© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Transplant Infectious Disease, ISSN 1398-2273
Case report
Successful treatment with fumagillin of the first pediatric case of digestive microsporidiosis in a liver-kidney transplant Bailly, F. G. Desoubeaux, Z. Maakaroun-Vermesse, C. Lier, E. Morio, F. Labarthe, L. Bernard, J. Chandenier. Successful treatment with fumagillin of the first pediatric case of digestive microsporidiosis in a liver-kidney transplant. Transpl Infect Dis 2013: 15: E250–E259. All rights reserved Abstract: We report the first successful use, to our knowledge, of fumagillin alone in a pediatric patient to cure intestinal microsporidiosis in a liver-kidney transplanted child. Detection of Enterocytozoon bieneusi in stool became negative from the first posttherapeutic control, while digestive symptoms disappeared in 4 days. During a 9-month follow-up, polymerase chain reaction and direct examinations remained negative for microsporidia in her feces. No major undesirable effects were noted during the antimicrosporidial therapy.
G. Desoubeaux1,2, Z. MaakarounVermesse3,4, C. Lier1, E. Bailly1, F. Morio5,6, F. Labarthe4,7, L. Bernard2,3, J. Chandenier1,2 Service de Parasitologie – Mycologie – Medecine tropicale, CHU de Tours, Tours, France, 2Faculte de Medecine, Universite Francßois Rabelais, CEPR UMR INSERM U1100/E.A. 6305, Tours, France, 3Service de Medecine Interne et Maladies Infectieuses, CHU de Tours, Tours, France, 4Service de Medecine Pediatrique, CHU de Tours, Tours, France, 5Laboratoire de Parasitologie et Mycologie Medicale, CHU de Nantes, Nantes, France, 6 IICiMed/E.A. 1155, Universite de Nantes, Nantes, France, 7 Faculte de Medecine, Universite Francßois Rabelais, N2C INSERM U1069, Tours, France 1
Key words: fumagillin; pediatrics; Enterocytozoon bieneusi; liver-kidney transplant; PCR; intestinal microsporidiosis Correspondence to: Dr Guillaume Desoubeaux, CHU de Tours, Service de Parasitologie – Myologie – Medecine tropicale, H^opital Bretonneau, P^ole de Biologie Medicale, B^atiment B2A – 1er etage, 2 boulevard Tonnelle, 37044 CHU de TOURS, Cedex 9 France Tel: +33(0)2 34 37 89 26 Fax: +33(0)2 47 47 80 82 E-mail:
[email protected] Received 23 May 2013, revised 13 August 2013, accepted for publication 17 September 2013 DOI: 10.1111/tid.12158 Transpl Infect Dis 2013: 15: E250–E259
Although it came to prominence during the acquired immunodeficiency syndrome (AIDS) pandemic (1, 2), microsporidiosis remains a rare infectious etiology of diarrhea, but it should always be considered in immunocompromised patients (2). Nowadays, microsporidial infections also occasionally occur in solid organ transplant (SOT) patients (3, 4). Treatment is often difficult because of the lack of effective therapeutic agents (5),
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and because of many drug interactions in such a heavily treated population. In this short report, we focus on 2 cases of microsporidiosis occurring in children from the same family. Because of congenital hyperoxaluria, the young sisters had both undergone double liver-kidney transplantation. After other anti-microsporidial therapies failed, we finally decided to assess fumagillin as a
Desoubeaux et al: Fumagillin in a transplanted child
treatment for microsporidiosis, although this drug is still not indicated in pediatric patients. Only the older sister was eligible for the fumagillin treatment. We present here the clinical and biological results of the first pediatric use, to our knowledge, of fumagillin for microsporidiosis.
Case report We report the cases of 2 sisters, ages 6 and 4 years, who were admitted to Tours University Hospital, France, for exploration of intermittent diarrhea lasting for 6 months, associated with weight loss and stagnation of growth. Both children had approximately 5 aqueous feces per day, but no fever or major abdominal pain. The 2 sisters were born from a consanguineous marriage. In their respective medical history, primary hyperoxaluria type 1 resulted in liver-kidney transplantation when each girl was 1 year of age. Immunosuppressive therapy initially consisted of tacrolimus (FK506), cyclosporine (CsA), and prednisone. In the younger sister, kidney transplant was immediately complicated by thrombosis leading to renal graft explantation within 4 days. As her renal function was subsequently quite satisfactory (i.e., creatinine clearance at 30 mL/min without dialysis), a second kidney transplantation was not immediately considered, and prednisone was consequently stopped. After quasiconcomitant Epstein–Barr virus (EBV) primary infections, CsA was also withdrawn in both patients, to substantially decrease their level of immunosuppression. At the time of present hospitalization, defined as day zero (D0) (the date of entrance into our hospital facility for diarrheal symptoms), lymphocyte counts were respectively 2.99 and 2.30 9 109/L. Neither bacteria nor pathogenic viruses were isolated from stools or blood. The therapeutic drug monitoring of FK506 was normal, making iatrogenic toxicity an unlikely cause of diarrhea. Only the Uvitex-2Bâ fluorescence (Polysciences, Inc, Warrington, Pennsylvania, USA) on each individual fecal smear (Fig. 1A) – confirmed by the trichrome-blue staining (Fig. 1B) – showed tiny spores suggestive of microsporidia (on D6). Polymerase chain reaction assay characterized the species as Enterocytozoon bieneusi, by means of designed primers Eb1 (5′-GACAGCCTGTGTGTGAGAATAC) and Eb5 (5′-AACGAATGACTTGACCCTGGTAA) targeting SSU-rDNA, plus a specific fluorescent probe EbS2 (6FAM-TGCTTAATTTAACTCAACGCGGGAAAA-TAMRA) (LightCycler 480â Roche, Meylan, France) (on D10). Sequencing the ITS1-region revealed the human genotype C for both isolates (on D58). No
epidemic contagion among relatives, or source of environmental contamination was noted. Further tapering of the immunosuppressive therapy could not be considered for fear of graft rejection (CsA had already been discontinued because of EBV, and prednisone had been withdrawn in the younger sister only). After a synchronous 2-week course of albendazole alone (400 mg/day for the older sister, and a halfdose for the younger one) (from D13 to D27), followed by 10 days of metronidazole alone (10 mg/kg 3 times daily) (from D41 to D51), the digestive symptoms did not improve, and stools were still positive on microbiological examination. Therefore, fumagillin was finally considered, although this drug is not clearly indicated in pediatric patients. Because of the thrombotic renal complication in the younger sister, only the older one was actually eligible to receive oral treatment at 20 mg/day for 7 days (from D112 to D119).
Results During treatment with fumagillin alone (D112–119), a single hepatic cytolysis appeared in the older sister (aspartate aminotransferase 130 UI/L, alanine aminotransferase 164 UI/L) (D114), without any proof for fumagillin involvement. Indeed, a few sporadic episodes of cytolysis had formerly been observed in the treated child, even before the use of fumagillin. Furthermore, this latter cytolytic episode resolved spontaneously, despite continuation of treatment. No blood disorder was reported: twice weekly platelet counts always stayed within the normal range (Fig. 2). Interestingly, we noticed an improvement of digestive symptoms over the first 4 days of therapy (D116). Whereas all the monthly pre-fumagillin fecal explorations had remained constantly positive ﴾(at D41, D82, D103, and D113, which is just before the initiation of fumagillin﴿, the first post-therapeutic control was, by contrast, negative (on D194). No adjustment of the immunosuppressive regimen was necessary: FK506 and prednisone were maintained at 0.9 mg twice daily and 5 mg/day respectively. Long-term follow-up was quite difficult, because the girls were from a nomadic community, and consequently travelled a great deal. Until they were definitively lost to follow-up (on D369), no acute digestive events were specifically reported by the parents in their treated daughter. Quarterly post-therapeutic clinical examinations exhibited a nontender abdomen without guarding. Moreover, her body mass increased steadily since the day of treatment initiation, from 17.2 to
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Desoubeaux et al: Fumagillin in a transplanted child
A
B
Fig. 1. (A) Methanol-fixed fecal smear impregnated by brightener agent according to Van Gool’s method (brightener Uvitex 2B 200%â CibaGeigy, magnification 9 1250) and (B) colored by blue trichrome stain according to Weber’s method (Para-PAKâ stool system, magnification 9 1000). With Uvitex 2Bâ, microsporidia appear in blue fluorescence (A) due to chitin in their cell wall. The shape is ovoid, measuring about 2 9 0.5 lm. The fluorescent polar filament is clearly visible in the middle of the spore. Through trichrome staining, microsporidia are colored in pink (B) on a blue-greenish background. Here, 5 specimens are grouped in clusters. The size is slightly larger than bacteria. Note the large colorless vacuole at the center of each spore.
recurrent abdominal pain. Before being lost to follow-up (on D369), her weight had fallen from 14.6 to 14.3 kg over 9 months. Unfortunately, her immunosuppressive treatment (FK506 alone at 1 mg twice daily) could not have been stopped or tapered further, with the aim of trying to eradicate microsporidia. Unlike her older sister, spores and DNA of E. bieneusi were still detected in her feces during the whole period of quarterly follow-up (at D113, D194, D265, and D369). To date, the younger sister is still awaiting her second kidney transplant. She most likely is still chronically infected with E. bieneusi. Fig. 2. Platelet count of the older sister. At day 112 (D112), treatment of the microsporidiosis with fumagillin was finally initiated for 7 days at the dose of 20 mg/day, only in the older sister. Throughout the whole period of use of fumagillin (D112–119), no thrombocytopenia was noted, with platelet counts ranging between 259 and 331 9 109/L.
20.7 kg in 9 months. Microbiological investigations of the stools subsequently remained negative over 9 months of regular follow-up (at D194, D265, and D369). Concomitantly, the non-treated sister still had intermittent diarrhea throughout the medical observation period (until D369). A noteworthy episode of profuse diarrhea occurred on D194. She also complained of
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Discussion Although recently attached to the Fungi group, microsporidia are ubiquitous unicellular microorganisms with an obligate intracellular stage, like some parasites. The first human case of microsporidiosis was described in the mid-20th century (6), but most cases have occurred since the AIDS pandemic, owing to the opportunistic skills of microsporidia (2). Previously unknown, new microsporidial species infectious for humans (e.g., E. bieneusi, Encephalitozoon intestinalis) have recently been added to the 1300 types already known (1, 7). The incidence of microsporidiosis among
Transplant Infectious Disease 2013: 15: E250–E259
Liver
Liver
Kidney
Kidney
Germany, 2001 (20)
South Africa, 2001 (21)
Canada, 2002 (22)
US, 1999 (15)
Germany, 2001 (19)
Heart
France, 1999 (14)
Kidney (6); Liver (1); Heart-lung (1)
Kidney (2)
France, 1996 (13)
France, 2001 (17, 18)1
Heart–lung
US, 1995 (12)
Kidney
Liver
Case country, year (reference)
France, 2000 (16)
Type of transplant (number of patients)
Transplant Infectious Disease 2013: 15: E250–E259 45/F
39/M
36/F
36/F
NA/NA
38/M
IS (+ high-dose Methylprednisolone and Muromonab-CD3 for acute graft rejection)
CsA, AZA, Prednisone
FK506
FK506
NA
FK506, MMF, Prednisone
CsA, AZA, Methylprednisone
CsA, AZA, MMF, Prednisone (+ ATG for acute graft rejection)
24/M
48/M
CsA, AZA, MMF, Prednisone
CsA, AZA (+ Methylprednisone and ATG for acute graft rejection)
FK506, Prednisone
Immunosuppressive regimen
46/M
48/M
48/F
Age (years)/Gender
2 months
1 year
5 years
5 years
NA
1.5 years
4 years
2 years
7 years
3 years
1.5 years
Interval time post transplant
Characteristics of reported cases of microsporidiosis occurring in solid organ transplant recipients
Disseminated (fever, lung infiltrate, keratoconjunctivitis, renal failure, general seizure)
Disseminated (fever, rigors, renal impairment)
Intestinal (chronic diarrhea and colicky abdominal pain, fever)
Intestinal (watery unbloody chronic diarrhea)
Intestinal (diarrhea)
Intestinal (acute diarrhea, vomiting, weight loss)
Intestinal (chronic diarrhea, vomiting, dyspepsia, fatigue, weight loss)
Intestinal (chronic diarrhea, weight loss)
Intestinal (chronic diarrhea, weight loss)
Intestinal (diarrhea, massive weight loss)
Intestinal (diarrhea, weight loss)
Clinical course
Recovery Initial symptomatic improvement (and microsporidial clearance) then death (related to neurological involvement)
Albendazole + topical Fumagillin in eyes (IS withdrawal)
Symptomatic improvement (but microsporidial persistence)
Mild symptomatic improvement (but microsporidial persistence)
Albendazole
Albendazole (FK506 tapered)
Albendazole (FK506 tapered)
NA
Recovery
Albendazole + Metronidazole (FK506 and prednisone tapered, MMF withdrawal) NA
Temporary symptomatic improvement (but microsporidial persistence)
Recovery
Recovery
Symptomatic improvement (but microsporidial persistence)
Recovery
Outcomes
Metronidazole
None (MMF withdrawal and AZA tapered)
Albendazole (MMF withdrawal and AZA tapered)
Albendazole
Metronidazole
Anti-microsporidial treatment
Desoubeaux et al: Fumagillin in a transplanted child
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E254 38/F, 58/F 33/M, 52/M, 68/M
Kidney
Pancreas; Kidney
Kidney (2)
Kidney (2)
Kidney (3)
US, 2003 (25)
US, 2004 (26)
Transplant Infectious Disease 2013: 15: E250–E259
France, 2006 (27)
Netherlands, 2009 (28)1
FK506, Prednisone
71/F
NA
NA
FK506, MMF, Prednisone
FK506, MMF, Prednisone
IS (+ high-dose Steroids for acute graft rejection)
mTOR (Sirolimus), CsA, Prednisone (+ high-dose Methylprednisolone for acute graft rejection)
FK506, CsA, AZA, MMF ( ATG for acute graft rejection)
FK506, Prednisone
FK506
Immunosuppressive regimen
49/M
43/M
45/F
42/M
Kidney
Mexico, 2003 (24)
NA/F (7), M (16)
34/M
NA/NA
Age (years)/Gender
Kidney (14); Liver (5); Heart and/or Lung (4)
Kidney
Liver
Type of transplant (number of patients)
France, 2003 (23)1
France, 2002 (11)
Case country, year (reference)
Table 1 Continued
NA
NA
8 years
2 months
2 months
2 months
5 months
NA
3 years
2 years
Interval time post transplant
Intestinal
Intestinal
Intestinal (diarrhea, weight loss)
Intestinal (diarrhea, nausea)
Disseminated (fever, left lower quadrant abdominal pain, bilateral infiltrated pneumonia, urinary tract infection, then peritonitis and multi-organ failure)
Disseminated (fever, bilateral keratoconjunctivitis, generalized seizures and developed left-sided numbness and motor weakness)
Disseminated (fever, ocular discomfort, diarrhea and colon wall thickening, abdominal and thoracic pain)
Asymptomatic or intestinal (diarrhea, weight loss)
Intestinal (chronic diarrhea)
Intestinal (chronic diarrhea)
Clinical course
Temporary symptomatic improvement, then death
Death
None
None (FK506 tapered and completed by basiliximab, MMF switched to AZA)
NA
NA
Fumagillin
NA
NA
NA
NA
Symptomatic improvement (but no microsporidial eradication), and relapse (1 year later)
Albendazole + topical Fumagillin in eyes (mTOR and CsA withdrawal)
Fumagillin
Variable
Recovery
Recovery
Outcomes
Albendazole or Fumagillin
Fumagillin
Fumagillin
Anti-microsporidial treatment
Desoubeaux et al: Fumagillin in a transplanted child
Spain, 2011 (4)
Kidney (2)
Kidney (10)
France, 2010 (10)1
Transplant Infectious Disease 2013: 15: E250–E259 mTOR (Sirolimus), MMF
FK506, MMF, Prednisone (+ Rituximab for acute graft rejection)
66/M
48/M
FK506, MMF
CsA, AZA, Prednisone
48/M
54/M
FK506, MMF
37/F
FK506, MMF, Prednisone
CsA, MMF, Prednisone
57/M
66/M
FK506, MMF, Prednisone
25/F
FK506, MMF, Prednisone
53/F FK506, MMF, Prednisone (+ Muromonab-CD3 for acute graft rejection)
FK506, Prednisone
36/F
23/M
FK506, MMF, Prednisone
CsA, MMF, ATG
FK506, MMF
CsA, mTOR (Everolimus)
Immunosuppressive regimen
19/M
38/F
64/M
Kidney
Kidney
45/M
Age (years)/Gender
Liver
Type of transplant (number of patients)
France, 2010 (29)
France, 2009 (8)
Case country, year (reference)
Table 1 Continued
15 years
3 months
3 years
14 years
2.5 years
1.5 years
3.5 years
8 years
7.5 years
3 years
7 years
6 years
1 month
3 years
3 years
Interval time post transplant
Intestinal (persistent liquid diarrhea, epigastric pain)
Intestinal (abundant liquid diarrhea)
Intestinal (diarrhea weight loss)
Intestinal (diarrhea, weight loss, abdominal cramps)
Intestinal (diarrhea, weight loss, abdominal cramps)
Diarrhea
Intestinal (diarrhea, weight loss, abdominal cramps)
Intestinal (diarrhea, weight loss)
Intestinal (diarrhea, abdominal cramps)
Intestinal (diarrhea)
Intestinal (diarrhea, weight loss)
Intestinal (diarrhea)
Disseminated (fever, cough, abdominal pain, anorexia)
Intestinal (persistent major diarrhea, nausea, weight loss)
Intestinal (persistent diarrhea, weight loss, fever)
Clinical course
Recovery Recovery (but microsporidial persistence)
Metronidazole then Albendazole
Recovery
Recovery
Recovery
Recovery
Recovery
Recovery
Recovery
Recovery
Recovery
Recovery
Filgrastrim
Fumagillin
Fumagillin (MMF withdrawal relayed by AZA)
Fumagillin (AZA withdrawal)
Fumagillin
Fumagillin
Fumagillin
Fumagillin (MMF withdrawal)
Fumagillin (MMF tapered)
Fumagillin
Fumagillin
Recovery
Recovery
Fumagillin
Albendazole (MMF switched to AZA)
Recovery
Outcomes
Fumagillin (mTOR withdrawal)
Anti-microsporidial treatment
Desoubeaux et al: Fumagillin in a transplanted child
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Transplant Infectious Disease 2013: 15: E250–E259 6/F
4/F
Lung
Kidney
Liver; Kidney
Liver; Kidney (then kidney explantation)
US, 2013 (33)
US, 2013 (34)
France, 2013 (current report)
56/M
57/M
FK506
FK506, Prednisone
FK506, MMF, Prednisone (+ ATG, Methylprednisolone for acute graft rejection)
FK506, MMF, Prednisone (+ ATG, Methylprednisolone and Rituximab for acute graft rejection)
FK506, MMF, Prednisolone, Basiliximab (+ Methylprednisolone for acute graft rejection)
AZA
AZA, Prednisone
Prednisolone
Immunosuppressive regimen
3 years
5 years
6 months
5 months
1 year
9 years
28 years
17 years
Interval time post transplant
Intestinal (intermittent aqueous diarrhea, weight loss)
Intestinal (intermittent aqueous diarrhea, weight loss)
Disseminated (fever, chills, cough, and acute kidney injury)
Disseminated (fever, diarrhea, altered mental status, cough and lung infiltrate, acute kidney injury)
Disseminated (fever, tachycardia, lung infiltrate)
Disseminated (chronic diarrhea, myalgia, fever and progressive weakness and neurological symptoms)
Intestinal (chronic diarrhea)
Intestinal (abundant liquid diarrhea, dehydratation and acute renal failure)
Clinical course
None
Fumagillin
Albendazole (MMF withdrawal)
Albendazole (IS tapered)
Albendazole (IS withdrawal except prednisolone)
None
Albendazole + Nitazoxanide then Fumagillin
Nitazoxanide
Anti-microsporidial treatment
Symptomatic persistence (and microsporidial persistence)
Recovery
Death
Death
Symptomatic improvement (but microsporidial persistence)
Death
Recovery
Recovery
Outcomes
Table 1
Retrospective study. F, female; M, male; FK506, tacrolimus; CsA, cyclosporine; AZA, azathioprine; ATG, anti-thymocyte globulin; MMF, mycophenolate mofetil; NA, not available; IS, immunosuppressive drugs; mTOR, mammalian target of rapamycin.
1
68/F
Kidney
Australia, 2012 (32)
49/M
Lung
Australia, 2012 (31)
59/F
49/M
Age (years)/Gender
Kidney
Kidney
Type of transplant (number of patients)
France, 2012 (30)
France, 2012 (9)
Case country, year (reference)
Table 1 Continued
Desoubeaux et al: Fumagillin in a transplanted child
Desoubeaux et al: Fumagillin in a transplanted child
the human immunodeficiency virus (HIV)-positive population has currently decreased, thanks to highly active anti-retroviral therapies restoring immunity (2). Nowadays, SOT recipients are considered as a new risk group for microsporidiosis. To date, only 74 cases have been reported in SOT (Table 1) (4, 8–34, and the present report), mainly in renal transplant recipients (n = 53), in whom the intensity of symptoms is not proportionally related to CD4+ count (unlike in the HIVpositive population) (3, 4, 8). Within this limited cohort, FK506, mycophenolate mofetil, and CsA have been used mainly as immunosuppressive agents. Diagnosis of microsporidiosis has been established between 1 month and 28 years after transplantation. As in our patients, E. bieneusi genotype C was involved in the majority of reported cases (9). Produced by Aspergillus fumigatus, fumagillin was initially developed for veterinary purposes (35). It expresses an anti-methionine-aminopeptidase activity that is particularly effective against the genus Enterocytozoon (10, 11). Side effects are mainly hematologic (especially thrombocytopenia), requiring regular monitoring of blood counts, which may consequently limit its more wide-ranging medical use. Fumagillin was approved in France in 2005 for the treatment of digestive microsporidiosis caused by E. bieneusi in HIV-positive adult patients (36). The commercial fumagillin pill (Flisint 20â; Sanofi-Aventis, Paris, France) is also available occasionally for E. bieneusi infections occurring in HIV-negative patients (e.g., in adult transplant patients or in adult patients with hematologic malignancies), by means of an expanded access program. However, fumagillin has not yet been approved by the U.S. Food and Drug Administration. Because of the total lack of clinical trials, no data are available in pediatric patients (37). To our knowledge, our reported case provides the first evidence of the successful use of fumagillin in a pediatric patient to eradicate E. bieneusi with clinical, microbiological, and molecular proofs. Within our cases of intestinal microsporidiosis occurring in 2 sisters with SOT, fumagillin could only be administered to the older sister. As the 2 cases were concomitantly monitored in our hospital facility, the non-treated younger sister could be regarded as serving as a kind of control subject compared with the older one. Seven days of fumagillin oral therapy alone rapidly disabled molecular detection of microsporidia in the older sister’s feces, and she remained negative during 9 months of follow-up. Furthermore, diarrheal symptoms disappeared in