8
Electron micrograph of tubular epithelial cell showing all stages of Encephalitozoon cuniculi development in a parasitophorous vacuole.

Case 1 - Figure 9
Electron micrograph showing E. cuniculi spores with characteristic posterior vacuole, electron-lucent chitin endospore and coiled polar tubule.
Case 1 - Figure 10
Case 1 - Figure 11
Case 1 - Figure 12

Renal Biopsy Findings
The renal biopsy showed an acute granulomatous interstitial nephritis. The 6 glomeruli present were all normocellular; light microscopic, immunofluorescent and electron microscopic studies of glomeruli were unremarkable. The interstitium showed significant morphologic changes including severe fibrosis, focal edema and a moderate inflammatory cell infiltrate composed of lymphocytes, plasma cells, occasional aggregates of neutrophils and few eosinophils. There was a marked granulomatous infiltrate with poorly formed granulomas composed of epithelioid histiocytes admixed with lymphocytes and a few neutrophils; occasional well-formed granulomas were identified. Some granulomas contained numerous pleomorphic rod-shaped organisms in histiocytes. In some foci, granulomas and inflammatory cells extended into and disrupted the tubules, resulting in partial to complete tubular necrosis. Some tubular epithelial cells contained numerous pleomorphic rod-shaped organisms, with some organisms identified in tubular lumens. In some tubules, the epithelial cells showed enlarged, hyperchromatic nuclei. Some arterioles showed thickened walls with beaded hyalinization. Two arterioles showed fibrinoid necrosis. A small muscular artery was surrounded by granulomatous inflammation and showed focal necrosis of the wall. The trichrome stain showed the severe interstitial fibrosis. An immunohistochemical stain for polyoma virus was negative. Special stains including GMS, AFB, PAS-fungal and Gram stain were performed. The gram stain showed the presence of irregularly stained gram-positive pleomorphic rod-shaped organisms, approximately 2 μm in diameter, with a transverse "belt-like stripe." On PAS-fungal stain, each organism showed a single PAS-positive dot ("dot-like pattern"). The organisms were negative on GMS and AFB stains. After observing organisms in the biopsy, the urine sediment was re-examined. On modified trichrome and Gram-chromotrope stains, organisms with a belt-like stripe were identified similar to those seen in the kidney biopsy. Electron microscopic (EM) studies of tubular epithelial cells were diagnostic for microsporidia, specifically Encephalitozoon species. The Centers for Disease Control (CDC) confirmed that the EM was diagnostic of either E. cuniculi or E. hellem which appear similar but are distinct from other microsporidia species by EM. Diagnostic features included: organisms in tubular epithelial cells developing in a parasitophorous vacuole and spores (2-3 μm) with a posterior vacuole and a polar tubule with 5-7 coils. Some spores also showed an extruded polar tubule used to infect host cells. The transverse "belt-like stripe" on gram stain corresponds to the spore's polar tubule and the "pink dot" on PAS-fungal stain corresponds to the spore's posterior vacuole.

The presence of microsporidia in the kidney prompted re-evaluation of previous lung biopsies (negative for GMS and AFB). The biopsy at 4 months post-transplant showed a few collections Gram-positive pleomorphic organisms confined to bronchial epithelial cells similar to those identified in the kidney. On Warthin-Starry stain, they appeared as solid black rods consistent with microsporidia. Interestingly, no inflammatory cell infiltrate was present in the lung tissue.

Differential Diagnosis of Renal Biopsy Findings
Few studies have described the pathology of microsporidia infection in detail. A spectrum of changes have been reported with E. cuniculi infection in the kidney in both HIV negative and positive cases including: interstitial infiltrates with or without a granulomatous reaction, abscesses, tubular necrosis and spores in tubules [1, 2, 3, 4]. In some cases, no inflammatory response in tissues has been reported, particularly in immunosuppressed patients. In animals, microsporidia commonly cause granulomatous interstitial nephritis (GIN) and may show vasculitis with fibrinoid necrosis in various tissues [5, 6]. GIN is rare in native and organ transplant recipients, comprising only 0.5-1.7% of all renal lesions [7, 8, 9]. The differential diagnosis is broad and includes: drugs, sarcoidosis, ANCA-associated vasculitis, crystal deposits with foreign body giant cell reaction, intravesical bacillus Calmette-Guerin therapy, xanthogranulomatous pyelonephritis, malakoplakia and tubulointerstitial nephritis with uveitis (TINU syndrome). Infectious etiologies include bacterial (Mycobacteria, Rhodococcus, E. coli), fungi (Histoplasma, Cryptococcus, Candida species) and viruses (CMV, adenovirus, polyoma virus) [9, 10, 11, 12]. Of these infections, the ones with initial lung involvement that disseminate to the kidney include: Mycobacteria, Rhodococcus, Histoplasma, Cryptococcus, CMV and adenovirus.

In three retrospective studies, GIN was most commonly due to drugs and sarcoidosis in over 50% of cases, ANCA vasculitis or infection in 12% of cases and rarely due to other causes [7, 13, 14]. Clinicopathologic correlation is important for making the correct diagnosis. In our case, these disorders were eliminated. Drug-induced interstitial nephritis was unlikely due to lack of an eosinophilic infiltrate. Our patient lacked clinical and morphologic features of sarcoidosis that is usually associated with well-formed granulomas containing giant cells. Foreign body giant cell reaction was not identified on renal biopsy and there was no history of intravesical bacillus Calmette-Guerin therapy or TINU. ANCA-associated vasculitis was unlikely in our case due to negative ANCA serology and absence of necrotizing or crescentic glomerulonephritis. Of the infectious etiologies, cultures and tissue special stains were negative for bacteria and fungus including Mycobacteria, E. coli, Histoplasma, Cryptococcus and Candida. Although Rhodococcus was first considered in our patient since it is acquired by animal contact and shows granulomas with gram-positive pleomorphic coccobacilli, this infection was excluded since it would have been detected on routine culture [10]. Viral infections were also excluded in our patient: CMV serology was negative and CMV intranuclear inclusions were not identified; polyoma virus studies on urine and blood as well as polyoma virus immunostain on renal biopsy were negative. Adenovirus was not tested in our case but this entity is more often associated with focal interstitial hemorrhage, tubulo-centric granulomas, smudgy intranuclear inclusions and positive immunostaining for adenovirus [12].

Microsporidia infection in our patient was confirmed in the urine specimen by the Centers for Disease Control. PCR using specific primer sets confirmed the presence of E. cuniculi DNA in the urine sediment. Urine was also cultured by inoculating it into mammalian cell cultures. Immunofluorescent staining of the culture supernatant using an E. cuniculi antibody confirmed the presence of E. cuniculi spores; calcofluor white that binds to the chitin coat of spores was also positive.

Clinical Course
Once E. cuniculi was identified by EM in the kidney biopsy, the patient was immediately treated with Albendazole 400 mg twice daily. Immunosuppression had already been drastically reduced in the weeks before the kidney biopsy. Urine and sputum were monitored for spores using calcofluor white staining and, after 5 days, the patient responded to therapy with a reduction in symptoms and spore numbers. However 1 week later, the patient succumbed to Aspergillus fumigatus pneumonia and at autopsy was found to have disseminated E. cuniculi involving the lung, kidney, brain, liver and spleen.

Final Diagnosis:
Acute granulomatous interstitial nephritis due to Encephalitozoon cuniculi

Infection-related arterial and arteriolar focal necrosis

Interstitial fibrosis and tubular atrophy, severe, due to ongoing interstitial nephritis

Arteriolar hyalinosis, beaded, moderate, probably due to tacrolimus effect

Case Discussion:
The phylum microsporidia comprises obligate intracellular spore-forming parasites that infect a broad range of hosts including mammals (cattle, dogs). Initially, microsporidia emerged as a cause of opportunistic infections in AIDS patients, but they are being increasingly reported in immunocompromised patients without AIDS including organ transplant recipients, patients with malignancies and patients with diabetes [15, 16]. Immunocompetent individuals including children, elderly and travelers may also develop microsporidiosis. Studies indicate that microsporidiosis may exist as a latent infection, raising the question whether healthy individuals carry persistent infections that may reactivate under conditions of immunosuppression and whether asymptomatic carriers may transmit infections to those at risk. Transmission of microsporidia occurs primarily through fecal-oral route with sources of infection including contaminated water or food. Infection may also result from inhalation of spores, through person-to-person sexual contact or through direct contact with infected animals (zoonotic transmission) [15, 16]. There are 14 microsporidia species known to infect humans and clinical presentation varies depending on the site of infection. Enterocytozoon bieneusi, the most common cause of microsporidia infection in humans, is mainly confined to the intestine, resulting in diarrhea and rarely disseminates. In contrast, the Encephalitozoon species (E. intestinalis, E. cuniculi and E. hellem) typically disseminate in immunocompromised HIV or non-HIV patients and give rise to clinical features including pneumonitis, interstitial nephritis, cystitis, keratoconjunctivitis and encephalitis. The kidney is a common site for disseminated infection and screening of urine is key to detect systemic infections [15].

Few cases of microsporidiosis have been reported in transplant recipients who are HIV negative - to date, only 70 cases including our case are documented in organ transplant recipients [17, 18]. These patients received one or more immunosuppressive agents that likely contributed to the pathogenesis of microsporidiosis. Studies suggest that lack of INF-γ resulting from CD4+ T cell depletion induced by mycophenolic acid (MMF) or other transplant immunosuppressive drugs may be responsible, in part, for the onset of microsporidiosis. The importance of CD4+ T cells has also been shown in SCID mice infected with E. cuniculi. Survival was improved by adoptive transfer of CD4+ T cells from wild type mice or by administration of INF-γ. Moreover, survival of these mice reconstituted with T cells or treated with INF-γ was further prolonged by co-administration of anti-E.cuniculi antibody, indicating that both T cell and humoral immunity are important for preventing microsporidia infection [19]. Of the 70 cases reported, the highest incidence was in renal allografts (67%), followed by liver, heart/lung, bone marrow, cornea and pancreas/kidney transplant. Chronic diarrhea was the main clinical feature and E. bieneusi the most common species in over 50% of cases, followed by E. cuniculi. Infection occurred from 19 days up to 7 years after transplant. Interestingly, only 5 cases of E. cuniculi infections in non-HIV organ transplant recipients have been reported in addition to our case [1, 2, 3, 20, 21, 22]. Disseminated infection occurred in 4 patients with renal transplants and one patient with bone marrow transplant had only respiratory distress. E. bieneusi is the only species reported in lung transplant recipients; our case is the first report of E. cuniculi in a lung transplant recipient.

Diagnosis of microsporidia is dependent on detecting spores in clinical samples. Organisms can be easily missed but there are a number of histochemical methods that facilitate diagnosis. Screening of body fluids is best accomplished using calcofluor white and modified trichrome stains [1]. In paraffin embedded tissue, gram stain and PAS stains are preferred. Species-specific antibodies against microsporidia are available that can be used to detect organisms in fluids and tissues using immunofluorescence and immunohistochemical staining. Recently, serologic testing has been used to detect carriers of microsporidiosis; however, it is not routinely used for diagnostic purposes in immunocompromised patients due to variable expression of antibodies in these individuals [15, 16].

EM is the gold standard for identifying microsporidia species [23]. E. cuniculi spores (2-3 μm) are surrounded by an outer electron dense layer composed of glycoprotein and an inner electron lucent layer composed of chitin. The cytoplasm contains a posterior vacuole and the polar tubule that is unique to microsporidia. The number and arrangement of coils of the polar tubule vary among species. The E. cuniculi polar tubule has 5-7 coils in a single row. In body fluids, only spores are visible, whereas in tissues all stages of the life cycle are present. Within host cells, E. cuniculi and E. hellem appear similar and develop within a nonseptated parasitophorus vacuole; these can be distinguished from E. intestinalis which develops in a septated parasitophorous vacuole. All developmental stages including meronts, sporonts, sporoblasts and matures spores are visualized. Enterocytozoon bieneusi can be distinguished from Encephalitizoon speciessince it develops in direct contact with the cell cytoplasm without a parasitophorous vacuole. Species identification is further confirmed using PCR-based methods. Specific primer sets are available for at least 5 species including E. cuniculi and are used to amplify the species specific rDNA gene in DNA extracts of fluids or tissues. The genotype of E. cuniculi can be further defined by DNA sequencing of the internal transcriber spacer region (ITS) of the rDNA [24]. Microsporidia cannot be detected on routine culture. Sediments of urine or fluids must be inoculated into mammalian cell cultures. After 2-4 weeks the infected cells are examined by EM and PCR to identify species and culture supernatants containing spores are examined by special stains [23]. Thus, the paradigm for laboratory diagnosis of microsporidia is to screen by calcofluor white and modified trichrome stain followed by confirmation using EM, PCR and culture.

In our case, microsporidia was an unexpected finding and EM was the most rapid method that confirmed the diagnosis of E. cuniculi, allowing immediate therapeutic intervention with Albendazole. PCR and culture studies required an additional 1-2 weeks to obtain results. Clearly, our patient was at risk for developing microsporidia due to heavy immunosuppressive therapy and history of direct contact with cattle and dogs. Four months post-transplant he presented with fever, pulmonary symptoms. In retrospect, he had a positive lung biopsy for E. cuniculi that was probably due to an acquired zoonotic infection via direct contact with animals and/or inhalation of spores from the soil. Over the next 3 months, symptoms worsened and renal failure developed. Although serologic studies and all cultures were negative, the urinalysis showed rod-shaped organisms. The clinical impression was tacrolimus toxicity, ATIN or unusual infection. Arteriolar hyalinosis due to tacrolimus may have contributed to renal dysfunction. However, it was predominantly due to severe granulomatous interstitial nephritis and vascular necrosis due to E. cuniculi. Infection likely spread from the lung to the kidney. Albendazole was immediately initiated and immunosuppressives were decreased. Compromised lung status due to E. cuniculi may have predisposed to Aspergillosis and 8 months post-transplant the patient expired due to Aspergillus pneumonia and disseminated E. cuniculi. Detection of E. cuniculi in the 4 month post-transplant lung biopsy, before the patient developed advanced kidney involvement, would have facilitated earlier treatment and potentially prevented disseminated microsporidia infection. A high index of suspicion for disseminated infection and examination of the urine earlier in the course of the patient's disease might have also helped to improve the outcome.

Treatment Options
The CDC guidelines recommend Albendazole for therapy of intestinal and disseminated microsporidiosis caused by microsporidia other than E. bieunusi [25]. Albendazole is a broad-spectrum antiparasitic agent that inhibits microtubule assembly and interferes with microsporidia cell division. Albendazole leads to clinical improvement and parasite eradication in patients with E. cuniculi, E hellem and E intestinalis, but it is less effective against E. bieunusi. The efficacy and safety of Albendazole in treating Encephalitozoon species was shown in a randomized double blind placebo controlled study involving 8 patients with AIDS and E. intestinalis infection [26]. Side effects of Albendazole are rare and include: hypersensitivity, reversible neutopenia and thrombocytopenia. Albendazole may need to be continued since relapse is common in patients who responded when therapy is stopped. No long term safety problems were encountered during maintenance therapy with Albendazole. Fulmagillin is more effective in clearing E. bieunusi infection.

Conclusion(s):
Our case is the first report of disseminated E. cuniculi infection in an HIV negative lung transplant recipient. In addition to HIV patients, immunocompromised non-HIV transplant recipients should be considered a risk group for microsporidia. Microsporidia should be considered in cases of FUO and/or multiorgan infection especially with renal insufficiency after other causes have been excluded. The urine sediment should be carefully examined and, if organisms are identified but cultures are negative, screen for microsporidia using calcofluor white and modified trichrome stain. Specimens may need to be screened multiple times as shedding may be intermittent (urine is #1 screen for disseminated disease). In certain cases, serotesting of donors may be useful to detect carriers of microsporidiosis to prevent donor-related infection. Further studies to elucidate the cellular/molecular mechanisms by which microsporidia infect tissues will be important and may lead to novel therapeutic strategies and improved clinical management.

References:
  1. Mohindra AR, Lee MW, Visvesvara G, Moura H, Parasuraman R, Leitch GJ, Xiao L, Yee J, del Busto R: Disseminated microsporidiosis in a renal transplant recipient. Transpl Infect Dis 4:102-107, 2002.

  2. Gamboa-Dominguez A, de Anda J, Donis J, Ruiz-Maza F, Visvesvara GS, Diliz H: Disseminated Encephalitozon cuniculi infection in a Mexican kidney transplant recipient. Transplantation 75:1898-1900, 2003.

  3. Mahmood MN, Keohane ME, Burd EM: Pathologic quiz case: a 45-year-old renal transplant recipient with persistent fever. Arch Pathol Lab Med 127:e224-e226, 2003.

  4. Tosoni A, Nebuloni M, Ferri A et al: Disseminated microsporidiosis caused by Encephalitozoon cuniculi III (Dog Type) in an Italian AIDS patient: a retrospective study. Mod Pathol 15:577-583, 2002.

  5. McCully RM, Van Dellen AF, Basson PA, Lawrence J: Observations on the pathology of canine microsproidiosis. Onderstepoort J Ves Res 45:75-91, 1978.

  6. Kunzel F, Joachim A: Encephalitozoonosis in rabbits. Parasitol Res 106:299-309, 2010.

  7. Bijol V, Mendez GP, Nose V, Rennke HG: Granulomatous interstitial nephritis: a clinopathologic study of 46 cases from a single institution. Int J Surg Pathol 14:57-63, 2006.

  8. Ozdemir BH, Sar A, Uyar P, Suren D, Demirhan B, Haberal M: Posttransplant tubulointerstitial nephritis: clinicopathologic correlation. Transplantation Proceedings 38:466-469, 2006.

  9. Meehan SM, Josephson MA, Haas M: Granulomatous tubulointerstitial nephritis in the renal allograft. Am J Kidney Dis 36:1-6, 2000.

  10. Tse KC, Chan TM, Wong S, Chan KW, Lam MF, Chan K, Li FK, Choy BY, Lai KN: Granulomatous interstitial nephritis of the allograft kidney associated with rhodococcal pulmonary infection. Nephrol Dial Transplant 19:486-490, 2004.

  11. Qian Q, Humayun H, Humayun Y, Sethi S: Granulomatous interstitial nephritis associated with disseminated histoplasmosis in an immunocompromised patient. Am J Kidney Dis 58:1018-1021, 2011.

  12. Asim M, Chong-Lopez A, Nickeleit V: Adenovirus infection in a renal allograft. Am J Kidney Dis 41:696-701, 2003.

  13. Viero RM, Cavallo T: Granulomatous interstitial nephritis. Hum Pathol 26:1347-1353, 1995.

  14. Mignon F, Mery JPh, Mougenot B et al: Granulomatous interstitial nephritis. Adv Nephrol 13:219-245, 1984.

  15. Didier ES, Weiss LM: Microsporidiosis: current status. Curr Opin Infect Dis 19:485-492, 2006.

  16. Didier ES, Weiss LM: Microsporidiosis: not just in AIDS patients. Curr Opin Infect Dis 24:490-495, 2011.

  17. Galvan AL, Martin Sanchez AM, Perez Valentin MA, Henriques-Gil, Izquierdo F, Fenoy S, del Aguila C: First cases of microsporidiosis in transplant recipients in Spain and review of the literature. J Clin Microbiol 49:1301-1306, 2011.

  18. A, Lang P, Delahousse M, Chauvet C, Sarfati C, Glotz D, Molina J-M: Fumagillin for treatment of intestinal microsporidiosis in renal transplant recipients. Am J Transpl 10:1925-1930, 2010.

  19. Salat J, Jelinek J, Chmelar J, Kopecky J: Efficacy of gamma interferon and specific antibody for treatment of microsporidiosis caused by Encephalitozoon cuniculi in SCID mice. Antimicrob Agents Chemother 52:2169-2174, 2008.

  20. Talabani H, Sarfati C, Pillebout E, van Gool T, Derouin F, Menotti J: Disseminated infection with a new genovar of Encephalitozoon cuniculi in a renal transplant recipient. J Clin Microbiol 48:2651-2653, 2010.

  21. Orenstein JM, Russo P, Didier ES, Bowers C, Bunin N, Teachey DT: Fatal pulmonary microsporidiosis due to Encephalitozoon cuniculi following allogeneic bone marrow transplantation for acute myelogenous leukemia. Ultrastruct Pathol 29:269-276, 2005.

  22. Teachey DT et al: Pulmonary infection with microsporidia after allogeneic bone marrow transplantation. Bone Marrow Transplant 33:299-302, 2004.

  23. Visvesvara GS: In vitro cultivation of microsporidia of clinical importance. Clin Microbiol Rev 15:401-413, 2002.

  24. Ghosh K, Weiss LM: Molecular diagnostic tests for microsporidia. Interdisciplinary Perspectives on Infectious Diseases, p. 1-13, 2009.

  25. Mofenson LM, Brady MT, Danner SP et al: Guidelines for the prevention and treatment of opportunistic infections among HIV-exposed and HIV-infected children. CDC MMWR. September 4, 2009.

  26. Molina JM, Chastang C, Goguel J et al: Albendazole for treatment and prophylaxis of microsporidiosis due to Encephalitozoon intestinalis in patients with AIDS: a randomized double-blind controlled trial. J Infect Dis 177:1373-1377, 1998.