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Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in Kuwait
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MYCMED-843; No. of Pages 5 Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
Case report
Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in Kuwait A. Jeragh a, S. Ahmad c, Z. Khan c,d,*, R.Y. Tarazi b, S. Ajmi a, L. Joseph c, S. Varghese c, S. Vayalil d a
Departments of Microbiology, Al Adan Hospital, Kuwait Cardiac surgery, Al Adan Hospital, Kuwait c Department of Microbiology, Kuwait d Mycology Reference Laboratory, Faculty of Medicine, Kuwait University, Kuwait b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2018 Received in revised form 10 September 2018 Accepted 29 October 2018 Available online xxx
The recently described genus Amesia encompasses four species but only Amesia atrobrunnea (= Chaetomium atrobrunneum) is known to be pathogenic to humans. Here, we describe a case of subcutaneous phaeohyphomycosis in Kuwait in an apparently immunocompetent patient diagnosed by direct microscopy of the infected tissue and culture. The identity of A. atrobrunnea was established by typical morphological characteristics and by sequencing of internally transcribed spacer (ITS) region and D1/D2 domains of rDNA. To the best of our knowledge, this is the first report documenting etiologic role of this species in causing a locally invasive subcutaneous infection.
Keywords: Amesia atrobrunnea Subcutaneous phaeohyphomycosis Surgical site infections Molecular identification
1. Introduction The genus Chaetomium comprises over 400 species which are ubiquitous in nature [1]. They can grow on a variety of substrates, particularly in damp or water-damaged buildings, and thus can be an important source of inhalant allergens contributing to asthma, rhinitis, sinusitis and other respiratory disorders in susceptible/genetically predisposed individuals [1–3]. Some Chaetomium species are also capable of causing invasive diseases [4–7]. Recently, the taxonomy of the genus has been revised on the basis of morphological characteristics and phylogenetic analyses of six loci [1]. Currently, the family Chaetomiaceae encompasses 10 genera which include five newly established genera and seven new species. Consequently, Chaetomium atrobrunneum has been included in the newly erected genus Amesia (X. Wei Wang, Samson & Crous, gen. nov, MycoBank MB818829) with type species Amesia atrobrunnea (Ames) X. Wei Wang & Samson (= Ch. atrobrunneum). The genus Amesia now includes three other species, namely, Amesia cymbiformis, Amesia nigricolor and Amesia gelasinospora. Within this genus, A. atrobrunnea is the only species that has been recognized as a human pathogen causing deep-seated infections [8–14]. Here, we describe the first case of subcutaneous phaeohyphomycosis caused by A. atrobrunnea in Kuwait and diagnosed by detection of the fungus by direct microscopic examination of the infected tissue, culture of the organism from the tissue sample and its
* Corresponding author. Department of Microbiology, Faculty of Medicine, Kuwait University, P. O. Box 24923, Safat 1311, Kuwait. E-mail address: [email protected] (Z. Khan).
C
2018 Elsevier Masson SAS. All rights reserved.
phenotypic features and DNA sequence comparisons of the internally transcribed spacer (ITS) region and D1/D2 domains of rDNA with fungal sequences in the databanks.
2. Observations A 45-year-old male was admitted to the Casualty in April, 2017 with sudden onset of chest pain and diaphoresis. On admission, his blood pressure was 220/110 mmHg and ECG revealed myocardial infarction with first degree AV block. Troponin I was > 100 ng/mL. Emergent CT scan and ECHO showed intimal flap in the ascending aorta, moderate aortic regurgitation, normal LV contractility and hypokinetic right ventricular function with no tricuspid regurgitation. There was mild pericardial effusion which required immediate placement of epidural catheter to monitor CSF pressure. Median sternotomy was performed and pericardium was opened. The right ventricle was edematous and had a large hematoma in its free wall with very poor contractile function. The left ventricle was hyper contractile with normal function. Axillary arterial and right atrial cannulation were performed. The aorta was cross clamped. A transverse aortotomy was performed opening the false and then the true lumen. Cardioplegia was given antegradely and retrogradely. The ostium of the right coronary artery (RCA) was severed completely due to the dissection. The distal RCA was grafted with saphenous vein graft. Resuspension of the aortic valve was done. Under a 40 min period of circulatory arrest a Frozen Elephant Trunk operation was performed. At the completion of the operation the patient could
10.1016/j.mycmed.2018.10.004 C 2018 Elsevier Masson SAS. All rights reserved. 1156-5233/$ – see front matter
Please cite this article in press as: Jeragh A, et al. Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in KuwaitSubcutaneous Amesia atrobrunnea infection–>. Journal De Mycologie Me´dicale (2018), doi:10.1016/j.mycmed.2018.10.004
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MYCMED-843; No. of Pages 5 2
A. Jeragh et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
Fig. 1. Potassium hydroxide (KOH)-calcofluor mount of digested tissue biopsy specimen showing septate hyphae of A. atrobrunnea.
Fig. 2. Six-day old colony of A. atrobrunnea on oatmeal agar showing white wooly texture and a large number of dark-colored ascomata.
not be weaned off the cardiopulmonary bypass due to severe right ventricular dysfunction because of the massive pre-operative inferior wall myocardial infarction. An extracorporeal membrane oxygenator (ECMO) was used to support the right heart for two days. With adequate oxygenation it was converted to temporary right ventricular assist devices (RVAD) with in-flow to the right common femoral vein and outflow to a 10 mm Hemasheild graft sutured to the main pulmonary artery. After one week without any recovery of the right ventricle, the temporary RVAD was removed and a permanent RVAD using a heart ware pump was inserted with the inflow the right atrium and the outflow the main pulmonary artery. The chest was closed. The patient was intubated and on mechanical ventilation during the course of the special intensive care unit (SICU) stay. His liver enzymes and lactate dehydrogenase were elevated and he also developed acuter kidney injury requiring continuous peritoneal/hemodialysis. Three days later, he was shifted to Intensive Care Unit and was put on ECMO and mechanical ventilation to support his lung functions. During the course of dialysis, the patient developed leukocytosis (range 15.2 109/L to 26.2 109/L), 15 days after the surgical procedure. Although no bacterial pathogens were isolated in blood cultures, he received short courses of broad-spectrum antibiotics as a preemptive
measure which included meropenem, vancomycin, tazocin, and acyclovir during his stay in ICU for 4 weeks. He also received a 2week course of caspofungin. There was no serological evidence of HBV, HCV, HIV or Treponema pallidum infection. As his condition continued to deteriorate, he was shifted back to coronary care unit on Day 32 in a comatose condition and declared brain dead on Day 36 of admission. The tissue biopsy collected from the infected site near sternum, where RVAD device was implanted was sent to Mycology Reference Laboratory for routine culture and diagnosis. The direct microscopic examination of the KOH-calcofluor digested tissue showed pigmented septate hyphae (Fig. 1) and culture yielded a filamentous fungus on blood agar and Sabouraud dextrose agar. On Sabouraud dextrosae agar, the isolate grew very well at 30 ?C as well as 37 ?C attaining a diameter of 2.5 cm and 3.5 cm in 4 days, respectively. The growth was substantially reduced at 45 ?C and showed only minimal growth at 47 ?C. Growth was much faster on potato dextrose agar reaching a diameter of 3.5 cm and 5 cm in 4 days, respectively. The colony first appeared white in color with wooly texture and then became grayish with appearance of dark-colored ascomata on the surface. The reverse was dark yellow or olivaceous green. The growth on oatmeal agar produced a large number of ascomata (Fig. 2). The ascomata were subspherical, dark brown in
Please cite this article in press as: Jeragh A, et al. Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in KuwaitSubcutaneous Amesia atrobrunnea infection–>. Journal De Mycologie Me´dicale (2018), doi:10.1016/j.mycmed.2018.10.004
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Fig. 3. A) Lactophenol-cotton blue mount prepared from six-day old culture showing a mature ascoma of A. atrobrunnea with dark brown ascomal hairs (setae) and clavate shaped asci with ascospores; B) large number of fusiform-shaped ascospores with apical germpores.
color, ranging between 50 to 130 mm and produced clavate shaped asci that contained 8 brown-colored, smooth-walled ascospores with subapical germ pores (Fig. 3A, B). The ascospores were fusiform in shape and 8.5–10 4.5–6 mm in size. The ascomal hairs (setae) were dark brown, long and straight. The DNA from the patient’s isolate (Kw77-5-17) was prepared as described previously and used as the template for PCR amplifications [15]. The ITS region (ITS1, 5.8S rRNA, and ITS2) was amplified with ITS1 and ITS4 primers while the divergent D1/ D2 domains of rDNA were amplified with the NL-1 and NL-4 primers, the amplicons were purified and both strands of the two amplicons were sequenced by using internal (ITS1FS, ITS2, ITS3 and ITS4RS for ITS region and NL-1FS, NL-2, NL-3 and NL-4RS for the D1/D2 domains) primers, as described previously [16,17]. GenBank basic local alignment search tool (BLAST) searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi?) were performed for species identification. Our isolate was identified as A. atrobrunnea as both, the ITS region (GenBank accession no. LT969523) of our isolate (Kw77-5-17) and D1/D2 domain (GenBank accession No. LT969522) of rDNA sequences showed 100% identity with the corresponding ITS region (GenBank accession No. JX280771) and D1/D2 domain (GenBank accession No. JX280666) sequences from A. atrobrunnea type strain
(CBS379.66). The ITS region sequence also showed 100% identity with several other (CGMCC 3.3594, MRDS12, NBRC 6542, C64 and MRDS8) strains of A. atrobrunnea. For determining antifungal susceptibility, the inoculum was prepared as described previously [18]. The growth was harvested in normal saline and vigorously vortexed for 15s to disperse the clumps mainly consisting of ascospores. These suspensions were adjusted with a hemacytometer to 107 ascospores/mL and spread all over the surface of RPMI medium. The Etest strips (bioMe´rieux, Marcy l’E´toile, France) of itraconazole, voriconazole, posaconazole, amphotericin B, anidulafungin, and caspofungin were implanted on the surface of the medium and incubated for 48 h at 35 ?C. Minimum inhibitory concentrations for itraconazole, voriconazole, posaconazole, amphotericin B, anidulafungin and caspofungin were read as follows: 2 mg/mL, 0.094 mg/mL, 0.38 mg/mL, 1 mg/ mL, 1.5 mg/mL, and 6 mg/mL, respectively.
3. Discussion The present report is noteworthy as it describes the first case of subcutaneous infection caused by A. atrobrunnea in an apparently immunocometent patient. No other bacterial pathogens were
Please cite this article in press as: Jeragh A, et al. Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in KuwaitSubcutaneous Amesia atrobrunnea infection–>. Journal De Mycologie Me´dicale (2018), doi:10.1016/j.mycmed.2018.10.004
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Table 1 Summary of published cases of A. atrobrunnea, (= C. atrobrunneum) infections. Case Reference No
Associated underlying conditions
Type of infection
Source of isolation Identification method
Antifungal treatment regimen
Outcome
Renal transplant Bone marrow transplant recipient
Brain abscess Cerebral abscess
Brain biopsy Brain biopsy
Not known
Died
Amphotericin B, itraconazole
Died
Cerebral abscess
Brain biopsy
Not available
Died
Cellulitis (left eye) and conjunctivitis
Eye discharge
Itraconazole, fluconazole
Resolved
Voriconazole
Improved
Topical natamycin, ketoconazole
Resolved
Fluconazole, amphotericin B, caspofungin Caspofungin
Died
Anandi et al. [8] Guppy et al. [9]
India
1989
32/M
USA
1998
31/M
3
Thomas et al. [10]
USA
1999
4
Zhang et al. [11]
China
2010
Not available Bone marrow transplant recipient 1.5/M Nil
5
Tabbara et al. [12]
USA
2010
11/M
Hodgkin lymphoma
Retinitis and Vitreous aspirate subhyaloid abscess
6
Balne et al. [13]
India
2012
44/M
Nil
Keratitis
7
Wang et al. [14]
China
2016
89/M
Bed-ridden for long Pneumonia term
Tracheal aspirates
8
Present case
Kuwait
2017
45/M
None
Tissue biopsy
1 2
a
Country Year Age/sex of report
Corneal scrapings
Subcutaneous infection
Morphological characteristicsa Morphological methods and electron microscopy No (conventional morphological methods Morphological characteristic and (ITS & D1/D2 sequencing No (conventional morphological methods) Morphological characteristic, ITS sequencing Morphological characteristic, ITS & D1/D2 sequencing Morphological characteristic, ITS & D1/D2 sequencing
Died
This isolate was initially identified as C. globosum but was subsequently re-identified as C. atrobrunneum [20].
isolated in culture. The diagnosis was unequivocally established by demonstrating septate hyphae of the fungus in direct microscopic examination of the infected tissue. Species-specific identity of the isolate was based on typical morphological characteristics and by obtaining 100% identity of the ITS region and D1/D2 domain sequences with the corresponding sequences from reference strain (CBS379.66) of A. atrobrunnea available in GenBank [14]. This report reinforces that dematiceous fungi have the potential to cause localized infections at surgical sites. So far, human infections due to A. atrobrunnea have been reported in seven patients [8–14], most of them were immunocompromised individuals (Table 1). Three of the patients had central nervous system involvement and died [8–10]. Our patient had no history of intravenous drug abuse or evidence of cerebral involvement. Although our patient expired due to brain death, it is unlikely that dissemination from primary subcutaneous lesion was the cause of mortality as repeated blood cultures were negative for the fungus. Recently, A. atrobrunnea (C. atrobrunneum) pneumonia has recently been implicated as a cause of pneumonia in an immunocompromised patient along with Aspergillus fumigatus [14]. Despite combination therapy of amphotericin B and caspofungin, the patient died. It is not clear if A. atrobrunnea was the primary cause of pneumonia or both had any symbiotic association for their growth in tracheal aspirates [14]. Isolation of A. fumigatus and Chaetomium homopilatum from tracheal secretion of a patient with leukemia, has also been reported previously, but clinical significance of the latter species remained uncertain [19]. Like other Chaetomium spp., such as C. strumarium [20], A. atrobrunnea (C. atrobrunneum) can be regarded as neurotropic species causing serious and life-threatening infections [8,9,21]. Little is known about the pathogenic attributes of Amesia species. Since Amesia/Chaetomium species can survive in high environmental temperatures in their natural habitat [7], they have inherent ability to resist hosts’ body temperature and can grow in vivo. Further studies are warranted to elucidate the role of
various virulence factors and bioactive metabolites produced by these species, which may have a role in the pathogenesis and neurotropism [1,3]. Surgical site infections due to other dematiaceous fungi, such as Bipolaris species and Exserohilum species, have also been reported in patients following cardiothoracic surgical interventions [22,23]. There is paucity of information on antifungal susceptibility of Chaetomium species [24,25]. Based on the published reports [14,15,21], voriconazole exhibited highest in vitro activity, followed by amphotericin B and itraconazole. Echinocandins showed minimal activity against our A. atrobrunnea isolate. They were also found to be less active against other members of the genus Chaetomium [25]. So far, there is no recommended treatment regimen for subcutaneous infections caused by A. atrobrunnea or other closely related species. Conclusion This report documents etiologic role of A. atrobrunnea in causing subcutaneous phaeohyphomycosis and highlights importance of molecular methods in establishing accurate identification. To the best of our knowledge, this is the first report documenting etiologic role of this species in causing a locally invasive subcutaneous infection. Contribution of authors AJ, RYT and SAA provided clinical case details, SA and ZK supervised laboratory work and wrote the manuscript, LJ, SV and SV provided technical support. Disclosure of interest The authors declare that they have no competing interest.
Please cite this article in press as: Jeragh A, et al. Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in KuwaitSubcutaneous Amesia atrobrunnea infection–>. Journal De Mycologie Me´dicale (2018), doi:10.1016/j.mycmed.2018.10.004
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Acknowledgement The authors are thankful to Omar Al-Musallam for technical support. References [1] Wang XW, Houbraken J, Groenewald JZ, Meijer M, Andersen B, Nielsen KF, et al. Diversity and taxonomy of Chaetomium and chaetomium-like fungi from indoor environments. Stud Mycol 2016;84:145–224. [2] Aru A, Munk-Nielsen L, Federspiel BH. The soil fungus Chaetomium in the human paranasal sinuses. Eur Arch Otorhinolaryngol 1997;254:350–2. [3] Miller JD, McMullin DR. Fungal secondary metabolites as harmful indoor air contaminants: 10 years on. Appl Microbiol Biotechnol 2014;98:9953–66. [4] Hoppin EC, McCoy EL, Rinaldi MG. Opportunistic mycotic infection caused by Chaetomium in a patient with acute leukemia. Cancer 1983;52:555–6. [5] Yeghen T, Fenelon L, Campbell CK, et al. Chaetomium pneumonia in a patient with acute myeloid leukaemia. J Clin Pathol 1996;49:184–6. [6] Lesire V, Hazouard E, Dequin PF, Delain M, Therizol-Ferly M, Legras A. Possible role of Chaetomium globosum in infection after autologous bone marrow transplantation. Inten Care Med 1999;25:124–5. [7] Ahmed SA, Khan Z, Wang X-W, Tarek A, Moussa A, Al-Zahrani HS, et al. 2016 Chaetomium-like fungi causing opportunistic infections in humans: a possible role for extremotolerance. Fungal Divers 2016;76:11–26. [8] Anandi V, John TJ, Walter A, Shastry JC, Lalitha MK, Padhye AA, et al. Cerebral phaeohyphomycosis caused by Chaetomium globosum in a renal transplant recipient. J Clin Microbiol 1989;27:2226–9. [9] Guppy KH, Thomas C, Thomas K, Anderson D. Cerebral fungal infections in the immunocompromised host: a literature review and a new pathogen–Chaetomium atrobrunneum: case report. Neurosurgery 1998;43:1463–9. [10] Thomas C, Mileusnic D, Carey RB, Kampert M, Anderson D. Fatal Chaetomium cerebritis in a bone marrow transplant patient. Hum Pathol 1999;30:874–9. [11] Zhang H, Ran Y, Li D, Liu Y, Xiang Y, Zhang R, et al. Clavispora lusitaniae and Chaetomium atrobrunneum as rare agents of cutaneous infection. Mycopathologia 2010;169:373–80. [12] Tabbara KF, Wedin K, Al Haddab S. Chaetomium retinitis. Retin Cases Brief Rep 2010. [13] Balne PK, Nalamada S, Kodiganti M, Taneja M. Fungal keratitis caused by Chaetomium atrobrunneum. Cornea 2012;31:94–5.
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[14] Wang H, Liu Y, Chen SC, Long Y, Kong F, Xu YC. Chaetomium atrobrunneum and Aspergillus fumigatus in multiple tracheal aspirates: Copathogens or symbiosis. J Microbiol Immunol Infect 2016;9:281–5. [15] Ahmad S, Khan ZU, Theyyathel AM, Diagnostic value of DNA. (1-3)-beta-dglucan, and galactomannan detection in serum and bronchoalveolar lavage of mice experimentally infected with Aspergillus terreus. Diagn Microbiol Infect Dis 2007;59:165–71. [16] Khan ZU, Ahmad S, Mokaddas E, Chandy R, Cano J, Guarro J. Actinomucor elegans var. kuwaitiensis isolated from the wound of a diabetic patient. Antonie van Leeuwoenhoek 2008;94:343–52. [17] Khan ZU, Ahmad S, Hagen F, Fell JW, Kowshik T, Chandy R, Boekhout T. Cryptococcus randhawai sp. nov., a novel anamorphic basidiomycetous yeast isolated from tree trunk hollow of Ficus religiosa (peepal tree) from New Delhi. India Antonie van Leeuwoenhoek 2010;97:253–9. [18] Serena C, Ortoneda M, Capilla J, Pastor FJ, Sutton DA, Rinaldi MG, et al. In vitro activities of new antifungal agents against Chaetomium spp. and inoculum standardization. Antimicrob Agents Chemother 2003;47:3161–4. [19] Schulze H, Aptroot A, Grote-Metke A, Balleisen L. Aspergillus fumigatus and Chaetomium homopilatum in a leukemic patient. Pathogenic significance of Chaetomium species. Mycoses 1997;40 Suppl1:104–9. [20] Abbott SP, Sigler L, McAleer R, McGough DA, Rinaldi MG, Mizell G. Fatal cerebral mycoses caused by the ascomycete Chaetomium strumarium. J Clin Microbiol 1995;33:2692–8. [21] Barron MA, Sutton DA, Veve R, Guarro J, Rinaldi M, Thompson E, et al. Invasive mycotic infections caused by Chaetomium perlucidum, a new agent of cerebral phaeohyphomycosis. J Clin Microbiol 2003;41:5302–7. [22] Pauzner R, Goldschmied-Reouven A, Hay I, Vered Z, Ziskind Z, Hassin N, et al. Phaeohyphomycosis following cardiac surgery: case report and review of serious infection due to Bipolaris and Exserohilum species. Clin Infect Dis 1997;25:921–3. [23] Vallabhaneni S, Purfield AE, Benedict K, Luvsansharav U, Lockhart SR, Pham CD, et al. Cardiothoracic surgical site phaeohyphomycosis caused by Bipolaris mould, multiple US states, 2008-2013: a clinical description. Med Mycol 2016;54:318–21. [24] Guarro J, Soler L, Rinaldi MG. Pathogenicity and antifungal susceptibility of Chaetomium species. Eur J Clin Microbiol Infect Dis 1995;14:613–8. [25] Chowdhary A, Meis JF, Guarro J, de Hoog GS, Kathuria S, Arendrup MC, et al. ESCMID and ECMM joint clinical guidelines for the diagnosis and management of systemic phaeohyphomycosis: diseases caused by black fungi. Clin Microbiol Infect Suppl 2014;3:47–75.
Please cite this article in press as: Jeragh A, et al. Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in KuwaitSubcutaneous Amesia atrobrunnea infection–>. Journal De Mycologie Me´dicale (2018), doi:10.1016/j.mycmed.2018.10.004
MYCMED-843; No. of Pages 5 Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
Case report
Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in Kuwait A. Jeragh a, S. Ahmad c, Z. Khan c,d,*, R.Y. Tarazi b, S. Ajmi a, L. Joseph c, S. Varghese c, S. Vayalil d a
Departments of Microbiology, Al Adan Hospital, Kuwait Cardiac surgery, Al Adan Hospital, Kuwait c Department of Microbiology, Kuwait d Mycology Reference Laboratory, Faculty of Medicine, Kuwait University, Kuwait b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2018 Received in revised form 10 September 2018 Accepted 29 October 2018 Available online xxx
The recently described genus Amesia encompasses four species but only Amesia atrobrunnea (= Chaetomium atrobrunneum) is known to be pathogenic to humans. Here, we describe a case of subcutaneous phaeohyphomycosis in Kuwait in an apparently immunocompetent patient diagnosed by direct microscopy of the infected tissue and culture. The identity of A. atrobrunnea was established by typical morphological characteristics and by sequencing of internally transcribed spacer (ITS) region and D1/D2 domains of rDNA. To the best of our knowledge, this is the first report documenting etiologic role of this species in causing a locally invasive subcutaneous infection.
Keywords: Amesia atrobrunnea Subcutaneous phaeohyphomycosis Surgical site infections Molecular identification
1. Introduction The genus Chaetomium comprises over 400 species which are ubiquitous in nature [1]. They can grow on a variety of substrates, particularly in damp or water-damaged buildings, and thus can be an important source of inhalant allergens contributing to asthma, rhinitis, sinusitis and other respiratory disorders in susceptible/genetically predisposed individuals [1–3]. Some Chaetomium species are also capable of causing invasive diseases [4–7]. Recently, the taxonomy of the genus has been revised on the basis of morphological characteristics and phylogenetic analyses of six loci [1]. Currently, the family Chaetomiaceae encompasses 10 genera which include five newly established genera and seven new species. Consequently, Chaetomium atrobrunneum has been included in the newly erected genus Amesia (X. Wei Wang, Samson & Crous, gen. nov, MycoBank MB818829) with type species Amesia atrobrunnea (Ames) X. Wei Wang & Samson (= Ch. atrobrunneum). The genus Amesia now includes three other species, namely, Amesia cymbiformis, Amesia nigricolor and Amesia gelasinospora. Within this genus, A. atrobrunnea is the only species that has been recognized as a human pathogen causing deep-seated infections [8–14]. Here, we describe the first case of subcutaneous phaeohyphomycosis caused by A. atrobrunnea in Kuwait and diagnosed by detection of the fungus by direct microscopic examination of the infected tissue, culture of the organism from the tissue sample and its
* Corresponding author. Department of Microbiology, Faculty of Medicine, Kuwait University, P. O. Box 24923, Safat 1311, Kuwait. E-mail address: [email protected] (Z. Khan).
C
2018 Elsevier Masson SAS. All rights reserved.
phenotypic features and DNA sequence comparisons of the internally transcribed spacer (ITS) region and D1/D2 domains of rDNA with fungal sequences in the databanks.
2. Observations A 45-year-old male was admitted to the Casualty in April, 2017 with sudden onset of chest pain and diaphoresis. On admission, his blood pressure was 220/110 mmHg and ECG revealed myocardial infarction with first degree AV block. Troponin I was > 100 ng/mL. Emergent CT scan and ECHO showed intimal flap in the ascending aorta, moderate aortic regurgitation, normal LV contractility and hypokinetic right ventricular function with no tricuspid regurgitation. There was mild pericardial effusion which required immediate placement of epidural catheter to monitor CSF pressure. Median sternotomy was performed and pericardium was opened. The right ventricle was edematous and had a large hematoma in its free wall with very poor contractile function. The left ventricle was hyper contractile with normal function. Axillary arterial and right atrial cannulation were performed. The aorta was cross clamped. A transverse aortotomy was performed opening the false and then the true lumen. Cardioplegia was given antegradely and retrogradely. The ostium of the right coronary artery (RCA) was severed completely due to the dissection. The distal RCA was grafted with saphenous vein graft. Resuspension of the aortic valve was done. Under a 40 min period of circulatory arrest a Frozen Elephant Trunk operation was performed. At the completion of the operation the patient could
10.1016/j.mycmed.2018.10.004 C 2018 Elsevier Masson SAS. All rights reserved. 1156-5233/$ – see front matter
Please cite this article in press as: Jeragh A, et al. Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in KuwaitSubcutaneous Amesia atrobrunnea infection–>. Journal De Mycologie Me´dicale (2018), doi:10.1016/j.mycmed.2018.10.004
G Model
MYCMED-843; No. of Pages 5 2
A. Jeragh et al. / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
Fig. 1. Potassium hydroxide (KOH)-calcofluor mount of digested tissue biopsy specimen showing septate hyphae of A. atrobrunnea.
Fig. 2. Six-day old colony of A. atrobrunnea on oatmeal agar showing white wooly texture and a large number of dark-colored ascomata.
not be weaned off the cardiopulmonary bypass due to severe right ventricular dysfunction because of the massive pre-operative inferior wall myocardial infarction. An extracorporeal membrane oxygenator (ECMO) was used to support the right heart for two days. With adequate oxygenation it was converted to temporary right ventricular assist devices (RVAD) with in-flow to the right common femoral vein and outflow to a 10 mm Hemasheild graft sutured to the main pulmonary artery. After one week without any recovery of the right ventricle, the temporary RVAD was removed and a permanent RVAD using a heart ware pump was inserted with the inflow the right atrium and the outflow the main pulmonary artery. The chest was closed. The patient was intubated and on mechanical ventilation during the course of the special intensive care unit (SICU) stay. His liver enzymes and lactate dehydrogenase were elevated and he also developed acuter kidney injury requiring continuous peritoneal/hemodialysis. Three days later, he was shifted to Intensive Care Unit and was put on ECMO and mechanical ventilation to support his lung functions. During the course of dialysis, the patient developed leukocytosis (range 15.2 109/L to 26.2 109/L), 15 days after the surgical procedure. Although no bacterial pathogens were isolated in blood cultures, he received short courses of broad-spectrum antibiotics as a preemptive
measure which included meropenem, vancomycin, tazocin, and acyclovir during his stay in ICU for 4 weeks. He also received a 2week course of caspofungin. There was no serological evidence of HBV, HCV, HIV or Treponema pallidum infection. As his condition continued to deteriorate, he was shifted back to coronary care unit on Day 32 in a comatose condition and declared brain dead on Day 36 of admission. The tissue biopsy collected from the infected site near sternum, where RVAD device was implanted was sent to Mycology Reference Laboratory for routine culture and diagnosis. The direct microscopic examination of the KOH-calcofluor digested tissue showed pigmented septate hyphae (Fig. 1) and culture yielded a filamentous fungus on blood agar and Sabouraud dextrose agar. On Sabouraud dextrosae agar, the isolate grew very well at 30 ?C as well as 37 ?C attaining a diameter of 2.5 cm and 3.5 cm in 4 days, respectively. The growth was substantially reduced at 45 ?C and showed only minimal growth at 47 ?C. Growth was much faster on potato dextrose agar reaching a diameter of 3.5 cm and 5 cm in 4 days, respectively. The colony first appeared white in color with wooly texture and then became grayish with appearance of dark-colored ascomata on the surface. The reverse was dark yellow or olivaceous green. The growth on oatmeal agar produced a large number of ascomata (Fig. 2). The ascomata were subspherical, dark brown in
Please cite this article in press as: Jeragh A, et al. Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in KuwaitSubcutaneous Amesia atrobrunnea infection–>. Journal De Mycologie Me´dicale (2018), doi:10.1016/j.mycmed.2018.10.004
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Fig. 3. A) Lactophenol-cotton blue mount prepared from six-day old culture showing a mature ascoma of A. atrobrunnea with dark brown ascomal hairs (setae) and clavate shaped asci with ascospores; B) large number of fusiform-shaped ascospores with apical germpores.
color, ranging between 50 to 130 mm and produced clavate shaped asci that contained 8 brown-colored, smooth-walled ascospores with subapical germ pores (Fig. 3A, B). The ascospores were fusiform in shape and 8.5–10 4.5–6 mm in size. The ascomal hairs (setae) were dark brown, long and straight. The DNA from the patient’s isolate (Kw77-5-17) was prepared as described previously and used as the template for PCR amplifications [15]. The ITS region (ITS1, 5.8S rRNA, and ITS2) was amplified with ITS1 and ITS4 primers while the divergent D1/ D2 domains of rDNA were amplified with the NL-1 and NL-4 primers, the amplicons were purified and both strands of the two amplicons were sequenced by using internal (ITS1FS, ITS2, ITS3 and ITS4RS for ITS region and NL-1FS, NL-2, NL-3 and NL-4RS for the D1/D2 domains) primers, as described previously [16,17]. GenBank basic local alignment search tool (BLAST) searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi?) were performed for species identification. Our isolate was identified as A. atrobrunnea as both, the ITS region (GenBank accession no. LT969523) of our isolate (Kw77-5-17) and D1/D2 domain (GenBank accession No. LT969522) of rDNA sequences showed 100% identity with the corresponding ITS region (GenBank accession No. JX280771) and D1/D2 domain (GenBank accession No. JX280666) sequences from A. atrobrunnea type strain
(CBS379.66). The ITS region sequence also showed 100% identity with several other (CGMCC 3.3594, MRDS12, NBRC 6542, C64 and MRDS8) strains of A. atrobrunnea. For determining antifungal susceptibility, the inoculum was prepared as described previously [18]. The growth was harvested in normal saline and vigorously vortexed for 15s to disperse the clumps mainly consisting of ascospores. These suspensions were adjusted with a hemacytometer to 107 ascospores/mL and spread all over the surface of RPMI medium. The Etest strips (bioMe´rieux, Marcy l’E´toile, France) of itraconazole, voriconazole, posaconazole, amphotericin B, anidulafungin, and caspofungin were implanted on the surface of the medium and incubated for 48 h at 35 ?C. Minimum inhibitory concentrations for itraconazole, voriconazole, posaconazole, amphotericin B, anidulafungin and caspofungin were read as follows: 2 mg/mL, 0.094 mg/mL, 0.38 mg/mL, 1 mg/ mL, 1.5 mg/mL, and 6 mg/mL, respectively.
3. Discussion The present report is noteworthy as it describes the first case of subcutaneous infection caused by A. atrobrunnea in an apparently immunocometent patient. No other bacterial pathogens were
Please cite this article in press as: Jeragh A, et al. Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in KuwaitSubcutaneous Amesia atrobrunnea infection–>. Journal De Mycologie Me´dicale (2018), doi:10.1016/j.mycmed.2018.10.004
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Table 1 Summary of published cases of A. atrobrunnea, (= C. atrobrunneum) infections. Case Reference No
Associated underlying conditions
Type of infection
Source of isolation Identification method
Antifungal treatment regimen
Outcome
Renal transplant Bone marrow transplant recipient
Brain abscess Cerebral abscess
Brain biopsy Brain biopsy
Not known
Died
Amphotericin B, itraconazole
Died
Cerebral abscess
Brain biopsy
Not available
Died
Cellulitis (left eye) and conjunctivitis
Eye discharge
Itraconazole, fluconazole
Resolved
Voriconazole
Improved
Topical natamycin, ketoconazole
Resolved
Fluconazole, amphotericin B, caspofungin Caspofungin
Died
Anandi et al. [8] Guppy et al. [9]
India
1989
32/M
USA
1998
31/M
3
Thomas et al. [10]
USA
1999
4
Zhang et al. [11]
China
2010
Not available Bone marrow transplant recipient 1.5/M Nil
5
Tabbara et al. [12]
USA
2010
11/M
Hodgkin lymphoma
Retinitis and Vitreous aspirate subhyaloid abscess
6
Balne et al. [13]
India
2012
44/M
Nil
Keratitis
7
Wang et al. [14]
China
2016
89/M
Bed-ridden for long Pneumonia term
Tracheal aspirates
8
Present case
Kuwait
2017
45/M
None
Tissue biopsy
1 2
a
Country Year Age/sex of report
Corneal scrapings
Subcutaneous infection
Morphological characteristicsa Morphological methods and electron microscopy No (conventional morphological methods Morphological characteristic and (ITS & D1/D2 sequencing No (conventional morphological methods) Morphological characteristic, ITS sequencing Morphological characteristic, ITS & D1/D2 sequencing Morphological characteristic, ITS & D1/D2 sequencing
Died
This isolate was initially identified as C. globosum but was subsequently re-identified as C. atrobrunneum [20].
isolated in culture. The diagnosis was unequivocally established by demonstrating septate hyphae of the fungus in direct microscopic examination of the infected tissue. Species-specific identity of the isolate was based on typical morphological characteristics and by obtaining 100% identity of the ITS region and D1/D2 domain sequences with the corresponding sequences from reference strain (CBS379.66) of A. atrobrunnea available in GenBank [14]. This report reinforces that dematiceous fungi have the potential to cause localized infections at surgical sites. So far, human infections due to A. atrobrunnea have been reported in seven patients [8–14], most of them were immunocompromised individuals (Table 1). Three of the patients had central nervous system involvement and died [8–10]. Our patient had no history of intravenous drug abuse or evidence of cerebral involvement. Although our patient expired due to brain death, it is unlikely that dissemination from primary subcutaneous lesion was the cause of mortality as repeated blood cultures were negative for the fungus. Recently, A. atrobrunnea (C. atrobrunneum) pneumonia has recently been implicated as a cause of pneumonia in an immunocompromised patient along with Aspergillus fumigatus [14]. Despite combination therapy of amphotericin B and caspofungin, the patient died. It is not clear if A. atrobrunnea was the primary cause of pneumonia or both had any symbiotic association for their growth in tracheal aspirates [14]. Isolation of A. fumigatus and Chaetomium homopilatum from tracheal secretion of a patient with leukemia, has also been reported previously, but clinical significance of the latter species remained uncertain [19]. Like other Chaetomium spp., such as C. strumarium [20], A. atrobrunnea (C. atrobrunneum) can be regarded as neurotropic species causing serious and life-threatening infections [8,9,21]. Little is known about the pathogenic attributes of Amesia species. Since Amesia/Chaetomium species can survive in high environmental temperatures in their natural habitat [7], they have inherent ability to resist hosts’ body temperature and can grow in vivo. Further studies are warranted to elucidate the role of
various virulence factors and bioactive metabolites produced by these species, which may have a role in the pathogenesis and neurotropism [1,3]. Surgical site infections due to other dematiaceous fungi, such as Bipolaris species and Exserohilum species, have also been reported in patients following cardiothoracic surgical interventions [22,23]. There is paucity of information on antifungal susceptibility of Chaetomium species [24,25]. Based on the published reports [14,15,21], voriconazole exhibited highest in vitro activity, followed by amphotericin B and itraconazole. Echinocandins showed minimal activity against our A. atrobrunnea isolate. They were also found to be less active against other members of the genus Chaetomium [25]. So far, there is no recommended treatment regimen for subcutaneous infections caused by A. atrobrunnea or other closely related species. Conclusion This report documents etiologic role of A. atrobrunnea in causing subcutaneous phaeohyphomycosis and highlights importance of molecular methods in establishing accurate identification. To the best of our knowledge, this is the first report documenting etiologic role of this species in causing a locally invasive subcutaneous infection. Contribution of authors AJ, RYT and SAA provided clinical case details, SA and ZK supervised laboratory work and wrote the manuscript, LJ, SV and SV provided technical support. Disclosure of interest The authors declare that they have no competing interest.
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Please cite this article in press as: Jeragh A, et al. Subcutaneous phaeohyphomycosis caused by Amesia atrobrunnea in KuwaitSubcutaneous Amesia atrobrunnea infection–>. Journal De Mycologie Me´dicale (2018), doi:10.1016/j.mycmed.2018.10.004