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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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.
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
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
GIN is rare in native and organ transplant recipients,
comprising only 0.5-1.7% of all renal lesions
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)
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
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 .
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 .
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.
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.
Acute granulomatous interstitial nephritis due to Encephalitozoon
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
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
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)
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 .
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
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 . 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
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 . 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
EM is the gold standard for identifying microsporidia species .
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
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 . 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.
The CDC guidelines recommend Albendazole for therapy of intestinal and disseminated microsporidiosis
caused by microsporidia other than E. bieunusi
. 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
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
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.
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