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Human Fungal Pathogen Identification and Diagnosis: (elementary to

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Contents
Human Fungal Pathogen Identification and Diagnosis ،
(elementary to advanced) ........................................................ 3
Abstract ..................................................................................... 3
Key words ................................................................................. 4
Introduction .............................................................................. 5
Table 1. Overview of laboratory methods available for the
diagnosis of invasive fungal diseases ........................................ 8
Table 2. Specimens applied for diagnosing fungal infections .. 9
Table 3. Rapid and histology stains available for fungal
examination and identification ................................................. 11
Direct Microscopic Examination and Histopathology........ 14
Figure 1. Pseudo-hypha, Yeast-like cells, Blastospore and
Chlamidospore in Candida albicans, grown on cornmeal agar
medium with 1% Tween80. ..................................................... 15
Figure 2. Aspergillus proliferates with septated, 2.5–4.5 μm
broad hyphae and branching dichotomously ........................... 16
Figure 3. Photomicrograph of Cryptococcus neoformans using
a light India ink staining preparation ....................................... 17
Figure 4. Systemic Phaeohyphomycosis (pulmonary) ............ 18
Figure 5. KOH preparation of skin scraping shows septate
branching mycelium................................................................. 19
Figure 6. Malassezia demonstration clusters of thick-walled,
round budding yeast and short, straight or angular mycelia
fragments (spaghetti and meatball appearance). ...................... 20
Figure 7. KOH preparation of skin scraping shows pseudo-
hypha and round-elliptical budding yeast (Candidiasis). ......... 21
Culture Techniques................................................................ 24
Serological Tests ..................................................................... 25

1
Table 4. Advantages and disadvantages of the various methods
available ................................................................................... 26
Table 5. Serodiagnosis of fungal diseases ............................... 27
Table 6. Molecular Detection of Fungi in Clinical Specimens
.................................................................................................. 28
Identification of fungal species ............................................. 29
Fungal Species Identification by MALDI-TOF Mass
Spectrometry .......................................................................... 30
Figure 8. MALDI biotyping and DNA-based identification
complement each other. ........................................................... 32
Table 7. MALDI-TOF- and DNA sequencing-based
approaches complement each other Properties and comparison
of MALDI biotyping and DNA sequencing-based approaches
(of barcodes/PCR products or genomic DNA) methods for the
identification of fungi. ............................................................. 33
Figure 9. The process of MALDI-TOF mass spectrometry .... 34
Immunological Identification of Fungal Species ................. 35
The Molecular Blueprint of a Fungus by Next-Generation
Sequencing (NGS) .................................................................. 37
Figure 10. Comparison between Sanger sequencing and next-
generation sequencing (NGS) technologies. ............................ 38
Microarray Technologies in Fungal Diagnostics ................ 39
Inertial Focusing on a Chip-Based Platform ....................... 41
Figure 11. Fungal cells were FITC stained to differentiate from
calcein stained WBCs. ............................................................. 43
AIE-based theranostic systems ............................................. 44
Figure 12. Flowchart for fungal identification using molecular
phylogenetic analysis. .............................................................. 46
Concluding Remarks ............................................................. 47
Reference ................................................................................ 48
Abbreviation ........................................................................... 54

2
Human Fungal Pathogen Identification and Diagnosis
، (elementary to advanced)

Abstract

Although fungal infections participate significantly to

human morbidity and mortality, the efficacy of these

diseases on human health is not appreciated.

Diagnosing fungal infections is a challenge,

particularly in the immunocompromised host. Signs

and symptoms are nonspecific, colonization is difficult

to identify from invasive disease, blood cultures (BC)

are commonly negative, and patients are often unable

to undergo invasive diagnostic procedures. Culture and

microscopic examination remain the “gold standard”

but are insensitive. Antigen assays such as the glucan

and galactomannan detection systems are mostly used.

However, these tests vary in sensitivity and specificity,

3
depending on the patient population involved.

Molecular-based assays are not yet clinically

validated ،however Advances in molecular technology

show great potential for the quick detection and

identification of fungi for medical, scientific and

commercial purposes.However, some new methods

Such as MALDI-TOF MS has become the standard

method for routine identification of most microbial

organisms in clinical laboratories and have largely

replaced biochemical assays.

Key words : Fungal infections, Galactomannan assay, Glucan

assay, Molecular-based assays, Microscopic examination,
Culture

4
Introduction

Funguses are widely distributed in the environment(1),

and of the 100,000 fungal species present, only 300

have been linked to diseases in humans and animals(2).

Opportunistic fungal infections cause considerable

morbidity and mortality in immunocompromised

patients(3). Human mycoses are caused by true or

opportunistic fungal pathogens. Primary (True) fungal

pathogens (Histoplasma capsulatum, Blastomyces

dermatitidis, Coccidioides immitis or posadasii,

Paracoccidioides brasiliensis) attack healthy,

immunocompetent hosts and are distributed in

identifiably geographic regions. Presently, fungal

infections account for 10 percent of all nosocomial

infections(4). Deep, systemic infections are less

common but can affect any organ.

Immunocompromised hosts such as organ-transplant

5
recipients and humans with underlying conditions such

as diabetes or lung diseases are at an increased risk of

suffering from fungal infections(5). The most

important fungal pathogens are Aspergillus species,

Cryptococcus neoformans, Candida sp, Pneumocystis

jirovecii, and Mucormycetes (Rhizopus, Mucor,

Lichtheimia sp) being the main representatives(5, 6).

Fungal infections are a problem in the "public health

community" because of the increasing number of

patients with weakened immune systems and the

advances in health care practices. Fungal infections are

difficult to diagnose because clinical manifestations are

unspecific.

6
A conclusive detection requires direct identification of

fungi from the site of infection(7). Genus or species

identification is not possible based on microscopic

characteristics. Culture techniques are often

insusceptible and time-consuming.

Fungi are part of the human microbiome (The human

microbiota is the aggregate of microorganisms that

resides on or within any of a number of human tissues

and bio-fluids, including the skin, mammary glands,

placenta, seminal fluid, uterus and ,etc) which limits the

role of antigen and antibody assays; fungal colonization

may lead to false positive test results. For some

emerging fungi such as Mucormycetes, no routine

serologic tests are at the moment available. Knowing

the fungal pathogen guides suitable anti-fungal

treatment, "dose and duration of therapy" (5-7).

7
Diagnosing fungal diseases in the laboratory consist of

microscopic examination, culture, antigen or antibody

(Ag - Ab) and MIC testing, or molecular assays,

typically (see Table 1).

Table 1. Overview of laboratory methods available for the

diagnosis of invasive fungal diseases

Conventional methods

Direct microscopy (KOH /LP/ Calcofluor white stains, Giemsa)

Histopathology
Culture
Serology
Galactomannan test for Aspergillus species
Mannan test for Candida species
Antibody test for aspergillosis in immunocompetent hosts
ß-1-3 D glucan panfungal test
Lateral device flow assays for Aspergillus and Cryptococcus species
Antigen and antibody based assays for non-European mycoses
Molecular methods
Fluorescence in situ hybridization test
PCR assays

Sample collection depends on the infection site

involved and consist of skin scrapings, Hair and nails,

tissue biopsies, and body fluids. Interpretation often

requires skill in mycology, and results must be

8
 attentively considered along with signs and symptoms

as well as the medical history of patients(8) (see Table

2).

Table 2. Specimens applied for diagnosing fungal infections

Assay Specimens Goal Results Time

Potassium Skin scrapings, KOH dissolves Screening tool
hydroxide hair or nail non-fungal which detects
solution clippings, elements and fungal

40 min
Rapid,
(KOH) tissue, vaginal reveals fungi on elements; does
secretions, body the microscope not inform
fluids, sputum Slide about genus
or species
Calcofluor Skin scrapings, CWS binds to Screening tool
white stain hair or nail fungal elements which detects
(CWS) clippings, body and fluoresces fungal
fluids, sputum, Under ultraviolet elements; does
40 min
Rapid,
BALs light; allows not inform
visualization about genus or
sensitive species
visualizing
fungal elements
Cultures Skin, nail, hair, The specimen is Tool to
body fluids, placed on culture diagnose
tissue, swabs, media Fungal
sputum, blood infections, to
Days to
Weeks

perform
species
identification
and antifungal
susceptibility
testing
Antifungal Fungal cultures Follow-up to To guide
susceptibil fungal culture; antifungal
3–4 days

ity susceptibility treatment, to

testing testing may be monitor fungal
ordered to guide epidemiology
antifungal therapy

9
 Antigen Blood, urine, Detects antigens To diagnose
testing CSF, body fluids associated with and monitor

Rapid
specific fungi, a infection
variety of tests is
available
Antibody Blood, CSF, Detects immune Diagnose
Testing other body fluids response to a current or
specific fungus; recent
may be ordered on infection by

Days
a single sample or specific
on acute and fungus; to
convalescent monitor
samples collected treatment
2–3 weeks apart
Molecular Fungus isolated Detects genetic Detects fungal
tests for in culture, or material of a DNA or RNA;
DNA and blood, CSF, specific fungus not yet widely

Days
RNA body fluids, available, not
tissue yet validated,
preferable in
research
settings

10
 Table 3. Rapid and histology stains available for fungal
examination and identification

Stain Use Comment

Calcofluor Detection of all fungi Rapid. Alone or in combination with

white 10 -20% KOH. Need for
fluorescence microscope at 420
nm(9).

Fontana - Histological stain for Stains the cell wall of Cryptococcus

masson melanin neoformans. Especially useful for
silver differentiation of Capsule-deficient
stain(FMS) C neoformans. Confirmation of
melanin in lightly pigmented cells of
Dematiaceous (dark colored) fungi.
Cell walls stain brown/black

Giemsa Staining of bone- marrow Useful for Histoplasma and

and peripheral blood smears Pneumocystis species identification
or sputum and BAL

Grocott- Histological stain Provides optimal contrast for the

Gomori detection of fungi in tissue. Specific
methenami for fungal cell walls. Fungi stain
ne silver black, dark brown or grey. Stains old
stain(GMS) and non-viable fungal elements most
effectively. Erythrocytes can mimic
yeasts
H&E General purpose histological Stains most fungi. Allows
stain demonstration of host tissue
reaction. Demonstrates natural
pigment in Dematiaceous fungi. Not
a specific fungal stain and fungal
elements are easily missed. Cartilage
and calcium deposits dark blue,
cytoplasm and other components
shades of red

India ink Negative staining. Rapid. Detection of Cryptococcus

Especially useful in CSF species

KOH Clearing of specimen to Rapid. With or without incubation at

allow improved Visibility of 56 oC May produce artefacts due to
fungal elements. crystallization of the KOH especially
Mostly with solid specimens on drying

11
Lacto Lacto phenol: preserves Rapid. Clear and specific images of
phenol fungal structures and fungal elements. Mostly used at the
Lacto Kills the fungus. Cotton identification steps
phenol- blue: stains the fungal
cotton blue elements

Mayer’s Histological stain Specifically stains

mucicarmin mucopolysaccharide capsular
e stain material of several fungi. May help
in confirmation of cryptococcal
infection

Papanicola Cytological stain mainly Stains most fungal elements

ou stain used to detect malignant
cells

Periodic Histological stain Stains yeasts and hyphae. Possible

Acid-Schiff artefacts with yeasts. Fungi appear
stain red-purple. Zygomycetes hyphae
may stain poorly

Verification of diagnosis requires microscopic

examination or cultural evidence of fungi at sterile

body specimens, usually(6). “For many superficial skin

and yeast infections, a clinical examination of the

patient and a microscopic examination of the specimen

may be adequate to determine a fungal disease”. For

systemic infections, additional diagnostic tests need to

be applied. Susceptibility testing may support

12
antifungal treatment options. The identification of

fungal antigens - antibodies (Ag) / (Ab) is used to

assess whether a patient has or had a fungal infection

(6, 7). These tests are quicker than fungal cultures

however are demonstrate for particular fungal

pathogens only. Many patients produce fungal

antibodies (Ab) from any exposure to the organism so

that a single antibody (Ab) test may not confirm the

tendance of a last infection(2). “Molecular tests” may

be performed to specify fungi grown in culture and/or

directly from the specimens collected (10).

13
Direct Microscopic Examination and Histopathology

Dyes binding to the fungal cell wall (chitin) will diffuse

green or blue-white fluorescence, therefore providing

rapid detection of fungi. The morphological

characteristics seen on microscopy are diverse and rely

on the pathogens involved; hyphae (septated, rarely

septated, aseptated), yeast cells (Blastospores),

budding cells, pseudo-hyphae, may be present.

Candida sp illustrates polymorphic yeasts (10–12 μm)

and shows species-related variations, Candida

(Torulopsis) glabrata lacking hyphal elements.

14
 Figure 1. Pseudo-hypha, Yeast-like cells, Blastospore and
Chlamidospore in Candida albicans, grown on cornmeal agar
medium with 1% Tween80, shown here at 200x magnification.
(Source: Yeast Infection – Alternative Health Practices,
http://kwtc.org/category/yeast-infection/).

15
 Figure 2. Aspergillus proliferates with septated, 2.5–4.5 μm
broad hyphae and branching dichotomously.

Mucorales demonstration thick-walled, distorted and

refractile hyphae (6–15 μm) and swollen cells (up to 50

μm), typically. India ink or Nigrosin staining of

cerebrospinal fluid for diagnosing cryptococcal

meningitis has a sensitivity of 60 % and shows the

characteristic capsules (Fig, 3).

16
Figure 3. Photomicrograph of Cryptococcus neoformans using
a light India ink staining preparation.

17
Figure 4. Systemic Phaeohyphomycosis (pulmonary). Biopsy of
the endo-bronchial lesion. A: presence of pseudohyphae and
regular short hyphae (Grocott methenamine silver. 40 X). B:
tissue section demonstrated the combination of yeast cells,
hyphae and pseudo-hyphae with positive melanin reaction
(Fontana-Masson staining: 100 X).(11)

Typically, dermatophytes demonstration branching

hyaline septate (non-pigmented) hyphae and

arthroconidia may also be seen (Fig, 5).

18
Figure 5. KOH preparation of skin scraping shows septate
branching mycelium

19
Figure 6. Malassezia demonstration clusters of thick-walled,
round budding yeast and short, straight or angular mycelia
fragments (spaghetti and meatball appearance).

20
Figure 7. KOH preparation of skin scraping shows pseudo-
hypha and round-elliptical budding yeast (Candidiasis).

21
P. jirovecii is easily detected by microscopy; “Silver

stains” display the cyst walls and Giemsa staining

demonstrates the nuclei of trophozoites and intracystic

stages. Immunofluorescence (IF) microscopy using

monoclonal antibodies can identify the organisms with

higher sensitivity than conventional microscopy (6).

Fungal elements can be missed as artifacts, fibrin and

etc, by H&E staining. Hyphae are best visualized with

periodic acid Schiff reaction (PAS), Gridley or the

Gomori Methenamine Silver protocol. Fontana-

Masson (FMS) is used to visualize fungal cell wall

melanin pigmented dematiaceous fungi, and aids in

differentiating capsule- negative C.neoformans

(melanin-positive) from other yeasts (melanin-

negative) in tissue. Muricarmine identifies C.

neoformans by staining the polysaccharide capsule (6,

12). Immunohistochemistry (IHC) with monoclonal

22
and polyclonal fluorescent- antibody reagents have

been developed for differentiating the genera of

Aspergillus, Fusarium, and Scedosporium in situ.

Unfortunately, the high percent of antigenic

relatedness among these and other fungal pathogens

has resulted in significant cross-reactivity and low

specificity (6). Microscopic examination does not

permit fungal genus or species identification , as

several fungi have same microscopic-histopathology

manifestation (e.g., Aspergillus and Fusarium sp);

however, it allows differentiation between infections

caused by septate moulds (non - Mucorales ) or aseptate

moulds (Mucorales), which affects the choice of

antifungal treatment(6, 7, 13-15).

23
Culture Techniques

Cultures represent the gold standard in diagnosing

fungal diseases. They facilitate detection of the specific

etiological agent and support antifungal susceptibility

testing (MIC method). Detection of fungemia is useful

in diagnosing opportunistic infections caused by

Cryptococcus neoformans, Candida sp, Fusarium sp,

Malassezia sp, Trichosporon sp, Scedosporium sp,

Aspergillus terreus and, etc. Blood cultures (BC) have

a low sensitivity for diagnosing candidemia (~50 %),

and several days may be required until they become

positive. Identification of Candida sp from non-sterile

body sites (Mucous membranes, mouth, and vagina) is

not special (6, 8, 13-15).

24
Serological Tests

Non-invasive diagnostic tests include detection of

fungal cell wall ingredients and antibodies. However,

the detection of serum antibodies (Ab) is ineffective, as

immunocompromised patients lack specific antibody

response (8, 16).

25
 Table 4. Advantages and disadvantages of the various methods
available

Method Indication Advantages Disadvantages

Galactomannan Early detection A screening test to No good data for non-neutropenic
(GM) Lateral of Invasive Accompany conventional patients
flow Assay aspergillosis diagnostic methods in Mould-active antifungal drugs
2 Serum samples/ patients at high risk of have an impact on sensitivity
Week IA Persistent GM antigenemia
during therapy is a poor
prognostic sign and should
prompt a reassessment
In some cases the stick is not
easy to read, hence lack of
result
“B- D –glucan” Diagnosis of The site of infection may False-(+) results when
(BG) invasive fungal be important: patients bacteremia
infections 2 with tissue infections Limited experience (less widely
Serum failed to show a used than GM)
samples/week significant drop in BG
(minimum) levels despite successful
outcomes
“Mannan plus” Candidemia Good sensitivity and C. parapsilosis and C.
anti-mannan specificity when guilliermondii fungemias
combined in ICU we’re not detected by the
patients Early diagnosis Platelia Candida Ag Plus
prior to blood culture Assay
results
Molecular” DNA detection High sensitivity Non-mycological
Methods”(PCR) mainly of (multicopy genes), Limited to reference
Aspergillus capacity for rapid laboratories (low availability)
species less speciation and ability to High costs, improve technical
experience for quantitate fungal burden equipment
Candida species Technical difficulties of efficient
Suitable for fungal DNA extraction from
blood, BAL , complex clinical samples
tissue

26
 Table 5. Serodiagnosis of fungal diseases

 The detection of “Candida species antigens” has been shown to have

Limited value.

 False positive reactions due to the rheumatoid factor (RF) and renal
insufficiency have been observed.

Antigen Assays  “Mannan” is rapidly cleared from the blood and occurs at low levels,
for Yeasts necessitating frequent sampling for sensitive detection.

 Enolase (ENO) is another antigen holding promise for the diagnosis

of invasive candidiasis.
 The detection of cryptococcal capsular polysaccharide of C.
neoformans and C. gattii allows rapid and sensitive diagnosis of
Serum and cerebrospinal fluid (CSF) infection (17, 18).

Galactomannan
(GM)  Galactomannan (GM) is a cell-wall polysaccharide detectable in
Immunoassay serum and other body fluids during invasive aspergillosis.
For Acute
Aspergillosis
 “Beta glucan” is a cell-wall constituent of many pathogenic fungi,
and is detectable in patient serum during infections with Candida,
Beta Glucan Aspergillus, Fusarium , Trichosporon , Saccharomyces ,
(BG) Acremonium species, and P. jirovecii
Pan fungal
Assay  The test does not detect Cryptococcus species or Mucormycetes.

 Antibody testing is limited to the diagnosis and monitoring of

treatment of coccidioidomycosis, chronic pulmonary aspergillosis,
Antibody allergic broncho-pulmonary aspergillosis (ABPA), and allergic,
Testing chronic, and granulomatous Aspergillus rhinosinusitis.

 It is supportive for the diagnosis of “Aspergillus bronchitis” in non-

immunocompromised patients, for the diagnosis of acute
(seroconversion) and chronic histoplasmosis, and diagnosis of
paracoccidioidomycosis(19).

27
Table 6. Molecular Detection of Fungi in Clinical Specimens

C. albicans The FISH method uses PNA probes for the

Peptide Nucleic diagnosis of C.albicans directly from positive
Acid (PNA) blood culture (18, 20).
Fluorescence
In Situ
Hybridization
( FISH ) Test

Polymerase Detection of fungal DNA by means of PCR has

Chain Reaction been studied in detail
(PCR) For candidemia and invasive aspergillosis (IA).
The potential usefulness of DNA detection is
evident, given that many opportunistic fungal
pathogens grow slowly or are difficult to
isolate(21, 22).

28
Identification of fungal species

 Fungal Species Identification by MALDI-TOF

Mass Spectrometry
 Immunological Identification of Fungal Species
 The Molecular Blueprint of a Fungus by Next-
Generation Sequencing(NGS)
 Microarray Technologies in Fungal Diagnostics
 Inertial Focusing on a Chip-Based Platform
 AIE-based theranostic systems

29
Fungal Species Identification by MALDI-TOF Mass
Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) is

an ionization technique that uses a laser energy

absorbing matrix to create ions from large molecules

with minimal fragmentation MALDI-TOF mass

chromatography has become the standard method for

identification of most microbial organisms in clinical

laboratories and has almost replaced biochemical

assays(23).

“Classification according to wide-spreading well

curated databases, covering the complete spectrum of

microorganisms encountered in the specimens at

hands”(24).

The protocols for harvesting cells and procuring

material suitable for downstream “MALDI-TOF” MS

analyses vary in special details between the different

30
groups of organisms, e.g., gram- negative or - positive

bacteria, mycobacteria, or fungi.

With respect to fungi, methods further vary between

moulds and yeasts; and even among diverse mould

genera if they do not lyse in a similar fashion.

Purification of microbial materials from clinical

specimen allows the direct recognition of bacteria;

however this is not yet exactly adapted to fungi(25).

31
Figure 8. MALDI biotyping and DNA-based identification
complement each other. A MALDI biotyping is the method of
choice for the identification of a defined number of species. B
Generating MALDI-TOF MS reference spectra for fungal
species identified by DNA-based methods allows the rapid and
economical detection of this species by MALDI biotyping in the
future(26).

32
Table 7. MALDI-TOF- and DNA sequencing-based approaches
complement each other Properties and comparison of MALDI
biotyping and DNA sequencing-based approaches (of
barcodes/PCR products or genomic DNA) methods for the
identification of fungi. The advantages of each method are
shown in italics(26).

33
Figure 9. The process of MALDI-TOF mass spectrometry
(Clark A. E., et al.; 2013)

34
Immunological Identification of Fungal Species

Immunodetection is described as a technique for

producing specific antibodies for antigen detection of

the major human fungal pathogens. In the case of

Candida sp, heat-killed cells are used to immunize

mice over 2 weeks and then splenocytes are isolated

and fused with myelomas to easily increase the

antibodies produced in the mice subsequently(27). The

resulting antibodies follow a purification process where

antibody levels and concentrations are determined.

Fungal cells are also lysed to obtain whole cell extracts

as an early step for identification of antigens using

immunoprecipitation. Fungal cells are also lysed to

obtain whole cell extracts as a primary step for

identification of antigens using immunoprecipitation.

Finally, this method permits the production of specific

35
antibodies against fungi and the identification of the

respective antigens in an in vivo model(28).

36
The Molecular Blueprint of a Fungus by Next-
Generation Sequencing (NGS)

Sequencing the total genome of an organism is

inestimable for its comprehensive molecular

characterization and has been drastically facilitated by

the appearance of high-throughput sequencing

techniques. Exclusively in clinical microbiology the

impact of sequenced strains increases as "resistance and

virulence" markers can easily be detected (29, 30).

37
Figure 10. Comparison between Sanger sequencing and next-
generation sequencing (NGS) technologies. Sanger sequencing
is limited to determining the order of one fragment of DNA per
reaction, up to a maximum length of *700 bases. NGS platforms
can sequence millions of DNA fragments in parallel in one
reaction, yielding enormous amounts of data. To see this
illustration in color, the reader is referred to the web version of
this article at www.liebertpub.com/wound (31).

38
Microarray Technologies in Fungal Diagnostics

Microarray technologies (DNA chip or biochip) have

been a major research tool in the last decades. In

addition they have been presented into several fields of

diagnostics including diagnostics of infectious

diseases. Microarrays are highly parallelized assay

systems that at first were developed for multi-

parametric nucleic acid detection. From there on they

rapidly developed towards a tool for the detection of all

kind of biological compounds (DNA, RNA, proteins,

carbohydrates, nucleic acids, cells, etc.) or their

modifications (methylation, phosphorylation, etc.).

The combination of closed-tube systems and lab on

chip devices with microarrays further enabled a higher

automation degree with a reduced contamination risk.

Microarray-based diagnostic applications currently

complement and may in the future replace classical

39
methods in clinical microbiology like blood cultures,

resistance determination, microscopic and metabolic

analyses as well as biochemical or

immunohistochemical (IHC) assays.

In addition, novel diagnostic markers appear, like

noncoding RNAs and miRNAs providing additional

room for novel nucleic acid based biomarkers. Here

authors focus an microarray technologies in diagnostics

and as research tools, based on nucleic acid-based

arrays(32).

40
Inertial Focusing on a Chip-Based Platform

Systemic Candida infections remain a leading cause of

nosocomial infections in the United States and

worldwide. Many challenges remain in achieving rapid,

direct diagnosis of fungal bloodstream infections due to

limitations of conventional diagnostic methods that

continue to demonstrate poor sensitivity, prolonged

culture times that lead to delayed treatment, and

detection variability between tests that compromises

result reproducibility. Despite advancements in

technology, mortality, and cost of care presented by

blood stream infection with Candida spp. (candidemia)

continues to rise and there is an urgent need for the

development of novel methods to accurately

detect Candida species present within the blood. This

is especially true when patients are infected with drug

41
resistant strains of Candida where accurate and

immediate therapeutic treatment is of the importance.

In study by Fuchs et al (2019) presents a method of

separating fungal cells from lysed blood using inertial

forces applied through microfluidics in order to

abbreviate the time required to achieve a diagnosis by

mitigating the need to grow blood cultures. We found

that C. albicans can segregate into a focused stream

distinct from white blood cells isolated within the

Inertial Fungal Focuser (IFF) after red blood cell lysis.

As a result of the focusing process, the collected cells

are also concentrated 2.86 times.

The same IFF device is applicable to non-

albicans species: Candida parapsilosis, Candida

glabrata, and Candida tropicalis, providing both

isolation from lysed blood and a reduction in solution

volume. Thus, the devised platform provides a means

42
to isolate medically significant fungal cells from blood

and concentrate the cells for further interrogation(33).

Figure 11. Fungal cells were FITC stained to differentiate from

calcein stained WBCs. (A) Device schematic. (B) A mixture of
cells that includes stained WBCs and fungal cells enters the IFF
without prior separation and are subjected to the microfluidic
forces and specified flow rate. (C) As cells travel through the
path of the coil, at a specific flow rate of 400 μl/min, C.
albicans are focused into a stream. (D) By the time they reach
the outlet, the cell form two distinct streams. (E) The lateral
separation of cell types was tested over a range to detect the
optimal applied flow rate. (F) The distribution observed at
device 100w-5-20-75 is shown in black and distribution for IFF
device design 100w-10-20-70 is shown in gray. Error bars
indicated standard deviation(33).

43
AIE-based theranostic systems

Pathogenic bacteria, fungi and viruses pose serious

threats to the human health under appropriate

conditions. There are many rapid and sensitive

approaches have been developed for identification and

quantification of specific pathogens, but many

challenges still exist. Culture/colony counting and

polymerase chain reaction are the classical methods

used for pathogen detection, but their operations are

time-consuming and laborious. On the other hand, the

emergence and rapid spread of multidrug-resistant

pathogens is another global threat. It is thus of utmost

urgency to develop new therapeutic agents or

strategies. Luminogens with aggregation-induced

emission (AIEgens) and their derived supramolecular

systems with unique optical properties have been

developed as fluorescent probes for turn-on sensing of

44
pathogens with high sensitivity and specificity. In

addition, AIE-based supramolecular nanostructures

exhibit excellent photodynamic inactivation (PDI)

activity in aggregate, offering great potential for not

only light-up diagnosis of pathogen, but also image-

guided PDI therapy for pathogenic infection(34).

45
Figure 12. Flowchart for fungal identification using molecular
phylogenetic analysis.

46
Concluding Remarks

An accurate mycological diagnosis is only possible

with the help of laboratory testing. In last years, only a

positive culture could reliably define a fungal infection.

With currently available improved diagnostics,

validation of the cause of fungal infection is possible

by serological and molecular testing. Conventional

microbiological and microscopic techniques remain the

basis of diagnosis but lack sensitivity. The combined

implementation of other available tools is therefore

obligatory for proper diagnosis.

47
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53
Abbreviation

 Ag: Antigen
 Ab: Antibody
 AIEgens: Luminogens with Aggregation-
Induced Emission
 BC: Blood Cultures
 BG: B- D –glucan
 BAL: Broncho Alveolar Lavage
 CWS: Calcofluor White Stain
 CSF: Cerebrospinal fluid
 DNA: Deoxyribonucleic acid
 FMS: Fontana - Masson Silver
 FISH: Fluorescence In Situ Hybridization
 GMS: Grocott-Gomori Methenamine Silver
 GM: Galactomannan
 H&E: Hematoxylin and Eosin
 IF: Immuno-Fluorescence
 IHC: Immuno-Histo Chemistry
 ICU: Intensive Care Unit
 IFF: Inertial Fungal Focuser
 KOH: Potassium Hydroxide

54
  MALDI-TOF MS: Matrix-Assisted Laser
Desorption/Ionization Mass Spectrometry
 MIC: Minimum Inhibitory Concentration
 NGS: Next-Generation Sequencing
 PAS: Periodic Acid-Schiff
 PCR: Polymerase Chain Reaction
 PNA: Peptide Nucleic Acid
 PDI: Photo Dynamic Inactivation
 RNA: Ribonucleic acid

55

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