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Examining The Importance of Laboratory and Diagnostic Testing When Treating and Diagnosing Onychomycosis
Examining The Importance of Laboratory and Diagnostic Testing When Treating and Diagnosing Onychomycosis
Examining The Importance of Laboratory and Diagnostic Testing When Treating and Diagnosing Onychomycosis
1
Case Western University, Cleveland, OH, Abstract
2
Icahn School of Medicine at Mount Sinai, Onychomycosis is a fungal nail infection caused primarily by dermatophytes. Several other
New York, NY, 3Touro University Nevada,
nail disorders, including psoriasis, can simulate onychomycosis. Accurate diagnosis is
Henderson, NV, and 4PharmaDerm a
division of Fougera Pharmaceuticals Inc.,
therefore vital for the ongoing treatment and management of onychomycosis and to avoid
Princeton, NJ, USA misdiagnosis and treatment delay, which can be both lengthy and costly. Often, a
combination of histologic and laboratory techniques is used to obtain an accurate
Correspondence diagnosis. The potential diagnostic challenges associated with the differential diagnosis of
Mahmoud Ghannoum, PhD
onychomycosis caused by dermatophytes and the most common techniques used to
Case Western University
confirm the diagnosis are discussed.
10900 Euclid Avenue
Cleveland, OH 44106
E-mail: mahmoud.ghannoum@case.edu
doi: 10.1111/ijd.13690
is critical when treating onychomycosis in order to avoid misdi- Samples for the latter two types can be collected with a nail drill
agnosis and treatment delay.4 This article discusses the impor- to enhance precision.11 Moreover, the type of sample collected
tance of obtaining an accurate diagnosis and reviews the also depends on the laboratory test being used. Periodic acid–
different laboratory techniques available to confirm the diagno- Schiff (PAS) staining requires nail plate clippings but will not
sis of onychomycosis caused by dermatophytes. distinguish fungal species. On the other hand, subungual debris
sent for fungal culture may provide viable fungal elements of
dermatophytes, nondermatophytic molds, and yeasts that can
Differential Diagnosis of Onychomycosis
be identified to genus and species.12,13
As mentioned previously, only 50% of nail dystrophy is because
of fungal infection, with several other conditions, including
Diagnostic Techniques
inflammatory disorders such as psoriasis and lichen planus,
presenting nail changes that clinically mimic onychomycosis.1,5,6 A diagnosis of onychomycosis is confirmed when fungal hyphae
Additionally, the clinical appearances of onychomycosis caused and fungal viability are demonstrated and the fungal species
by different fungal species are often indistinguishable, thus indi- identified.14 A variety of laboratory and diagnostic techniques,
cating that laboratory testing is required for identification of the described below, are currently used, all of which have benefits
infecting organism.7 and limitations; thus accurate diagnosis is often achieved using
History and clinical evaluation are not sufficient to make the a combination of these techniques.14
diagnosis of onychomycosis, and empiric treatment of pre-
sumed onychomycosis may lead to lower than expected cure 10% Potassium hydroxide
rates and missed diagnoses. A diagnosis based solely on physi- The potassium hydroxide (KOH) test is a popular method used
cal examination/clinical signs can be incorrect,1,8 and various to confirm onychomycosis, with accuracy dependent on proper
laboratory assessments would be useful for confirmation.5 specimen collection, preparation, and examiner experience.
Physicians should be aware that laboratory confirmation, Specimen obtained from the nail bed and undersurface of the
when used in conjunction with their clinical expertise, can avoid nail plate is allowed to dissolve in 10% KOH solution after
delays in treatment.9 Traditional diagnostic techniques are placement on a glass slide for examination by light microscopy
based on microscopy, fungal culture, and histological examina- (Figure 1).10 The test is inexpensive,15 and the results are
tion. New technologies such as molecular assays and mass quickly available.1,10 The simplicity of the test enables it to be
spectrometry are expanding the clinical laboratory capabilities to performed in a clinician’s office.10 This test allows the examiner
correctly identify dermatophyte to the species level. to observe septate hyphae indicating that the patient is infected
with a dermatophyte, but no definitive information on the causa-
tive agent (i.e., genus and species) is obtained, and fungal via-
Optimal Specimen Collection Technique
bility cannot be determined.10,15 As such, positive results from
Obtaining clean samples is vital for the diagnosis of onychomy- KOH tests can be misleading if used as a test of cure.
cosis, as sample contamination is common. Simple steps to Inconsistent sensitivity has also been associated with KOH
prevent or minimize sample contamination include the use of a preparations.10,15 Sensitivity can be affected by a number of dif-
sterilizing solution such as 70% isopropanol to cleanse the nail ferent factors, including the experience of the examining clini-
plate and surrounding soft tissue prior to sample collection.1,5 A cian/technical staff, the preparation of the sample, and
sterile nail clipper should be used to clip the nail plate, and a differences in how samples are obtained. Moreover, a high rate
sterile curette or blade should be used to obtain subungual deb- of false-negatives, ranging between 5% and 15%, has also
ris from underneath the nail plate (i.e., the exposed nail bed been reported because of low visibility and sparse distribution
where the infection resides).1 of hyphae on the slide.5,16
Another critical factor is the collection of enough sample for
microscopic examination and culture,10 as too often, inadequate Calcofluor white
nail samples (either in quantity or quality) have led to failure of Calcofluor white is a fluorescent agent that is mixed with KOH
fungal diagnosis. The location from which the sample should be in order to stain the chitin in the fungal cell wall, thus making
collected varies depending on the category/type of onychomy- fungal elements more easily visible against the background of
cosis:1 for distal and lateral subungual onychomycosis the sam- host cellular material; however, as with KOH, the identity and
ple should be taken from the most proximal location after viability of the infecting microorganisms cannot be determined.17
clipping of the distal onycholytic nail plate;11 in proximal subun- Calcofluor white binds to the beta 1-3 and beta 1-4 polysaccha-
gual onychomycosis, the upper nail plate should be debrided rides in cellulose and chitin and fluoresces when exposed to UV
and a sample containing the underlying nail debris should be radiation. Peak excitation and emission wavelengths for
collected; and for superficial white onychomycosis, the speci- calcofluor white solution are 365 and 435 nm, respectively
men should be obtained from affected superficial nail plate.1 (Figure 1).10,18 The sensitivity issue associated with the
(a) (b)
traditional KOH test is overcome using calcofluor white if a removed from the site of infection.20 In order to reduce the like-
proper specimen is collected, although both techniques have lihood of obtaining false-negative results, the specimen should
shown similar effectiveness.18 However, the significant barrier be collected as proximally to the infection as possible – an area
to the regular use of this technique in office practice remains most likely to contain viable organisms and least likely to con-
the need for a fluorescence microscope.18 tain contaminants.5 False-positive results may very rarely be
caused by contamination from organisms present as transient
Fungal culture-based methods flora or contamination of growth media.16
Fungal culture has been considered to be the ‘gold standard’
technique in the diagnosis of onychomycosis.10,14 Clinical sam- Histopathology
ples are plated onto a properly selected general media such as Histopathological techniques use direct microscopic examination
potato dextrose agar with added antibiotics to inhibit overgrowth of tissue sections that have been stained with specific dyes to
by bacterial contaminants and a selective media such as Myco- visualize fungal growth patterns, which may suggest the pres-
sel containing cycloheximide, which inhibits the growth of sapro- ence of a dermatophyte.10,14 They cannot, however, determine
phytic fungi.10 Using a curette to obtain adequate subungual fungal viability.10
debris or multiple smaller fragments of nail plate and nail bed
has been suggested to improve the chances of obtaining a posi- Periodic acid–Schiff stain
tive result. Distal nail clippings should not be submitted for fun- The periodic acid–Schiff (PAS) stain is a histopathology stain of
gal culture because they often carry bacterial or saprophytic fungal polysaccharides performed on a nail plate biopsy speci-
mold contaminants that can easily overgrow dermatophytes on men. It is highly sensitive and, when coupled with fungal cul-
culture media. Although the cultures are then incubated for at ture, increases the overall sensitivity to 96%.1,15,21 The sample
least 4 weeks at 30 °C before being considered negative, the is taken using a nail clipper which removes as proximally as
culture could become positive for a dermatophyte within a possible the full thickness nail plate and hyperkeratotic nail bed
week. A significant advantage to using fungal culture is that it is if the latter is easily accessible. The sample is placed in forma-
able to identify the causative agent,5 thus being more specific lin and subsequently sectioned and stained with PAS stain,
than KOH testing.15 which reacts with the aldehyde groups in the fungal cell walls to
However, saprophytic mold or bacterial overgrowth may produce a magenta-colored stain (Fig. 1).10 PAS staining
occur, and there is a relatively long delay (e.g., 2–4 weeks) allows spores, hyphae, pseudohyphae, and yeast to be visual-
associated with obtaining the results, which may lead to frustra- ized. In addition to high sensitivity, PAS staining results are
tion for clinicians and their patients.10,15 The sensitivity of fungal available quickly, usually within 24 to 48 hours.10 However, as
culture is also lower than a properly performed microscopy with KOH testing, the main disadvantages of PAS staining are
test,10,19 and false-positive or false-negative results reduce the that the specific causative agent is unable to be confirmed and
reliability. that viable and nonviable organisms are indistinguishable.15
False negatives can occur for a number of reasons, including Furthermore, the cost associated with PAS is often higher than
insufficient nail material or improper sample collection too far other diagnostic techniques.22
67 patients with suspected dermatophytosis. Real-time PCR (m/z) ratios. These ratios measure how quickly charged ions
detected and correctly identified the causative agent in speci- from the fungal sample move through the time of flight (TOF)
mens from which T. rubrum, T. interdigitale, M. audouinii, or tube. Once spectra are generated, comparison of the m/z ratios
T. violaceum were cultured and also identified a dermatophyte to a reference database leads to fungal identification. Protein
species in an additional seven specimens that were negative by compositions differ between fungal species, which allows for
microscopy and culture.27 This highly sensitive assay also discrimination between closely related organisms.29
proved to have high positive and negative predictive values Several researchers have reported good correlation between
(95.7 and 100%, respectively), facilitating the accurate, rapid dermatophyte identification by conventional or gene sequencing
diagnosis conducive to targeted rather than empirical therapy and MALDI-TOF analysis.30–32 However, all have had to supple-
for dermatophytosis. ment current commercial databases with data generated from
their own reference dermatophyte strains. Alshawa et al.31
PCR-reverse line blot reported a correlation rate of 91.9% of dermatophyte strains
In a recent study, Bergmans et al.28 developed and successfully using conventional methods, while Jensen et al.33 matched
used a PCR-reverse line blot (PCR-RLB) for rapid detection 99.3% of identifications derived by gene sequencing. Clearly,
and identification of nine dermatophyte species in nail, skin, and MALDI-TOF will prove an invaluable methodology for rapid
hair samples. The developed method was based on ITS1 identification of dermatophyte isolates once commercial data-
sequences using genus and species-specific probes in isolates bases become more robust. However, another current limitation
obtained from 819 clinical samples (596 nail, 203 skin, and 20 of this method is the necessity for isolating pure dermatophyte
hair). In this method, membranes containing immobilized colonies from clinical samples before MALDI-TOF assay can be
oligonucleotide probes were exposed to denatured PCR prod- attempted. Further research to enable detection of dermato-
ucts, allowed to hybridize for 30 minutes, then subjected to phytes directly from clinical samples is required.
stringency washes and detection using streptavidin-peroxidase
and chemiluminescence. The investigators reported a positive
Mixed testing results
PCR-RLB reaction in 93.6% of 172 culture-positive and micro-
28
scopy-positive samples. The frequency of inconsistent results (i.e., opposing results from
different diagnostic tests) remains a challenge in onychomyco-
MALDI-TOF sis diagnosis (Table 1). A study conducted by Clayton et al.
Matrix-assisted laser desorption ionization–time of flight showed 11% of 2113 toenails were KOH positive but fungal cul-
(MALDI-TOF) mass spectrometry (MS) is being adapted for use ture negative.19 A negative result obtained from fungal culture
in microbiology laboratories. The ability to analyze large biologi- can be misleading, as it has been reported that almost half of
cal molecules by MS is made possible by the application of soft all onychomycosis samples fail to yield a positive culture.5 Prob-
ionization techniques that generate a spectrum of components. lems with obtaining a suitable sample contribute to the high
In a MALDI-TOF analysis, a saturated solution of an organic false-negative rate for fungal culture, with results as high as
compound, or matrix, is added to a portion of a fungal colony, 60%.34 Despite this, it is widely regarded that onychomycosis is
and the mixture is then applied to a metal plate. Upon drying, confirmed most accurately by positive results from fungal cul-
the crystallized mixture is subsequently irradiated using a laser ture.5 In addition, false-positive results from KOH tests do not
beam to force sublimation into a gas phase, followed by ioniza- ascertain that the observed fungi are viable and may accurately
tion of the fungal sample.29 reflect the state of the infection. Examples of differences
Ionized proteins within the sample are analyzed by a mass between KOH and culture results have been observed in many
spectrometer analyzer to produce a spectrum of mass-to-charge clinical trials of new antifungals being developed to treat
32,37
Table 1 Diagnostic Tests for Onychomycosis.
Visuala 77 47 No No $
KOH 67–93 38–78 No No $ 5–30 min
Culture 31–59 83–100 Yes Yes $$ Up to 4 weeks
Periodic acid–Schiff 92 72 No Yes $$ 24–48 h
stain
PCR 95 100 No Yes $ Within 24 h
a
Culture and periodic acid–Schiff staining.
5 Why is it important not to use microscopy alone for a test of 5 Elewski BE. Onychomycosis: pathogenesis, diagnosis, and
cure? management. Clin Microbiol Rev 1998; 11: 415–429.
6 Koshnick RL, Lilly KK, St Clair K, et al. Use of diagnostic tests
a. The hyphae may be nonviable, leading to a false-positive
by dermatologists, podiatrists and family practitioners in the
diagnosis. United States: pilot data from a cross-sectional survey. Mycoses
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10 Alberhasky RC. Laboratory diagnosis of onychomycosis. Clin
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12 Chaya AK, Pande S. Methods of specimen collection for
a. Cost of the procedure
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9 What is the main pitfall in using PCR to identify dermato-
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22 Lilly KK, Koshnick RL, Grill JP, et al. Cost-effectiveness of
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