Mycetoma caused by Microascus gracilis: a novel agent of human eumycetoma in Sudan

Mycetoma caused by Microascus gracilis: a novel agent of human eumycetoma in SudanOR IG IN AL
AR TI CL E
Trans R Soc Trop Med Hyg 2021; 115: 426–430 doi:10.1093/trstmh/trab010 Advance Access publication 30 January 2021
Mycetoma caused by Microascus gracilis: a novel agent of human eumycetoma in Sudan
Najwa A. Mhmouda,b, Emmanuel Edwar Siddig a,b,c, Bertrand Nyuykongec, Sahar Mubarak Bakhieta,d, Wendy W. J. van de Sandec, and Ahmed Hassan Fahala,∗
aMycetoma Research Centre, University of Khartoum, PO Box 102, Khartoum, Sudan; bFaculty of Medical Laboratory Sciences, University of Khartoum, Khartoum, Sudan; cErasmus MC, University Medical Centre Rotterdam, Department of Medical Microbiology and Infectious Diseases, Rotterdam, The Netherlands; dInstitute of Endemic Diseases, University of Khartoum, Khartoum, Sudan
∗Corresponding author: Tel: 00249912346703; E-mail [email protected], [email protected]
Received 23 October 2020; revised 06 December 2020; editorial decision 23 December 2020; accepted 8 January 2021
Species of the genus Microascus are uncommon agents of human diseases despite their ubiquitous presence in the environment. In this communication, the first case ofwhite grain eumycetoma caused by the fungusMicroas- cus gracilis is reported. The patient was initially misdiagnosed as having actinomycetoma based on the grains morphological and cytological features and was treated with antimicrobial therapy with no clinical improve- ment. She underwent wide local surgical excision to improve the response to medical treatment and further grain cultural, molecular and taxonomy techniques were conducted and the diagnosis of mycetoma due to M. gracilis was established. The antifungal susceptibilities of this isolate to nine drugs were tested in vitro and they showed poor activity. Combination therapy with surgery and itraconazole led to complete recovery. A medical literature search revealed no previous report onM. gracilis as a causative agent of eumycetoma and hencewe are reporting this new causative agent of human eumycetoma. Also, the difficulty in themanagement of this patient emphasizes the need for accurate and appropriate diagnostic tests for the identification ofmycetoma-causative organisms and thus proper management.
Keywords: antifungal susceptibility, eumycetoma, filamentous fungi, Microascus gracilis, mycetoma
Introduction The patient was a 50-year-old housewife from western Sudan. She presented to the Mycetoma Clinic at the Mycetoma Research Centre (MRC) of the University of Khartoum, Khartoum, Sudan in 2017 with a recurrent painless left foot swelling. Her condi- tion started 2 y prior to presentation with a small swelling of the left foot that had gradual onset and progress and was painless. Multiple sinuses and seropurulent discharge containing white grains then developed. The patient underwent two surgical exci- sions under local anaesthesia elsewhere and no treatment was given. At her presentation to the MRC in 2017 she gave a history of
local trauma and a family history of mycetoma. Her gynaecolog- ical, social, geographic and drug history was not contributory to her problem. On examination, she looked well. All vital measurements
and systemic examination were also normal. Local examination revealed a painless firmmass 8×9 cm in size involving the dorsal and plantar parts of the left foot. There were multiple active and healed sinuses and discharge (Figure 1A).
Her renal and hepatic function tests as well as her full blood count were normal. The left foot X-ray revealed evi- dence of soft tissue swelling and periosteal reaction (Figure 1B). Fine-needle aspiration for cytology was performed and showed an intense inflammatory infiltrate with grains suggestive of actinomycetoma. The patient was started on amoxicillin/clavulanate potassium
in a dose of 1 g twice a day, co-trimoxazole 1920 mg twice a day and folic acid 0.05 mg/d. She was followed up regularly at the outpatient clinic, however, there was no clinical response to this treatment. She underwent wide local surgical excision under spinal
anaesthesia to improve the response to medical treatment. Numerous white grains were collected that were hard in consis- tency and of different sizes. Cultures of the grains grew Microascus gracilis. Susceptibility
testing of the fungus and minimum inhibitory concentrations (MICs) were determined using the Sensititre YeastOne Alamar Blue assay (Thermo Fisher, Waltham, MA, USA) as previously described.1 The result indicated that the fungus had a high MIC to nine antifungals tested, including amphotericin B (>4 µg/ml),
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Figure 1. (A) The foot lesion with multiple sinuses and white grains. (B) The left foot X-ray with soft tissue swelling, periosteal reaction and bone involvement.
flucytosine (>64 µg/ml), terbinafine (>16 µg/ml), caspofungin (>16 µg/ml) and azole compounds. To date, no interpretive breakpoints for in vitro antifungal susceptibility are available to guide mycetoma treatment.2 Therefore the patient had received the same treatment given to eumycetoma patients caused by Madurella mycetomatis, namely 200 mg itraconazole twice a day combined with surgery. She also received folic acid 5 mg daily according to the guidelines of the MRC and received daily wound dressing. The patient’s overall condition improved over 30 months while she was on itraconazole and regularly followed up in the outpatient clinic.
Fungal isolation The grains were washed twice in physiological saline and inoc- ulated onto yeast extract agar (YEA; Oxoid, Basingstoke, UK), Sabouraud dextrose agar (SDA; Oxoid), malt extract agar (MEA; Oxoid) and potato dextrose agar (PDA; Oxoid) at 37°C and cul- tured for 4 weeks. The isolate was identified morphologically as M. gracilis. It
grew rapidly at 37°C on SDA and formed white colonies that became powdery and/or granular as they matured. Under such conditions the fungus normally grows rapidly, expanding as much as 4.5–5.5 cm within 10 d. From the surface, the colour was initially white and later became dark green or smoky brown (Figure 2A). From the reverse, it was pale with brownish-black zones. After 2 weeks of incubation, small black structures were seen growing on the surface of the SDA. Microscopic exami- nation of these structures revealed perithecia (100–300 µm in diameter) (Figure 2B) having lunate ascospores measuring 4.5–6.5 by 2–4 μm (Figure 2C). The hyphae of M. gracilis are hyaline (transparent) and septate (Figure 2D). The anamorph has conidia (Figure 2D) that are flattened at the base and tapered at the apex.
DNA extraction, amplification and sequencing DNA from the fungal isolates was extracted as described else- where3 using the ZR Fungal/Bacterial DNA MiniPrep Kit (Zymo Research, Irvine, CA, USA). Amplification and sequencing of the
recombinant DNA internal transcribed spacer (ITS) region was performed as described elsewhere4 using the BigDye Termina- tor v3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA) according to themanufacturer’s instructions. The sequenceswere analysed using Chromas software (Technely- sium, South Brisbane, QLD, Australia). Following ITS amplifica- tion and sequencing, upon blast the ITS sequence of isolates was 100% identical to M. gracilis. The surgical biopsy was fixed in 10% formal saline. After
processing the biopsy and preparation of the histopathological block, slides were cut using a rotary microtome and stained with haematoxylin and eosin (H&E) and by Fontana-Masson for melanin. Within the tissue, multiple pale-stained grains were seen. The grains themselves looked pale in H&E, with fungal hyphae scattered at the periphery that were thickened cell walls. They were surrounded by inflammatory cells such as histiocytes, neutrophils and lymphocytes, in linewith type I and II tissue reac- tions5 (Figure 3). Fontana-Masson-stained section was negative.
Discussion Mycetoma is one of themore prevalent implantation fungal infec- tions. It is a progressive and destructive chronic granulomatous subcutaneous inflammatory disease6–9 caused by certain true fungi or bacteria and classified as eumycetoma and actinomyce- toma, respectively.10,11 The infection most probably occurs fol- lowing traumatic inoculation of the aetiological causative agent into the subcutaneous tissue. Once implanted, the organisms adapt to the host tissue environment and protect themselves by the formation of grains that are aggregates of the organism and a matrix component or cement.7 The disease is endemic in many tropical and subtropical
regions, where it is considered a major health problem.6,7 In Sudan, eumycetoma is usually caused by the fungus M. myce- tomatis, which forms black grains in human tissue.6–8 It is characterised by local swelling and draining sinuses that release grains of different colours, consistencies and sizes depending on the causative agent.8 Early and proper identification of the causative agents is the major factor for successful management of mycetoma patients and better prevention of complications and disability.12–14
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Figure 2. Photograph showing (A) Microascus gracilis colonies after 2 weeks incubation in SDA, (B) cleistothecia, (C) asci and ascspores and (D) conid- iophores and conidia.
Figure 3. Photomicrograph showing the white grains with pale colour surrounded and infiltrated by neutrophils and lymphocytes in line with type I and II tissue reactions (H&E, magnification 40×).
The Microascus species (order Microascales, family Microas- caceae) can be found worldwide and have been recovered from a wide geographical range. Currently there are close to 40 recognised species of Microascus.15 Although their isolation from
clinical specimens has been reported, their role as pathogens of human disease has been unclear. Recently, however, infection with this organism has been
recorded in immunocompromised as well as immunosuppressed patients.9–13 Theymay also cause foot infections and onychomy- coses6,7 and keratitis following eye trauma.8 The fungi are also associated with deep tissue infections, mainly in immunocom- promised and occasionally in immunocompetent patients, caus- ing invasive diseases.9–23 The genus has been reported to be resistant in vitro to almost
all the currently used drugs, including amphotericin B, flucyto- sine, terbinafine, caspofungin and azole compounds.24 Microascus species are ubiquitous environmental saprotrophic
Ascomycota fungi that are usually isolated from soil and plant material, but can also occur in indoor environments.20 The most common species in this genus are Microascus niger, Microascus cinereus,Microascus cirrosus,Microascusmanginii andMicroascus trigonosporus.7,15,16 M. graciliswas first isolated in 1962 from food in Japan by Ina-
gaki22 In the past it was known as Scopulariopsis gracilis, which has been recategorized and designated as M. gracilis.23 It has never been reported in human infections or isolated
from clinical specimens. Here we present the first case of white grainmycetoma caused by this fungus (Table 1). In this report we showed the identification of this fungus required a combination
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Table 1. White grain eumycetoma causative agents
Species Order Family Grain Reference
Phaeoacremonium krajdenii Diaporthales Togniniaceae White 26
Phaeoacremonium parasiticum Diaporthales Togniniaceae White 27
Aspergillus flavus Eurotiales Aspergillaceae White 28
Aspergillus hollandicus Eurotiales Aspergillaceae White 29
Aspergillus nidulans Eurotiales Aspergillaceae White 30
Acremonium recifei Hypocreales Nectriaceae White 31
Cylindrocarpon cyanescens Hypocreales Nectriaceae White 32
Cylindrocarpon destructans Hypocreales Nectriaceae White 33
Fusarium falciforme Hypocreales Nectriaceae White 34
Fusarium solani Hypocreales Nectriaceae White 34
Fusarium verticillioides Hypocreales Nectriaceae White 34
Acremonium kiliense Hypocreales Incertae sedis White 31
Acremonium potronii Hypocreales Incertae sedis White 31
Phialemonium obovatum Hypocreales Cephalothecaceae White 35
Scedosporium boydii Microascales Microascaceae White 36
Microsporum canis Onygenales Arthrodermataceae White 37
Trichophyton Onygenales Arthrodermataceae White 38
Neotestudina rosatii Pleosporales Testudinaceae White/black 39,40
Microascus gracilis Microascales Microascaceae White This study
of techniques, and was not an easy task, as it was first misiden- tified on histologically as an actinomycete. DNA sequencing of the reported fungus proved necessary
for accurate identification and differentiation from other pale grain eumycetoma and actinomycetoma organisms, as the clin- ical appearance of these pathogens is similar. Furthermore, their appearance in histopathological sectionsmay be similar and con- siderable expertise is needed to identify them to avoid inappro- priate treatment. To date, no interpretive breakpoints for in vitro antifungal sus-
ceptibility are available to guidemycetoma treatment.22,25 There- fore the patient received itraconazole for 30 months, but new, more potent antifungals are needed. In conclusion, we believe that the reported isolate is a true
causative organism, as the lesion had persisted for years and was cured by surgical intervention in combination with medical treatment.
Authors’ contributions: NAM, EES, BN, SMB, WVS and AHF conceived and designed the study, performed the study, analysed the data and wrote and revised themanuscript. All authors read and approved the final manuscript.
Acknowledgements None.
Funding: None.
Competing interests: None declared.
Ethical approval: The study was approved by the Mycetoma Research Centre Institutional Review Board. The patient gave written informed consent.
Data availability: Data available upon request.
References 1 Nyuykonge B, Klaassen CHW, Zandijk WHA, et al. Diagnostic implica- tions of mycetoma derived from Madurella pseudomycetomatis iso- lates from Mexico. J Eur Acad Dermatol Venereol. 2020;34(8):1828– 34.
2 Ahmed SA, de Hoog GS, Stevens DA, et al. In vitro anti- fungal susceptibility of coelomycete agents of black grain eumycetoma to eight antifungals. Med Mycol. 2015;53(3):295– 301.
3 Lim W, Eadie K, Horst-Kreft D, et al. VNTR confirms the hetero- geneity of Madurella mycetomatis and is a promising typing tool for this mycetoma causing agent. Med Mycol. 2019;57(4):434– 40.
4 Ahmed AO, Mukhtar MM, Kools-Sijmons M, et al. Development of a species-specific PCR-restriction fragment length polymorphismanaly- sis procedure for identification ofMadurella mycetomatis. J Clin Micro- biol. 1999;37(10):3175–8.
5 Fahal AH, el Toum EA, el Hassan AM, et al. The host tissue reac- tion to Madurella mycetomatis: new classification. J Med Vet Mycol. 1995;33(1):15–7.
6 Siddig EE, Mhmoud NA, Bakhiet SM, et al. The accuracy of histopathological and cytopathological techniques in the identifi- cation of the mycetoma causative agents. PLoS Negl Trop Dis. 2019;13(8):e0007056.
429
niversiteit R otterdam
N. A. Mhmoud et al.
7 Siddig EE, Mohammed Edris AM, Bakhiet SM, et al. Interleukin-17 and matrix metalloprotease-9 expression in the mycetoma granuloma. PLoS Negl Trop Dis. 2019;13(7):e0007351.
8 Fahal AH. Mycetoma: a thorn in the flesh. Trans R Soc Trop Med Hyg. 2004;98(1):3–11.
9 Fahal AH, Suliman SH, Hay R. Mycetoma: the spectrum of clinical pre- sentation. Trop Med Infect Dis. 2018;3(3):97.
10 Sandoval-Denis M, Sutton DA, Fothergill AW, et al. Scopulariopsis, a poorly known opportunistic fungus: spectrum of species in clinical samples and in vitro responses to antifungal drugs. J Clin Microbiol 2013;51(12):3937–43.
11 Agarwal GP, Singh SM. Microascus cinereus infection of human nail. Indian J Med Sci. 1980;34(11):263–5.
12 de Hoog GS, Guarro J, Gené J, et al. Atlas of clinical fungi, 2nd ed. Utrecht, The Netherlands: Centraalbureau voor Schimmelcultures; 2001.
13 Ragge NK, Hart JC, Easty DL, et al. A case of fungal keratitis caused by Scopulariopsis brevicaulis: treatment with antifungal agents and pen- etrating keratoplasty. Br J Ophthalmol 1990;74(9):561–2.
14 Baddley JW, Moser SA, Sutton DA, et al. Microascus cinereus (anamorph Scopulariopsis) brain abscess in a bonemarrow transplant recipient. J Clin Microbiol. 2000;38(1):395–7.
15 Miossec C, Morio F, Lepoivre T, et al. Fatal invasive infection with fungemia due toMicroascus cirrosus after heart and lung transplanta- tion in a patientwith cystic fibrosis. J ClinMicrobiol 2011;49(7):2743–7.
16 Krisher KK, Holdridge NB, Mustafa M, et al. Disseminated Microascus cirrosus infection in pediatric bone marrow transplant recipient. J Clin Microbiol. 1995;33(3):735–7.
17 Célard M, Dannaoui E, Piens MA, et al. Early Microascus cinereus endo- carditis of a prosthetic valve implanted after Staphylococcus aureus endocarditis of the native valve. Clin Infect Dis. 1999;29(3):691–2.
18 Petit A, Levine E, Epaud R, et al. Scopulariopsis brevicaulis abscess in a child treated for myeloblastic leukaemia. Lancet Infect Dis 2011;11(5):416.
19 Baxter M, Murray IG, Taylor JJ. A case of mycetoma with serological diagnosis of Allescheria boydii. Sabouraudia. 1966;5(2):138–40.
20 Mohammedi I, Piens MA, Audigier-Valette C, et al. Fatal Microas- cus trigonosporus (anamorph Scopulariopsis) pneumonia in a bone marrow transplant recipient. Eur J Clin Microbiol Infect Dis. 2004;23(3):215–7.
21 Iwen P, Schutte SD, Florescu DF, et al. Invasive Scopulariopsis brevi- caulis infection in an immunocompromised patient and review of prior cases caused by Scopulariopsis and Microascus species. Med Mycol 2012;50(6):561–9.
22 Inagaki N. On some fungi isolated from foods (I). Trans Mycol Soc Japan 1962;4(1):1–5.
23 Sandoval-Denis M, Gené J, Sutton DA, et al. Redefining Microascus, Scopulariopsis and allied genera. Persoonia. 2016;36:1–36.
24 van de SandeWW, Fahal A, Verbrugh H, et al. Polymorphisms in genes involved in innate immunity predispose toward mycetoma suscepti- bility. J Immunol. 2007;179(5):3065–74.
25 Cuenca-Estrella M, Gomez-Lopez A, Mellado E, et al. Scopulari- opsis brevicaulis, a fungal pathogen resistant to broad-spectrum antifungal agents. Antimicrob Agents Chemother. 2003;47(7):2339– 41.
26 Gilgado F, Serena C, Cano J, et al. Antifungal susceptibilities of the species of the Pseudallescheria boydii complex. Antimicrob Agents Chemother. 2006;50(12):4211–3.
27 Hemashettar BM, Siddaramappa B, Munjunathaswamy BS, et al. Phaeoacremonium krajdenii, a cause of white grain eumycetoma. J Clin Microbiol. 2006;44(12):4619–22.
28 Colombier M-A, Alanio A, Denis B, et al. Dual invasive infection with Phaeoacremonium parasiticum and Paraconiothyrium cyclothyrioides in a renal transplant recipient: case report and comprehensive review of the literature of Phaeoacremonium phaeohyphomycosis. J Clin Microbiol. 2015;53(7):2084–94.
29 Ahmed SA, Abbas MA, Jouvion G, et al. Seventeen years of sub- cutaneous infection by Aspergillus flavus; eumycetoma con- firmed by immunohistochemistry. Mycoses. 2015;58(12):728– 34.
30 Mhmoud NA, Ahmed SA, Fahal AH, et al. Pleurostomophora ochracea, a novel agent of human eumycetoma with yellow grains. J Clin Micro- biol. 2012;50(9):2987–94.
31 Prasanna S, Grover N, Bhatt P, et al. A case of Aspergillus nidu- lans causing white granule mycetoma. Med J Armed Forces India. 2016;72(1):88–90.
32 Koshi G, Padhye AA, Ajello L, et al. Acremonium recifei as an agent of mycetoma in India. Am J Trop Med Hyg. 1979;28(4):692– 6.
33 Hemashettar BM, Siddaramappa B, Padhye AA, et al. White grain mycetoma caused by a Cylindrocarpon sp. in India. J Clin Microbiol. 2000;38(11):4288–91.
34 Zoutman DE, Sigler L. Mycetoma of the foot caused by Cylindrocarpon destructans. J Clin Microbiol. 1991;29(9):1855–9.
35 Summerbell RC, Schroers H-J. Analysis of phylogenetic relationship of Cylindrocarpon lichenicola and Acremonium falciforme to the Fusar- ium solani species complex and a review of similarities in the spec- trumof opportunistic infections caused by these fungi. J Clin Microbiol. 2002;40(8):2866–75.
36 Lomax LG, Cole JR, Padhye AA, et al. Osteolytic phaeohyphomycosis in a German shepherd dog caused by Phialemonium obovatum. J Clin Microbiol. 1986;23(5):987–91.
37 Cortez KJ, Roilides E, Quiroz-Telles F, et al. Infections caused by Sce- dosporium spp. Clin Microbiol Rev. 2008;21(1):157–97.
38 Kramer SC, Ryan M, Bourbeau P, et al. Fontana-positive grains in mycetoma caused by Microsporum canis. Pediatr Dermatol 2006;23(5):473–5.
39 Akiba H, Motoki Y, Satoh M, et al. Recalcitrant trichophytic granuloma associated with NK-cell deficiency in a SLE patient treated with corti- costeroid. Eur J Dermatol 2001;11(1):56–58.
40 van de Sande WW. Global burden of human mycetoma: a systematic review and meta-analysis. PLoS Negl Trop Dis. 2013;7(11):e2550.
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