Lynne Sigler

DNA and the classical way: Identification of medically important molds in the 21st century

The advent of the 21st century has seen significant advances in the methods and practices used for identification of medically important molds in the clinical microbiology laboratory. Historically, molds have been identified by using observations of colonial and microscopic morphology, along with tables, keys and textbook descriptions. This approach still has value for the identification of many fungal organisms, but requires expertise and can be problematic in determining a species identification that is timely and useful in the management of high-risk patients. For the increasing number of isolates that are uncommon, atypical, or unusual, DNA-based identification methods are being increasingly employed in many clinical laboratories. These methods include the commercially available GenProbe assay, methods based on the polymerase chain reaction such as single-step PCR, RAPD-PCR, rep-PCR, nested PCR, PCR-RFLP, PCR-EIA, and more recent microarray-based, Luminex technology-based, and real-time PCR-based methods. Great variation in assay complexity, targets, and detection methods can be found, and many of these methods have not been widely used or rigorously validated. The increasing availability of DNA sequencing chemistry has made comparative DNA sequence analysis an attractive alternative tool for fungal identification. DNA sequencing methodology can be purchased commercially or developed in-house; such methods display varying degrees of usefulness depending on the breadth and reliability of the databases used for comparison. The future success of sequencing-based approaches will depend on the choice of DNA target, the reliability of the result, and the availability of a validated sequence database for query and comparison. Future studies will be required to determine sequence homology breakpoints and to assess the accuracy of molecular-based species identification in various groups of medically important filamentous fungi. At this time, a polyphasic approach to identification that combines morphologic and molecular methods will ensure the greatest success in the management of patients with fungal infections.

Mycorrhizal and root endophytic fungi of containerized Picea glauca seedlings assessed by rDNA sequence analysis.

Fungi colonizing fine roots of containerized Picea glauca seedlings were assessed in four large conifer nurseries in northern Alberta. PCR amplification of fungal rDNA (internal transcribed spacer and a portion of the 5? end of the large subunit gene) from random samples of fine feeder roots gave between 1 and 4 amplicons per seedling. Amplicons were either separated by electrophoresis and sequenced directly, or cloned and sequenced. The resulting sequences were compared to sequences obtained from cultures established from seedling roots and from GenBank by maximum parsimony analysis. ITS sequences formed 11 distinct clades, each including at least one reference sequence. The ectomycorrhizal basidiomycetes Thelephora americana and Amphinema byssoides were dominant, whereas ascomycetes were less common. Fungi with sequences similar to members of the Heleotiales which form ericoid mycorrhizas were also present. Correspondence analysis revealed strong positive and negative associations among fungal taxa as well as an influence of applied fertilizer level on fungal diversity and species composition.

The Ajellomycetaceae, a new family of vertebrate-associated Onygenales

Phylogenies inferred from the analysis of DNA sequence data have shown that the Onygenales contains clades that do not correspond with previously described families. One lineage identified in recent molecular phylogenetic studies includes the dimorphic pathogens belonging to the genera Ajellomyces, Emmonsia and Paracoccidioides. To evaluate the degree of support for this lineage and determine whether it includes additional taxa, we examined relationships among the members of this clade and selected saprobic onygenalean taxa based on maximum-parsimony analyses of partial nuclear large RNA subunit (LSU) and internal transcribed spacer (ITS) sequences. A clade distinct from the Onygenaceae was found to encompass Ajellomyces (including the anamorph genera Blastomyces, Emmonsia and Histoplasma) and Paracoccidioides brasiliensis. The members of this lineage are saprobic and pathogenic vertebrate-associated taxa distinguished by their globose ascomata with coiled appendages, muricate globose or oblate ascospores, and lack of keratinolytic activity. Anamorphs are solitary aleurioconidia or irregular alternate arthroconidia. Based on molecular data and on morphological and physiological similarities among these taxa, we propose the new family, Ajellomycetaceae.

Molecular Characterization of Reptile Pathogens Currently Known as Members of the Chrysosporium Anamorph of Nannizziopsis vriesii Complex and Relationship with Some Human-Associated Isolates

ABSTRACT In recent years, the Chrysosporium anamorph of Nannizziopsis vriesii (CANV), Chrysosporium guarroi, Chrysosporium ophiodiicola, and Chrysosporium species have been reported as the causes of dermal or deep lesions in reptiles. These infections are contagious and often fatal and affect both captive and wild animals. Forty-nine CANV isolates from reptiles and six isolates from human sources were compared with N. vriesii based on their cultural characteristics and DNA sequence data. Analyses of the sequences of the internal transcribed spacer and small subunit of the nuclear ribosomal gene revealed that the reptile pathogens and human isolates belong in well-supported clades corresponding to three lineages that are distinct from all other taxa within the family Onygenaceae of the order Onygenales. One lineage represents the genus Nannizziopsis and comprises N. vriesii, N. guarroi, and six additional species encompassing isolates from chameleons and geckos, crocodiles, agamid and iguanid lizards, and humans. Two other lineages comprise the genus Ophidiomyces, with the species Ophidiomyces ophiodiicola occurring only in snakes, and Paranannizziopsis gen. nov., with three new species infecting squamates and tuataras. The newly described species are Nannizziopsis dermatitidis, Nannizziopsis crocodili, Nannizziopsis barbata, Nannizziopsis infrequens, Nannizziopsis hominis, Nannizziopsis obscura, Paranannizziopsis australasiensis, Paranannizziopsis californiensis, and Paranannizziopsis crustacea. Chrysosporium longisporum has been reclassified as Paranannizziopsis longispora. N. guarroi causes yellow fungus disease, a common infection in bearded dragons and green iguanas, and O. ophiodiicola is an emerging pathogen of captive and wild snakes. Human-associated species were not recovered from reptiles, and reptile-associated species were recovered only from reptiles, thereby mitigating concerns related to zoonosis.

Geosmithia argillacea: An Emerging Cause of Invasive Mycosis in Human Chronic Granulomatous Disease

BACKGROUND Chronic granulomatous disease (CGD) is an inherited disorder of the nicotinamide adenine dinucleotide phosphate oxidase that leads to defective production of microbicidal superoxide and other oxidative radicals, resulting in increased susceptibility to invasive infections, especially those due to fungi. METHODS Geosmithia argillacea was identified from cultured isolates by genomic sequencing of the internal transcribed spacer region. Isolates previously identified as Paecilomyces variotii, a filamentous fungus closely resembling G. argillacea, were also examined. RESULTS We identified G. argillacea as the cause of invasive mycosis in 7 CGD patients. In 5 cases, the fungus had been previously identified morphologically as P. variotii. All patients had pulmonary lesions; 1 had disseminated lesions following inhalational pneumonia. Infections involved the chest wall and contiguous ribs in 2 patients and disseminated to the brain in 1 patient. Four patients with pneumonia underwent surgical intervention. All patients responded poorly to medical treatment, and 3 died. CONCLUSIONS We report the first cases of invasive mycosis caused by G. argillacea in CGD patients. G. argillacea infections in CGD are often refractory and severe with a high fatality rate. Surgical intervention has been effective in some cases. G. argillacea is a previously underappreciated and frequently misidentified pathogen in CGD that should be excluded when P. variotii is identified morphologically.

Pathogenicity of the Chrysosporium anamorph of Nannizziopsis vriesii for veiled chameleons (Chamaeleo calyptratus)

Veiled chameleons (Chamaeleo calyptratus) were experimentally challenged with the fungus Chrysosporium anamorph of Nannizziopsis vriesii (CANV). Chameleons were exposed to conidia in their captive environment, or were inoculated by direct application of a conidial suspension inoculum on intact and on abraded skin. The CANV induced lesions in all experimental groups and was recovered from infected animals, fulfilling Kochs postulates and confirming that it may act as a primary fungal pathogen in this species of reptile. A breach in cutaneous integrity, as simulated by mild scarification, increased the risk of infection but was not required for the CANV to express pathogenicity. Initial hyphae proliferation occurred in the outer epidermal stratum corneum, with subsequent invasion of the deeper epidermal strata and dermis. A spectrum of lesions was observed ranging from liquefactive necrosis of the epidermis to granulomatous inflammation in the dermis. CANV dermatomycosis appears to be contagious and can readily spread within a reptile collection, either directly through contact with infective arthroconidia or indirectly via fomites. Dense tufts of arthroconidiating hyphae were demonstrated histologically on the skin surface of many animals that developed dermatomycosis, and these arthroconidia may act as infective propagules involved in the transfer of disease between reptiles.

Cutaneous Mycobiota of Captive Squamate Reptiles with Notes on the Scarcity of Chrysosporium Anamorph of Nannizziopsis vriesii

ABSTRACT The Chrysosporium anamorph of Nannizziopsis vriesii (CANV) is a fungus that has been implicated in several recent cases of reptile dermatomycoses. A survey was conducted to investigate whether this fungus was present on the skins of healthy squamate reptiles. Skin was collected as aseptically as possible from actively shedding lizards (n = 36) or from freshly shed snake exuvia (n = 91) and placed on fungal culture media for selective recovery of cycloheximide-tolerant fungi. The CANV was cultured from only one animal, an African rock python, Python sebae. Fungi belonging to 50 genera were identified from 127 reptiles: Aspergillus spp., Penicillium spp., and Paecilomyces lilacinus were most frequently isolated. Keratinophilic fungi isolated from reptiles did not belong to zoophilic or anthropophilic species, inferring that the potential for acquisition of dermatophytosis from handling squamate reptiles is low.

Disseminated fungal infection in a renal transplant recipient involving Macrophomina phaseolina and Scytalidium dimidiatum: case report and review of taxonomic changes among medically important members of the Botryosphaeriaceae

We report the first case of human infection with the fungal plant pathogen Macrophomina phaseolina in a Sri Lankan-born Canadian man following a renal transplant in India. The patient subsequently succumbed to invasive infection with Scytalidium dimidiatum. Molecular sequence analysis confirmed the identification of both fungi and revealed that they are related species within the ascomycete family Botryosphaeriaceae. We review the rationale for the recent reclassification of S. dimidiatum as Neoscytalidium dimidiatum and of Nattrassia mangiferae (formerly considered a synanamorph of S. dimidiatum) as Neofusicoccum mangiferae. This and other recent cases illustrate the potential for plant pathogenic fungi to cause invasive human diseases which are refractory to antifungal therapy.

Yeasts associated with termites: a phenotypic and genotypic characterization and use of coevolution for dating evolutionary radiations in asco- and basidiomycetes

Summary Thirtynine yeast isolates or dimorphic fungi were obtained from the hindgut of the lower termites Mastotermis darwiniensis (Mastotermitidae), Zootermopsis angusticollis, Z. nevadensis (Hodotermitidae), Neotermes jouteli (Kalotermitidae), Reticulitermes santonensis, Heterotermes indicola (Rhinotermitidae) and the roach Cryptocercus punctulatus. Using RAPD-PCR the 39 yeast isolates were assigned to 13 different species. Commonly yeast species were specific to the termite species isolates from. There were only two yeast species which were found in different species of lower termites. Based on phenotypic characters Debaryomyces hansenii showed a high score in four species. The qualitative and quantitative yeast cell wall monosaccharide composition, the ubiquinone system, partial sequencing of 18S ribosomal DNA (bases 1273 to 948; numbering according to the gene of Saccharomyces cerevisiae), and the ultrastructure of septal pores indicate that 11 yeast species belong to the Endomycetales. Although ascospores were lacking, two of these species were identified to belong to the genus Debaryomyces. One remaining yeast isolate was identified as a Sporothrix anamorph representative for the filamentous Ascomycetes (Ophiostomataceae s. str.); the second species showed affinities to the Basidiomycetes in particular to the genus Trichosporon. Comparing an additional 18S rDNA fragment (bases 595 to 993) and RAPD-PCR data using different species type strains of the genus Sporothrix, the filamentous ascomycete was genotypically identified as Sporothrix albicans. Sporothrix. albicans, although phylogenetically closely related to S. schenckii var. schenckii and Ophiostoma stenoceras remains genotypically distinct. An emended species description of S. albicans is presented. Evidence is provided that the yeasts isolated from the hindgut can be considered symbionts.

Toenail infection caused by Onychocola canadensis gen. et sp. nov.

Three cases of great toenail infection are described in which a slow-growing arthroconidial hyphomycete was isolated repeatedly and in pure culture. Direct microscopy revealed hyaline, round to barrel-shaped arthroconidia, hyaline hyphae of varying width, and broad thick-walled brownish hyphae. Three additional isolates were obtained from clinical specimens, for which the results of direct microscopy were unknown or negative. The fungus was resistant to cycloheximide, sensitive to common antifungal drugs by susceptibility tests in vitro and sensitive to benomyl. It was urease positive, hydrolysed casein and tyrosine but not xanthine or hypoxanthine, showed no specific nutritional requirements but grew better on carbohydrate-free media, assimilated 12 carbohydrates and potassium nitrate, and failed to perforate hair. The fungus is described as Onychocola canadensis Sigler gen. et sp. nov., and it is compared to Scytalidium lignicola, Scytalidium hyalinum and the Scytalidium synanamorph of Nattrassia mangiferae (Hendersonula toruloidea).