Medical Mycology, 2019, 57, S259–S266

doi: 10.1093/mmy/myy159
Review Article

Review Article
Recent advances and novel approaches in laboratory-based

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diagnostic mycology
P. Lewis White∗
Mycology Reference Laboratory, Public Health Wales, Microbiology Cardiff, Cardiff, United Kingdom
∗
To whom correspondence should be addressed. Dr. P. Lewis White, PhD, Mycology Reference Laboratory, Public Health Wales,
Microbiology Cardiff,Cardiff, UK. CF14 4XW. Tel: +44 029 2074 6581; E-mail: lewis.white@wales.nhs.uk
Received 8 August 2018; Revised 29 November 2018; Accepted 20 December 2018; Editorial Decision 9 December 2018

Abstract
The field of diagnostic mycology represents much more than culture and microscopy and is rapidly em-
bracing novel techniques and strategies to help overcome the limitations of conventional approaches. Com-
mercial molecular assays increase the applicability of PCR testing and may identify markers of antifungal
resistance, which are of great clinical concern. Lateral flow assays simplify testing and turn-around time,
with potential for point of care testing, while proximity ligation assays embrace the sensitivity of molecular
testing with the specificity of antibody detection. The first evidence of patient risk stratification is being
described and together with the era of next generation sequencing represents an exciting time in mycology.

Key words: fungi, proximity ligation assay, T2 Candida, lateral flow assay, next generation sequencing, risk stratification.

Introduction acceptance has been hindered by lack of methodological stan-

dardization and a limited range of commercially available assays.
In recent years there have been many developments in the field
In recent years, a range of commercial polymerase chain reaction
of mycology. The standardization of molecular tests and the
(PCR) tests to aid in the diagnosis of aspergillosis, candidiasis,
development of novel commercial assays permit the introduc-
and Pneumocystis pneumonia (PcP) have been released.1 Fur-
tion of such tests outside of specialist reference facilities. The
thermore, the efforts of the European Aspergillus PCR initiative
appearance of antifungal resistance, in fungi most commonly as-
(EACPRI) have standardized nucleic acid extraction protocols
sociated with disease (e.g., Candida and Aspergillus spp.), is of
for Aspergillus PCR testing, and the group has expanded (re-
great concern, and strategies to monitor for this are emerging.
branded as the Fungal PCR initiative (FPCRI) www.fpcri.eu)
Predictive models attempting to stratify patients according to the
to standardize molecular methods for the detection of Candida,
risk of developing disease are being described, with the poten-
PcP, Mucorales species, and for the testing of tissue samples.2
tial to provide personalized medicine and preempt overt disease.
By combining methodological recommendations with commer-
Novel technology in the form of next generation sequencing is
cially manufactured and quality controlled assays, testing can be
providing us with a greater understanding of the mycobiome,
fully standardized, and performance monitored by participation
evolution of the organism, interactions with the host, resistance
in external quality assurance schemes (e.g., quality control for
mechanisms, and potential novel antifungal targets. This review
molecular diagnostics [QCMD] for Aspergillus, Pneumocystis,
will describe some of the recent developments with potential in
and Candida PCR). A summary of the performance of commer-
the field of laboratory based diagnostic mycology.
cially available assays for the detection of Aspergillus is shown
in Table 1.
Molecular diagnostics The release of the T2Candida assay represented a major step
While molecular assays for the detection of fungal DNA in clin- forward for the diagnosis of candidosis. The fully automated
ical samples have been described for over two decades, their process detects C. albicans/C. tropicalis, C. glabrata/C. krusei,


C The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. S259
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S260
Table 1. A summary of the performance of various commercial PCR tests for the detection of Aspergillus DNA.
Assay Sample type
Microgen MycAssay Aspergillus∗ BAL
Serum/Plasma
Pathonostics Aspergenius BAL
Serum/Plasma
Ademtech Mycogenie BAL
Serum/Plasma
Bruker Fungiplex Aspergillus Serum/Plasma
OLM AspID BAL
∗
Previously Myconostica MycAssay Aspergillus.
and C. parapsilosis, accountable for >95% of the cases of can-
didemia and tests EDTA blood using the T2Dx platform. It has
the potential to overcome the limitations of culture, providing a
rapid time to results (<5 h) and high sensitivity when testing con-
trived samples (92%).3 In the initial DIRECT study there was
only a limited number of clinical cases (n = 7: 6 candidemias
and 1 intra-abdominal abscess), the T2Candida assay missed
two cases, both of which had received prior antifungal therapy
(AFT). Conversely, there were 29 T2Candida positive cases that
were negative by routine blood culture, all but one was consid-
ered false positive, when using culture as the reference method.
Given the specificity of the T2Candida assay (98%) and limited
sensitivity of culture, it is feasible that these represented false
negative culture results.3 Indeed, for sepsis patients in intensive
care unit (ICU) (>3 days) the positive predictive values of a
positive T2Candida result would be 67%, based on a pretest
probability (incidence) of 3%.4 The follow-up DIRECT2 trial
evaluated performance on 152 patients with candidemia across
14 US centers, sensitivity was 89% when testing samples taken,
on average, 56 hours after the initial diagnosis, and T2Candida
positivity (45%) was significantly greater than the companion
secondary blood culture sample (24%).5 A similar finding was
seen in the STAMP study, where post initiation of therapy for
documented candidemia, T2Candida positivity exceeded that of
blood culture when monitoring clearance.6 Both of these stud-
ies indicate a role for the T2Candida in determining prognosis,
adequate source control, treatment de-escalation and optimal
duration. Although being a molecular test the T2Candida as-
say will detect nonviable candidal cells, and the implications
of this must be considered. However, the more likely role for
the test will be for excluding candidosis based on its excel-
lent negative predictive value (NPV: 98–>99.9%).4 This can
be used to limit unnecessary AFT, which is commonplace in
ICU settings and help offset the high cost associated with per-
forming the T2Candida test, albeit this may be limited with the
availability of generic caspofungin, and in centers that primar-
ily use fluconazole.7 Furthermore, performance of the assay in
the MADRID prospective observational study showed limited
Medical Mycology, 2019, Vol. 57, No. S3
Sensitivity range (%) Specificity range (%) References
80–100 88–99 49–52
25–60 83–100 53–55
42–84 80–91 10, 56
79–80 78–91 11, 57
54–93 90 58, 59
100 85 58
95 90 60
94 77 61
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sensitivity (36%)/NPV (80%) under the influence of empirical
AFT, whereas specificity/positive predictive value was excellent
(100%), indicating a role better suited to confirming a diagnosis
or persistent infection. To make testing cost-effective, stratifi-
cation of high-risk patients through risk-prediction modeling is
essential to achieve a sufficient pre-test probability. Irrespective
of the prevalence of disease the negative predictive value of the
T2 test is >98%, but a prevalence of around 10% may be opti-
mal, providing PPV and NPV of approximately 82% and 99%,
respectively.4
Antifungal resistance
Of great clinical concern is the emergence of fungal disease that
is resistant to AFT. Whether this is cases caused by a relatively
novel pathogen (e.g., Candida auris), species that are inherently
resistant to a particular AFT (e.g., Aspergillus terreus and am-
photericin B) or the appearance of resistant strains of commonly
encountered species (e.g., azole resistance in A. fumigatus) the
use of culture has, until lately, provided the only route in identi-
fying possible resistance. The limited sensitivity of culture, cou-
pled with the delay in turn-around time (TAT) has the potential
to negatively affect patient prognosis, and delays in initiating
appropriate AFT have been associated with increased mortal-
ity.8,9 Molecular techniques are the only nonculture procedures
with the potential to identify markers associated with resistance.
The US Centers for Disease Control and Prevention (CDC) in
collaboration with T2 Biosystems have developed an assay spe-
cific for the detection of C. auris, with the aim of modifying the
system to process skin and environmental swabs while maintain-
ing full automation. Commercial assays that detect mutations in
genes (e.g., cyp51A in A. fumigatus, or dihydropteroate synthase
[DHPS] in Pneumocystis jirovecii) that encode the proteins tar-
geted by AFT, permit potential resistance to be detected without
the need for culture, possibly enhancing sensitivity and TAT, al-
beit in the absence of MIC data. For azole resistant aspergillosis,
the Pathonostics Aspergenius R
is capable of detecting the most
prevalent mutations conferring azole resistance (TR34/L98H,
White
TR46/Y121F, and TR46/T289A) in the CYP51A gene of
A. fumigatus. When testing bronchoalveolar lavage (BAL) fluid
from 11 proven/probable cases, eight mutation profiles were de-
termined direct from the clinical sample.10 When testing serum
seven cases (50%) of invasive aspergillosis (IA) had at least
one genetic region potentially associated with azole-resistance
successfully amplified, although no resistance markers were de-
tected, and all were culture negative.11 For PcP, the commer-
cially produced Pathonostics PneumoGenius R
has been devel-
oped for the detection of Pneumocystis jirovecii and DHPS
mutations associated with resistance to sulfa-based drugs such
as sulfamethoxazole and dapsone.12 While the sensitivity and
specificity of 70% and 82%, respectively, were disappointing
for a PcP PCR system, the assay was able to screen for sulfa-
resistance direct from 89 samples and showed a 4.5% resis-
tance rate.12 However, the clinical utility of such testing is yet to
proven, given that most cases many cases with DHPS mutations
have been successfully treated with high-dose trimethoprim-
sulfamethoxazole.13
Novel assays
Lateral flow devices for the detection of fungal
disease
The lateral flow device (LFD) for the detection of Cryptococ-
cal antigen (CrAg) has significantly improved the microbiolog-
ical diagnosis of cryptococcosis. Quick to perform, requiring
basic laboratory equipment and limited training its clinical per-
formance epitomizes what a near patient or point of care test
(POCT) should be. A meta-analyses generated excellent sensitiv-
ity and specificity of 98% when testing serum, and 99% when
testing cerebral spinal fluid, meaning the assay can be used to
both confidently rule in or exclude a diagnosis of cryptococcosis
using either sample type.14
The development of a LFD of aspergillosis represented an ex-
citing development.15 The assay utilizes the specific monoclonal
antibody MAb JF5 that is specific for an antigen secreted by
the growing Aspergillus hyphal tip. As with the CrAg LFD it
is a technically simplistic and rapid, limiting the need for spe-
cialist equipment and expertise, but to date the performance
has been variable. A meta-analytical review of the LFD gener-
ated pooled sensitivity and specificity of 68% and 87%, respec-
tively, when testing serum.16 When testing BAL fluid, perfor-
mance was improved with a pooled sensitivity and specificity
and 86% and 93%, respectively.16 The improved performance
when testing BAL fluid complements the likely optimal strategy
for using the LFD to provide rapid confirmation of clinically sus-
pected disease, suited to a POCT approach. The testing of serum
has provided less consistent performance and requires sample
pre-treatment that limit its use as a POCT. More so, the test-
ing of serum/plasma is better integrated into a high frequency
screening pathway of at-risk patients, and the LFD is not suited
S261
to testing large batches of samples, where automated enzyme-
linked immunosorbent assay (ELISA) platforms should be used.
One important fact when considering LFD performance, is that
most evaluations to date have been performed using the proto-
type version, and it is hoped that consistency will be gained
by the recent (2017) release of a CE marked version of the
assay.
The release of the galactomannan (GM) lateral flow as-
say has also shown good agreement with the GM ELISA as-
say when testing serum (95%) and BAL fluid (92%), but
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clinical validity is lacking (http://www.immy.com/aspgergillus
galactomannan lfa). The detection of urine antigens specific to
Aspergillus using a lateral flow assay optimized with the galacto-
furanose specific monoclonal antibody (mAb476) generated sen-
sitivity and specificity of 80% and 92%, respectively.17
Proximity ligation assay
A proximity ligation assay (PLA) to aid in the diagnosis of as-
pergillosis has been developed.18 PLA combines the specificity of
antibody detection (utilizing the Aspergillus specific monoclonal
antibody JF5, already exploited in the Aspergillus LFD) with the
sensitivity of a molecular approach. Two biotinylated proxim-
ity probes (A + B) based on the JF5 antibody, target the anti-
genic mannoproteins and are linked to two different, noncom-
plementary streptavidin labeled DNA oligonucleotides. Binding
of probes to adjacent epitopes brings the oligonucleotides to-
gether to form a single DNA strand that can be amplified and
detected by qPCR.18
Analytical analysis confirmed the accuracy for Aspergillus
species and generated a limit of detection of 4 ng/ml. Using con-
trived samples, spiked with Aspergillus culture filtrate prepa-
ration the detection limit of the PLA was 100- and 10-fold
greater than the LFD and GM, respectively, when testing serum.
A limited clinical evaluation testing BAL fluid from four cases
of proven IA and 10 control patients generated sensitivity and
specificity of 100%.18 When released as a commercially avail-
able product it is hoped that performance will meet expectations.
Until then we will not know whether the PLA will help overcome
the limitations of other current biomarkers (e.g., false negativity
associated with prior AFT usage or false positivity associated
with enhanced analytical sensitivity).
Pan-fungal biomarker
The discovery of a pan-fungal dihexasaccharide is particularly
important given its potential as a biomarker for cases of mu-
cormycosis.19 Of further benefit is that detection of the disac-
charide is accomplished using matrix-assisted laser desorption
ionization (MALDI) mass spectrometry (MS). Despite being an
expensive piece of equipment, MALDI-MS is commonplace in
microbiology departments in developed countries, and testing is
S262
cheap and simple to perform, although evaluation across the
range of manufacturers is required. For the diagnosis of as-
pergillosis detection of the disaccharide generated a sensitivity
and specificity of 93% and 76%, respectively, and in 40% (6/15)
of patients the MALDI-MS was positive prior to GM.19 The
sensitivity and specificity of the MALDI-MS for the diagnosis
of invasive candidosis was 83% and 69%, respectively, and for
mucormycosis the sensitivity was 90%. Further evaluations to
determine the robustness of this approach and determine accu-
rate clinical performance are underway.
Exhalation breath tests for aspergillosis
The detection of volatile organic compounds (VOC) in exhaled
air using electronic nose (eNose) technology represents nonin-
trusive mycological test to aid in the diagnosis of aspergillosis
and possibly other respiratory fungal pathogens. It cannot be
considered a novel approach, given in 2008 it was confirmed
that the VOC 2-Pentylfuran was consistently produced by As-
pergillus fumigatus, A, flavus, A. terreus, and Fusarium spp.
but not produced by most bacteria, with the exception of Step-
tococcus pneumoniae.20 When evaluating exhaled air-samples
from cystic fibrosis (CF) patients, 2-Pentylfuran was detected
from 4/4 patients with known Aspergillus colonization, 3/7 pa-
tients with no evidence of Aspergillus colonization/infection, and
was absent from 10/10 healthy control subjects.20 A follow-
up study confirmed that 2-Pentylfuran was absent from the
breath of healthy individuals (0/14) and was more evident in
patients with known lung disease (16/32).21 When using per-
sistent culture of Aspergillus from sputum or BAL fluid as a
reference, the sensitivity and specificity of 2-Pentylfuran detec-
tion was 77% and 78%, respectively. While these two stud-
ies showed that 2-Pentylfuran is not readily detected in breath
from a healthy individual, it is present in food products, such
as asparagus and coffee, and is a by-product of linoleic acid
degradation, which itself is present in olive and canola oil
and 2-Pentylfuran could contaminate human breath post inges-
tion/digestion.21 Furthermore, its presence in human cell mem-
branes could provide a source in breath, after release from hu-
man cells as a result of lung infection/inflammation not caused by
aspergillosis.21
In 2013, the application of eNose technology, with the capac-
ity to generate a pattern of sensor signals (breathprint) unique to
an odor, and subsequent VOC, was evaluated for the diagnosis
of aspergillosis.22 When evaluating a limited number (five cases
of probable IA and six control subjects) of neutropenic hema-
tology patients the overall accuracy of the eNose system was
91% (Se: 100%, Sp: 83%). In a second study of 27 CF patients,
where nine patients were colonized with Aspergillus, the overall
accuracy was 89% (Se: 78%, Sp: 94%).23 While eNose technol-
ogy can identify the presence of Aspergillus within the patient,
through generation of a unique breathprint it cannot elucidate
Medical Mycology, 2019, Vol. 57, No. S3
the individual VOC that are used to differentiate cases from
controls. Studies of eNose technology to assist in the diagno-
sis of aspergillosis are also limited, and the subsequent number
of patients evaluated is too low to provide a definitive under-
standing of their role in the diagnosis of fungal disease. Given
the noninvasive nature and simplicity of testing, it is unclear why
this process has not been evaluated further but may be a result of
inconsistent nature of individual breathprints that cannot easily
be replicated across centers.
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Novel approaches
Environmental surveillance for azole resistance in
Aspergillus fumigatus
With Aspergillus fumigatus primarily an environmental organ-
ism, knowing the level of environmental driven azole resistance
becomes critical to management of aspergillosis. The current
recommended front-line therapy remains voriconazole, and effi-
cacious prophylactic regimens utilize posaconazole.24 The major
environmentally mutation associated with azole resistance in A.
fumigatus (TR34 /L98H) infers pan-azole resistance, and the mor-
tality with resistant IA disease is high (88%).25,26 Even recently
licensed azole formulations, such as isavuconazole, have shown
reduced efficacy on cases caused by A. fumigatus isolates har-
boring these resistance mechanisms.27,28
In cases of IA the recovery of A. fumigatus culture is low,
with most cases diagnosed using nonculture technology (GM/(1-
3)-β-D-Glucan (BDG)) in combination with typical clinical man-
ifestations. This results in a clinical dilemma, where identifying
resistance in precluded by the absence of an organism for suscep-
tibility testing. While molecular techniques can identify a limited
range of potential mutations by direct testing of the sample, per-
formance outside the testing of BAL fluid is limited.
Awareness of the local azole resistance rates through envi-
ronmental monitoring is critical and centres caring for patients
at risk of aspergillosis should perform surveillance to determine
their local epidemiology.25 This can be performed through soil
sampling of agricultural, rural, urban, and healthcare settings,
while also performing air-sampling in healthcare settings. Iso-
lates can be screened for azole resistance using VIP agar plates
supplemented with itraconazole (4 mg/l), voriconazole (2 mg/l),
and posaconazole (0.5 mg/l).29,30
Internationally defined thresholds of environmental resistance
have been proposed to adjust primary therapy accordingly.31
Where resistance is low (<5%), voriconazole should be admin-
istered, unless subsequent investigations yield an azole resistant
clinical isolate. If environmental resistance rates were 5–<10%,
then opinion was divided on persisting with voriconazole alone,
or adding an echinocandin, or changing to liposomal ampho-
tericin. In a scenario where environmental resistance rates were
≥10% therapy, in the in the absence of clinical susceptibility
White
data, a switch to voriconazole plus an echinocandin or to lipo-
somal amphotericin is recommended.31
Combination testing
It would appear that the best strategy for the optimal diagnosis
of invasive fungal disease involves combining mycological tests.
For IA, it was clear that no individual test was optimal when per-
forming PCR, GM, and BDG on blood from an animal model.32
While BDG did generate 100% specificity, its sensitivity was
only 46%, and BDG positivity did not occur until later into the
course of the disease. By combining a range of two diagnostic
tests, disease could be accurately diagnosed if both tests were
positive, but only the combination of whole blood PCR with
GM could confidently exclude disease if both were negative.32
The combination of PCR, with GM and BDG permitted dis-
ease to be confirmed or excluded if all were positive or negative,
respectively.
For the combined GM/PCR diagnosis of IA in the adult
haematology setting there is significant evidence, including
multicenter randomized controlled trials and evaluations of
prospective routine practice.33,34 When comparing combined
GM/Aspergillus PCR against culture and histology the non-
culture arm provided an effective strategy for excluding dis-
ease, reducing the use of empirical AFT (17%, P = 0.002).33
Combined Aspergillus PCR/GM surveillance, compared with
GM alone, reduced unnecessary empirical AFT, reduced time
to diagnosis and cases of proven/probable IA.35 Meta-analysis
confirmed that if both PCR and GM were consistently neg-
ative, then the sensitivity (99%) was sufficient to exclude to
IA, whereas the specificity when both assays were positive was
98%.36
Guidelines for the diagnosis of PcP in the adult haematol-
ogy population suggest a diagnostic algorithm involving real-
time PCR and immunofluorescent microscopy (IF) of BAL.13
When both are positive, a diagnosis of PCR is confirmed and
vice versa.13 If the tests are discordant, then the PCR result is
favoured, questioning the relevance of IF. When BAL samples
are not available, (1-3)-β-D-Glucan (BDG) testing of serum is
recommended, where excellent sensitivity allows negativity to
exclude PcP, but positivity should be confirmed by PCR (or IF)
testing of less invasive respiratory samples.13 A combination of
PCR on upper respiratory tract samples with BDG on serum
may permit PcP to be both confidently diagnosed or excluded
without the need for bronchoscopy, but prospective evaluation
of this strategy is required.
For candidiasis a range of tests are available (PCR, BDG,
Candida antibody, Candida antigen, and conventional mycol-
ogy). While there have been several studies evaluating certain
combination of tests, there is insufficient data to confirm the opti-
mal combination strategy, particularly involving the T2Candida
assay.37–39 The recently funded UK Health Technology Assess-
S263
ment, the A-STOP study, may go some way in providing an
answer.
Predicting the probability of invasive aspergillosis
Many patients share clinical risk and host factors, receive similar
clinical management and with exposure to Aspergillus inevitable,
it is not clear why only certain patients develop IA. Genetic risk
likely plays a role, and single-nucleotide polymorphisms (SNPs)
in genes that code for the C-type lectin receptors have been
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associated with IA.40 Clinical risk factors for IA are well de-
scribed (e.g., allogeneic SCT, long term neutropenia, graft vs
host disease, prolonged high-dose corticosteroid use), but little
has been done to assign a definitive probability of disease asso-
ciated with an individual marker or a combination of clinical,
genetic, and biomarker tests (e.g., GM-EIA, Aspergillus PCR). A
definitive probability of risk could provide thresholds necessary
to commence AFT or to classify patients for different manage-
ment strategies.
A recent proof of concept study combined clinical risk with
genetic markers associated with IA in Dectin-1 and DC-SIGN
and Aspergillus PCR assay in an attempt to stratify 322 neu-
tropenic haematology patients according to the probability of
developing IA.41 Approximately 50% of all patients were at low
risk of developing IA (≤5% probability), and 8% were con-
sidered at high risk (>50%). Only 2% of patients who had not
received an allogeneic SCT had a risk of IA exceeding 50%, com-
pared to 44% of patients who had received an allogeneic SCT.
In patients with no risk factors, the probability of developing
IA was 2.4%, compared to 79.1% in patients with four or more
factors.41 Further research is critical, involving more clinical and
genetic risk factors, along with prospective evaluation. Advances
in both genomic technology (e.g., next generation sequencing)
and IT will permit the development of responsive, portable, and
easy to use software applications that could provide real-time
personal medicine.
The role of next generation sequencing
Next generation sequencing (NGS) is advancing all fields of sci-
entific research. The field of mycology research is focusing on
epidemiological and evolutionary investigations but also investi-
gating mechanisms of resistance and the mycobiome.42–44 NGS
has been used for high-resolution analysis of the cyp51A gene
in order to identify SNPs associated with resistance to azole an-
tifungals in A. fumigatus and perform evolutionary analysis.43
SNP analysis confirmed that the TR34 /L98H mutation to be the
most common cause of azole resistance. Evolutionary investi-
gations showed that degree of genetic diversity in A. fumiga-
tus within a single country was significant, indeed comparable
to that found between continents, with the exception of India
where it appeared that all resistant isolates were significantly
S264
related, suggesting a selective sweep of a highly fit resistant
genotype containing the mutation.43 NGS has also been used to
investigate strains of azole and echinocandin resistance in Can-
dida species.45 Six genes commonly associated with antifungal
resistance (ERG11, ERG3, TAC1, CgPDR1, FKS1, and FKS2)
were analyzed by NGS, and 91 SNPs were detected, including six
novel coding SNPs were identified. Confirming the applicability
of NGS for genome wide detection of resistance mechanisms in
clinical strains subjected to multidrug pressure.45
Determining an accurate mycobiome in the respiratory tract
will aid us in understanding the interactions between the host and
the microbiome, and organisms within the microbiome, whether
fungal, bacterial, or viral. Molecular assays, including NGS can
overcome some of the limitations of culture based approaches
which fail to detect the entire microbiological population and
limit our understanding.46 In a review of investigations of the
mycobiome of the lower respiratory tract using molecular meth-
ods, Candida species were the dominant fungi, something that
was already determined by cultured based investigations.46 With
Candida species accepted as commensals of the respiratory mu-
cosal membranes, their clinical significance has been questioned
in this setting, despite their abundance. The performance of pan-
fungal molecular methods may be affected by population diver-
sity, where an overwhelming presence of a single species may use
up excessive amounts of molecular reagents, limiting the detec-
tion of less evident but clinically relevant fungi. While pan-fungal
detection is critical to mycobiome studies, these strategies may
need to be adjusted to allow application of NGS in the clinical
setting, while taking into account the knowledge that a reduc-
tion in mycological diversity/richness may be associated with
disease.47
A further application of NGS will involve the genetic screen-
ing susceptible patients for mutations that are associated with a
specific fungal disease. The predictive modeling mentioned above
involved screening for five SNPs previously associated with IA.41
The number of host mutations associated with aspergillosis is far
in excess of this, and it is likely that this list is far from com-
plete. Microarrays and genome wide SNP analysis have been
performed to identify genetic risk factors, but the development
of NGS provides a more convenient and encompassing method
that could be applied to clinical practice and enhance personal-
ized medicine.
Concluding comments
The availability of commercial molecular tests to assist in the
diagnosis of IFD will enhance the appeal of fungal PCR, making
it comparable with antigen testing in respect to methodological
standardization, performance robustness, and quality control.
Given PCR performance in the absence of commercial options
was comparable to that of commercial antigen tests, it is feasible
that the overall performance of molecular testing may be en-
Medical Mycology, 2019, Vol. 57, No. S3
hanced in the commercial era.48 The development of commercial
molecular tests for the detection of resistant species (e.g., Can-
dida auris) or genetic markers of resistance (e.g., TR34/L98H
in A. fumigatus) may enhance the identification of difficult to
manage cases by overcoming the limitations of conventional my-
cological techniques. Nevertheless, the diagnosis of IFD will be
best achieved through the combination of diagnostics tests, in-
cluding culture, and together with risk factor stratification and
screening for genetic markers of host susceptibility may allow
us to predict individuals at greatest risk of disease. The initial
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clinical validity of most novel tests can be biased by the test-
ing of patient cohorts with artificially large case numbers (i.e.,
pretest probability), and most assays still require validation in
real-life prospective settings. NGS, whether applied direct to the
host, to specimens from the host, or directly to the organism
will, no doubt, prove invaluable over the next decade. With
many fungi being primarily environmental organisms and with
the knowledge that clinical resistant disease can be driven by
environmental/agricultural practices, we must not solely focus
research on the clinic.
Declaration of interest
The author reports no conflict of interest. The author alone is responsible
for the content and the writing of the paper.
References
1. White PL, Barnes RA. Molecular diagnosis of fungal disease. In Kibbler CC,
Barton R, Gow NAR, Howell S, MacCallum DM, Manuel RJ, eds. Oxford
Textbook of Medical Mycology. Oxford: Oxford University Press, 2018.
2. Barnes RA, White PL, Morton CO et al. 2018. Diagnosis of aspergillosis by PCR:
clinical considerations and technical tips. Med Mycol. 56: 60–72.
3. Mylonakis E, Clancy CJ, Ostrosky-Zeichner L et al. T2 magnetic resonance assay
for the rapid diagnosis of candidemia in whole blood: a clinical trial. Clin Infect
Dis. 2015; 60: 892–899.
4. Clancy CJ, Nguyen MH. T2 magnetic resonance for the diagnosis of bloodstream
infections: charting a path forward. J Antimicrob Chemother. 2018; 73: iv2–iv5.
5. Clancy CJ, Pappas PG, Vazquez J et al. Detecting infections rapidly and easily
for Candidemia Trial, Part 2 (DIRECT2): a prospective, multicenter study of the
T2Candida Panel. Clin Infect Dis. 2018; 66: 1678–1686.
6. Mylonakis E, Zacharioudakis IM, Clancy CJ, Nguyen MH, Pappas PG. Efficacy
of T2 magnetic resonance assay in monitoring candidemia after initiation of
antifungal therapy: the Serial Therapeutic and Antifungal Monitoring Protocol
(STAMP) Trial. J Clin Microbiol. 2018; 56. pii: e01756–17.
7. Harrison D, Muskett H, Harvey S et al. Development and validation of a risk
model for identification of non-neutropenic, critically ill adult patients at high
risk of invasive Candida infection: the Fungal Infection Risk Evaluation (FIRE)
study. Health Technol Assess. 2013; 17: 1–156.
8. Hope WW, Petraitis V, Petraitiene Rx, Aghamolla T, Bacher J, Walsh TJ. The
initial 96 hours of invasive pulmonary aspergillosis: histopathology, comparative
kinetics of galactomannan and (1→3) β-d-glucan and consequences of delayed
antifungal therapy. Antimicrob Agents Chemother. 2010; 54:4879–4886.
9. Garey KW, Rege M, Pai MP et al. Time to initiation of fluconazole therapy
impacts mortality in patients with candidemia: a multi-institutional study. Clin
Infect Dis. 2006; 43: 25–31.
10. Chong GL, van de Sande WW, Dingemans GJ et al. Validation of a new As-
pergillus real-time PCR assay for direct detection of Aspergillus and azole resis-
tance of Aspergillus fumigatus on bronchoalveolar lavage fluid. J Clin Microbiol.
2015; 53: 868–874.
White
11. White PL, Posso RB, Barnes RA. Analytical and clinical evaluation of the
PathoNostics AsperGenius Assay for detection of invasive aspergillosis and resis-
tance to azole antifungal drugs during esting of serum samples. J Clin Microbiol.
2015; 53: 2115–2121.
12. Montesinos I, Delforge ML, Ajjaham F et al. Evaluation of a new commercial
real-time PCR assay for diagnosis of Pneumocystis jirovecii pneumonia and iden-
tification of dihydropteroate synthase (DHPS) mutations. Diagn Microbiol Infect
Dis. 2017; 87: 32–36.
13. Alanio A, Hauser PM, Lagrou K et al. 5th European Conference on Infections
in Leukemia (ECIL-5), a joint venture of The European Group for Blood and
Marrow Transplantation (EBMT), The European Organization for Research and
Treatment of Cancer (EORTC), the Immunocompromised Host Society (ICHS)
and The European LeukemiaNet (ELN). ECIL guidelines for the diagnosis of
Pneumocystis jirovecii pneumonia in patients with haematological malignancies
and stem cell transplant recipients. J Antimicrob Chemother. 2016; 71: 2386–
2396.
14. Huang HR, Fan LC, Rajbanshi B, Xu JF. Evaluation of a new cryptococcal
antigen lateral flow immunoassay in serum, cerebrospinal fluid and urine for the
diagnosis of cryptococcosis: a meta-analysis and systematic review. PLoS One.
2015; 10: e0127117.
15. Thornton CR. Development of an immunochromatographic lateral-flow device
for rapid serodiagnosis of invasive aspergillosis. Clin Vaccine Immunol. 2008;
15:1095–1105.
16. Pan Z, Fu M, Zhang J, Zhou H, Fu Y, Zhou J. Diagnostic accuracy of a novel
lateral-flow device in invasive aspergillosis: a meta-analysis. J Med Microbiol.
2015; 64: 702–707.
17. Marr KA, Datta K, Mehta S et al. Urine antigen detection as an aid to diagnose
invasive aspergillosis. Clin Infect Dis. 2018; 67: 1705–1711.
18. Johnson G, Shannon M, Thornton C et al. Proximity ligation assay for the
early detection of invasive aspergillosis. 25th European Congress of Clinical
Microbriology and Infectious Diseases; 25–28 April 2015. Copenhagen. 2015.
19. Mery A, Sendid B, François N et al. Application of mass spectrometry technology
to early diagnosis of invasive fungal infections. J Clin Microbiol. 2016; 54: 2786–
2797.
20. Syhre M, Scotter JM, Chambers ST. Investigation into the production of 2-
Pentylfuran by Aspergillus fumigatus and other respiratory pathogens in vitro
and human breath samples. Med Mycol. 2008; 46: 209–215.
21. Chambers ST, Syhre M, Murdoch DR, McCartin F, Epton MJ. Detection of
2-pentylfuran in the breath of patients with Aspergillus fumigatus. Med Mycol.
2009; 47: 468–476.
22. de Heer K, van der Schee MP, Zwinderman K et al. Electronic nose technology
for detection of invasive pulmonary aspergillosis in prolonged chemotherapy-
induced neutropenia: a proof-of-principle study. J Clin Microbiol. 2013; 51:
1490–1495.
23. de Heer K, Kok MG, Fens N et al. Detection of airway colonization by Aspergillus
fumigatus by use of electronic nose technology in patients with cystic fibrosis. J
Clin Microbiol. 2016; 54: 569–575.
24. Patterson TF, Thompson GR, 3rd, Denning DW et al. Practice guidelines for
the diagnosis and management of aspergillosis: 2016 update by the Infectious
Diseases Society of America. Clin Infect Dis. 2016; 63: e1–e60.
25. van der Linden JW, Arendrup MC, Warris A et al. Prospective multicenter inter-
national surveillance of azole resistance in Aspergillus fumigatus. Emerg Infect
Dis. 2015; 21: 1041–1044.
26. van der Linden JW, Snelders E, Kampinga GA et al. Clinical implications of azole
resistance in Aspergillus fumigatus, The Netherlands, 2007-2009. Emerg Infect
Dis. 2011; 17: 1846–1854.
27. Lepak AJ, Marchillo K, Vanhecker J, Andes DR. Isavuconazole (BAL4815)
pharmacodynamic target determination in an in vivo murine model of in-
vasive pulmonary aspergillosis against wild-type and cyp51 mutant isolates
of Aspergillus fumigatus. Antimicrob Agents Chemother. 2013; 57: 6284–
6289.
28. Seyedmousavi S, Brüggemann RJ, Meis JF, Melchers WJ, Verweij PE, Mouton
JW. Pharmacodynamics of isavuconazole in an Aspergillus fumigatus mouse
infection model. Antimicrob Agents Chemother. 2015; 59: 2855–2866.
29. Van der Linden JWM, Arendrup MC, Van der Lee HAL, Melchers WJG,
Verweij PE. Azole containing agar plates as a screening tool for azole resistance
of Aspergillus fumigatus. Mycoses. 2009; 52, 19–28.
S265
30. Buil JB, van der Lee HAL, Rijs AJMM et al. Single-center evaluation of an agar-
based screening for azole resistance in Aspergillus fumigatus by using VIPcheck.
Antimicrob Agents Chemother. 2017; 22; 61. pii: e01250–17.
31. Verweij PE, Ananda-Rajah M, Andes D et al. International expert opinion on the
management of infection caused by azole-resistant Aspergillus fumigatus. Drug
Res Updates. 2015; 21: 30–40.
32. White PL, Wiederhold NP, Loeffler J et al. Comparison of nonculture blood-
based tests for diagnosing invasive aspergillosis in an animal model. J Clin Mi-
crobiol. 2016; 54: 960–966.
33. Morrissey CO, Chen SC, Sorrell TC et al. Galactomannan and PCR versus culture
and histology for directing use of antifungal treatment for invasive aspergillosis
in high-risk haematology patients: a randomised controlled trial. Lancet Infect
Downloaded from https://academic.oup.com/mmy/article-abstract/57/Supplement_3/S259/5530951 by guest on 06 April 2020
Dis. 2013; 13: 519–528.
34. Barnes RA, Stocking K, Bowden S, Poynton MH, White PL. Prevention and
diagnosis of invasive fungal disease in high-risk patients within an integrative
care pathway. J Infect. 2013; 67: 206–214.
35. Aguado JM, Vázquez L, Fernández-Ruiz M et al. Serum galactomannan versus
a combination of galactomannan and PCR-based Aspergillus DNA detection
for early therapy of invasive aspergillosis in high-risk hematological patients: a
randomized controlled trial. Clin Infect Dis. 2014; 60: 405–414.
36. Arvanitis M, Anagnostou T, Mylonakis E. Galactomannan and polymerase
chain reaction-based screening for invasive aspergillosis among high-risk hema-
tology patients: a diagnostic meta-analysis. Clin Infect Dis. 2015; 61: 1263–
1272.
37. Held J, Kohlberger I, Rappold E, Busse Grawitz A, Häcker G. Comparison
of (1→3)-β-D-glucan, mannan/anti-mannan antibodies, and Cand-Tec Candida
antigen as serum biomarkers for candidemia. J Clin Microbiol. 2013; 51: 1158–
1164.
38. Dichtl K, Seybold U, Wagener J. Serological biomarkers of candidemia: a retro-
spective evaluation of three assays. Infection. 2018. doi: 10.1007/s15010-018-
1224-3 [epub ahead of print].
39. León C, Ruiz-Santana S, Saavedra P et al. Cava Trem Study Group. Contribu-
tion of Candida biomarkers and DNA detection for the diagnosis of invasive
candidiasis in ICU patients with severe abdominal conditions. Crit Care. 2016;
20: 149.
40. Hardison SE, Brown GD. C-type lectin receptors orchestrate antifungal immu-
nity. Nat Immunol. 2012; 13: 817–822.
41. White PL, Parr C, Barnes RA. Predicting invasive aspergillosis in hematology
patients by combining clinical and genetic risk factors with early diagnostic
biomarkers. J Clin Microbiol. 2017; 56: pii: e01122–17.
42. Hagiwara D, Takahashi H, Watanabe A et al. Whole-genome comparison of
Aspergillus fumigatus strains serially isolated from patients with aspergillosis. J
Clin Microbiol. 2014; 52: 4202–4209.
43. Abdolrasouli A, Rhodes J, Beale MA et al. Genomic context of azole resistance
mutations in Aspergillus fumigatus dtermined using whole-genome sequencing.
MBio. 2015; 6: e00536.
44. Bittinger K, Charlson ES, Loy E et al. Improved characterization of medically
relevant fungi in the human respiratory tract using next-generation sequencing.
Genome Biol. 2014; 15: 487.
45. Garnaud C, Botterel F, Sertour N et al. Next-generation sequencing offers new
insights into the resistance of Candida spp. to echinocandins and azoles. J An-
timicrob Chemother. 2015; 70: 2556–2565.
46. Krause R, Moissl-Eichinger C et al. Mycobiome in the lower respiratory tract: a
clinical perspective. Front Microbiol. 2017; 7: 2169.
47. Delhaes L, Monchy S, Fréalle E et al. The airway microbiota in cystic fibrosis: a
complex fungal and bacterial community–implications for therapeutic manage-
ment. PLoS One. 2012; 7: e36313.
48. White PL, Wingard JR, Bretagne S et al. Aspergillus polymerase chain reaction:
systematic review of evidence for clinical use in comparison with antigen testing.
Clin Infect Dis. 2015; 61: 1293–1303.
49. Torelli R, Sanguinetti M, Moody A et al. Diagnosis of invasive aspergillosis by a
commercial real-time PCR assay for Aspergillus DNA in bronchoalveolar lavage
fluid samples from high-risk patients compared to a galactomannan enzyme
immunoassay. J Clin Microbiol. 2011; 49: 4273–4278.
50. Guinea J, Padilla C, Escribano P et al. Evaluation of MycAssayTM aspergillus for
diagnosis of invasive pulmonary aspergillosis in patients without hematological
cancer. PLoS One. 2013; 8: e61545.
S266
51. Orsi CF, Bettua C, Pini P et al. Detection of Pneumocystis jirovecii and As-
pergillus spp. DNA in bronchoalveolar lavage fluids by commercial real-time
PCR assays: comparison with conventional diagnostic tests. New Microbiol.
2015; 38: 75–84.
52. Orsi CF, Gennari W, Venturelli C et al. Performance of 2 commercial real-time
polymerase chain reaction assays for the detection of Aspergillus and Pneumo-
cystis DNA in bronchoalveolar lavage fluid samples from critical care patients.
Diagn Microbiol Infect Dis. 2012; 73: 138–143.
53. White PL, Perry MD, Moody A, Follett SA, Morgan G, Barnes RA. Evaluation
of analytical and preliminary clinical performance of Myconostica MycAssay
Aspergillus when testing serum specimens for diagnosis of invasive aspergillosis.
J Clin Microbiol. 2011; 49: 2169–2174.
54. Pini P, Bettua C, Orsi CF et al. Clinical performance of a commer-
cial real-time PCR assay for Aspergillus DNA detection in serum sam-
ples from high-risk patients: comparison with a galactomannan enzyme
immunoassay. Eur J Clin Microbiol Infect Dis. 2015; 34: 131–136.
55. Danylo A1, Courtemanche C2, Pelletier R2, Boudreault AA3. Performance of
MycAssay Aspergillus DNA real-time PCR assay compared with the galac-
tomannan detection assay for the diagnosis of invasive aspergillosis from serum
samples. Med Mycol. 2014; 52: 577–583.
Medical Mycology, 2019, Vol. 57, No. S3
56. Chong GM, van der Beek MT, von dem Borne PA et al. PCR-based detection of
Aspergillus fumigatus Cyp51A mutations on bronchoalveolar lavage: a multicen-
tre validation of the AsperGenius assay R in 201 patients with haematological
disease suspected for invasive aspergillosis. J Antimicrob Chemother. 2016; 71:
3528–3535.
57. White PL, Posso RB, Barnes RA. Analytical and clinical evaluation of the
PathoNostics AsperGenius Assay for detection of invasive aspergillosis and resis-
tance to azole antifungal drugs directly from plasma samples. J Clin Microbiol.
2017; 55: 2356–2366.
58. Dannaoui E, Gabriel F, Gaboyard M et al. Molecular diagnosis of invasive
aspergillosis and detection of azole resistance by a newly commercialized PCR
kit. J Clin Microbiol. 2017; 55: 3210–3218.
Downloaded from https://academic.oup.com/mmy/article-abstract/57/Supplement_3/S259/5530951 by guest on 06 April 2020
59. Guegan H, Robert-Gangneux F, Camus C et al. Improving the diagnosis of
invasive aspergillosis by the detection of Aspergillus in broncho-alveolar lavage
fluid: comparison of non-culture-based assays. J Infect. 2018; 76: 196–205.
60. White PL. Molecular diagnosis of fungal disease. Bruker Symposium “Expansion
in to unmet clinical need,” ECCMID April 22, 2017.
61. Prattes J, Hoenigl M, Zinke SE, Heldt S, Eigl S, Johnson GL, Bustin S, Stelzl E,
Kessler HH. Evaluation of the new AspID polymerase chain reaction assay for
detection of Aspergillus species: a pilot study. Mycoses. 2018; 61: 355–359.