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Journal of Clinical Microbiology, April 2003, p. 1722-1725, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1722-1725.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

New Filamentous Fungus Sagenomella chlamydospora Responsible for a Disseminated Infection in a Dog

Unitat de Microbiologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, 43201 Reus,¹ Laboratorio de Micología Clínica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain²

Received 30 September 2002/ Returned for modification 27 November 2002/ Accepted 2 January 2003

	ABSTRACT

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A filamentous fungus that caused a fatal systemic infection in a dog has been identified as the new species Sagenomella chlamydospora. When the case was initially reported, the fungus was identified as Paecilomyces sp. This study emphasizes how difficult can be the identification of the causative agent of an infection when an uncommon microorganism is involved. This is the first time that this genus has been involved in animal infections, including humans.

	TEXT

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Recently García et al. (7) reported and illustrated a case of disseminated mycosis in a dog (a German shepherd cross). The fungus was isolated in the postmortem examination from lesions in numerous organs, i.e., the kidneys, mitral valve, abdominal aorta, and vertebral discs. The diagnosis was difficult because the clinical symptoms were very unspecific. The first signs were multiple discospondylitis of unknown origin and moderate cervical rigidity. A complete blood test, antibacterial serology, and urine analysis showed no abnormalities. However, the animal had a history of depression, anorexia and weight loss, listlessness, and slight spasticity in the walk. The fungal etiology of the infection was recognized when an aspergillosis enzyme-linked immunosorbent assay, using a crude extract of Aspergillus fumigatus mycelium as an antigen, was positive. A presumptive diagnosis of systemic aspergillosis was established, and the dog was treated with oral ketoconazole (200 mg/8 h) with clinical improvement, although the enzyme-linked immunosorbent assay for anti-Aspergillus remained positive. The dog died suddenly. The postmortem examination of tissue sections from various organs stained with hematoxylin and eosin revealed the presence of abundant branched, septate, hyaline hyphae (see Fig. 6 to 8 of reference 7). A single fungus was isolated from all those examined organs. It was initially identified as Paecilomyces sp., and the case was published (7). Later, the case strain was sent to the Faculty of Medicine of the Rovira i Virgili University in Reus, Spain, for specific identification.

The fungus was inoculated on malt extract agar (MEA) (Oxoid, Basingstoke, England) and oat meal agar (30 g of oat flakes, 1 g of MgSO₄ · 7H₂O, 1.5 g of KH₂PO₄, 15 g of agar, 1,000 ml of tap water), and incubated at 25, 37, and 40°C in the dark. Growth ranges and colony morphology at 25°C were very similar on both media. The colonies reached 11 to 13 mm in diameter after 7 days. They were white to cream colored, cottony at the center, and membranous towards the edge (Fig. 1A). The reverse was colorless, but became pale brown at the center with age. The vegetative hyphae were hyaline, 1 to 2 µm wide, smooth walled, and usually formed strands from which simple conidiogenous cells (phialides) and chlamydospores rose perpendicularly (Fig. 1B and 2A). The phialides were predominantly lateral, hyaline, smooth walled, cylindrical, sometimes centrally swollen, and usually tapered towards the apex, 3 to 15 µm long by 1 to 2.5 µm wide, with a short cylindrical collarette and internal wall thickening at the tip (Fig. 1C and 2B). The conidia were hyaline, smooth walled or slightly rough, pyriform, fusiform, or ellipsoidal, 2.5 to 6.5 µm long by 1.5 to 4 µm wide, and with distinct connectives (narrowed and thickened structures formed by a differentiation of the conidial wall) on both ends and formed coherent chains (Fig. 1C and D and 2C). An abundant production of chlamydospores, often more abundant than conidia, was a typical feature of this fungus. The chlamydospores were solitary, usually arising on short stalks, hyaline, smooth and thick walled, unicellular, globose, subglobose, or obpyriform, occasionally broadly ellipsoidal, and 5 to 8 µm long by 4 to 7 µm wide (Fig. 1D and 2D).

View larger version (137K): [in this window] [in a new window]   FIG. 1. S. chlamydospora IMI 387422. (A) Colonies growing on MEA at 25°C. (B to D) Chlamydospores and phialidic conidiogenous cells with conidia in cohering chains.

View larger version (29K): [in this window] [in a new window]   FIG. 2. S. chlamydospora IMI 387422. (A) Chlamydospores and phialidic conidiogenous cells with conidia in cohering chains on a hyphal strand. (B) Phialidic conidiogenous cells on a vegetative hypha. (C) Conidia with connectives in cohering chains. (D) Chlamydospores arising on short stalks from a vegetative hypha.

Living cultures of the case isolate were deposited in the Faculty of Medicine in Reus (Spain) as FMR 7371, in the CABI Bioscience in Edgham (England) as IMI 387422, and in the Centraalbureau voor Schimmelcultures in Utrecht (The Netherlands) as CBS 109945.

The members of the hyphomycetous genus Sagenomella are mainly characterized by their slow growth on MEA, the predominance of simple phialidic conidiogenous cells, often centrally swollen, and their conidia with connectives on both ends, which form coherent chains. Sagenomella was reported by Gams (5) to accommodate some new species morphologically similar to the genera Acremonium. Additionally, other known Acremonium and Paecilomyces species (4, 10) were transferred to that genus. Some of these species had been previously included in the genus Sagrahamala by Subramanian (13, 14) on the basis of how phialidic conidia cohere to each other to form chains. However, after an accurate study of the conidial chain ontogeny of different hyphomycetes, Gams (5) considered Sagrahamala a synonym of Acremonium. Sagenomella currently encompasses 12 species, including the anamorphs of Sagenoma ryukyuensis and Talaromyces ocotl, two ascomycetes which belong to the family Trichocomaceae (Eurotiales) (3, 5, 8, 15). However, none of the species of Sagenomella already described has the conjunction of morphological features observed in this case strain, and it is therefore described below as a new species. It is so named because of its abundant production of chlamydospores.

Sagenomella chlamydospora Gené et Guarro, sp. nov. Coloniae in vitro albae vel cremeae, gossypinae. Hyphae vegetativae leves, 1 to 2 µm latae. Phialides orthotropicae, plerumque simplices, cylindrico cum apice attanuatum, 3 to 15 by 1 to 2.5 µm. Conidia in catenulis cohaerentibus, hyalina, levia vel leniter aspera, pyriformia, fusiformia vel ellipsoidea, 2.5 to 6.5 by 1.5 to 4 µm. Chlamydosporae copiosae, laterales, hyalinae, aseptatae, plerumquam globosae vel subglobosae, 5 to 8 by 4 to 7 µm.

Holotype. IMI 387422, from disseminated infection in a dog.

Sagenomella alba, Sagenomella sagenomatis, and Sagenomella sclerotialis (5, 12) are the morphologically closest species to S. chlamydospora, but they have smaller conidia and no chlamydospores. In addition, S. alba does not grow at 37°C, S. sagenomatis can grow above 40°C, and S. sclerotialis, although its growth at 37°C is luxuriant, always produces globose sclerotia. The latter structures have never been observed in the new species. The only Sagenomella species that develop chlamydospores are Sagenomella humicola and Sagenomella verticillata, but both form phialides grouped in verticils. The chlamydospores in S. humicola are dark brown, and the ones in S. verticillata are subhyaline to brown and often disposed in chains. Furthermore, neither S. humicola nor S. verticillata grows at 37°C.

The initial identification of the case strain as Paecilomyces sp. seems logical because some of the morphological features (conidia and chlamydospores) of the most common species of the genus, Paecilomyces variotii, are similar to those observed in the present species. Although simple phialides are not typical of P. variotii, the phialidic arrangement in some strains of such species can be greatly influenced by the growth conditions. Occasionally, conidiophores can be reduced to simple phialides. The possibility that the case strain is a host-adapted disgonic P. variotii, analogous to the host-adapted Aspergillus fumigatus often seen in chronic infections, was also considered. P. variotii colonies are typically buff or tan colored and very different from those observed in the present fungus. However, it is possible that if the fungal conidiation is sparse because of host adaptation, the normal colony color, which derives from profuse conidiation, might not be detectable. Several cases of disseminated infection by Paecilomyces spp. in dogs (7) and other animals (2) have been reported. The recognition of a species different from P. variotii, or other fungi of clinical relevance, was confirmed by molecular analysis. The internally transcribed spacer (ITS) regions and 5.8S ribosomal DNA gene of the clinical isolate were sequenced using the ITS-5 and ITS-4 primers (17), and a BLAST sequence homology search was performed in the GenBank database (1). Surprisingly, the closest sequence belonged to a strain of Eupenicillium lassenii (NRRL 5272), another ascomycete of the Trichocomaceae but with a Penicillium anamorph that is morphologically completely different from Sagenomella spp. However, the similarity between the two sequences was very low (91%). The sequence of the case strain was also compared with the sequences of two strains of P. variotii (AF033395 and AF291870) deposited in GenBank, and the similarity values were lower than 86.06%. The sequence of the case strain was deposited in GenBank with the accession number AJ519984.

The in vitro activity of amphotericin B, flucytosine, ketoconazole, itraconazole, ravuconazole, terbinafine, and voriconazole against the case strain was determined by a broth microdilution method (11), mainly following to the guidelines of the National Committee for Clinical Laboratory Standards for molds (9). The test was carried out using RPMI 1640 medium buffered to pH 7 with 0.165 M morpholinepropanesulfonic acid, an inoculum of 10⁴ CFU/ml, an incubation temperature of 30°C, an incubation time of 96 h, and an additive drug dilution procedure. MICs were 1 µg of amphotericin B per ml, 0.12 µg of ketoconazole per ml, 0.25 µg of itraconazole per ml, 128 µg of fluconazole per ml, 0.03 µg of terbinafine per ml, 0.03 µg of ravuconazole per ml, 1 µg of voriconazole per ml, and >128 µg of flucytosine per ml. The dog was unsuccessfully treated with ketoconazole, even though its in vitro antifungal activity was good. When the fungal infection was diagnosed, the infection may already have been widely disseminated in the organism, creating an almost certainly irreversible situation (7).

The detection of fungal infections in animals is difficult. It requires expert clinicians with plenty of experience and considerable knowledge of mycology. Most mycoses can be easily confused with other conditions, since the clinical picture of the animal systemic mycoses is very unspecific, making clinical diagnosis very difficult. In dogs, the first signs are usually peripheral nervous signs, mainly lameness, spinal pain, and lethargy, all as consequences of the mold settling in the spinal column (6, 7). The causative agent needs to be promptly diagnosed and identified, usually by a joint microbiological and histopathological study, if the infection is to be correctly treated and resolved. This is also very important because it reduces the risk of the infection spreading to humans (16).

This study emphasizes how complex and difficult it can be to diagnose and identify the etiologic agent of an infection. In animals, this is still more complicated than in humans because there is less experience and the information available on fungal infections is scarce. In the present case, the fungal origin of the infection was recognized only through a cross-reaction by an anti-Aspergillus ELISA. The symptoms were unspecific, and the fungal origin was not confirmed until the postmortem examination, when a unique fungus was repeatedly isolated from different organs and their histopathological examination revealed the presence of compatible fungal structures. The identification of the causative agent has also been complex because it is the first time that this species has been found and that the genus Sagenomella has been involved in animal infections, including those of humans. Indeed, it is a very rare fungus, characterized by poorly differentiated reproductive structures, which can be easily confused with degenerated better-known fungi, such as species of Paecilomyces or Acremonium. Molecular analysis was useful in reconsidering the initially wrong mycological diagnosis.

	ACKNOWLEDGMENTS

 
We are indebted to R. Summerbell and W. Gams (Centraalbureau voor Schimelcultures, Utrecht, The Netherlands) for their comments on identifying the fungus.

	FOOTNOTES

 
* Corresponding author. Mailing address: Unitat de Microbiologia, Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Carrer Sant Llorenç 21, 43201-Reus, Tarragona, Spain. Phone: 34 977759359. Fax: 34 977759322. E-mail: jgd{at}fmcs.urv.es.

	REFERENCES

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1. Altschul, S. F., T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
2. De Hoog, G. S., J. Guarro, J. Gené, and M. J. Figueras. 2000. Atlas of clinical fungi, 2nd ed. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, and the Rovira i Virgili University, Reus, Spain.
3. Fassatiová, O., and M. Pková. 1990. Sagenomella bohemica Fassatiová et Pková sp. n. (Moniliales). eská Mykol. 44:240-242.
4. Gams, W. 1971. Cephalosporium—artige schimmelpilze (hyphomycetes). Gustav Fischer Verlag, Stuttgart, Germany.
5. Gams, W. 1978. Connected and disconnected chains of phialoconidia and Sagenomella gen. nov. segregated from Acremonium. Persoonia 10:97-112.
6. García, M. E., and J. L. Blanco. 2000. Principales enfermedades fúngicas que afectan a los animales domésticos. Rev. Iberoam. Micol. 17:S2-S7.[Medline]
7. García, M. E., J. Caballero, P. Toni, I. García, E. Martínez de Merlo, E. Rollan, M. González, and J. L. Blanco. 2000. Disseminated mycoses in a dog by Paecilomyces sp. J. Vet. Med. A 47:243-249.[CrossRef]
8. Heredia, G., M. Reyes, R. M. Arias, and G. K. Bills. 2001. Talaromyces ocotl sp. nov. and observations on T. rotundus from conifer forest soils of Veracruz State, Mexico. Mycologia 93:528-540.
9. National Committee for Clinical Laboratory Standards. 1998. Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Proposed standard M 38-P. National Committee for Clinical Laboratory Standards, Wayne, Pa.
10. Onions, A. H. S., and G. L. Barron. 1967. Monophialidic species of Paecilomyces. Mycol. Papers 107:1-25.
11. Pujol, I., J. Guarro, C. Llop, L. Soler, and J. Fernández. 1996. Comparison study of broth macrodilution and microdilution antifungal susceptibility tests for the filamentous fungi. Antimicrob. Agents Chemother. 40:2106-2110.[Abstract]
12. Stolk, A. C., and G. F. Orr. 1974. Sagenoma, a new genus of Eurotiaceae. Mycologia 66:676-680.
13. Subramanian, C. V. 1972. Conidial chains, their nature and significance in the taxonomy of hyphomicetes. Curr. Sci. 41:43-49.
14. Subramanian, C. V. 1977. Revisions of hyphomycetes-I. Kavaka 5:93-98.
15. Ueda, S., and S. Udagawa. 1984. Sagenoma ryukyuensis, a new thermotolerant ascomycete. Mycotaxon 20:499-504.
16. van Cutsem, J., and F. van Rochette. 1991. Mycoses in domestic animals. Janssen Research Foundation, Beerse, Belgium.
17. White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. 315-322. In M. A. Innis, D. H. Geldfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols: a guide to the methods and applications. Academic Press, New York, N.Y.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Gené, J. Articles by Guarro, J. Search for Related Content PubMed PubMed Citation Articles by Gené, J. Articles by Guarro, J.