Human Blastomycosis in South Africa Caused by Blastomyces percursus and Blastomyces emzantsi sp. nov., 1967 to 2014

Abstract

We reevaluated 20 cases of blastomycosis diagnosed in South Africa between 1967 and 2014, with Blastomyces dermatitidis considered to be the etiological agent, in light of newly described species and the use of more advanced technologies. In addition to histopathological and/or culture-based methods, all 20 isolates were phenotypically and genotypically characterized, including multilocus typing of five genes and whole-genome sequencing. Antifungal susceptibility testing was performed as outlined by Clinical and Laboratory Standards Institute documents M27-A3 and M38-A2. We merged laboratory and corresponding clinical case data, where available. Morphological characteristics and phylogenetic analyses of five-gene and whole-genome sequences revealed two groups, both of which were closely related to but distinct from B. dermatitidis, Blastomyces gilchristii, and Blastomyces parvus The first group (n = 12) corresponded to the recently described species Blastomyces percursus, and the other (n = 8) is described here as Blastomyces emzantsi sp. nov. Both species exhibited incomplete conversion to the yeast phase at 37°C and were heterothallic for mating types. All eight B. emzantsi isolates belonged to the α mating type. Whole-genome sequencing confirmed distinct species identities as well as the absence of a full orthologue of the BAD-1 gene. Extrapulmonary (skin or bone) disease, probably resulting from hematogenous spread from a primary lung infection, was more common than pulmonary disease alone. Voriconazole, posaconazole, itraconazole, amphotericin B, and micafungin had the most potent in vitro activity. Over the 5 decades, South African cases of blastomycosis were caused by species that are distinct from B. dermatitidis Increasing clinical awareness and access to simple rapid diagnostics may improve the diagnosis of blastomycosis in resource-limited countries.

Keywords: Blastomyces; South Africa; blastomycosis; emergomycosis; histoplasmosis; mycoses; tuberculosis.

FIG 1

A case of disseminated blastomycosis caused by Blastomyces emzantsi. Shown is purulent discharge from a cutaneous sinus tract at the T5 vertebral level (case 8). (Republished from reference with permission of the publisher.)

FIG 2

Morphology of B. emzantsi. Shown are the saprophytic phase (25°C) on Sabouraud agar after 2 weeks (a, c to k, and o to q) and the intermediate phase (30°C) after 3 weeks on BHI agar with 5% horse blood (b) or Sabouraud dextrose agar (l to n and r to u). (a) Floccose, white colony, with a narrow margin peripheral to the crumpled central areas. (b) At 30°C on BHI agar with 5% horse blood, colonies were gray (darker gray on the reverse), with numerous aerial hyphal tufts and a flattened margin. (c) Two terminal conidia, a developing clavate cell (white arrow), and a clavate cell with secondary septation (black arrow). (d) Hyphal septation (arrowheads) in conidiogenesis and clavate cell formation. (e) Clavate, complanate cells (arrows) clearly distinguishable from developing conidia. (f) Complanate terminal cells (arrows) and a developing terminal conidium (arrowhead). (g) Hyphal cell subtending single, lateral conidia and a terminal, basally septate, clavate cell. (h) The apparent clusters of conidia are usually due to the short pedicels of many of the laterally positioned conidia. (i) Multiple conidia on short secondary conidiophores can be subtended from a single basal cell. (j) Conidial ornamentation is variable, from glabrous to papillate. (k) The single conidium may be lateral or clustered on vesiculate primary conidiophores. (l) Clavate cell between conidiophores, with variously arranged conidia. Some inflation of subtending cells is apparent at an intermediate temperature. (m) Increase in temperature results in conidiophores becoming increasingly ampulliform and vesiculate, with distended hyphal cells. Light microscopy creates the illusion that one of each of the laterally paired conidia is sessile. (n) Expanded primary conidiophore with two conidia on extremely short secondary conidiophores. (o) Helical hyphae, one with both developing clavate cells (black arrow) and conidia (white arrow). (p) Numerous conidia developing on hyphal gyres, with bundles of older hyphae behind them. (q) Abundant verrucose conidia and hyphae aggregated into rope-like structures. (r) Development of an adiaspore-like cell (inflated terminal conidia). (s) Sectioned material providing accurate imaging of the diversity of cell forms, including the development of giant cells (*) from enlarged, fragmented hyphal cells; increased vacuolation of enlarging cells; numerous thin hyphal profiles; and some thick-walled cells (arrow) (tangentially sectioned). (t) Section through the hyphal bundles seen by light and scanning microscopy revealing numerous endo/intrahyphal profiles. (u) An inflated intercalary cell suggestive of a chlamydospore. Bars, 5 μm (c, d, k to o, q, s, and u) and 2 μm (e to j, p, r, and t).

FIG 3

Morphology of B. emzantsi at 37°C. (a to e) Cultured isolates on BHI agar at 4 weeks; (f) Grocott’s stain of a paravertebral pus smear; (g and i to l) hematoxylin-and-eosin-stained section through the sinus tissue of a vertebral abscess; (h) potassium hydroxide stain of pus. (a) A beige, butyrous, cerebriform colony still with some white, aerial hyphal tufts. (b) Incomplete transformation in culture typified by large, thick-walled, yeastlike cells juxtaposed to hyphal fragments. (c) Thick-walled giant cell filled with cytoplasm (indicative of an active, viable cell). The slight disruption in the cell wall (arrow) may be the beginning of a budding process. (d) Section through one of the large, thick-walled cells filled with cytoplasm. A number of thin areas in the cell wall (arrows) are suggestive of the initiation of multiple budding sites. Note the many vegetative hyphal profiles surrounding the giant cell. (e) Budding yeastlike cells with a smaller lateral cell, from a cultured isolate. (f) Similar configuration of cells as in panel e but photographed from the clinical specimen. (g) Section through infected sinus tissue, again illustrating yeastlike budding cells with a smaller laterally positioned cell. (h) Image showing a typical broad-based budding yeast cell. (i) A number of budding, thick-walled cells, sectioned through various planes. It appears that one of the daughter cells (arrow) has penetrated the tissue around the phagocytic vacuole. (j) Although budding cells are apparent in the section (arrow), other budding-cell profiles appear to contain hyphal cells (arrowheads), as the cytoplasm is delimited by a cell wall on the inside of the host vacuole. (k) Hyphal invasion of surrounding tissue from a daughter cell within the phagocytic vacuole. (l) Hypha-like extrusion of a thicker-walled cell into surrounding sinus tissue. Bars, 2 μm (b, d to i, and k) and 3 μm (c, j, and l).

FIG 4

Multigene phylogenies of B. percursus (group 1) (SA-NICD-01 to SA-NICD-08, SA-NICD-17, and SA-NICD-18) and B. emzantsi (group 2) (SA-NICD-09 to SA-NICD-16). (a and b) Maximum likelihood tree inferred from the ITS gene and concatenated gene alignment of ITS2–LSU–PRP8–β-tubulin–actin based on 1,000 replicates. (a) Sequences of Paracoccidioides lutzii, Paracoccidioides brasiliensis, Blastomyces dermatitidis, Histoplasma capsulatum, and Emergomyces africanus from Kenyon et al. (22) and of Blastomyces silverae, Blastomyces helicus, Blastomyces parvus, and Emmonsia sola from Jiang et al. (8). (c) Maximum likelihood tree inferred from SNPs, including three annotated genome assemblies of Blastomyces species (B. dermatitidis ER3, B. parvus UAMH 130, and B. percursus BP222). All nodes were supported by 100% of bootstrap replicates. The boxes indicate the mating type for each Blastomyces species. (d) Geographic distribution of B. percursus and B. emzantsi cases in South African provinces.