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TABLE 19-4.
GROWTH MEDIUM COMMENTS AND USES
Primary Media Without Antibacterials or Antifungals
agar
Primary Media with Antibacterials or Antifungals
Any of the above media
Selective/Differential Media
Microsporum, Trichophyton, Epidermophyton
fungi and Aspergillus
screening medium)
medium)
Candida
a
Histo plasma capsulatum
and Blastomyces dermatitidis
Candida krusei
more Candida
Candida agar
a
Candida albicans versus other
Specialized Media
Canavanine glycine
agar
isolation of Cryptococcus gattiiCryptococcusCryptococcus
neoformans
Trichophyton
used for the cultivation and differentiation of Candida
Trichophyton rubrum
CHAPTER 19 • InfECTIous DIsEAsEs: fungI, VIRusEs, AnD MyCobACTERIA 457
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TABLE 19-4.
GROWTH MEDIUM COMMENTS AND USES
and esculin base medium
a
17CandidaCandida
Candida
Source
for the growth rates and identication of fungi. It is generally recommended that fungal cultures be incubated for 4 weeks before
being considered negative (no growth for fungus). Several factors
inuence the length of incubation including the choice of media
(eg, yeasts on chromogenic [48hours] versus routine media [5 to
7 days]) and type of fungus suspected (eg, slow- growing dimorphic systemic fungi may need 8 weeks).
Once the organism has been cultured and isolated, the following approach has usually been conducted: (1) determine the
morphology of the unknown fungus and determine if it is consistent with any of the groups listed in Table19-1 or lamentous
bacterium (some of the aerobic actinomycetes [eg, Nocardia]
resemble fungi and must be ruled out) and (2) note the rate of
growth, colony, and microscopic morphologies of the possible
organism(s) (Table19-2) and refer to necessary textbooks to
compare descriptions, drawings, color plates, discussions of characteristics, and other test results to assist in dierentiating the
likely organism.
15,18
In the case of yeasts and yeast- like organisms,
additional testing, such as the germ tube test, biochemical testing using commercially available systems, or the urease test, may
allow species identication of isolates from various body sites.
Both NAAT and MALDI-TOF MS are increasingly being used to
modernize clinical microbiology laboratories. ese rapid, inexpensive, and accurate methods for identication of fungal organisms allow less dependency on performing time- consuming
biochemical procedures and/or needing visual expertise for
detection of microscopic and colonial morphology.
CryptococcusC neoformans
and C gattii
some strains of Cformans
France]) is available to detect circulating galactomannan antigen
in serum or bronchoalveolar lavage (BAL) uid and has been
shown to be an earlier diagnostic marker for invasive aspergillosis in neutropenic patients with hematologic malignancies
and hematopoietic stem cell transplantation.
18,27
e monitoring of antigen titers has also been shown to correlate with the
response to antifungal therapy, patient survival, and autopsy
ndings in neutropenic patients. Other genera of fungi, including Histoplasma, Penicillium, Alternaria, Geotrichum, and Paeci-
lomyces, have shown reactivity to the assay kit. Several causes of
false- positive results have also been reported, including patients
receiving specific antibiotics (eg, piperacillin- tazobactam,
amoxicillin- clavulanic acid), certain foods (eg, pasta, vegetables, milk), and Plasma-Lyte A. e detection of galactomannan is also reduced in patients receiving antifungal agents active
against molds and patients with chronic granulomatous disease.
A nongalactomannan antigen method has recently been developed as a point- of- care (POC) test for rapid detection of invasive pulmonary aspergillosis. is rapid detection method uses
a monoclonal antibody (JF5) and a lateral- ow device (LFD)
(OLM Diagnostics, Newcastle upon Tyne, UK) to detect an
extracellular glycoprotein antigen produced by A. fumigatus.
is test has a high negative predictive value and good sensitivity and specicity, especially with BAL uid. U.S. Food and Drug
(FDA) approval of this test for diagnostic use is still pending.
Antigen testing is considered the primary diagnostic test
10,18-24,26
in screening cerebrospinal uid (CSF) for suspected cases of
cryptococcal meningitis because the India ink procedure has
Antigen Detection
Cell wall components of various invasive fungi have been used
as diagnostic markers for antigen testing. Galactomannan is a
polysaccharide component of the cell wall of Aspergillus and it
is released by growing hyphae. A commercial enzyme- linked
immunosorbent assay (ELISA) (Platelia Aspergillus Galactomannan Test [Bio-Rad Laboratories, Marens-La-Coquette,
a low sensitivity. e combination of antigen detection test
and an India ink stain of the CSF are recommended for the
primary evaluations of suspected cases of cryptococcal meningitis. Several commercial kits are available for the detection
of cryptococcal antigen in serum and CSF.18 Galactoxylomannan is a polysaccharide capsular component of Cryptococcus
and is the antigen detected for infections caused by serotypes of
458 BASIC SKILLS IN INTERPRETING LABORATORY DATA
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C neoformans, C gattii, and C deneoformans. Latex agglutination
(LA) and enzyme immunoassay (EIA) methods are sensitive
(93% to 100%) and specic (93% to 100%) diagnostic tests for
the detection and quantitation of circulating cryptococcal antigen in serum and CSF. e reported titer determinations of the
two testing methods (eg, LA versus EIA testing) or from different commercial latex kits are not numerically similar. us,
the same testing method and latex kit should be used to monitor serial samples for a patient. Numerous causes have been
responsible for false- positive results, with both testing methods including rheumatoid factor, soaps, disinfectants, hydroxyethyl starch, malignancy, and infections associated caused by
bacteria, Trichosporon, Capnocytophage, Rothia, or Geotrichum
beigelii. Both low and high antigen titers can result in falsenegative results. Recently, LFD device has become available for
measuring cryptococcal antigen from serum and CSF samples.
e advantages of LFD include similar specicity and increased
sensitivity as LA and EIA for testing serum samples, ease of use,
lower costs compared with other test kits, and the similar accuracy between whole blood samples and blood obtained from nger pricks. False- positive results with LFD have been reported at
low titers in patients without a history of cryptococcal infection.
Enzyme immunoassay can be used to detect a polysaccharide antigen from H capsulatum in body uids (eg, serum or
plasma, urine, CSF, or BAL uid). It has been recommended that
the antigen screening test be validated by antibody testing (eg,
immunodiusion [ID] and complement xation [CF]).18 e
diagnosis of histoplasmosis should be based on a combination of
diagnostic test results because antigen testing is associated with
cross- reactivity to other fungal infections (eg, blastomycosis,
coccidioidomycosis, paracoccidioidomycosis) and false- positive
results (eg, rheumatoid factor, rabbit antithymocyte globulin).
e test sensitivity varies with disease presentation (eg, 77% for
acute pulmonary histoplasmosis, 34% for subacute pulmonary
histoplasmosis, 21% for chronic pulmonary histoplasmosis, 92%
for progressively disseminated histoplasmosis), patient groups
(eg, HIV infection, immunocompromised, disseminated diseases), and specimen type (60% to 86% in serum, 80% to 95%
in urine, 25% to 50% in CSF, 93.5% in BAL). A monoclonal antibody ELISA has also been developed and has improved sensitivity (98%) and specicity (97%).
Antigen detection tests for H capsulatum, B dermatitidis,
and Coccidioides species are performed by the clinical reference laboratory, MiraVista Diagnostics (Indianapolis, IN), on
a fee- for- service basis. Antigen detection is generally not used
as a diagnostic tool and has a limited role for blastomycosis and
coccidioidomycosis because of low levels of detection in antigenemia and antigenuria, false- positive reactions, and/or crossreactions are common in patients with other mycoses.
Mannin, a major component of the Candida cell wall, has
been the main diagnostic marker used in antigen detection tests
of Candida species.18 For serology testing, antimannin antibodies can be monitored because mannin can induce a strong antibody response toward oligomannose epitopes. A wide range in
assay sensitivity and specicity has been reported when either
antigen or antibody detection tests are used alone for the diagnosis of candidemia and disseminated candidiasis. e combined
detection of circulating mannan and antimannan antibodies in
serum or plasma by immunoenzymatic assays (Platelia Candida
Ag Plus and Platelia Candida Ab Plus, Bio-Rad Laboratories,
Marens-La-Coquette, France) has been recommended to maximize the early diagnosis of invasive candidiasis. Concomitant
detection of mannan and antimannan antibodies for Candida
species has a median sensitivity of >80%, particularly for C albi-
cans, C glabrata, and C tropicalis.
Serology
Several dierent serology methodologies (eg, ID, countercurrent
immunoelectrophoresis, ELISA, CF tests, uorescent- enzyme
immunoassay) have been investigated for the detection of specic fungal pathogens.
fungal infections requires knowledge of the laboratory tech
nique used to perform the antibody testing. Serologic assays
are most useful as diagnostic testing of fungal infections in the
immunocompetent host because a poor antibody response is
common in immunosuppressed patients, resulting in a falsenegative result.
Serologic tests (ie, ID and CF) play an important role in the
clinical diagnosis of infections caused by H capsulatum.
ese tests have been the most useful in patients with chronic
pulmonary or disseminated histoplasmosis. e ID test is more
specic than CF test and can serve as a useful screening procedure with and without the CF test. e CF test is more sensitive than ID but has shown cross- reactivity and positive results
in patients with various other types of infections, including
bacterial, viral, mycobacterial, and fungal (eg, aspergillosis,
blastomycosis, candidiasis, coccidioidomycosis, paracoccidioidomycosis). CF test can also have false- negative results in the
presence of rheumatoid factor or cold agglutinins. Other serologic assays (eg, LA, EIA, ELISA) have also been evaluated and
may be useful for the diagnosis of specic types of H capsulatum
infections. e potential of cross- reactivity or lack of commercial availability limits the current use of these assays.
e ID and CF tests are reliable serologic methods for the
diagnosis of invasive infections of coccidioidomycosis and
paracoccidioidomycosis.
noglobulin M (IgM) antibodies (heated coccidioidin) and is
useful in the diagnosis of active disease. e ID has replaced
the historical use of a tube precipitin test. e CF detects IgG
antibodies (unheated coccidioidin) and is useful in diagnosing
acute or chronic diseases and predictive for monitoring treatment response and a poor prognosis. LA and a highly sensitive EIA are also available; however, false- positive results have
been noted. ese tests should only be used as a screening
tool, in which a positive result must be conrmed by another
method.
Finally, serology testing methods (ie, immunoelectrophoresis, ELISA, and uorescent- enzyme immunoassay) for
Aspergillus- specic antibodies are useful for the diagnosis of
noninvasive diseases such as allergic bronchopulmonary aspergillosis, aspergilloma, and chronic cavitary aspergillosis.18 Low
sensitivity and/or specicity currently limit the use of serology testing as denitive diagnosis of invasive fungal infections
caused by species of Blastomyces, Candida, and Cryptococcus.
18,24
Interpretation of results for most
18,27
e ID test mainly detects immu-
18,24,27
-
CHAPTER 19 • InfECTIous DIsEAsEs: fungI, VIRusEs, AnD MyCobACTERIA 459
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(1,3)-β-D-Glucan Detection
Commercial assays for the detection of (1,3)-b- - glucan, a poly-
saccharide present in cell wall of common pathogenic yeasts,
have been used as a panfungal diagnostic tool for invasive fungal infections such as aspergillosis, Fusarium infection, trichosporonosis, and candidiasis.
18,27
is assay has also been used to
detect (1,3)-β- - glucan from P jirovecii, in both HIV- positive
and HIV- negative patients. is diagnostic assay is not useful for
mucoraceous molds (eg, Zygomycetes such as Rhizopus, Mucor,
and Absidia), which do not produce (1,3)-β- - glucan, or Cryp-
tococcus species and B dermatitidis, because they produce only
low levels of (1,3)-β- - glucan. Limited evaluations have assessed
this diagnostic assay for the detection of histoplasmosis and
coccidioidomycosis.
In the United States, Fungitell assay (Associates of Cape Cod
Inc., East Falmouth, MA) is widely available and the only FDAapproved screening test for detecting (1,3)-β- - glucan in serum.
e manufacturer’s recommended guidelines for a positive
serum (1,3)-β- - glucan value is ≥80 pg/mL and a negative value
<60 pg/mL; values between 60 and 79are considered indeterminate (http://www.acciusa.com). Repeat testing (eg, twice weekly)
is recommended to improve the predictive value and specicity
of the test. False- positive results have been observed in patients
receiving hemodialysis (with cellulose membranes), treated with
certain blood products (eg, albumin, immunoglobulins), having bacterial bloodstream infections, mucositis, or gra- versushost disease, and/or exposed to glucan- containing materials (eg,
gauze or swabs). Concurrent β- lactam therapy, such as piper-
acillin–tazobactam or amoxicillin–clavulanate, and antitumoral
polysaccharides have also been associated with false- positive
results. Because of these potential risks of a false- positive result,
the test may be more useful in excluding a diagnosis of invasive
fungal infection when results are reported negative. Because this
assay is nonspecic with varying levels of sensitivity and specicity, its use should be in conjunction with clinical examination
of the patient and other diagnostic tests and procedures to make
a conclusive diagnosis of invasive fungal infection.
Molecular Diagnosis
Molecular diagnostic tests have a signicant and increasing role
in the detection and identication of fungi.
of these techniques include organisms being observed microscopically but not grown on culture; a more rapid and objective
identication of molds with unrecognizable or unproductive
structures or yeasts not included in commercial databases; the
ability to dierentiate fungi with similar characteristics; and the
precise genotyping for epidemiology studies and updates to taxonomy, classication, and nomenclature of fungi.18 e reader is
referred to a glossary of common molecular terms for comprehending information in this rapidly evolving eld.
e ribosomal targets and internal transcribed spacer regions
have been the main target used for molecular identication
of fungi. Procedural steps that are commonly involved with
molecular identication techniques include extraction of DNA,
amplication of DNA segment of interest, and DNA analysis.
Amplication is most oen performed by polymerase chain
15,18-24
e advantages
15,18
reaction (PCR) with non–sequencing-
based or sequencingbased identication methods. A wide variety of methodologies
are available, including local and in- house laboratory- developed
PCR tests. Clinicians need to contact their laboratory to determine which tests are available and which molecular diagnostic
tests may need to be sent to a reference laboratory. In addition,
selection of appropriate primers and/or probes for molecular
testing oen relies on the initial impressions and/or characteristics of the isolate, particularly for less robust molecular
identication methods. In many situations, a combination of
morphologic and molecular testing methods is best used for
species identication.
Many evaluations have been ongoing for dierent PCR assays
for invasive Candida spp. and Aspergillus infections.
15,19,27
Limited and variable sensitivity and specicity have been some of
the main issues restricting the routine use of this method. Additional issues that need to be addressed include specimen type,
sample volume, best method of DNA extraction, target range,
and denitions of positive results. However, further evaluation
with standardized methodology and decreased inconsistencies
between tests should allow PCR to become a promising method
for detection of Candida and Aspergillus spp. Like other fungal infections, nucleic acid detection has been extensively used
as a research- based tool with a slower progression to routine
use by clinical microbiology laboratories and/or commercial
availability.
27
Several molecular- based diagnostic assays have been cleared
by the FDA and are commercially available for clinical use in the
United States.
15,18-22,27
Even more devices have been marked Conformitè Europèene InVitro Diagnostic (CE-IVD) and marketed
for use in Europe.19 ese devices have incorporated detection
technologies such as DNA amplication followed by magnetic
resonance, peptide nucleic acid uorescent insitu hybridization
(PNA-FISH), chemiluminescent labeled with single- stranded
DNA probes, nested multiplex PCR or real- time PCR with
DNA melt- curve analysis, and a system platform involving PCR
amplication, ow cytometry, and dual- lasers detection. Most
of the available commercial devices have focused on the detection and identication of fungi species commonly associated in
infections, such as Aspergillus, Cryptococcus, and several Can-
dida spp. (ie, C albicans, C glabrata, C krusei, C parapsilosis,
and C tropicalis). e following brief discussion highlights a few
examples of molecular devices that have FDA clearance for clinical use in the United States.
e T2Candida Panel and automated T2Dx Instrument
(T2Biosystems, Lexington, MA) uses novel technologies to allow
rapid (eg, 3 to 5hours) and accurate diagnosis of invasive candidiasis directly from whole blood samples (no need for blood
culture and isolation of Candida spp.).
15,18-22,27
e instrument is
a fully automated as a clinical multiplex benchtop diagnostic system using PCR and miniaturized magnetic resonance technology. e T2Candida panel can identify C albicans, C tropicalis,
C parapsilosis, C glabrata, and P kudriavzevii. Other panels are
in development, including T2Cauris panel (research use only)
for the detection of the emerging multidrug- resistant Candida
auris. A T2Bacteria panel has FDA clearance and is available for
use on the same instrument.
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PNA-FISH (Yeast Trac Light PNA FISH, OpGen, Gaithersburg, MD) provides rapid identication of up to ve Candida
spp. directly from yeast- positive blood cultures.
15,18-22
is device
has a fast turnaround time (eg, approximately 90 minutes) with
high sensitivity (92% to 100%) and specicity (94% to 100%).
e rapid identication methodology used is hybridization of
uorescent PNA probes to organism- specic rRNA, with detection via uorescent microscopy. Aer the Gram stain and the
hybridization process are completed, C albicans and C parap-
silosis are identied microscopically as bright green uorescing cells, while Candida tropicalis uoresces bright yellow, and
C glabrata and C krusei uoresce bright red. Other yeasts do not
uoresce. e colors of the light probes also provide an indication about the potential use of uconazole in these patients
because C albicans and C parapsilosis are generally susceptible
to uconazole (green light for go), C glabrata can be resistant
to uconazole, and C krusei is intrinsically resistant to u-
conazole (red light for stop). e yellow signal produced by
C tropicalis indicates that caution should be used because uconazole susceptibility is variable for this organism. is method
has a signicant impact over traditional identication methods, which could take up to 3 or more days for identication of
Candida spp., as well as guiding the most eective antifungal
drug therapy. e PNA-FISH methodology is also used for rapid
identication of bacteria, including gram- positive (ie, Staphy-
lococcus aureus, coagulase- negative staphylococci, enterococci)
and gram- negative (ie, Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa) organisms. A single automated system using FISH technology (Accelerate Pheno BC kit, Accelerate Diagnostics, Inc., Tucson, AZ) has also been developed
and can identify C albicans, C glabrata, and gram- positive and
gram- negative organisms.
Probe- based assays for culture identication of fungi have
become commercially available.
15,18-22
AccuProbe (Hologic, Inc.,
Mississauga, ON, Canada) uses luminometer to detect hybridization of a chemiluminescent labeled, single- stranded DNA
probe to target rRNA present in a fungal culture. ree separate probes have FDA clearance and are available for the culture identication of dimorphic fungi, including Blastomyces
dermatitidis, Coccidioides immitis, and Histoplasma capsulatum.
Performance data have demonstrated high sensitivity (>98%)
and specicity (>99%) for each pathogen. e B dermatitidis
probe has the potential to cross- react with other fungi, including
Emmonsia species, Paracoccidiodes brasiliensis, and Gymnascella
spp. In addition, the Coccidioides probe is unable to distinguish
between species, namely Coccidioides immitis and Coccidioides
posadasii. AccuProbe tests are also available for culture identication of bacteria (ie, Neisseria gonorrheae, S aureus, Listeria
monocytogenes, Streptococcus pneumoniae) and mycobacteria.
BioFire FilmArray (BioFire, Salt Lake City, UT) is an automated invitro diagnostic device that detects multiple nucleic
acid targets by using nested multiplex PCR with DNA melting curve analysis.
15,18-22
Six dierent identication panels
are currently available, with yeast being included on two of
these panels. e Blood Culture ID (BCID) Panel can test
for 43 targets associated with bloodstream infections, including gram- positive and gram- negative bacteria, yeast, and
10antimicrobial resistance genes. e yeast detected by the
BCID Panel includes six Candida spp. (ie, C albicans, C auris,
C glabrata, C krusei, C parapsilosis, and C tropicalis) and Cryptococcus neoformans/gatti. is panel requires a positive blood
culture sample. e overall sensitivity and specicity for the
BCID Panel are 99% and 99.8%, respectively. Cryptococcus neo-
formans/gatti, along with 13 common bacterial and viral pathogens, are included on the Meningitis/Encephalitis (ME) Panel.
e ME Panel can directly detect pathogens from a 0.2 mL sample of CSF, and has also demonstrated a high sensitivity (94.2%)
and specicity (99.8%).
Luminex (xMAP and xTAG, Luminex Molecular Diagnostics, Inc., Austin, TX) is a commercially available multianalyte
proling platform that can provide detection and identication of clinically important pathogens directly from positive
blood culture bottles and other types of clinical samples.
15,18-22
e platform has combined PCR amplication, ow cytometry,
and dual- laser detection system to provide multiplexed assay
capabilities. e xMAP (x = analyte or unknown; MAP = MultiAnalyte Proling) hybridization technology can detect an antigen (target) by using a capture antibody attached to the surface
of a color- coded microbeads (microsphere) and a detection
antibody that incorporates a uorescent label. Luminex- based
technology has allowed rapid and reliable identication of clinically important fungal pathogens, including up to 10 genus- and
29 species- specic diagnoses. xTAG (the TAG name is derived
from three nucleic acid bases being used: T, A, G) consists of the
MagPlex-TAG microsphere (that are precoupled with anti-TAG
sequence) and the user designator primer and TAG sequence
that complements the x-TAG sequence on the bead. e xTAG
analyte- specic reagents can be combined with xMAP instruments for amplication and detection. Evaluations of the xTAG
Fungal ASR assay have demonstrated that multiple yeast species
could be identied with 100% sensitivity, 99% specicity, and
99% positive and 100% negative predictive values when compared with traditional fungal culture results.
MALDI-TOF Mass Spectrometry
Matrix- assisted laser desorption- ionization time- of- flight
(MALDI-TOF) mass spectrometry (MS) is ideal for genus and
species identication and has the potential for accurate strain
typing and identication for fungi, bacteria, and mycobacte-
5,10,15,18-20,26
rium.
for identifying yeasts and molds recovered on culture media
and using sample preparation for MALDI-TOF MS. Reports
on the use of MALDI-TOF MS for routine rapid identication
have focused on clinically important yeasts (eg, Candida spp.,
including Candida auris, C neoformans, and C gattii spp.) and
dermatophyte species (eg, Neoscytalidium spp., Trichophyton,
Microsporum, Epidermophyton, and Arthroderma). Identica
tion of dimorphic and lamentous fungi as well as molds (eg,
Aspergillus spp., Fusarium spp., Pseudallescheria–Scedosporium
complex, Penicillium spp., Lichtheimia spp.) have been more
challenging because of dierent developmental forms on agar
media and the inuences of the phenotype. MALDI-TOF MS is
becoming the primary diagnostic method for rapid identication of fungus isolates in the clinical microbiology laboratory.
is technology is a rapid and accurate method
-
CHAPTER 19 • InfECTIous DIsEAsEs: fungI, VIRusEs, AnD MyCobACTERIA 461
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However, its initial use for fungal identication has moved at a
slower pace than the current use of MALDI-TOF MS for bacterial identication.
e advantages of the MALDI-TOF MS for fungal identication are low cost of materials (a few cents) for each organism
identication, ease of performance, and rapid, accurate results
(approximately 11 minutes if just one isolate is tested; 2.5 minutes per isolate in a batch of 96 isolates, with the average time per
isolate in published reports being 4 to 6 minutes). e simplicity of MALDI-TOF MS removes the specic skills and ability to
visually identify fungi macroscopic and microscopic characteristics. Current limitations include the initial costs of instrumentation for the system, lack of sample preparation techniques, and
inadequate fungal spectra in commercial database and soware
of manufacturers (ie, two commercial systems currently exist in
the United States: Bruker, Billerica, MA and Vitek MS, bioMérideux, Inc, Durham, NC). e expansion of database libraries
and developments in sample preparation are rapidly evolving
to establish validated and routine procedures for large numbers
of clinically important fungal strains and species. MALDI-TOF
MS is also becoming a reliable and reproducible method for
antifungal susceptibility testing (eg, caspofungin for isolates of
Candida spp.).
20,28
Finally, MALDI-TOF MS is also being investigated for epidemiologic testing of fungal isolates for outbreak
investigations and as an early surveillance test and/or screening
tool for antifungal drug resistance.
20
Antifungal Susceptibility Testing
e importance of antifungal susceptibility testing has become
increasingly recognized as a useful component in the treatment optimization of invasive infections caused by Candida
spp. because of the increasing number of available antifungal
agents, emerging resistance issues to standard therapy, and the
changing epidemiology of invasive fungal disease. Obtaining
antifungal susceptibility testing is particularly important when
azole- resistant isolate is suspected, when failure to respond
to antifungal therapy has occurred, and in species in which
acquired resistance oen exists (ie, Candida glabrata, Candida
auris, and Aspergillus fumigatus).
e CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) has developed standardized
reference methods for macrodilution and microdilution susceptibility testing of yeasts and molds and broth microdilution
method for dermatophyte.
ologies, including agar dilution, disk diusion, E- test methods,
and semisolid agar, have also been applied to susceptibility tests
of yeasts and molds. e commercial availability of simplied
and/or automated testing methods (eg, E- test and other gradient
strip testing; Vitek 2; Sensititre YeastOne) consistent with CLSI
reference methods is allowing an increasing number of clinical
laboratories to routinely perform antifungal susceptibility testing. Molecular testing methods for the detection of resistance
have also been expanding.
Interpretive MIC breakpoints based on CLSI- and
EUCAST- recommended invitro susceptibility testing methods have been recommended for Candida spp.
hensive reviews regarding the microbiological, molecular,
28-30
Agar- based alternative method-
28-30
28-30
Compre-
pharmacokinetic- pharmacodynamic, and clinical antifungal data for Candida spp. provide species- specic interpretive
clinical breakpoints for azole agents and the echinocandins
(Table19-5).28 ese data have been used to establish epidemiologic cuto values, detect emerging resistance among Candida
spp., and harmonize antifungal susceptibility testing standards
by CLSI and EUCAST.
28-30
In addition, tentative breakpoints
for the multidrug- resistant pathogen Candida auris have been
proposed (https.//www.cdc.gov/fungal/candida- auris/c- auris
- antifungal.html).
Interpretive breakpoint criteria for amphotericin B and some
of the triazole agents against selected Aspergillus spp. have been
reported; other fungal pathogens remain to be standardized.
28-30
e recommended EUCAST clinical breakpoints for Asper-
gillus fumigatus include ≤1 mg/L (susceptible) and >2 mg/L
(resistant) for amphotericin, itraconazole, and voriconazole,
and ≤0.125 mg/L (susceptible) and >0.25 mg/L (resistant) for
posaconazole (provided sucient drug concentrations can be
achieved).
VIRUSES
More than 650 viruses are known to cause infection in humans
and other vertebrate animals.31 e three major properties that
classify viruses into families include (1) the nucleic acid (NA)
core (either DNA or RNA but not both); (2) whether the viral
NA is single- stranded or double- stranded (https://viralzone
.expasy.org/656); and (3) the presence or absence of a lipopro-
tein envelope (Tables19-6 and 19-7).
13,31,32
Viruses also dier
based on their genome topology (eg, linear, circular, single versus multiple segments). Virus families can be further categorized
based on morphology (eg, size, shape, and substructure), mode
of replication, and molecular and genomic characteristics. e
most recent information on the rapidly changing classication
and taxonomy of viruses can be obtained from the website database (http://ictv.global/report/) that has been established by e
International Committee on Taxonomy of Viruses (ICTV). e
2019 ICTV report now recognizes ve hierarchical ranks consisting of 55 orders, 168 families, 103 subfamilies, 1421 genera,
and 6,590 species of viruses; however, a larger number of viruses
remain unclassied.
The Identification of Viruses
e ability to detect and accurately identify viruses in the clinical laboratory has increased during the last 30 years as a result
of wider applicability of diagnostic laboratory techniques with
increased sensitivity and decreased turnaround time, the availability of newer reagents and rapid commercial diagnostic kits,
and the addition of new antiviral drugs for specic viral infec-
1,13,32-37
tions.
specically PCR and real- time PCR, are allowing routine clinical laboratories to provide virology services for the increasing frequency of infectious diseases that depend on rapid viral
diagnosis.
It is important to note that all diagnostic tests for the iden-
tication of viruses are not available at each institution, and
In addition, several NA amplication tests (NAATs),
1,13,32
462 BASIC SKILLS IN INTERPRETING LABORATORY DATA
https://t.me/med1917
TABLE 19-5.In VitroCandida
MIC BREAKPOINT (mg/L)
CLSI EUCAST
ANTIFUNGAL AGENT CANDIDA spp.
Anidulafungin C albicans
C krusei, C tropicalis
C parapsilosis
a
C glabrata
C albicans
C krusei, C tropicalis
C parapsilosis
a
C glabrata
C albicans, C tropicalis, C parapsilosis
C glabrata
a
C albicans, C tropicalis, C parapsilosis
C albicans,C tropicalis, C parapsilosis
C krusei
a
C parapsilosis to anidulafungin and micafungin and C glabrata
b
C glabrata
≤Candida albicans
Source
Manual of Clinical Microbiology
S R S R
≤ > ≤ >
≤ > ≤ >
≤ >4 ≤ >4
≤ > ≤ >
≤ > ≤ >
≤ >
≤ >4 ≤ >
≤ > ≤ >
≤ >4 ≤ >4
b≤ b> ≤ >
≤ >
≤ > ≤ >
≤ >1
the clinician must establish a relationship with the laboratory
that will be performing viral testing. In certain clinical situations, samples may need to be sent out for diagnostic testing at
either large reference or public health laboratories because they
are able to provide the necessary methods that are dicult or
impossible to routinely perform in the clinical virology laboratory. In addition, certain viruses (eg, arboviruses, arenaviruses,
loviruses, Variola virus, and rabies virus) require testing at biosafety level (BSL) 3 or 4 facilities and are oen sent to the Centers for Disease Control and Prevention (CDC) or the CDC’s
Division of Vector-Borne Infectious Diseases.
13,32,33
e ability to accurately diagnose a viral infection is highly
dependent on appropriate selection, timing, collection, and handling of biological specimens.
32-34
In general, the highest titers
of viruses are present early in the course of illness and decrease
as the duration of illness increases. erefore, it is important
to collect specimens for the detection of viruses in the early
course of an infection. In most cases, identication of viruses is
a specimen- driven process. Because collection procedures are
highly dependent on viruses being suspected, attention needs to
be taken regarding collection containers and devices, and transport systems (eg, whether a viral transport medium is needed).
e dierent types of clinical specimens that can be collected
for viral culture and antigen detection include respiratory
specimens (eg, nasopharyngeal swabs, aspirates, and washes;
throat swabs; BAL and bronchial washes), blood, bone marrow,
CSF, stool, biopsy tissue, urine, ocular specimens, vesicles and
other skin lesions, and amniotic uid. In addition, specimens
for molecular diagnostic testing (eg, PCR and other nucleic
amplication techniques) must be obtained following specic
guidelines so that the stability and ampliability of the NAs are
ensured.
CHAPTER 19 • InfECTIous DIsEAsEs: fungI, VIRusEs, AnD MyCobACTERIA 463
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TABLE 19-6.
Humans
to
EXAMPLES
OF SPECIES
FAMILY NATURE ENVELOPE SHAPE
NUCLEOCAPSID,
SYMMETRY
COMMONLY
INFECTING
HUMANS
METHODS COMMONLY
USED FOR DETECTION OF
a
VIRUS
Adenoviridae Human
mastadenovirus
Hepadnaviridae
Yes
transcribing
Herpesviridae Yes
macacine
to monitor viral load in
serology
skin and mucous membrane
increasingly used)
determine immunity and
sensitive)
Human
cytomegalovirus
monitoring viral load in blood
Histology
determine viral load)
continued)
464 BASIC SKILLS IN INTERPRETING LABORATORY DATA
https://t.me/med1917
TABLE 19-6.
EXAMPLES
OF SPECIES
FAMILY NATURE ENVELOPE SHAPE
NUCLEOCAPSID,
SYMMETRY
COMMONLY
INFECTING
HUMANS
METHODS COMMONLY
USED FOR DETECTION OF
a
VIRUS
measure cell- mediate
γ
Human
associated
Papillomaviridae Human
Parvoviridae Human
bocavirus
Adenoassociated
Human
Human
1
not routinely available for
infection in children
Tissue histology
bocaviruses included on
to monitor viral load in
CHAPTER 19 • InfECTIous DIsEAsEs: fungI, VIRusEs, AnD MyCobACTERIA 465
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TABLE 19-6.
EXAMPLES
OF SPECIES
FAMILY NATURE ENVELOPE SHAPE
NUCLEOCAPSID,
SYMMETRY
COMMONLY
INFECTING
HUMANS
METHODS COMMONLY
USED FOR DETECTION OF
a
VIRUS
Polyomaviridae
Poxviridae Yes Brick-
oval
Variola virus
contagiosum
virus
Yaba monkey
tumor virus
immunochemistry and
detection)
has limited availability
Cell culture
b
a
b
Source
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