Molecular Detection of Human Fungal Pathogens Are Separated from Hyphae by a Septum and Taper Toward Immunosuppressive Therapy, and Anatomic Disorders

Dongyou Liu, Xianghong Du, and Song Weining

Contents 46.1 Introduction...... 385 46.1.1 Classification and Morphology...... 385 46.1.2 Clinical Features and Pathogenesis...... 386 46.1.3 Laboratory Diagnosis...... 387 46.2 Methods...... 387 46.2.1 Sample Preparation...... 387 46.2.2 Detection Procedures...... 388 46.2.2.1 Sequencing Analysis of ITS and Partial 28S rRNA Gene...... 388 46.2.2.2 PCR-RFLP of ITS2 Region...... 388 46.3 Conclusion...... 388 References...... 388

46.1 Introduction Haematonectria, and Neocosmospora), and the new combination Fusarium falciforme was proposed for Acremonium 46.1.1 Classification and Morphology falciforme [4]. The genus Acremonium is classified within the mitosporic Teleomorphs of Acremonium are found in several gen- Hypocreales group, order Hypocreales, class Sordariomycetes, era of ascomycetes such as Emericellopsis, Hapsidospora, subphylum Pezizomycotina, phylum Ascomycota, and king- Nectria, Nectriella, Neocosmospora, Pronectria, and dom Fungi. The mitosporic Hypocreales group encompasses Thielavia (Sordariales), reflecting the polyphylic nature of 24 genera: Acremonium, Acrostalagmus, Cephalosporium, the Acremonium genus. For example, Acremonium alabam- Chaetopsina, Cylindrocladiella, Escovopsis, Fusarium, ense is the anamorph of Thielavia terrestris, a member of Gliocladiopsis, Gliocladium, Hobsonia, Illosporium, Sordariales. It has been proposed that the name Acremonium Myrothecium, Parasarcopodium, Polycephalomyces, should be restricted to only the anamorphs of the family Rotiferophthora, Sesquicillium, Solheimia, Stachybotrys, Hypocreaceae, and a new genus should be established to Stilbella, Trichothecium, Tubercularia, Ustilaginoidea, accommodate other species of Acremonium, such as grass Verticillium, and Xenocylindrocladium [1]. endophytes and others related to the Clavicipitaceae. In turn, the genus Acremonium consists of 33 recog- Acremonium spp. are filamentous fungi that are com- nized species: Acremonium alabamense, A. atrogriseum, monly isolated from soil, plant debris, rotting mushrooms, A. alcalophilum, A. alternatum, A. antarcticum, A. blochii, etc. in Europe, Asia, Egypt, and North and Central America. A. breve, A. brunnescens, A. butyri, A. collariferum, A. cro- Several Acremonium species have been implicated in human tocinigenum, Acremonium cucurbitacearum, Acremonium infections, including Acremonium falciforme, Acremonium curvulum, Acremonium exuviarum, A. fuci, A. falciforme kiliense, Acremonium strictum, and Acremonium recifei. (obsolete synonym: Cephalosporium falciforme), A. hya- Acremonium grows moderately. After 7 day incubation linulum, A. kiliense (obsolete synonyms: Cephalosporium at 25°C on potato glucose agar, colonies measure 1–3 cm in granulomatis, C. infestans, and C. madurae), A. murorum, diameter and are compact, flat or folded, and slightly raised A. nepalense, A. obclavatum, A. potronii, A. recifei (obso- in the center. Colonies are white to pale gray, velvety, pow- lete synonym: Cephalosporium recifei), A. restrictum, dery, and membrane-like initially and become cottony or fas- A. roseogriseum, A. rutilum, A. sclerotigenum, A. spinosum, ciculate (showing spiky aggregates of hyphae) with age. On A. strictum, A. stromaticum, A. thermophilum, A. zeae, and the front, colonies are white, pale gray, cottony, or pale pink Cephalosporium acremonium, in addition to over 30 unas- in color and, on the reverse, they are either uncolored or a signed species [1–3]. pink-to-rose-colored pigment production. A recent study showed that Acremonium falciforme Acremonium spp. possess hyaline, narrow, and sep- shares considerable sequencing similarity in the ribosomal tate hyphae, with vegetative hyphae forming hyphal ropes. large subunit (LSU) with members of the Fusarium solani Unbranched, solitary, awl-shaped erect phialides are located s pecies complex (i.e., the F. solani clade containing F. solani, on the hyphal tips, the hyphal ropes, or both. The phialides

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© 2011 by Taylor & Francis Group, LLC 386 Molecular Detection of Human Fungal Pathogens are separated from hyphae by a septum and taper toward immunosuppressive therapy, and anatomic disorders. The fact their apices. Conidia (1–3 × 3–8 μm) are usually one celled that A. kiliense infection often presents as a cutaneous papu- (ameroconidia), hyaline or pigmented, globose to cylindrical, lar eruption without other clinical signs in immunocompetent and mostly aggregated in slimy heads at the apex of each patients suggests its relatively low pathogenicity. phialide (which may or may not possess collarettes). Bound Acremonium strictum is another species commonly by a gelatinous material, conidia may be single or multicel- identified in human diseases [13,15,20–26]. For example, a lular, fusiform with a slight curve (crescent-like). 59-year-old male who had undergone nonmyeloablative con- A. falciforme often produces crescentic, nonseptate ditioning therapy and bone marrow transplantation after an conidia, sometimes being two or three celled; A. kiliense initial diagnosis of acute myelogenous leukemia presented forms solitary hyphae, chlamydospores (especially in oatmeal gastrointestinal graft-versus-host disease (GVHD) with agar), and short straight cylindrical conidia; A. recifei usu- severe gastrointestinal bleeding. A fungus was isolated in ally shows crescentic and nonseptate conidia; and A. strictum blood cultures and also a fungal organism was identified in colonies may appear moist, pink, or salmon color, resembling multiple organs by histopathology at autopsy. Gram stains of those of Lecythophora hoffmannii. positive blood culture bottles showed both yeast-like forms with hyphal elements and fully formed hyphal masses sug- gestive of a sporulating mold. On Sabouraud agar subculture 46.1.2 Clinical Features and Pathogenesis at 30°C, colonies measured 2.2 cm in 7 days. Young colo- Being mainly environmental saprophytes, Acremonium nies were smooth, moist, and pink, with a colorless reverse; species often gain entry to human host following penetrat- mature colonies were velvety and raised in the center. ing injuries or surgery procedures [5,6]. Besides being one Lactophenol aniline blue preparations revealed conidia and of the causative agents of eumycotic white grain mycetoma septate hyphae. The conidia (of 3–4 μm × 1–1.5 μm in size) (principally by A. falciforme, A. kiliense, and A. recifei), were one celled, cylindrical, smooth, hyaline to slightly pink, Acremonium spp. are also responsible for rare cases of ony- and grouped in slimy heads. The conidiophores were simple, chomycosis, keratitis, endophthalmitis, endocarditis, gastritis, slender, and erect phialides with basal septa arising from fungemia, meningitis, diffuse cerebritis, peritonitis, invasive the vegetative hyphae. Based upon micro- and macroscopic pulmonary disease, and osteomyelitis [7–13]. These infections characteristics, the fungus was identified as A. strictum [27]. are often diagnosed in patients with predisposing conditions The next common Acremonium species associated with (e.g., Addison’s disease, neutropenia, immune suppression, human infections is A. falciforme [7,9,10,28]. In addition, a burns, organ transplantation, artificial implants, intravenous number of clinical cases due to Acremonium spp. have also drug abuse, splenectomy, bone marrow transplantation, and been reported [29–31]. A patient with leukemia presented diabetes mellitus) and preterm infants [14,15]. Neonates who painful cutaneous nodules and severe myalgia indicative of receive mechanical ventilation, umbilical vein catheterization, pyomyositis. Culture of an aspiration grew an Acremonium previous treatment with antibacterial agents, and prior use of species organism. After surgical drainage and treatment with parenteral nutrition such as intravenous lipid show increased amphotericin B and granulocyte colony-stimulating factor susceptibility to Acremonium infections. Acremonium endo- (G-CSF), the patient recovered fully [32]. In another case, a phthalmitis may develop after penetrating keratoplasty and 64-year-old woman developed chronic uveitis in her left eye, 2 retinal detachment surgery, with intraocular inoculations weeks after an uncomplicated cataract extraction. Potassium from irrigation solutions, and display clinical symptoms of hydroxide (KOH)–Calcofluor staining and Gram stain- mild pain, redness, floaters, and slightly decreased visual acu- ing of the vitreous fluid uncovered septate fungal hyphae of ity. Invasive Acremonium infection is difficult to treat and the Acremonium species. Subsequent culture of the vitreous fluid outcome is generally poor. Acremonium species was reported grew an Acremonium organism. The patient was given intra- to cause invasive allergic fungal sinusitis (AFS) or eosino- venous ampotericin B daily for 5 days and oral voriconazole philic fungal rhinosinusitis (EFRS) in an immunocompetent medication for 4 weeks. During the postoperative 18 month patient, leading to unilateral visual loss [16]. follow-up, she was stable without significant relapse of uveitis, Among the Acremonium species of clinical interest, and her visual acuity improved from 20/40 to 20/20 [33]. Acremonium kiliense is most important and commonly Acremonium species are generally sensitive to ampho- described [17–19]. Examined by light microscopy, A. kiliense tericin B and ketoconazole. Amphotericin B therapy in is stained Gram-positive and is characterized by the appear- combination with ketoconazole or another new azole or ance of septate hyphae and chlamydospores. The fungus grows allylamine may be used for treatment [34]. Patients experi- slowly in specific culture (e.g., Sabouraud agar, glucose, and encing failure of amphotericin B treatment may be treated malt extract). A range of clinical diseases has been attributed with a triazole derivative voriconazole. Voriconazole to A. kiliense, including dermatophytoses, kerion, onycomyco- achieves a sufficient therapeutic level in aqueous and vit- ses, keratitis, and mycetomas in immunocompetent individuals reous liquids by oral administration for eradication of and pneumonia, arthritis, osteomyelitis, endocarditis, perito- Acremonium osteomyelitis [35]. nitis, meningitis, and sepsis in immunocompromised patients. Acremonium chrysogenum is the production host Risk factors for A. kiliense infection are prosthesis, catheters, employed in the fermentation process of manufacturing

© 2011 by Taylor & Francis Group, LLC Acremonium 387 cephalosporins with broad activity against Gram-positive 46.2 Methods and Gram-negative bacteria, while application of cephalosporins is preferred in a wide range of cases due to microbial 46.2.1 Sample Preparation resistance toward penicillins [36,37]. Culturef o mycetoma lesion is performed by the collection of the abscess or the fistula secretion or by tissue biopsy. The 46.1.3 Laboratory Diagnosis samples are cultured in media such as Sabouraud agar or mycobiotic agar to isolate fungi and/or blood agar to isolate Acremonium is a ubiquitous, saprophytic fungus that is char- bacteria. The etiologic agents are identified according to their acterized by the formation of narrow hyphae with solitary, macroscopic and microscopic features. slender (2 μm), unbranched, awl (needle)-shaped phialides Blood culturing is performed using the BACTEC 9240 (or weakly branched conidiophores) arising from vegetative automated blood-culturing system (Becton Dickinson), with hyphae and producing clusters (slimy messes) or chains of each culture consisting of one each Plus Aerobic/F, Lytic/10 small, one-celled conidia mostly aggregated at the apex of Anaerobic/F, and Myco/F Lytic bottles. Aerobic and anaer- each phialide. In tissue sections, Acremonium often shows obic media are held in the BACTEC cabinet for 5 days; hyaline, septate hyphae and characteristic reproductive Myco/F bottles are held for 28 days. Aerobic and anaerobic structures known as phialides and phialoconidia. The use of bottles positive for yeast-like fungi are subcultured to choco- specific fungal stains such as Grocott methenamine-silver late, bromcresol green, and inhibitory mold agar plates and and Giemsa stains aids the identification of Acremonium, incubated at 35°C supplemented with CO2 ot 5%. Other fun- although 10% KOH and Gram stain offer alternative detec- gal cultures are performed using Sabouraud dextrose agar tion methods. (SAB; Emmon’s modification); brain heart infusion agar Acremonium shows a high degree of morphologi- with blood, chloramphenicol, cycloheximide, and gentami- cal similarity to Fusarium, Verticillium Lecythophora, cin; and inhibitory mold agar incubated at 30°C. Subcultures Phialemonium, Gliomastix, and Cylindrocarpon as well as for morphological studies are made on potato dextrose agar Candida. Strains of Fusarium, which do not produce mac- and incubated at 30°C [27]. roconidia, is differentiated from those of Acremonium by For DNA extraction from fungal strains, a loopful from their faster growth and production of deeply woolly colo- individual colonies of each fungus culture is homogenized nies. In comparison with Acremonium, Lecythophora and in 500 μL of lyticase lysis buffer (LLB; 50 mM Tris pH Phialemonium phialides are not separated from hyphae by a 7.6, 1 mM EDTA pH 8.0, 0.2% β-mercaptoethanol, and septum; while Gliomastix generates olive-green to greenish- 1 U/100 μL recombinant lyticase [Sigma]) and incubated black colonies and chains or balls of dark conidia. at 37°C for 1 h. After incubation, acid-washed glass beads Acremonium is differentiated from hyaline isolates of 710–1180 μm in diameter (Sigma) are added and the solution Phialophora by the absence or very limited development of is vortexed thoroughly for 2 min. Amounts of 400 μL of the a collarette on the phialide and the predominant formation supernatant are used for DNA extraction on a MagNA Pure of awl-shaped phialides with a basal septum. Compared to compact instrument using a MagNA Pure compact nucleic Acremonium, microconidial Fusarium isolates usually grow acid isolation kit I (Roche Diagnostics). DNA concentrations faster and have colonies with a characteristic fluffy appear- are determined by using a PicoGreen double-stranded DNA ance. Potato dextrose agar and cornmeal agar are the most quantification kit (Molecular Probes) on an F-2500 fluores- suitable media for their identification, and exposure to day- cence spectrophotometer (Hitachi) [41]. light maximizes their culture color characteristics. For peripheral blood specimens, after hypotonic lysis of Given the length of time for fungal culture and special- the erythrocytes using red blood cell lysis buffer (10 mM Tris ist skills for macroscopic and microscopic identification pH 7.6, 5 mM MgCl2,0M 1 m NaCl), the leukocytes are pel- of Acremonium from other morphological similar fungal leted and resuspended in 470 μL LLB. The subsequent steps organisms, nucleic acid amplification and sequence analy- were identical to the extraction protocol described above. For sis have been increasingly applied for their characteriza- blood culture specimens, 200 μL aliquots derived from blood tion and speciation. In particular, small subunit (SSU) 18S cultures previously shown to be fungus positive are trans- and LSU 28S ribosomal RNA (rRNA) genes as well as their ferred to Falcon tubes and red blood cell lysis buffer is added. internal transcribed spacer (ITS) regions provide valuable The subsequent procedure is as described above. For plasma target for molecular detection of fungal organisms includ- containing white blood cells, peripheral blood specimens ing Acremonium spp. [38,39]. For example, PCR amplifica- anticoagulated with EDTA are kept at 4°C for at least 4 h to tion of partial 5S rRNA gene and internal transcribed spacer sediment the red blood cells. The entire supernatant, that is, region 2 (ITS2) followed by digestion of the amplicon with plasma containing white blood cells, is used for DNA extrac- restriction enzymes Hinfl and Sau3AI divides Acremonium tion. The samples are centrifuged at 15,000 × g for 10 min. spp. into distinct restriction fragment length polymorphism Most of the plasma is removed, leaving a residual volume of (RFLP) groups, with A. kiliense and A. strictum forming 100 and 430 μL of LLB is added. The DNA extraction is per- similar subgroups on the basis of morphological differences formed as described above. For specimens from respiratory and distinct RFLP patterns [40]. tract and lung biopsies, solid material is cut into small pieces

© 2011 by Taylor & Francis Group, LLC 388 Molecular Detection of Human Fungal Pathogens and homogenized in 430 μL of LLB. The ensuing steps are followed by the initial activation of DNA polymerase at 96°C as described above [41]. for 12 min; (ii) 40 cycles consisting of a denaturation step Alternatively, fungal DNA can be extracted from mature for 10 s at 96°C, an annealing step for 10 s at 58°C, and an colonies, grown on SAB agar with chloramphenicol and gen- extension step for 30 s at 72°C; and (iii) the final extension at tamicin (Remel) at 30°C, using the QIAmp Mini Kit (Qiagen) 72°C for 4 min. following the manufacturer’s tissue extraction protocol [27]. Restriction analysis of amplified PCR products is performed by HaeIII, endonuclease digestion (New England BioLabs). The restriction fragments are separated on a 3% (wt/vol) aga- 46.2.2 Detection Procedures rose gel, stained with ethidium bromide, visualized by UV 46.2.2.1 S equencing Analysis of ITS and transillumination (312 nm), and analyzed by ULTRA LUM Partial 28S rRNA Gene (Ultra-Lum, Inc.) gel detection and analysis system. Novicki et al. [27] employed the ITS1 (5′-TCC GTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTT ATTGATATGC-3′) primers to amplify the intergenic tran- 46.3 Conclusion scribed spacer 1 (ITS1) and ITS2 regions of the rRNA operon, The anamorphic genus Acremonium consists of a large num- flanking the 5.8S rRNA gene [42]. In addition, the D1–D2 vari- ber of saprophytic filamentous fungal species that have hya- able domain of the 28S rRNA gene is amplified with the NL-1 line, septate hyphae and reproduce by phialidic conidiation. (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL-4 Several Acremonium spp. are involved in eumycotic myce- (5′-GGTCCGTGTTTCAAGACGG-3′) primers. toma and other focal infections in otherwise healthy indi- PCR mixture (25 μL) is made up of GeneAmp 1× PCR viduals. In addition, they have been increasingly implicated buffer II (Applied Biosystems), 2.5 mM MgCl2, M 0.2 m in invasive systemic mycotic diseases in immunosuppressed deoxynucleotide triphosphate, 400 nM each of primers ITS1 individuals with underlying conditions such as malignancy and ITS4, or primers NL-1 and NL-4, 2.5 U AmpliTaq DNA and other illnesses. Given their similar morphological fea- polymerase (Applied Biosystems), and 5 μL of template tures to a number of fungal organisms that may display var- DNA. ied sensitivity to antifungal agents, it is important to identify PCR amplification is performed with an initial 95°C for Acremonium to species level. Unfortunately, identification of 10 min (polymerase activation); 35 cycles of 95°C for 30 s, Acremonium spp. by macroscopic and microscopic examina- 55°C for 30 s, and 72°C for 1 min; and a final extension at tion is not only time-consuming but also challenging. This 72°C for 10 min. is especially so with some Acremonium species that do not The resulting amplicons are sequenced and compared with develop a teleomorphic stage in vitro. Therefore, applica- those at the National Center for Biotechnology Information tion of molecular detection procedures such as PCR and GenBank nucleotide database using the nucleotide–nucleotide sequencing analysis is critical for accurate determination of BLAST program for homologous sequences. The sequences Acremonium species identity and assists effective treatment are then aligned and phylogenetic trees are drawn with and control of Acremonium infections. Clustal X using the neighbor-joining method. The aligned sequences are edited with Jalview version 1.3b. Phylogenetic trees are displayed using Treeview version 1.6.6. This allows References precise identification of fungal organisms of interest includ- 1. The UniProt Consortium. Available at http://www.uniprot. ing Acremonium spp. org/, accessed on August 1, 2010. 2. Glenn AE et al. Molecular phylogeny of Acremonium and its 46.2.2.2 PCR-RFLP of ITS2 Region taxonomic implications. Mycologia 1996;88:369–383. Keynan et al. [43] showed that by using primers ITS1 and 3. Weisenborn JLF, Kirschner R, Piepenbring M. A new ITS4 in the first round PCR and internal primers UNF1 darkly pigmented and keratinolytic species of Acremonium (5′-GCATCGATGAAGAACGCAGC-3) and UNF2 (Hyphomycetes) with relationship to the Plectosphaerellaceae (5′-TTGATATGCTTAAGTTCAGCGG-3′) with specificity from human skin and nail lesions in Panama. Nova Hedwigia 2010;90:457. for the ITS2 region in the second round PCR, a 285 bp product 4. Summerbell RC, Schroers HJ. Analysis of phylogenetic is generated. Subsequent digestion of the nested PCR product relationship of Cylindrocarpon lichenicola and Acremonium with endonuclease HaeIII results in 193 and 92 bp fragments, falciforme to the Fusarium solani species complex and which are characteristic of Acremonium species. a review of similarities in the spectrum of opportunis- The PCR mixture (50 μL) is composed of 1× HotStart tic infections caused by these fungi. J Clin Microbiol. Master Mix (Qiagen), 15 pmol of UNF1 mixture primers, 2002;40(8):2866–2875. 15 pmol of UNF2 primer, 100 μm of dUTP (Sigma), 0.5 U of 5. Fridkin SK, Jarvis WR. Epidemiology of nosocomial fungal uracil-DNA-gycosylase (UDG) (Sigma) for the elimination infections. Clin Microbiol Rev. 1996a;9:499–511. 6. Geyer AS et al. Acremonium mycetoma in a heart transplant of possible cross contamination, and 5 μL of template DNA recipient. J Am Acad Dermatol. 2006;55(6):1095–1100. produced after standard DNA isolation. 7. Lau YL et al. Invasive Acremonium falciforme infection in a The PCR reaction is conducted under the following condi- patient with severe combined immunodeficiency. Clin Infect tions: (i) incubation at 37°C for 10 min for UDG treatment Dis. 1995;20:197–198.

8. Roilides E et al. Acremonium fungemia in two immunocom- 27. Novicki TJ et al. Genetic diversity among clinical isolates of promised children. Pediatr Infect Dis J. 1995;14:548–550. Acremonium strictum determined during an investigation of a 9. Cameron JA et al., Chronic endophthalmitis caused by fatal mycosis. J Clin Microbiol. 2003;41(6):2623–2628. Acremonium falciforme. Can J Ophthalmol. 1996;31:367–368. 28. Van Etta LL, Peterson LR, Gerding DN. Acremonium fal- 10. Koç AN, Erdem F, Patiroglu, T. Case report: Acremonium falciforme (Cephalosporium falciforme) mycetoma in a renal ciforme fungemia in a patient with acute leukaemia. Mycoses transplant patient. Arch Dermatol. 1983;119:707–708. 2002;45:202–203. 29. Fincher RME et al. Infection due to the fungus Acremonium 11. Mattei D et al. Successful treatment of Acremonium fungemia (Cephalosporium). Medicine 1991;70:398–409. with voriconazole. Mycoses 2003;46:511–514. 30. Kan SF et al. Cutaneous hyalohyphomycosis caused by 12. Alfonso JF et al. Acremonium fungal infection in 4 patients Acremonium in an immunocompetent patient. Br J Dermatol. after laser in situ keratomileusis. J Cataract Refract Surg. 2004;150(4):789–790. 2004;30(1):262–267. 31. Yamazaki R et al. Systemic infection due to Acremonium after 13. Miyakis S et al. Invasive Acremonium strictum infection allogeneic peripheral blood stem cell transplantation. Intern in a bone marrow transplant recipient. Pediatr Infect Dis J. Med. 2006;45(16):989–990. 2006;25:273–275. 32. Chang HY et al. Acremonium pyomyositis in a pediat- 14. Jeffrey WR et al. Disseminated infection due to Acremonium ric patient with acute leukemia. Pediatr Blood Cancer species in a patient with Addison’s disease. Clin Infect Dis. 2005;44:521–524. 1993;16:170. 33. Joe SG et al. Case report of Acremonium intraocular infec- 15. Yalaz M et al. Fatal disseminated Acremonium strictum infection after cataract extraction. Korean J Ophthalmol. 2010 tion in a preterm newborn: A very rare cause of neonatal sep- April;24(2):119–122. ticaemia. J Med Microbiol. 2003;52:835–837. 34. Guarro J et al. Acremonium species: New emerging fungal 16. Mulwafu WK et al. Allergic fungal sinusitis secondary to opportunistis—In vitro antifungal susceptibilities and review. Acremonium species causing unilateral visual loss. East Clin Infect Dis. 1997;25:1222–1229. Central Afr J Surg. 2006;11:62–64. 35. Weissgold DJ, Maguire AM, Brucker AJ. Management of 17. Brabender W et al. Acremonium kiliense osteomyelitis of the postoperative Acremonium endophthalmitis. Ophthalmology calvarium. Neurosurgery 1985;16:554–556. 1996;103:749–756. 18. Fridkin SK et al. Acremonium kiliense endophthalmitis that 36. Weil J, Miramonti J, Ladisch MR. Cephalosporin-C—Mode occurred after cataract extraction in an ambulatory surgical of action and biosynthetic-pathway. Enzyme Microb Technol. care and was traced to an environmental reservoir. Clin Infect 1995;17:85–87. Dis. 1996b;22:222–227. 37. Barber MS, Giesecke U, Reichert A, Minas W. Industrial 19. Pastorino AC et al. Acremonium kiliense infection in a child enzymatic production of cephalosporin-based beta-lactams. with chronic granulomatous disease. Braz J Infect Dis. Adv Biochem Eng Biotechnol. 2004;88:179–215. 2005;9(6):529–534. 38. Chen YC et al. Identification of medically important yeasts 20. Schell WA, Perfect JR. Fatal, disseminated Acremonium using PCR-based detection of DNA sequence polymorphisms strictum infection in a neutropenic host. J Clin Microbiol. in the internal transcribed spacer 2 region of the rRNA genes. 1996;34:1333–1336. J Clin Microbiol. 2000;38:2302–2310. 21. Koç AN et al. Peritonitis due to Acremonium strictum in a 39. Chen YC et al. Polymorphic internal transcribed spacer region patient on continuous ambulatory peritoneal dialysis. Nephron 1 DNA sequences identify medically important yeasts. J Clin 1998;79:357–358. Microbiol. 2001;39:4042–4051. 22. Koç AN et al. Pleuritis caused by Acremonium stric- 40. Borazjani RN, Lott TJ, Ahearn DG. Comparison of 5.8S and tum in a patient with colon adenocarcinoma. Mycoses ITS2 rDNA RFLP patterns among isolates of Acremonium 2008;51(6):554–556. obclavatum, A. kiliense, and A. strictum from diverse sources. 23. Koç AN, Mutlu Sarigüzel F, Artiş T. Isolation of Acremonium Curr Microbiol. 1998;36(2):70–74. strictum from pleural fluid of a patient with colon adenocarci- 41. Landlinger C et al. Species-specific identification of a wide noma. Mycoses 2009;52(2):190–192. range of clinically relevant fungal pathogens by use of Luminex 24. Anadolu R et al. Indolent Acremonium strictum infec- xMAP technology. J Clin Microbiol. 2009;47(4):1063–1073. tion in an immunocompetent patient. Int J Dermatol. 42. White TJ et al. Amplification and direct sequencing of fungal 2001;40:451–453. ribosomal RNA genes for phylogenetics. In MA Innis, DH 25. Scott IU, Flynn HW Jr., Miller D. Delayed-onset endophthal- Gelfand, JJ Sninsky, and TJ White (eds.), PCR Protocols: A mitis following cataract surgery caused by Acremonium stric- Guide to Methods and Applications. Academic Press, San tum. Ophthalmic Surg Lasers Imaging. 2005;36:506–507. Diego, CA, pp. 315–322, 1990. 26. Guarro J et al. A case of colonization of a prosthetic 43. Keynan Y, Sprecher H, Weber G. Acremonium vertebral osteo- mitral valve by Acremonium strictum. Rev Iberoam Micol. myelitis: Molecular diagnosis and response to voriconazole. 2009;26(2):146–148. Clin Infect Dis. 2007;45(1):e5–e6.