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Alternaria infectoria phaeohyphomycosis in a renal transplant patient Article in Medical Mycology · July 2006 DOI: 10.1080/13693780500412004 · Source: PubMed
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Medical Mycology 2006, 1 /4, PrEview article
Case Report
Alternaria infectoria phaeohyphomycosis in a renal transplant patient ERIC NULENS*$, EMMANUEL DE LAERE%, HEIDI VANDEVELDE§, LUUK B. HILBRANDS’, ANTONIUS J. M. M. RIJS*$, WILLEM J. G. MELCHERS*$ & PAUL E. VERWEIJ*$ *Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, $Nijmegen University Centre for Infectious Diseases, Nijmegen, The Netherlands, %Laboratorium Medische Microbiologie, Heilig Hart Ziekenhuis, Roeselare, Belgium, §Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, and ’Department of Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
A male renal transplant patient developed a tumor on the dorsum of his right hand. After excision, histological examination of the tumor showed hyphal structures, but growth developed very slowly. Therapy consisted of surgery alone. A definitive identification of Alternaria infectoria was only possible with molecular techniques.
Introduction
Case report
Phaeohyphomycosis is a group of fungal infections that are found worldwide. The most important phaeohyphomycetes are dematiaceous fungi, for example Alternaria , Cladiophora , Exophiala, Scedosporium and Ulocladium [1]. Identification of these moulds may be labour intensive, and needs experienced laboratory technicians. Alternaria spp. are often difficult to differentiate with morphological and biochemical techniques, therefore other techniques are have been developed to detect and identify these moulds. Opportunistic infections by species of the genus Alternaria have thus far been rarely reported, but with the increase of immunosuppressive treatments, these infections are likely to be encountered more commonly in the future. In the present case a renal transplant patient developed a subcutaneous infection with Alternaria infectoria and we describe the difficulties we encountered during culture, identification and susceptibility testing of the mould.
A 64-year-old man had a history of long-standing hypertension complicated by renal insufficiency due to benign nephrosclerosis. In August 1998, peritoneal dialysis was started and in August 2002, he underwent a successful renal transplantation with a kidney from a cadaver donor. His immunosuppressive treatment consisted of tacrolimus (aiming at a trough level of 5/10 ng/ml), prednisolone (10 mg/d), and mycophenolate mofetil (1000 mg/d). He developed de novo diabetes mellitus, which was treated with oral medication. Three months after the transplantation, he noticed a slowly growing, blue-colored tumor on the dorsum of the right hand. There was no history of prior skin laceration at the same location. The patient did not have fever or leucocytosis. A few weeks later, when the tumor measured 12 mm in diameter, it was first punctured and later excised under local anesthesia. Without any additional treatment or changes in his immunosuppressive medication, the subsequent course was uneventful and the tumor did not recur. The excised tumor was sent to pathology and the medical microbiology laboratory for examination and culture. Microscopic examination revealed a centrally invaded dermis with inflammatory infiltration of histiocytes, multinuclear giant cells, and neutrophile granulocytes, with focal abscess formation, lympho-
Received 13 June 2005; Accepted 13 October 2005 Correspondence: Eric Nulens, Department of Medical Microbiology, General Hospital Sint-Jan Brugge, Ruddershove 10, 8000 Brugge, Belgium. Tel: / 32 50 45 26 04; Fax: /32 50 45 26 09; E-mail: eric.
[email protected]
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DOI: 10.1080/13693780500412004
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cytes, and plasma cells. In the same area an extended vascular proliferation was seen. Subsequently Haematoxylin-Eosin and Periodic Acid Schiff (PAS), and Grocott staining showed hyphal structures in the giant cells and also in the inflammatory infiltrate. Microscopy was negative for bacteria and mycobacterium and cultures remained sterile. The tumor was also inoculated in Sabouraud broth, Sabouraud agar with and without actidion, and diluted Sabouraud agar (diluted Sabouraud has the same composition as Sabouraud, but is 10 times less concentrated, to promote conidia formation). All media were incubated at 308C, and inspected for growth once a week. Fungal growth occurred only in Sabouraud broth and was subcultured on diluted Sabouraud agar and water agar. Identification was performed at the Department of Medical Microbiology, Radboud University Nijmegen Medical Centre. Finally after six weeks of incubation on water agar at 308C, a few conidia were detected in a lacto-phenol slide (Fig. 1). The mold presumptively belonged to the genus Alternaria . The strain and two clinical A. infectoria strains from our collection were also incubated on DRYES medium as previously described [1]. Growth was visible in less than 14 days, and all three strains had a whitish appearance. A presumptive differentiation of Alternaria infectoria was made, on the basis of poorer growth of A. infectoria on culture media than A. alternata, shorter conidiophores, the smooth-like appearance of the conidia, and the appearance on DRYES medium [1,2]. The strain was sent to the Centraal Bureau voor Schimmelculturen (CBS, Utrecht, The Netherlands) for confirmation; their conclusion was that the strain belonged to the genus Alternaria , presumptive to the species alternata.
To make a definitive identification of the fungus, a PCR and sequencing of the amplified DNA using primers ITS1 and ITS4 was performed as previously described [3]. The sequence obtained was analyzed by using the BLAST alignment program of the GenBank database (National Institutes of Health, USA). A 100% homology was found for: AIN549824 Lewia infectoria (teleomorph of Alternaria infectoria ) partial 18S rRNA gene, ITS1/ITS2 and partial 28S rRNA gene. The in vitro activity of amphotericin B, 5-Fluocytosine, voriconazole, fluconazole, itraconazole, terbinafine, and caspofungin against the strains were tested with a micro dilution technique, according to CLSI (clinical laboratory standards institute; former NCCLS) M38-A guidelines [4]. After incubation for 70 /74 h at 358C, final reading of the micro titration plate was performed visually. The MIC-endpoint for the antifungals was defined, as the lowest concentration at which there was 100% inhibition of growth, except for caspofungin for which the lowest drug concentration resulting in aberrant hyphal growth by examination with an inverted microscope or the minimum effective concentration (MEC) was determined. The MICs for different antifungals for our clinical strain are presented in Table 1. Review of the literature showed that the majority of infections due to Alternaria species are mainly soft tissue and skin and eye infections, often after traumatic injuries [5 /9]. We hypothesized that the predilection for a localized skin infection, was the inability of the strain to grow at 378C. Therefore growth curves were performed with Anthos Labtec HT3 (Anthos Labtec Instruments). The apparatus is a fully automated microplate absorption photometer. Fungal growth was measured by optical density of a fungal suspension at 208C, 308C and 378C every 30 min, for a total of 168 h of incubation. Briefly, a conidia suspension with a transmission between 68 /70% in a photometer is Table 1 Susceptibility pattern of clinical strain 9837. Susceptibility testing was performed according to CLSI guidelines, but due to very slow growth, with very poor conidia formation, susceptibility was performed with mycelium growth alone.
Fig. 1 Microscopic examination with lactophenol of the mold after six weeks incubation on water agar. Dark brown, short, primary conidiophores with no secondary conidiophores were seen. The conidia had transverse and few oblique septa, and the conidial wall was smooth.
Antifungal agent
MICa (mg/ml)
Amphotericin B 5-Fluocytosine Fluconazol Itraconazole Voriconazole Terbinafin Caspofunginb
2 /64 /64 0.5 4 8 4
a MIC: Minimum inhibitory concentration. concentration.
b
Minimum effective
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A. infectoria phaeohyphomycosis in a renal transplant patient
prepared in 0.9% NaCl solution, and diluted 50 times in RPMI. 100 ml of the conidia suspension is inoculated into a 96-well microtiter plate, with each well containing 100 ml RPMI. In our case, due to very slow growth of the mold, we prepared a suspension of mycelium instead of conidia. Besides our clinical strain (9837), we tested the growth rate of two other clinical A. infectoria strains from clinical skin specimens from our collection (v21-41, and 8686). In Fig. 2 the optimal growth temperature of the three clinical strains after 168 h of incubation is presented. After at least 32 h of incubation, initial growth was detected in the microwells. Strain v21-41 showed the highest, while our clinical strain showed the lowest growth rate (data not shown).
Discussion Opportunistic infections by species of the genus Alternaria have thus far been rarely reported, but with the increase of immunosuppressive treatments, these infections are likely to be encountered more commonly in the future. Alternaria spp. are usually involved in skin, mucosa, eye, and disseminated infections in immunocompromised patients, such as cancer patients, hematology patients, and transplant patients but also in injection drug use and patients suffering from diabetes mellitus and asthma [5,10/14]. Our patient had no history of a trauma, and he was unemployed since 1998. In immunocompetent patients, Alternaria infections have usually an occupational origin, such as carpenters, forest workers, and gardeners [1,7]. Alternaria spp. belongs to the Order Dothideales, Family of Pleosporaceae, together with other opportunistic molds such as Curvularia , Bipolaris, and Exserohilum [2,5,10]. The main habitat of A. infectoria is leaves and kernels of cereals and grasses [1,10]. 0.7
Optical density
0.6 0.5 0.4 0.3 0.2 0.1 0 20 9837
8683
30 Temperature (°C)
37
V21-41
Fig. 2 Optimal growth temperature measured by optical density at endpoint for three clinical Alternaria infectoria strains.
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A. infectoria usually grows more slowly on routine culture media, and sterile mycelia grow after several weeks of incubation and are therefore difficult to identify. Even in instances when conidia do occur, identification to species level may be difficult [8,15]. A. infectoria has shorter primary conidiophores, fewer secondary conidiophores, and has rougher conidia than A. alternata [1,2]. A more distinctive differentiation can also be made on the basis of macroscopic morphology, color appearance and sporulation on potato carrot agar and DRYES medium [1,15]. Even though A. infectoria belongs to dematiaceous molds, melanin, the brown pigment, which is characteristic of these fungi, is not always apparent in culture [8,15]. In our case it took several weeks before even a presumptive identification of the mold was possible, because growth was very slow, conidia did not develop on Sabouraud agar, and only sparsely developed on water agar, and the development of melanin was not observed. To induce sporulation and hence identification, inoculation on Potato Carrot Agar, with an alternating lightdark incubation cycle may be an alternative [1]. However, a more rapid molecular approach towards detection and identification of molds in high-risk patients is warranted. Molecular techniques have been developed for faster identification of molds with good results to adequately differentiate between A. infectoria and other Alternaria spp., mainly A. alternaria [6,15/ 17]. It has been shown that genetically, A. infectoria is only distantly related to the other Alternaria species, and thus is easy to differentiate from other Alternaria species, since A. infectoria has an insertion of 30 bp in the ITS-I region, which other Alternaria species lack [1,14,15,17]. An amplicon of 600 bp with an ITS domain of about 510 bp is amplified for A. infectoria [15]. On the other hand, it has been shown that ITS rDNA sequencing is poorly discriminative in separating the other Alternaria species, and even in separating them from related molds [1,15]. Itraconazole is the most active compound against the strain cultured from our patient, and itraconazole is often used for the treatment of infections due to dematiaceous fungi, although treatment failure with itraconazole has been reported [11]. However CLSI breakpoints for dematiaceous molds do not exist and in vitro susceptibility testing according to the CLSI guidelines is frustrated by lack of sporulation. Nevertheless this strain showed susceptibilities comparable with susceptibility reported in other publications [18,19]. We showed that optimal growth rate of A. infectoria in our case appeared at 308C, and that growth was much slower at room temperature or at 378C. This may explain the predominantly cutaneous
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infections caused by this mold. Other cases of A. infectoria superficial and invasive infections have been described, and these infections were treated with surgery and the admission of antifungals [6 /8,20]. In our case the patient was treated with surgical excision of the tumor, without further systemic antifungal treatment. In conclusion, a renal transplant patient is presented who developed a locally invasive A. infectoria infection. The patient was treated with surgery alone. Definitive identification was obtained after amplification and sequencing of fungal DNA.
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