Diagnostic methods for detection and characterisation of dimorphic fungi causing invasive disease in Africa: development and evaluation
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Date
2020-03
Authors
Maphanga, Tsidiso Gugu
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
Emergomyces, Histoplasma, Blastomyces and Sporothrix species complex are thermally
dimorphic fungi that have emerged as the causative agents of invasive fungal infections among
immunocompetent and immunocompromised patients for the past 12 years. Accurate
diagnoses of the mycoses caused by these fungi are challenging, not only because as there is a
lack of clinical awareness globally, but also because these thermally dimorphic fungi are
difficult to identify in the laboratory by conventional methods (i.e. culture, histology). In
addition, cultured isolates require specialised biosafety facilities, which pose a tremendous
challenge to resource-limited, diagnostic laboratories. As a result, estimates of the prevalence
and burden of disease are poor. In addition, the lack of standardised antifungal susceptibility
testing methods for these pathogens makes it difficult to determine the minimum inhibitory
concentration (MIC), which may jeopardise patient treatment. These challenges indicate the
need for alternative laboratory methods for identification. Furthermore, molecular methods are
required for the correct identification of these pathogens in clinical settings, so that efficient
antifungal agents may be used appropriately.
We conducted several studies to address these challenges. In the first study, 50 Emergomyces
africanus isolates from AIDS patients presenting with disseminated emergomycosis, from both
public and private laboratories in South Africa during a nine-year period (2008 to 2016), were
described and analysed. The Clinical Laboratory Standards Institute guideline (M27-A4 for
yeast phase and M38-A2 for mould phase) was used to determine the MICs for the yeast and
mould phase. Amphotericin B, itraconazole, voriconazole and posaconazole were more potent
in vitro, while fluconazole, echinocandins and flucytosine were less potent agents.
In the second study, we tested and analysed 212 urine samples from patients with suspected
invasive fungal disease submitted to the mycology reference laboratory from August 2014 to
December 2018 by public and private sector laboratories in South Africa. Corresponding fungal
culture and histopathology results from several electronic laboratory information systems were
obtained. Thirty-seven cultured fungal isolates were sent in parallel with the urine specimen
for species-level identification (confirmed by phenotypic and molecular identification
methods). The IMMY monoclonal Histoplasma capsulatum galactomannan enzyme
immunoassay (EIA) had a sensitivity and specificity of respectively 88% and 72% when
compared to culture-confirmed histoplasmosis. In contrast, the EIA showed a sensitivity and
specificity of 83% and 93% when compared to histologically confirmed histoplasmosis/
emergomycosis.
In the third study, we re-evaluated 20 cases of blastomycosis diagnosed among South African
patients between 1967 and 2014. All isolates, initially identified as Blastomyces dermatitidis
by histopathological and/or culture-based methods, were characterised phenotypically,
genotypically and by whole genome sequencing. Morphological characteristics and
phylogenetic analyses of multilocus sequence typing and whole genome sequences revealed
two groups, both of which were closely related to but distinct from B. dermatitidis, and other
related species. One group corresponded to the recently described Blastomyces percursus and
the other group was described as Blastomyces emzantsi sp. nov. Whole genome sequencing
confirmed distinct species identities and the absence of a full ortholog of the BAD-1 gene.
Clinically, extrapulmonary disease was more common than lung involvement.
In the fourth study, 156 dimorphic isolates from immunocompromised patients presenting with
systemic endemic mycoses, from both public and private laboratories in South Africa during
an 11-year period (2008 to 2019), were sent to the mycology reference laboratory for species-
level identification. All isolates were initially identified by deoxyribonucleic acid (DNA)
sequencing of the internal transcribed spacer (ITS) gene and those which failed sequencing
were identified by sequencing of the large subunit (LSU) gene. The identity of the Sporothrix
isolates was confirmed by sequencing the calmodulin gene. Only 63% of the dimorphic
pathogens were correctly identified to genus level by culture-based methods at the diagnostic
laboratories when compared to DNA sequencing methods. At the reference laboratory, culturebased methods correctly identified 95% of the dimorphic isolates. ITS sequencing correctly
identified Emergomyces africanus, Histoplasma capsulatum, Blastomyces percursus,
Emergomyces pasteurianus and Talaromyces marneffei. Calmodulin sequencing confirmed
Sporothrix species and these clinical isolates belonged to Sporothrix schenckii sensu stricto.
There was an increase in the number of cases of emergomycosis, sporotrichosis,
histoplasmosis, blastomycosis and talaromycosis above what was previously reported. Skin
lesions and pulmonary involvement were common.
In conclusion, the in vitro susceptibility data for E. africanus supported the management of
disseminated emergomycosis with amphotericin B, followed by itraconazole, voriconazole, or
posaconazole. Fluconazole was a relatively less potent agent. The Histoplasma EIA provided
a rapid alternative for diagnosis of histoplasmosis in an endemic setting. However, it should be
noted that it may cross-react with patients infected with Emergomyces africanus. We observed
that South African blastomycosis cases were caused by species that are distinct from B.
dermatitidis, B. gilchristii and B. parvus, which might account for clinical differences between
sub-Saharan and North American blastomycosis. Lastly, DNA sequencing was a valuable tool
for accurate identification of dimorphic fungi to species level when compared to culture in the
current setting, which could facilitate early diagnosis and initiation of proper treatment.
However, in settings where DNA sequencing is not available, training of laboratory staff is
necessary for the improvement of diagnosis of these pathogens in South Africa.
Description
Keywords
Thesis (Ph.D. (Medical Microbiology))--University of the Free State, 2020, Medical mycology, Mycoses, Fungi, Fungal infections