© 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

Transplant Infectious Disease 2013: 15: E250–E259

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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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E256

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

Successful treatment with fumagillin of the first pediatric case of digestive microsporidiosis in a liver-kidney transplant.

We report the first successful use, to our knowledge, of fumagillin alone in a pediatric patient to cure intestinal microsporidiosis in a liver-kidney...
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