Onychomycosis: a critical study of techniques and criteria for confirming the etiologic significance of nondermatophytes

Onychomycosis: a critical study of techniques and criteria

for confirming the etiologic significance of

nondermatophytes

RICHARD C. SUMMERBELL*, ELIZABETH COOPER$, URSULA BUNN%, FRANCES JAMIESON% &

ADITYA K. GUPTA$§

*Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands, $Mediprobe Laboratories, Toronto, %LaboratoriesBranch, Ontario Ministry of Health, Toronto, §Division of Dermatology, Department of Medicine, Sunnybrook and Women’sCollege Health Sciences Center and the University of Toronto, Toronto, Ontario, Canada

Nondermatophytic filamentous fungi (NDF) other than Scytalidium species are

variously said to cause between 0 and 50% of all toenail onychomycoses, though

most estimates are in the 2�/5% range. Due to the difficulty of obtaining ‘gold

standard’ control data for comparison, the accuracy of many laboratory evaluation

processes used to deal with potential NDF onychomycoses has never been

rigorously measured, thus allowing use of differing interpretations of the

significance of cultures. To allow valid comparison of these procedures and

interpretations, a large series of patients who declined treatment were sampled on

multiple occasions from all apparently onychomycotic toenails until adequate

certainty had been attained that all etiologic agents had been isolated and, where

necessary, confirmed as etiologic via consistent repeated isolation. This informa-

tion was used to evaluate results that had been obtained in the initial direct

microscopy and culture studies for the same patient population, as such results are

strongly relied on in routine diagnosis. Direct microscopy (KOH) was found to be

73.8% sensitive for dermatophytes and 67.2% sensitive for proven etiologic NDF

(difference not significant). Culture of the initial specimen coincidentally had a

sensitivity of 74.6% for both fungal groups. KOH and culture in tandem were

83.9% sensitive for indicating fungal etiology based on the first specimen. Different

evaluative frameworks currently used to interpret NDF isolations were contrasted.

The ‘classic’ evaluation procedure, in which all NDF considered etiologic must be

isolated from at least two successive nail specimens, at least one of which must be

microscopic positive for compatible fungal filaments, had a sensitivity of 59.5% but

a specificity of 100% for true NDF infections and mixed NDF/dermatophyte

infections. The most widely used ‘simple association’ evaluation criterion, in which

NDF outgrowth is considered etiologic whenever direct microscopy is positive for

fungal elements and no dermatophyte grows out from the initial specimen, had a

sensitivity of 53.6% and a specificity of 70.3% for NDF infections. A frequently

criticized, but in some quarters still advocated, variant of the simple association

criterion considers NDF outgrowth to be significant whenever the corresponding

specimen is positive for fungal filaments in direct microscopy; application of this

Correspondence: Richard C. Summerbell, Centraalbureau voor

Schimmelcultures, Uppsalalaan 8, 3584 CT, Utrecht, the

Netherlands. Tel: �/31 3021 22688; Fax: �/31 3025 12097; E-mail:

[email protected]

Received 16 September 2003; Accepted 8 January 2004

– 2005 ISHAM DOI: 10.1080/13693780410001712043

Medical Mycology February 2005, 43, 39�/59

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criterion yielded a sensitivity of 60.7% for true infections but a specificity of only

42%. With the aid of two standard notes soliciting repeat specimens, the classic

criterion was able to attain 92.7% accuracy in recognizing all NDF etiologic agents

and 100% accuracy in disregarding all contaminants from initial specimens that

were positive in direct microscopy and yielded a filamentous fungus in initial

culture. Even in exhaustive longitudinal study, only 20.2% of NDF infections were

found to be associated with a concurrent dermatophytosis. In auxiliary studies,

some nails remained NDF-infected after dermatophytes had been successfully

eliminated by therapy.

Introduction

Both dermatophyte fungi and some members of the

Scytalidium dimidiatum (synanamorph name Nattras-

sia mangiferae ) clade are specialized for pathogenesis of

human nails and skin. Other nondermatophytic fila-

mentous fungi (NDF) rarely cause skin infection, but

approximately 35 species have been rigorously impli-

cated in causation of onychomycosis [1]. The precise

role of these NDF in onychomycosis is imperfectly

understood, and there is considerable controversy

about how best to interpret clinical findings in many

cases in which an NDF grows from an abnormal nail.

This uncertainty has led to disparity in different

researchers’ interpretative frameworks, with the result

that statistics published in various studies on the

relative prevalence of nondermatophytes in onychomy-

cosis differ to an extreme degree. Selected examples are

shown in Table 1; others are reviewed by Greer [2]. The

same degree of discrepancy also applies to infections

attributed to yeasts, but this topic is beyond the scope

of this article.One major factor causing difficulty in NDF status

evaluation is that the reliability of dermatophyte

growth from true-positive samples is far from ideal:

various studies have found that dermatophytes causing

onychomycosis fail to grow from about 15�/50% of

true-positive samples, even though their filaments can

often be seen in the same samples in potassium

hydroxide (KOH)-based direct microscopy [3�/6]. Gen-

tles [7], in a study in which each patient yielding a

filament-positive sample was followed up with repeated

sampling until the corresponding dermatophyte was

grown, showed that 73% grew a culture on the first

sampling occasion, while another 15.7% first grew a

culture on the second sampling occasion. Disturbingly,

5.6% failed to grow a culture until the third sampling,

while another 5.6% grew a culture only from the fourth

or a later sample. The underlying reason, as seen when

vital stains are used on such samples [8], is that

dermatophyte growth in nails is modular and growth

fronts in nail tissue eventually die out, leaving dead

filaments behind in extensive tracts of nutritionally

depleted nail tissue. Needless to say, dermatophyte

cultures cannot be grown from these dead fronts. From

each such sample cultured, however, there is a risk that

a contaminating mold may be grown and misinter-

preted as the agent of the infection. This hazard

connects with another factor making NDF evaluation

vexatious, which is that many of the fungi most

commonly causing NDF onychomycoses are also

among the fungi most commonly obtained in culture

as innocuous contaminants of feet and nails [1]. Not

only are they common indoor fungi in general but also,

the same affinity for nail chemical constituents that

allows them to cause onychomycosis may facilitate

their growth on chemically related, environmentally

proximal materials such as dead skin flakes in shoes

and on floors (when these floors are damp or humid),

as well as moist, decaying shoe leather.

Table 1 Contribution of different groups of organisms to the etiology of onychomycosis as attributed in various studies (in % of total etiologic

agents isolated). Study [reference no.] and geographic area

Attributed etiology [43] UK [14] USA [10] Canada [11] UK [13] Italy

Dermatophytes only 80.5 15.6 90.5 55.7 23.2

Yeasts only 17.0 8.9 5.5 33.2 59.1

Moulds only 2.5 48 4 11.0 17.6

Mixed: dermatophyte�/other NS 19.1 NS NS NS

NS, not stated.

– 2005 ISHAM, Medical Mycology, 43, 39�/59

40 Summerbell et al.

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In all laboratories assessing onychomycosis, an NDF

is generally considered significant in the rare cases inwhich direct nail microscopy shows very distinctive

nondermatophytic structures (e.g. Scopulariopsis con-

idia or Aspergillus conidiophores) occurring in addi-

tion to fungal filaments in or connectedly emergent

from the affected nail tissue.

Laboratories may differ profoundly, however, in how

they interpret NDF growth from a nail infection in

which fungal filaments of a nondescript nature are seenin direct microscopy, as occurs in the majority of

dermatophytic and NDF infections. A taxonomic

approach to onychomycosis studies suggests that in

cases in which an NDF grows from a sample, authors

generally use one of three major interpretative criteria

to establish that the isolated NDF is a sole causal agent

and not a contaminant. First, the classic , rigorous

procedure for NDF onychomycosis confirmation de-mands at least one demonstration of compatible fungal

filaments in direct microscopy of the relevant nail

tissue, as well as at least two independent isolations

of the putative NDF causal agent from specimens

taken at different times at least a week apart [9,10]. In

the case of a possible mixed infection involving both a

dermatophyte and a nondermatophyte, an additional

(third) isolation of the nondermatophyte may berecommended as an ideal procedure. Second, in an

attempt to circumvent the troublesome need for a

second specimen in evaluating NDF outgrowth, Walshe

and English [11] proposed an inoculum-counting pro-

cedure (later slightly modified by English [12]) in which

an NDF was declared causal when (i) compatible

fungal filaments were present in direct nail microscopy;

(ii) no dermatophyte grew in culture; and (iii) the samenondermatophytic fungus grew from at least five of 20

nail fragments planted from scrapings or drillings of

the lesion. The possibility of recognizing mixed infec-

tions was generally excluded when this criterion was

used. Finally, peer-reviewed publications regularly

appear in which NDF etiology is evaluated with

minimal complexity by means of a simple association

criterion, that is by a criterion in which an NDF istreated as causal if it grows from a single nail specimen

in which fungal filaments are seen in direct microscopy,

but from which no dermatophyte grows (see, for

example, ref. 13). A variant version of this criterion

accepts all NDF that grow from filament-positive nail

specimens as causal, regardless of whether a dermato-

phyte also grows or not (see, for example, ref. 14). In

the former case, authors may be stated to use aconservative simple association criterion, and in the

latter a liberal variant of this criterion. Data generated

by studies using an explicit liberal simple-association

criterion are very similar to data generated in studies

that simply list all fungal isolations from onychomyco-

tic nails without definitively pronouncing on the

significance of the nondermatophytic isolations (see,

for example, ref. 15). Such agnostic simple association

studies tend to be presented in a way that causes them

to be routinely interpreted by later authors as if they

documented nondermatophytic infections rather than

mere isolations (e.g. see handling of such a study done

by Budak et al. [15] in a review by Greer [2]); they thus

form a category of publication that is de facto distinct

from studies that explicitly purport to document the

incidence of probably etiologically insignificant fungi in

association with nails or feet (see, for example, refs

16,17). The simple association criteria have long been

subjected to criticism, but this criticism is not universal;

indeed, one such study recently was awarded the 2001

William J. Stickel Bronze Award by the American

Podiatric Medical Association [14]. It generally agreed

that, as Ellis [18] stated, ‘there is considerable con-

troversy on the significance of nondermatophyte moulds

and yeasts when they are identified in the presence of a

dermatophyte.’A thorough review of past onychomycosis studies

and their relation to these analytical categories is

needed, but is beyond the scope of the present paper.

In addition to the above-listed common interpretive

criteria about which some controversy may exist, there

are also two definitive high-tech criteria, namely

differential flow cytometry and specific in-situ immu-

nohistochemistry of infected nail tissue [19,20]. Though

these techniques appear to give unequivocal results, the

labor and cost of setting them up has so far deterred

use in both routine and other reference laboratories.

The merits and deficiencies of inoculum-counting

techniques for analyzing NDF in onychomycosis have

been elaborated in a previous analysis [21]. The purpose

of the present study was to evaluate the classic and

simple-association NDF sole-agent and mixed-infec-

tion evaluation criteria with a data set derived from a

large longitudinal study of onychomycosis cases. In the

study, untreated onychomycosis patients yielded an

extended series of nail specimens over a period of up

to 3 years. The multiple specimens taken per nail, and

the long-term follow-up involved, provided gold stan-

dard data that could be used to distinguish both causal

and incidental fungi. Comparison of this decisive

information with the ways in which initial specimens

from the same patients would be interpreted by routine

laboratories using the various common confirmation

criteria allowed a determination of how well these

criteria perform as interpretative frameworks for


Onychomycosis 41

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determining etiologic significance in putative NDF

onychomycoses.

Materials and methods

Patient population

Details of the selection and composition of the patient

population have been given by Gupta et al. [21]. In

brief, patients were enrolled in the study on the basis of

having at least one abnormal, apparently lesional

toenail that had not been treated with antifungal drugs

at least 6 months prior to the study and that, by

consent, would not be treated over the course of the

study. In total, 473 patients (mean age 64, standarddeviation (SD)9/15 years) were included in the study, of

whom 341 yielded sufficient specimens to be fully

evaluated in the context of the present study. Each

fully evaluated patient was examined at least three

times within a 3-year interval, and the majority were

examined on five or more occasions.

Sampling and laboratory study

Sampling was done with a sterile #15 scalpel blade or

curette according to standard procedures [22]. The area

of nail to be sampled was disinfected with ethanol andsuperficial material was abraded away and discarded

before more deeply situated material was collected for

fungal analysis.

Laboratory studies

The techniques recommended by Kane and Summer-

bell [23] were followed for inoculation of nail scrapings.

To facilitate growth of dermatophytes, Sabouraud agar

amended with a mixture of 100 mg/ml cycloheximide,

100 mg/ml chloramphenicol, and 50 mg/ml gentamicin

(CCG) was used in combination with Casamino acids�/

erythritol�/albumin (CEA) medium [24] plus CCG. Tofacilitate mold outgrowth, a cycloheximide-free med-

ium, Littman oxgall agar (Difco Laboratories, Detroit,

MI) amended with 100 mg/ml streptomycin, was

employed. Media with no fungal growth were declared

negative at 21 days. In a deviation from routine

practice, all mold species growing from the specimens

were identified at least to genus level, and all potentially

causal molds except Acremonium spp. were identifiedto the species level. Molecular analysis of the Acre-

monium isolates involved is currently in progress.

Infection classification

Individually for each affected toe, lesional areas

compatible with superficial white onychomycosis were

sampled separately from areas compatible with distal�/

lateral subungual onychomycosis in order to assurethat all etiologic agents involved were elucidated. The

records obtained were pooled for each patient in the

present study, but a breakdown by disease type is given

by Gupta et al. [21]. Records for each patient were

examined and when a distinct pattern of fungal

isolation became clear the patient was classified as to

whether he or she supported an uncomplicated derma-

tophytosis, an NDF infection, a mixed NDF/dermato-phyte infection or another infection type. Those who

grew a dermatophyte at least once but did not

consistently grow any particular mold after two or

more visits were classed as uncomplicated dermatophy-

tosis patients. Those who (i) grew a particular mold

consistently on three successive occasions; (ii) yielded a

specimen showing consistent filaments in direct micro-

scopy at least once; and (iii) continued to grow the samemold consistently in most or all specimens during

follow-up of the same nail (e.g. left hallux) and nail

stratum (i.e. subungual or superficial), without growing

a dermatophyte on any occasion, were classified as

NDF onychomycosis patients. Those who grew a

dermatophyte on one or more occasions, and also

grew a mold with the same consistency, site specificity,

and (where occurring alone in an individual nail orstratum) direct microscopic verifiability as indicated

above for NDF onychomycosis, were classed as mixed-

infection patients. S. dimidiatum and Scytalidium

hyalinum sole-agent and mixed infections were consid-

ered to constitute a separate category; any outgrowth of

these species, which do not occur as contaminants in

the study area, was considered etiologically significant.

Patients who grew neither a dermatophyte nor aconsistent mold, and who were negative for fungal

filaments in direct microscopy, were classed as having

lesions of nonmycotic etiology. Cases in which a patient

yielded positive direct microscopy at least once but also

failed to grow a dermatophyte or a consistent mold in

multiple specimens were considered to be of unresolved

etiology.

Evaluation of laboratory practices

The cases studied were evaluated in terms of how they

would be understood based on reports generated in

normal laboratory practice, both in routine labora-

tories and in the reference laboratory of R.C.S. As

routine practice relies heavily on the results of the initial

patient specimen, which is often the only one taken, theresults of this specimen were focused on and were

contrasted with the overall conclusion about the

patient’s infection status after multiple samples had



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been analyzed. In the classic evaluation procedure for

NDF onychomycoses, the results of the first twospecimens are usually taken as the deciding point;

therefore, in evaluating the success of this criterion, the

results deriving from this pair of specimens were

considered. Only successive specimens from the same

nail were taken into consideration in this evaluation.

Sensitivities, specificities, and positive predictive values

were generated where appropriate both for discrete

types of laboratory results, such as elucidation of adermatophyte in a dermatophytosis patient, and for

judgment criteria used to make inferences about the

status of infections (e.g. criteria stating that, given

certain findings, patients should be deemed to have or

not have an NDF infection). Sensitivity was calculated

by the formula TP}/(TP�/FN), where TP�/true posi-

tives and FN�/false negatives. Specificity was TN}/

(TN�/FP), where TN�/true negatives and FP�/falsepositives. Positive predictive value was reckoned as

TP}/(TP�/FP). In the case of evaluation criteria

applied to NDF growing from specimens, a correct

affirmation of etiologic status was considered a true

positive, a correct exclusion of etiologic status a true

negative, and so on.

We also investigated the efficacy of two notes used in

the R.C.S.’s laboratory in an attempt to elicit themaximal number of NDF onychomycosis confirma-

tions according to classic criteria. These notes are

attached to outgoing report sheets for nail specimens

that have grown a potentially significant NDF.

‘Note 1’

Note 1 is similar to those sent out by many laboratories

for potentially significant NDF that grow from speci-

mens where direct microscopy has revealed the presence

of nonspecific (i.e. not recognizable with certainty as

dermatophytic or nondermatophytic) fungal filaments,

and reads as follows.

This common, normally nonpathogenic fungus, when isolated from

filament-positive nail material, is usually a contaminant overgrowing

nonviable dermatophyte inoculum. Uncommonly, it causes onycho-

mycosis and can be confirmed as an etiologic agent by consistently

repeated isolation in the absence of a dermatophyte. Rare mixed

infections will show consistently repeated isolation in successive

samples along with at least one isolation of a dermatophyte.

This note is not used for all NDF outgrowth, as

many such species are constantly occurring contami-

nants that never cause onychomycosis; its use isrestricted to cases of isolation of species well docu-

mented [1] via the classic confirmation procedure (often

supplemented with additional repeat specimens) as

being potentially significant (Table 2). A few potentially

significant species, such as Chaetomium globosum and

Alternaria alternata , are excluded from the list ofelicitors of Note 1, because they are constant nail

contaminants that only very rarely cause onychomy-

cosis, and because actual onychomycosis cases caused

by these fungi are often, though not always, associated

with dark or otherwise unusual filaments in direct

microscopy. An efficient way of dealing routinely with

such fungi in rare cases where they produce only

nondescript filaments in infected nail tissue is yet tobe designed.

‘Note 2’

Note 2 is as far as we know an innovation of our own

based on the observation that true NDF onychomy-

cosis cases are more likely than dermatophytoses to be

associated with patchy nail invasion leading to anunusually high chance of fortuitously negative direct

microscopic findings in readings of single specimens

[21]. It appears that these fortuitously negative readings

Table 2 Fungi routinely reported via Notes 1 and 2 (see main text)

as potential agents of opportunistic onychomycosis when encountered

in outgrowth from nail specimens. Corresponding teleomorph or

anamorph names are given where these alternative states are

commonly seen in clinical laboratory cultures and aid in routine

identification

Acremonium potronii

A. sclerotigenum

A. strictum

Aphanoascus fulvescens (anamorph Chrysosporium state of A.

fulvescens )

Aspergillus candidus

A. chevalieri (teleomorph Eurotium chevalieri )

A. flavus

A. fumigatus

A. nidulans (teleomorph Emericella nidulans )

A. niger

A. sydowii

A. terreus

A. ustus

A. versicolor

A. vitis (teleomorph Eurotium amstelodami )

Curvularia lunata

Fusarium oxysporum

F. proliferatum

F. solani

F. verticillioides (formerly called F. moniliforme )

Lasiodiplodia theobromae

Microascus cinereus (anamorph Scopulariopsis state of M. cinereus )

M. cirrosus (anamorph Scopulariopsis state of M. cirrosus )

Onychocola canadensis

O. kanei

Scopulariopsis brevicaulis

S. brumptii

S. candida


Onychomycosis 43

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are not generally based on reader error; in fact, the

reading is generally accurate but the nail fragmentschosen for reading happen to lack fungal elements by

chance alone. Exhaustive analysis shows, however, that

a minority of other fragments in the same specimen

packet generally do contain these elements [21]. Thus a

single negative direct microscopic reading cannot be

taken to rule out NDF onychomycosis when a Table 2

mold grows out in culture, especially when this growth

is at a high inoculum level [21].To cover this eventuality in routine practice where

the physician and patient must decide if submitting a

second specimen is worthwhile, we composed Note 2 as

follows.

In cases like the present one where direct microscopic examination

shows no mycelium, this isolation would ordinarily be considered

surface contamination. Since the species isolated does occasionally

cause onychomycosis, however, in addition to being a common

contaminant, this isolation is noted for your records. In a small

proportion of such cases, a follow-up specimen from the affected nail

will yield positive direct microscopy and regrow the same fungal

species, confirming opportunistic onychomycosis.

Results

Infections

Of 341 patients yielding three or more evaluable

specimens, 311 yielded results that allowed unequivocal

classification of the infection status of their nails (Table

3). The remainder showed fungal filaments in direct

microscopy on one occasion but grew neither a

dermatophyte nor a consistent mold in up to eight

successive specimens; these patients were considered tobe of unresolved status. Of the 311 patients with

resolved status, 177 (56.9%) were uncomplicated der-

matophytosis patients, 17 (5.5%) had dermatophytosis

mixed with NDF infection; 67 (21.5%) had NDF

infection and 47 (15.1%) were considered to have nail

lesions for which fungal etiology had been ruled out.

There was also one probable yeast infection, one

pure S. dimidiatum infection, and one mixedT. mentagrophytes �/Scytalidium hyalinum infection.

Species identification of most of the NDF implicated

in causing the infections scrutinized in this study is

given in our previous publication [21] in connection

with relevant inoculum count data. The dermatophytes

isolated in uncomplicated infections included 121

Trichophyton rubrum , 50 T. mentagrophytes complex,

one Microsporum gypseum, three mixtures of T. rubrum

and T. mentagrophytes complex, one mixture of

T. rubrum and T. violaceum , and one mixture of

T. rubrum , M. gypseum and T. mentagrophytes com-

plex. Mixed dermatophyte/mold infections included, as

dermatophyte components, nine T. rubrum , seven T.

mentagrophytes complex, and one mixture of T. rubrum

and T. mentagrophytes complex. Just one case could be

tentatively classed as a yeast infection, with persistent

colonization seen by Candida parapsilosis. In this case,

no pseudohyphae were seen in direct microscopy and

the nail disruption was concluded to have been caused

by adjacent paronychia.

Direct microscopy

In initial specimens, cases of dermatophytosis, includ-

ing mixed infections, were signaled by positive direct

microscopy (fungal filaments seen) in 144 of 195 cases

(test sensitivity of 73.8%). In the long-term, positive

direct microscopy was confirmed for 100% of derma-

tophytosis patients.In uncomplicated NDF infection, the initial speci-

men was microscopic positive in 45 of 67 cases (67.2%).

This proportion of microscopic positives is statistically

similar to that seen with dermatophytes in the initial

specimen (P�/0.68, chi-squared). In the remaining 22

cases, the initial specimen was microscopic-negative,

though in all cases multiple later specimens were found

to be positive. Including correctly negative readings fortrue nonmycotic nails along with positive readings for

both dermatophyte- and NDF-infected nails, direct

microscopy of the initial specimen gave an accurate

picture of the nail’s infection status in 235 of 311

evaluable cases (75.6%).

Some physicians may treat patients for dermatophy-

tosis on receiving or generating a report that fungal

filaments are present in direct microscopy of nails. Forthe resolved cases in the present study, however, only

131 of 190 instances of positive direct microscopy in the

initial specimen signaled dermatophytosis (i.e. this test,

if used as an indicator of uncomplicated dermatophy-

tosis, has a positive predictive value of only 70.0%, at

least in populations similar to the one studied here). If

mixed dermatophyte infections (including NDF and

Scytalidium mixtures) are included as valid diagnosesof dermatophytosis, despite any differences in thera-

peutic strategy that might be entailed by mixtures, the

positive predictive value of this test would still be just

75.8%.

Scopulariopsis brevicaulis cases are occasionally

associated with the appearance of recognizable Scopu-

lariopsis elements, usually conidia, in direct microscopy

along with fungal filaments [1]. In the present study,distinctive Scopulariopsis direct microscopic presenta-

tion was seen in only two of 13 S. brevicaulis cases in

direct examination of the initial specimen, and addi-



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Table 3 Correspondence of patient infection categories, as determined by multiple follow-up samples, with the results of initial nail specimens

Outgrowth from first

specimen

Direct

microscopy

for first

specimen

Infection category (final classification based on longitudinal study)

Dermatophytosis

alone

Dermatophyte-

mold mixed

infection

Dermatophyte-

Scytalidium

mixed

infection

Mold infection

alone

Scytalidium

infection

alone

Yeast

infection

Nonmycotic

nail

abnormality

Unresolved:

KOH�/once,

no significant

culture in four

or more

specimens

Dermatophyte only �/ 67 3 �/ �/ �/ �/ �/ �/

�/ 20 �/ �/ �/ �/ �/ �/ �/

yc 1 �/ �/ �/ �/ �/ �/ �/

Mold only �/ 17 3 �/ 43 (incl. 1 insignif.)* �/ �/ �/ 8

�/ 3 2 �/ 10 (incl. 2 insignif.)* �/ �/ 19 7

yc �/ �/ �/ �/ �/ �/ 1 �/

Dermatophyte plus mold �/ 28 6 �/ �/ �/ �/ �/ �/

�/ 7 �/ �/ �/ �/ �/ �/ �/

Dermatophyte plus yeast �/ 8 �/ �/ �/ �/ �/ �/ �/

�/ 1 �/ �/ �/ �/ �/ �/ �/

Scytalidium alone �/ �/ �/ 1 �/ �/ �/ �/ �/

Yeast alone �/ 1 �/ �/ 1 �/ �/ �/ 3

�/ 2 �/ �/ 2 �/ �/ 6 1

Mold plus yeast �/ 1 �/ �/ �/ �/ �/ �/ �/

�/ �/ �/ �/ �/ �/ 1 �/ �/

yc �/ �/ �/ �/ �/ �/ 1 �/

Negative (no fungus grown) �/ 9 �/ �/ 1 1 �/ �/ 4

�/ 12 3 �/ 10 �/ �/ 20 7

Total (n�/341) �/ 177 17 1 67 1 1 47 30

Direct microscopy: �/, fungal filaments present; �/, no fungal elements seen; yc, budding yeast cells seen; *insignificant in the number of cases specified, a mold grew in the initial specimen,

but it was etiologically insignificant and in fact a different mold species was ultimately shown to be the agent of infection (see comments in text about wrong mold isolations).

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tional examinations in connection with later samples

revealed such in-situ elements in only one additional

case (Table 4). Scopulariopsis conidia were not seen in

any of the three well-established mixed S. brevicaulis /

dermatophytosis cases examined.

Culture

To illustrate the types of diagnostic decisions that were

necessary in this study, a small number of selected

culture records from our patients are shown in Table 5.

A typical, relatively uncomplicated NDF case is pre-

sented (patient OH-1) as well as an uncomplicated

dermatophytosis yielding a few insignificant NDF

colonies (patient MA-14). In addition, more complex

types of cases are shown (e.g. patients OL-1, SI-1 and

BO-1) for whom initial specimen results gave little

evidence of the actual type of infection involved.

Dermatophytes

In uncomplicated dermatophytosis patients, the initial

specimen yielded 132 of the 177 etiologic dermato-

phytes that were ultimately detected (test sensitivity of

74.6%; Table 6). Another 14.1% of the infecting

dermatophytes first grew out in the second specimen,

while in 11.4% of cases the infecting dermatophyte

grew out only in the third or a later specimen. A similar

pattern was seen in mixed dermatophytosis/mold

infections, where a small majority of etiologic derma-

tophytes grew out from the initial specimen, but nearly

half first grew from a repeat specimen. In two cases,

dermatophyte growth was not seen until the fifth

successive specimen or later.

Specimens yielding positive direct microscopy and

growth of only a dermatophyte are generally inter-

preted as straightforward cases of dermatophytosis.

Longitudinal study in the present series of patients,

however, showed that three of 70 such initial specimen

records (4.3%) were actually derived from mixed

dermatophyte/NDF cases.

NDF

In uncomplicated NDF cases, the NDF species ulti-

mately judged to be etiologic in the case grew from the

first specimen in 50 of the 67 cases studied (test

sensitivity of 74.6%). In three additional cases in which

an NDF was judged causal, one or more NDF grew in

the initial specimen but the species ultimately found to

be causal did not do so; the outgrowing NDF from the

initial specimens in these cases were purely incidental.

To minimize the considerable confusion this minor

factor causes, these are noted hereafter as cases of

‘wrong mold’ outgrowth from initial specimens. If the

17 NDF from mixed dermatophytosis/NDF infection

are included along with the uncomplicated NDF cases

in the assessment of initial culture reliability, then

results show that overall, 61 etiologically significant

NDF (72.6%) grew in the initial specimen, while 23

were first grown from the second or a later specimen.In a total of 140 cases in which an NDF grew in the

initial specimen (Table 7), this mold was causally

significant in either an uncomplicated NDF infection

or mixed dermatophytosis/NDF infection in 61 cases

but was insignificant in 79 cases, giving initial-specimen

culture of an NDF a positive predictive value for

etiology of just 43.6%. Though most of the etiologically

significant initial-specimen NDF outgrowths were

combined with positive direct microscopy (51 of 61

cases; 83.6%), 10 cases showed negative direct micro-

scopy in the initial specimen. (As mentioned above,

positive direct microscopic readings were obtained in

later specimens for all such cases in which an NDF was

found to be causal). In cases in which all NDF growing

in the initial specimen were insignificant, direct micro-

scopy was positive in 47 cases, including 46 dermato-

phytosis cases and one of the three instances of wrong

mold outgrowth mentioned above. The causal derma-

tophyte itself grew from the initial specimen in 28 of the

46 dermatophytosis cases, leaving 18 true dermatophy-

tosis cases in which an insignificant NDF was the sole

filamentous fungus growing out from a specimen

microscopic positive for fungal hyphae.

Table 4 Appearance of Scopulariopsis -specific structures in direct microscopy in infections caused by S. brevicaulis

Infection category Direct microscopic

result classification

Finding in first

specimen

Finding in total number

of specimens taken

(at least three per patient)

Pure Scopulariopsis cases conidia present 2 3

conidia absent 8 7

Mixed dermatophytosis/Scopulariopsis infection cases conidia present �/ �/

conidia absent 3 3



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Table 5 Chronological sample series for six patients with onychomycosis. Records are arranged vertically under coded patient designators, with initial sample at top of column for each

patient and later samples towards the bottom. A final diagnosis for each patient is given at the bottom of the column of records

OH�/1* PH�/1 MA�/14 OL�/1 SI�/1 BO�/1

4/96$, L3R3%, SW§, �/,

Acremonium sp. (14) **

4/96 L1R1%, DL§, �/, Aspergillus terreus (7) 5/96, R1, DL, �/,

Trichophyton

rubrum

4/96, R1, DL, �/,

Paecilomyces

lilacinus (1),

Aspergillus nidu-

lans (1)

5/96, L1, DL, �/,

Aspergillus versicolor

(1), Arthrographis

kalrae (3)

5/96, R1, DL, �/, Candida albicans

6/96, L3R3, SW, �/,

Acremonium sp. (15�/)

4/96, L2, SW, �/, nfg%% 10/96, R1, DL, �/,

Fusarium solani (1)

6/96, R1, DL, �/,

A. nidulans (1)

11/96, L1, DL, �/,

Fusarium solani (8),

Aspergillus

fumigatus (1),

Alternaria alternata

(1)

6/96, R1, DL, �/, Scopulariopsis

brevicaulis (n.c. §§), Candida sp.

6/96, R1, DL, �/,

Acremonium sp. (2),

Scedosporium sp. (1)

8/96, L1, DL, �/, A. terreus (n.c.) 11/96, R1, DL, �/, T.

rubrum

10/96, R1, DL, �/,

A. nidulans (8)

4/97, L1, DL, �/,

Fusarium solani (10)

6/96, L4, SW, �/, Aspergillus candidus

(1), Alternaria alternata (1)

8/96, R3, SW, �/,

Acremonium sp. (15�/)

8/96, R1&2, SW, �/, Onychocola canadensis (9) 1/97, R1, DL, �/, T.

rubrum , Candida sp.

1/97, R1, DL, �/,

A. nidulans (1)

9/97, L1, DL, �/,

Fusarium solani (10),

A. fumigatus (7)

9/96, R1, DL, �/, S. brevicaulis (n.c.)

8/96, R1, DL, �/,

Acremonium sp. (15),

Candida sp.

10/96, L1R1, DL, �/, A. terreus (15�/) 3/97, R1, DL, �/, nfg 3/97, R1, SW, �/,

A. nidulans (3)

10/97, L1, DL, �/,

Fusarium solani (n.c.),

Penicillium sp. (n.c.)

9/96, L4, SW, �/, nfg

11/96, L3, SW, �/,

Acremonium sp. (5), Sco-

pulariopsis brevicaulis (1)

4/97, R2, DL, �/, O. canadensis (15�/) 6/97, R1, DL, �/,

Scopulariopsis

brumptii (4)

5/97, R1, DL, �/,

Acremonium sp.

(1)

12/97, L1, DL, �/,

Fusarium solani (15)

10/96, R1, DL, �/, S. brevicaulis (n.c.),

Candida sp.

11/96, R1, DL, �/, nfg 7/97, R1, DL, �/, O. canadensis (15�/), Alternaria

alternata (2)

12/97, R1, DL, �/,

nfg

10/97, R1, DL, �/,

A. nidulans (8)

2/97, R1, DL, �/, Onychocola

canadensis (15�/), Candida sp.

4/97, L3, SW, �/,

Acremonium sp. (3)

5/98, R2, DL, �/, O. canadensis (15�/) 5/98, R1, DL, �/, T.

rubrum

1/98, R1, DL, �/,

A. nidulans (7)

5/97, R1, DL, �/, C. albicans

4/97, R1, DL, �/,

Acremonium sp. (13)

5/98, R1, DL, �/, O. canadensis (15�/),

Trichophyton mentagrophytes

7/98, R1, DL, �/,

Penicillium sp. (2)

5/97, L4, SW, �/, A. candidus (1)

10/97, R3, SW, �/,

Acremonium sp. (1)

5/98, L1, DL, �/, A. terreus (15�/),

T. mentagrophytes

10/98, R1, DL, �/,

Phoma sp. (n.c.)

6/97, R1, DL, �/, O. canadensis (15),

Penicillium sp. (2), Chaetomium

globosum (1), S. brevicaulis (1)

10/97, R1, DL, �/,

Acremonium sp. (2),

Scopulariopsis brevicaulis

(1)

7/98, L1, DL, �/, A. terreus (10) 6/97, L4, SW, �/, nfg

2/98, L2&3, SW, �/,

Acremonium sp. (15�/),

11/98, R1, DL, �/, O. canadensis (15) 6/97, R1, DL, �/, O. canadensis (15),

C. globosum (1), S. brevicaulis (2),

Microascus cirrosus (3)

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Direct microscopy plus culture of first specimen

Studies comparing alternative techniques such as nail

histopathology to KOH-based direct microscopy and

culture (see Discussion below) often contrast the

sensitivities of various techniques for revealing the

presence of any and all etiologic filamentous fungi inabnormal nails in the first specimen taken. For

comparison with such studies, it should be noted

that in the present study, for the first specimen alone,

the overall sensitivity of KOH examination and culture

together to unequivocally demonstrate the involve-

ment of a filamentous fungal etiologic agent, whether

with a demonstration of fungal filaments present in

KOH or with culture positive for either a dermato-phyte or a Scytalidium , was 83.9% (219 definite

positives in first specimens for 263 patients with

known filamentous fungal infection; all relevant data

included in Table 3). The FN figure used in the

denominator of this calculation was based on gold-

standard knowledge of the total number of patients

who could ultimately be found to have at least one

etiologic filamentous fungus; this figure has not beendetermined in any previous study of which we are

aware.

Evaluation criteria evaluated

The existing evaluation criteria differ mainly in their

treatment of specimens in which nondermatophytic

fungi grow out. No dermatologic mycology laboratory

system provides physicians with information poten-

tially prejudicing an interpretation of specimens that

are both direct microscopic negative and culture

negative, or of specimens that are microscopic positive

and culture-negative. Thus analysis of the overallaccuracy of these interpretative systems per se must

generally be focused on specimens yielding various

categories of cultured organisms.

Simple association: conservative variant

Considering only cases with positive direct microscopy

and outgrowth of a filamentous fungus in culture, the

conservative variant of the simple association criterion

for judging initial specimens (all NDF considered

etiologic if growing in the absence of a dermatophyte

from a specimen microscopically positive for fungal

filaments; any dermatophytes growing always consid-ered to be sole agents of infection) would provide a

correct assessment in 145 cases (81.9%) and an

incorrect assessment in 32 cases (18.1%), consisting

of nine cases in which an etiologic NDF involved in a

mixed dermatophytosis infection would be missedTa

ble

5(C

on

tin

ued

)

OH� /

1*

PH�/

1M

A�/

14

OL�/

1S

I�/1

BO�/1

2/9

8,

R1

,D

L,�/,

Acr

emo

niu

msp

.(8

)

11

/98

,L

1,

DL

,�

/,A

.te

rreu

s(1

5�

/)9

/97

,L

4,

SW

,�/

,S

.b

revi

cau

lis

(1),

Asp

erg

illu

sve

rsic

olo

r(1

1),

Alt

ern

ari

a

alt

ern

ata

(1)

5/9

8,

L2&

3,

SW

,� /,

Acr

emo

niu

msp

.(1

5�

/)

5/9

9,

R4

,D

L,� /

,O

.ca

na

den

sis

(15

)6

/98

,R

1,

DL

,�/

,n

fg

5/9

8,

R1

,D

L,� /,

Acr

emo

niu

msp

.(1

5�

/)

11

/99

,R

4,

DL

,�

/,O

.ca

na

den

sis

(15

),

T.

men

tag

rop

hyte

s

6/9

8,

L4,

SW

,�/,

S.

brevi

cauli

s(1

3),

C.

alb

ica

ns

11

/99

,R

2,

DL

,�

/,O

.ca

na

den

sis

(15�

/),8

/98

,R

1,

DL

,�

/,O

nych

oco

laca

na

-

den

sis

(15

),C

an

did

asp

.

9/9

7,

L4

,S

W,�

/,n

fg

Dia

gn

osi

s

Acr

emo

niu

msp

.D

Lin

R1

an

dS

Win

L3

an

dR

3,

po

ssib

lya

lso

L2

A.

terr

eus

an

dT

.m

enta

gro

phy

tes

DL

inL

1;

O.

cana

den

sis

an

dT

.m

enta

gro

phy

tes

DL

inR

1a

nd

R4

;O

.ca

na

den

sis

DL

inR

2;

O.

can

ad

ensi

sS

Win

R1

an

dR

2

T.

rub

rum

DL

inR

1A

.n

idu

lan

sD

L

an

dS

Win

R1

F.

sola

ni

DL

inL

1S

.b

revi

cauli

sa

nd

O.

cana

den

sis

DL

inR

1;

SW

inL

4o

fu

nce

rta

inca

use

,

po

ssib

lyA

.ca

nd

idus

an

d/o

rS

.

brevi

cauli

s

*P

ati

ent

cod

ed

esig

nati

on

;$t

ime

sam

ple

tak

en(m

on

th/y

ear)

;%n

ail

(L,

toe

of

left

foo

t;R

,ri

gh

t);

§str

atu

min

volv

ed(D

L,

dis

tal�

/late

ral

sub

un

gu

al

on

ych

om

yco

sis;’

dir

ect

mic

rosc

op

icre

sult

;

**

gro

wth

den

sity

(nu

mb

ero

fin

ocu

law

ith

po

siti

ve

ou

tgro

wth

ou

to

f1

5p

lan

ted

);§§

n.c

.,n

oin

ocu

lum

cou

nt

ma

de;

%%n

.f.g

.,n

ofu

ng

us

gro

wn

;S

W,

sup

erfi

cia

lw

hit

eo

nych

om

yco

sis)

.



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(three of the nine cases) or incorrectly ignored (six

cases), 17 cases in which an etiologic dermatophyte

involved in uncomplicated dermatophytosis would be

missed and an incidental NDF considered etiologic

instead, and four cases in which an etiologic dermato-

phyte involved in a mixed infection would be missed

and sole etiology would be ascribed to the etiologic

NDF involved. There was also one case in which an S.

hyalinum isolate involved in a mixed infection with a

dermatophyte would be incorrectly judged a sole agent

as a result of the dermatophyte’s failure to grow in the

initial specimen, and one case in which NDF infection

would be attributed to an incidental wrong mold NDF

instead of the actual etiologic species. The figures for

correct and incorrect judgments in regard to the

conservative simple association criterion are based on

the premise that the user of the criterion would not

make a final judgment about cases in which positive

direct microscopy was accompanied by no fungal

growth or by growth of only a yeast. If the user

interpreted the outgrowing yeasts as etiologic, the

percentage of erroneous judgments involved would

increase.

When conservative simple-association is analyzed

specifically for its ability to assess the significance of

NDF outgrowth from microscopic-positive specimens

from which only an NDF grew in the initial specimen

(or an NDF plus a yeast, assuming the latter would be

ignored as not corresponding to the elements seen in

direct microscopy), it can be seen that with the data in

Table 3 this practice would generate correct attributions

of NDF etiology (to the correct NDF species rather

than to a wrong mold outgrowth) in 42 cases (65.6%),

whereas it would attribute causality to an insignificant

NDF in 19 cases (29.7%) and would attribute sole

causality to three molds actually involved in a mixedinfection with a dermatophyte (4.7%).

In order to obtain the overall sensitivity of con-

servative simple-association applied to etiologic NDF

(including sole-agent NDF and mixed dermatophytosis

components), one needs to determine the proportion of

the true cases that would be correctly recognized by a

criterion accepting only etiologic NDF growing from

microscopic positive initial specimens (i.e. 51), minussix cases of etiology incorrectly dismissed because of a

co-occurring dermatophyte, that is, 45 of 84 cases

(53.6%). The positive predictive value of this criterion

for our study population, 70.3%, is determined by

dividing its 45 correct calls into a total composed of

these calls plus the total of its false-positive calls,

including the above-mentioned 18 cases in which an

insignificant NDF was the sole filamentous fungusgrowing out from a microscopic-positive specimen, plus

one incorrect call based on wrong mold outgrowth

from a microscopic-positive specimen. The specificity

of conservative simple-association for correctly recog-

nizing NDF etiology is 76.5%, based on correctly

calling 62 nonetiologic NDF ‘negative’ and falsely

calling another 19 ‘positive’.

Simple association: liberal variant

The liberal simple-association criterion (all NDF

considered etiologic if growing from a specimen

microscopically positive for fungal filaments), when

applied to microscopic-positive, filamentous-fungal-

culture-positive initial specimens in our data, wouldprovide a correct judgment for 123 cases (69.5%),

including 75 uncomplicated dermatophytosis cases,

Table 6 Serial specimen first yielding dermatophyte in pure dermatophytosis and mixed-infection patients. Specimen first yielding dermatophyte

Infection category First Second Third Fourth Fifth or later

Dermatophytosis 132 (74.6)* 25 (14.1) 7 (4.0) 4 (2.3) 9 (5.1)

Mixed dermatophyte-mold infection 9 (52.9) 4 (23.5) 2 (11.8) �/ 2 (11.8)

Mixed dermatophyte-Scytalidium infection 1

*Percentages of total dermatophytes within each category (sole dermatophytosis/mixed dermatophytosis) are in parentheses.

Table 7 Non-Scytalidium molds growing from the initial specimen: direct microscopy reading and etiologic significance*

Filaments in initial specimen

direct microscopy

Cases in which a significant

NDF grew in 1st specimen

Cases in which one or more

insignificant NDF grew in

first specimen

Total

�/ 51 47 98

�/ 10 32 42

Total 61 79 140

*Cases with ‘unresolved’ final status and cases with only yeast cells in direct microscopy of initial specimen are excluded.


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six mixed infections (with both NDF and dermatophyte

component correctly recognized), and 42 uncompli-cated NDF infections. On the other hand, it would

generate incorrect judgments for 54 cases (30.5%),

including 18 cases of incidental NDF growing from

uncomplicated dermatophytosis patients being falsely

considered etiologic, 28 cases of incidental NDF

growing along with dermatophytes in initial specimens

falsely being considered participatory in mixed infec-

tions, three cases of NDF truly involved in mixedinfections with dermatophytes falsely being considered

sole agents, and three cases of NDF involved in mixed

infections with dermatophytes being missed with re-

sulting attribution of the infection to the dermatophyte

alone. Also, as in the case of the conservative simple-

association criterion, there would be one case in which

an S. hyalinum isolate involved in a mixed infection

with a dermatophyte would be incorrectly judged a soleagent, and one case in which NDF infection would be

attributed to the wrong mold.

Applied to cases of NDF outgrowth from micro-

scopic-positive specimens from which only an NDF

grew in the initial specimen, liberal simple-association

would generate judgments identical to those made by

its conservative counterpart (i.e. it would be 65.6%

accurate for our sample).However, in relation to all true NDF infections (pure

and mixed), regardless of their initial direct microscopic

result, this criterion, in which all true cases initially

presenting as microscopic-negative are missed, would

have a sensitivity (for NDF only, ignoring dermato-

phyte components of mixed infections) of 51/84

(60.7%). This is higher than the sensitivity of the

conservative variant; NDF occurring in mixed infec-tions are not ignored in the liberal variant. The liberal

variant, however, has a positive predictive value based

on a numerator of 51 correct calls divided by a

denominator including these calls plus 18 false sole-

agent NDF attributions, 28 false mixed NDF attribu-

tions, and one attribution of infection to a wrong mold;

this ratio equals 52.0%. In other words, in populations

similar to that studied here, this criterion is indis-tinguishably better than a coin toss for determining

whether an isolated NDF is etiologic. More dramati-

cally still, the specificity of this criterion in terms of

correctly evaluating NDF is 42%.

Classic evaluation

The classic criterion (NDF infection accepted whendirect microscopy is compatibly positive at least once

and the same NDF is isolated from at least two

distinctly temporally separated samples of the same

nail), when applied to microscopic-positive, filamen-

tous-fungal-culture-positive initial specimens in ourdata, would provide an appropriate judgment for 164

cases (92.7%), including 121 pure dermatophytoses, 36

uncomplicated NDF infections and six mixed-infection

cases. On the other hand, it would miss or fail to

confirm at least one involved pathogenic type in six

pure NDF cases and six mixed infections (including

five in which the NDF was unconfirmed or undetected

and one in which the dermatophyte was undetected), aswell as one Scytalidium -dermatophyte mix and one

case with wrong mold outgrowth in the initial speci-

men. The 121 successfully handled pure dermatophy-

toses include nine cases that are considered

appropriately handled because even though the derma-

tophyte failed to grow in the first specimen (or, in cases

where Note 1 was issued, in the first two specimens),

the NDF that grew were not reported in a mannersuggestive of etiologic significance. This refraining from

misattribution caused these specimens to have the same

status as common direct-microscopic-positive, culture-

negative specimens, which are generally interpreted as

highly likely to be dermatophytic.

An NDF (whether significant or not) grew from a

microscopic-positive initial specimen in 70 cases in the

present study. In a system using the classic confirma-tion criterion for NDF follow-up investigation, culture

and direct microscopic findings from the initial speci-

men would trigger the sending out of Note 1 (see

above) in all of these cases except those in which the

NDF species growing did not occur in Table 2. In fact,

Note 1, in our regular dermatologic mycology practice,

would have been sent out in response to 61 micro-

scopic-positive specimens from the present study grow-ing a fungus from Table 2 in culture. As seen in the top

four rows of Table 8, 51 true cases of NDF or mixed

NDF onychomycosis would have been included in the

number of specimens for which initial examination

generated this response. In the remaining cases, the

note would have been sent out in response to a

noncausal NDF; in all such cases in the present study,

the second specimen grew a dermatophyte, a discor-dant, incidental NDF, an incidental yeast or no fungus.

This would have allowed the NDF from the first

specimen to be correctly classed as noncausal. In

evaluation of the initial specimen plus the first addi-

tional specimen submitted in response to Note 1

(assuming an ideal response by patients and physicians,

i.e. assuming that such a confirmatory specimen was

sent in by the attending physician each time the notewas issued), this note-sending practice would have led

to confirmation of 36 uncomplicated NDF infections

and NDF components of seven mixed infections. These



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Table 8 True cases of nondermatophyte onychomycosis, including mixed infections: accuracy of diagnostic classification after study of two serial specimens, and effectiveness of two

informative notes used to solicit confirmatory specimens from physicians

Results of first two direct miroscopy

and culture tests

Conventional interpretation in classic system Infection category

D1 C1 D2 C2 Pure mold

infection

Mixed mold/dermatophyte

infection

�/ �/ �/ �/ Confirmed via note 1 22 6

�/ �/ �/ �/ Unconfirmed despite issue of note 1 �/ 2


�/ �/ �/ �/ Unconfirmed despite issue of note 1 6 �/

�/ �/ �/ �/ Unconfirmed; note 1 issued for common agents in report for 2nd specimen* 3 2

�/ �/ �/ �/ Unsuspected �/ 1


�/ �/ �/ �/ Unconfirmed despite issue of note 2 3 �/

�/ �/ �/ �/ Unconfirmed; note 1 issued for common agents in report for 2nd specimen 8 3

�/ �/ �/ �/ Unconfirmed; note 2 issued for common agents in report for 2nd specimen 3 �/

�/ �/ �/ �/ Unsuspected 3 �/

Totals a�/67 a�/17

a Confirmed by classic criterion 41 9

a Not confirmed by classic criterion within two specimens 26 8

C1, culture for specimen 1; C2, culture for specimen 2; D1, direct microscopy for specimen 1; D2, direct microscopy for specimen 2; ‘Confirmed’ means that the truly infecting

nondermatophytic agent or co-agent of the infection is confirmed as etiologically significant. The dermatophytic component of mixed infections is not referred to in this Table. For text of

Notes 1 and 2, see Materials and methods; *’Note 1 issued for common agents in report for second specimen’ means that though the infection in question would not be confirmed after two

successive specimens, the second specimen’s result, including a culture record of a species from Table 2, would trigger issuance of a note suggesting that an additional (third) specimen be

taken.

–2005

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numbers together constitute 84.3% of the 51 true NDF

or mixed infections eliciting the note. Six of the 42 trueuncomplicated NDF infections (14.3%) and two of the

nine mixed infections stimulating use of note one would

not be correctly confirmed due to chance failure of the

causal NDF to grow from the second specimen.

Note 2, used in cases where the initial specimen is

microscopic negative but grows a known potential

agent of onychomycosis (Table 2), would have been

issued in 21 cases in this study. This practice would haveresulted in second-specimen confirmation of uncom-

plicated NDF infection in five cases and mixed

dermatophytosis in two cases (Table 8, row 7), but

would falsely have missed three additional cases of

NDF infection. The missing of these cases would have

derived from the chance failure of the fungal elements

present in the infected areas to be seen in direct

microscopy in both the first and second specimens(Table 8, row 8). A further 17 true uncomplicated NDF

infections and six mixed infections would not have been

elucidated by procedures relying on use of Notes 1 and

2, as the results of the initial specimen would not have

triggered the issuing of either of these notes. If, in these

cases, the physician had spontaneously decided to

submit a second specimen despite not receiving a

laboratory note recommending this (as not infrequentlyoccurs in the Canadian health care system if onycho-

mycosis is strongly suspected but not confirmed by the

first specimen), 19 of these cases of missed infection

would have elicited Note 1 or Note 2 in examination of

the second specimen, and would thus have been set on

track for potential correct diagnosis despite the un-

helpful results of the first specimen. Only four of the

true NDF or mixed onychomycosis cases would stillhave been completely unsuspected after a second

specimen had been examined and cultured. In just

one of these four cases, a direct microscopic-positive

reading in one of the two specimens might have led the

physician to make a default clinical presumption of

dermatophytosis, in view of the two negative culture

trials that had occurred; in this case, in fact, a mixed

dermatophytosis/NDF infection was involved (Table 8,row 6). Importantly, no spurious, false attributions of

uncomplicated NDF or mixed NDF infection would

have been made as a result of reporting the outcomes of

the first two specimens, assuming that the submitting

physicians understood the classic confirmation criter-

ion.

It is salient to integrate the above results with the

remaining results for resolved cases in order to judgethe overall performance of the KOH-and-culture sys-

tem in conjunction with classic interpretive criteria and

the two advisory notes. Looking at the quality of first-

specimen results combined with any second-specimen

results directly elicited by use of the notes, one findsthat 230 of 311 cases (74%) would be judged completely

correctly, with all involved etiologic agents reported in

each case. These cases would break down as 136 of the

177 pure dermatophytoses ultimately recognized, in-

cluding four dermatophytoses culturally confirmed

only because Note 1 would have been sent out in

response to noncausal NDF, plus five cases in which

both the dermatophyte and the NDF components ofthe 17 total mixed infections would be fully confirmed

as a result of sending out Notes 1 or 2, 41 of 67

uncomplicated NDF cases, zero of two cases involving

Scytalidium, one yeast infection (detected as constant

C. parapsilosis growth only because Note 2 would have

been sent out in response to an insignificant S.

brevicaulis growth, eliciting a second specimen that

would not otherwise have been called for) and 47 of 47nonfungal cases. In addition, the dermatophyte com-

ponent but not the NDF component would be

recognized for six mixed infections, the NDF but not

the dermatophyte component for four mixed infections,

and the S. hyalinum but not the co-occurring derma-

tophyte in one mixed Scytalidium case, meaning that at

least one etiologic agent would be correctly recognized,

or the lack of fungal etiology correctly recognized, in241/311 (77.5%) of cases. It should be stressed that in

contrast to the 92.7% correctness figure given above for

microscopic-positive first specimens yielding a filamen-

tous fungal culture, this 77.5% correctness figure

reflects the dilution of accurate analysis by numerous

true-positive cases with negative direct microscopy and/

or a negative culture in the initial specimen.

Among cases with resolved status, there were 11microscopic-positive, culture-negative initial specimens

for which judgment would correctly remain pending in

the classic system, as well as in all other systems. Such

microscopic-positive, culture-negative reports, though

not resembling planned advisories like Notes 1 and 2,

may in themselves stimulate any physician wishing to

know the identity of an etiologic agent to consider

submitting a second specimen. If these 11 specimenswere subtracted from the number of inadequate initial

specimen results (i.e. if they were no longer considered

incorrect strictly because a true etiologic agent was left

unidentified), then 77.5% (241/311) of patients could be

said to have been adequately informed of their nails’

etiologic status by the methods used in this study,

exclusive of any results obtained for further specimens

spontaneously submitted by physicians. If the 11partially correct mixed-infection results were added,

as in the preceding paragraph, this would raise the

figure for what might be called ‘reasonably adequately



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handled cases’ to 81.0%. The most significant compo-

nent of the cases considering inadequately handledwould be the 12 dermatophytoses, three mixed infec-

tions and 10 NDF infections, together comprising 8.0%

of the 311 resolved cases examined, for which both

direct microscopy and culture were completely negative

in the initial specimen. This inadequacy, of course, is

intrinsic to the sampling and/or laboratory techniques

used and does not vary among the interpretive systems

compared here.For contrast with the evaluations given of the simple

association criteria with pure and mixed NDF infec-

tions, the sensitivity and specificity of the classic

criterion should also be summarized in this regard.

The use of Notes 1 and 2 would have resulted in the

confirmation of 41 of 67 uncomplicated NDF infec-

tions and the NDF component of nine of 17 mixed

infections, giving a sensitivity of 59.5%. This is slightlylower than the 60.7% sensitivity of liberal simple-

association, as stringent confirmation requirements

allow chance negative findings in the second specimen

to obstruct a small number of confirmation attempts.

This sensitivity level, however, is partnered with a

positive predictive value and a specificity of 100%, as

this criterion makes false positives extremely unlikely.

In no case was the same incidental, noncausal Table 2NDF isolated from two successive specimens in a way

that would lead to a false report of confirmed NDF or

mixed onychomycosis. To rule out the possibility that

the delineation of gold-standard reference data in our

study might have been compromised by our allowing

two successive, coincidental growths of the same NDF

from a nail region invested with effete, nongrowing

dermatophyte filaments to be falsely interpreted as atrue positive for NDF infection, a third growth of the

same NDF was made a minimal criterion for accep-

tance of gold-standard-level true NDF etiology, and

the long-term record of outgrowth was monitored to

ensure there was no unexplained loss of an apparent

infective component in these untreated patients. Any

dubious cases would have been excluded from the

analysis; in fact, however, this step was only taken ina small number of cases where an insufficient number

of specimens could be obtained to clarify the status of

the outgrowth.

Unresolved cases have been excluded from all

analysis so far. In classic methodology 15 of these 30

cases (i.e. those with negative direct microscopy in the

initial specimen) would be given normal negative

reports. The remainder would be considered mycosesof unknown type; however, as our later records showed,

further attempts to gain insight into the nature of these

cases would be unfruitful even after seven or eight

successive specimens. The cases would therefore remain

unclassified from the viewpoint of laboratory mycol-ogy, although clinically they would likely be treated as

dermatophytosis. In either type of simple-association,

any of these cases yielding an NDF in the initial

specimen (i.e. eight cases in the present study) would be

incorrectly classed as infections caused by the isolated

NDF. With this factor included, and assuming that

none of these unconfirmable attributions of NDF

infection were correct, the positive predictive value ofconservative simple association for correct calls in

regard to NDF outgrowth would fall from 70.3 to

62.5%. That of liberal simple-association would fall to

48.1%. The value for the classic criterion for NDF

infection would remain at 100%.

Discussion

In most laboratory testing, test sensitivities and specifi-

cities below 90% would be considered unreliable,

especially for a final reference test. The extreme

ecological complexity of onychomycosis, however, has

so far engendered a situation in which definitive tests

must generally be judged on the basis of sensitivities,

positive predictive values and/or specificities in the

70�/80% range or below. Some authors, indeed, ignorecriticism and continue to use interpretive frameworks

with specificities below 50%. It should be noted,

however, that in the absence of gold-standard data

from longitudinal studies like the present one, these

accuracy levels have not previously been precisely

specified.

The value of etiologic agent identification in onycho-

mycosis is sometimes questioned, with the reflectionthat this information is superfluous unless drug re-

sponses vary among the etiologic agents identified [25].

The emphasis given in the present study to accurate

identification of the etiologic agent is partially based on

the knowledge that drug susceptibilities and in-vivo

responses often do vary significantly among onycho-

mycotic agents [21,26�/28]. In addition, however, our

viewpoint is that physicians generally prefer accuratelaboratory identification in cases where significantly

varying responses among etiologic agents are possible

(because ethical considerations make inappropriate

therapy even of rare types of cases objectionable,

especially when significant expense or any risk of

adverse effects is involved), and any impulse to de-

emphasize this accuracy in regard to onychomycosis is

simply a practical accommodation to the difficultiesand costs currently experienced. This response thus

reflects a pragmatic strategy that remains compatible

with the ultimate goal of improving accuracy by


Onychomycosis 53

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gaining a more thorough scientific understanding of

the techniques, organisms and ecological relations thatbecome involved when onychomycosis is diagnosed in

the laboratory.

The patient population studied in the present analy-

sis was predominantly geriatric, and this group is well

known to have more NDF onychomycoses than are

found in younger groups [29]. The proportion of NDF

and mixed infections found in the present study should

not be expected to be duplicated in studies ondemographically different groups. One might even

find, as Ellis et al. [18] did, a stringently selected

clinical trial patient population of over 100 patients

with very few demonstrable NDF infections. In general

laboratory practice in our area, however, classically

confirmed NDF onychomycoses have been found to

make up about 3% of the total number of onychomy-

coses examined [10]. These data were obtained beforeNotes 1 and 2 were devised, and many cases for which

no second specimen had been obtained were excluded

from calculations of etiologic agent prevalence and

were considered to be merely ‘suggestive’ cases based

on initial specimen results. This may indicate that the

true proportion of NDF onychomycoses dealt with was

somewhat higher than that given in our 1989 study [10].

Also, the study in question [10] did not evaluate mixedinfections. A later study [30] facilitated such investiga-

tions by showing that sampling individual nails and

affected nail strata (subungual, superficial or, where

applicable, medial) separately greatly aids in clarifying

interpretation of mixed-infection results, as many

infecting NDF are confined to a single nail within a

single stratum, while other nails and strata may bear

other etiologic agents.The present study numerically confirms the long-

asserted judgment that simple association criteria have

unacceptable specificity in regard to NDF infections.

The great disparity of figures in the literature on

proportions of NDF onychomycosis (Table 1) is

primarily based on the ongoing use of simple-associa-

tion criteria by some authors. The liberal simple-

association criterion in particular greatly exaggeratesthe number of mixed dermatophyte/NDF infections

recognized, and studies employing this criterion can

often be immediately recognized in that numbers of

mixed infections will be nearly as large as, or even

greater than, one or both of the other major infection

categories, that is uncomplicated dermatophytosis and

uncomplicated NDF infection. Extension of the criter-

ion to incidental yeasts results in data showing largenumbers of spurious yeast infections. Delineating such

recognition criteria for these studies can be important,

as authors may state their methods and evaluation

criteria vaguely, leaving their methodology to be

partially inferred from the data collected. It may bethought to be self-evident that a criterion accepting

possible incidental NDF as etiologic would drastically

overestimate NDF infection numbers; however, the

ongoing appearance of studies using these criteria

shows that precise measurement of the degree of error

involved may be needed to convince the users of these

criteria, as well as reviewers and editors, that the

criteria are inadmissible.It should be pointed out that our study on mostly

geriatric patients to a considerable extent flatters the

simple association criteria, in that a reasonable propor-

tion of the NDF infections they recognize turn out to

be genuine on further analysis. In younger patient

populations, the overwhelming majority of NDF infec-

tions recognized by these criteria (e.g. infections judged

to be caused by NDF based on microscopic-positivespecimens yielding only an NDF in culture) would be

spurious.

The gold-standard data definitively resolve another

long-controversial area of onychomycosis, namely, the

matter of how frequently NDF infections are accom-

panied or facilitated by the presence of a dermatophyte.

As dermatophytes can indeed be isolated belatedly in

some mixed infections, after much sampling in whichonly the co-occurring NDF grows, the impression has

been given that perhaps an underlying dermatophyte is

always coextant in NDF infections. The well-known

Pseudeurotium ovale onychomycosis case of English et

al . [31], in which a single colony of a co-occurring T.

rubrum was isolated only after 200 nail fragments, 20

from each toe, had been plated out, may have appeared

to substantiate this impression. In fact, Table 6 clearlyshows that the isolation of dermatophytes from mixed

infections, like the isolation of dermatophytes from

uncomplicated infections, shows the approximate in-

verse-exponential distribution that is ubiquitously seen

in ecological sampling studies (see, for example, ref. 32).

By fortuity, in fact, the numbers of dermatophytes

isolated per sampling occasion in our data for derma-

tophytes from mixed infections falls off almost exactlyas powers of two. Our 17 mixed infections thus

represent all but a very small residual number of as

yet unelucidated dermatophytes growing in association

with NDF in the onychomycoses studied. The notion

that a significantly higher number of the remaining

NDF infections in our study coexist with a tinea

unguium can be excluded. Any impression that derma-

tophytes are always or usually present to facilitateNDF infections appears to be grossly erroneous.

The role of dermatophytes in facilitating mixed

infections can also be evaluated via studies in which



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these infections are treated. Though no patients

inducted into the present study were treated, some

other patients seen concomitantly were treated, and

some were also repeatedly sampled. Isolations from

three such patients are shown in Table 9. These patients

were treated 12 weeks per os with an antifungal drug or

drugs that cannot be named due to clinical trial-related

confidentiality agreements. As can be seen, patient 1

had a mixed S. brevicaulis/T. rubrum infection,

although the former component was not elucidated

until after treatment had begun. As the other data

collected in the present study show, the failure of the S.

brevicaulis component to grow out in the initial speci-

men can probably be ascribed to the approximately

25% probability of this happening due to chance alone

rather than to the species being absent in the pre-

therapeutic baseline sample. (The sampling physician

may also have become more oriented to S. brevicaulis -

affected lesional areas when purely dermatophyte-

affected areas began to resolve.) After 8 weeks of

treatment, the dermatophyte was apparently eliminated

and did not recur in 6 months of post-treatment follow-

up, but S. brevicaulis continued to be isolated and the

nail continued to be clinically abnormal. Clearly S.

brevicaulis had no dependence on T. rubrum for

ongoing nail colonization. In patient 3, a T. rubrum

isolate occurring concomitantly with S. brevicaulis

apparently recrudesced after treatment, but during the

period in which it was under maximal drug inhibition

the S. brevicaulis component of the infection continued

to grow abundantly. In patient 2, the NDF component

of a Fusarium solani/T. rubrum onychomycosis con-

tinued to grow 3 months after the end of therapy, while

T. rubrum appeared to have been eliminated by therapy.

Though the F. solani did not grow in the final two

follow-up specimens taken in this case, the nail

remained clinically abnormal with no sign of resolution

of infection, and assessors of the case concurred that it

was most likely the two isolation failures and one

negative direct microscopy in the final samples were

chance effects rather than an indication that F. solani

had been eliminated. Unfortunately, the patient did not

appear for the scheduled final follow-up sample. These

three treatment studies, then, reinforce the idea that

many NDF involved in mixed dermatophytosis can

continue to prosper in nails from which the dermato-

phyte has been eliminated, or in which dermatophyte

growth is temporarily strongly inhibited by treatment

effects. This conclusion accords with the finding of

many dermatophyte-free NDF infections in the main

study presented here. Though the sample size involved

in these treatment studies is minimal, even a single

definite case of an NDF participant in a mixed

infection prospering in the long-term absence of its

former dermatophyte cohabitant is sufficient to con-

firm that this possibility is realistic.

Data from the present study on the number of

specimens required to grow the etiologic dermatophytes

from uncomplicated tinea unguium are almost identical

to those acquired previously by Gentles [7]. A relatively

surprising finding of our study, however, is that

etiologic NDF were equally likely to fail to grow out

in the first specimen, and to appear only in the second

or a later specimen. (Though the near-exact equality,

differing only in the second, insignificant decimal place,

seen in percentages of first specimen dermatophyte and

NDF outgrowth in the present study can only be

considered coincidental). Indeed, in all respects, NDF

infections proved just as difficult as dermatophytoses to

detect reliably, whether in direct microscopy or in

culture, even when they were demonstrably established

in longitudinal analysis. The physician submitting a

Table 9 Serial evaluations of fungal outgrowth and direct microscopy for nails with mixed-agent onychomycosis treated 12 weeks per os with

oral antifungal therapy

Sample Patient 1 Patient 2 Patient 3

Direct Culture Direct Culture Direct Culture

Pre-treatment �/ Trichophyton rubrum �/ T. rubrum �/ T. rubrum, S. brevicaulis

1 month on therapy �/ T. rubrum, Scopulariopsis brevicaulis �/ T. rubrum, Fusarium solani �/ S. brevicaulis

2 months on therapy �/ S. brevicaulis �/ F. solani �/ S. brevicaulis

3 months on therapy �/ S. brevicaulis �/ F. solani �/ S. brevicaulis

1 month post therapy �/ S. brevicaulis �/ F. solani �/ S. brevicaulis

2 months post therapy �/ S. brevicaulis �/ �/ �/ S. brevicaulis

3 months post therapy �/ S. brevicaulis �/ F. solani �/ S. brevicaulis

4 months post therapy �/ S. brevicaulis �/ �/ �/ T. rubrum, S. brevicaulis

5 months post therapy �/ S. brevicaulis �/ �/ �/ S. brevicaulis

6 months post therapy �/ S. brevicaulis ND ND �/ S. brevicaulis

ND, not done.


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dermatologic mycology sample faces an unusually

difficult situation, in that as much as one-quarter ofthe single-specimen data returned by the laboratory,

though accurate (based on the samples submitted and

the use of recommended techniques) will ultimately

prove to have been incomplete or otherwise misleading.

In response to this difficulty, recent studies have

evaluated the sensitivity and overall diagnostic utility of

histopathological studies of nail biopsies vis-a-vis

conventional and modified KOH-and-culture techni-ques [25,33]. Though at least one small comparative

study has shown a significantly higher number of

positive results for histopathology than for KOH-and-

culture [33], no study so far has attempted to estimate

the overall sensitivity of histopathology against defini-

tive gold-standard data of the type used in the present

study. Also, the 28 cases in study [33] biopsy positive

for fungal hyphae yielded only 14 positive KOH resultsand seven cultures positive for nonyeast, noncontami-

nant fungi, including six dermatophytes and one

etiologically unconfirmed, unidentified Aspergillus.

Treating the biopsies positive for hyphae as signaling

known onychomycosis cases and tentatively accepting

the Aspergillus as causal, one finds that the 50% KOH-

positive rate and the 25% positive etiologic culture rate

are both highly significantly lower (P B/0.01, chi-squared) than parallel rates of over 70% positivity for

both individual techniques in the present study, sug-

gesting some methodological deficiency in the KOH

and culture techniques used.

The idea, advanced more or less tentatively by some

authors [25,33,34], that histopathological studies could

perhaps be used as a single method for the evaluation

of onychomycosis, is not to be recommended, in that itwould clearly lead to many NDF infections being

misdiagnosed as dermatophytoses. Though hyphae of

nondermatophytic fungi are often somewhat thicker,

thinner or more nodulose than those of dermatophytes

[23], it remains clear (contrary to assertions made by

Contet-Audonneau et al. [35] and others) that there is

as yet no truly reliable, objective way to distinguish

dermatophytic from most nondermatophytic elementsseen in histopathology conducted with the normally

used fungal stains (periodic acid-Schiff, Gomori methe-

namine silver). The accuracy of such histopathological

filament classification has never been rigorously tested

with either highly or normally experienced observers.

Our study shows that even the NDF most frequently

seen forming clearly nondermatophytic elements in nail

lesions, S. brevicaulis, only does so in a minority of thecases it causes (3/13 in the present study). With the

patient population seen in the present study, even if we

accorded histopathology a theoretical 100% sensitivity

for detecting fungal elements from all true cases in the

initial specimen, it would still, if it took all nondescriptfilaments to represent dermatophytosis, result in a

misdiagnosis of 17 mixed NDF infections, 64 of 67

pure NDF infections (assuming distinctive S.

brevicaulis elements were elucidated in three cases),

and both Scytalidium infections. Of our 263 patients

with filamentous fungal infection, 31.6% would be

incorrectly diagnosed. This is 26.7% of the whole

patient population; such a rate of misdiagnosis isscarcely acceptable. The same problem would occur if

the recently mooted technique of in-vivo confocal

microscopy [36] were used in the absence of culture to

diagnose presumptive dermatophyte onychomycosis.

Extended studies to determine if histopathology

partnered with culture, including follow-up of possible

NDF or mixed NDF patients for at least one additional

specimen, could significantly improve on conventionalKOH-and-culture would be of interest. Unfortunately,

in current literature, studies combining histopathology

with culture are just as likely as KOH-and-culture

studies to use dubious shortcuts for confirming NDF

etiology. Borkowski et al. [33], apparently adopting

liberal simple-association, accepted all outgrowths as

significant positive cultures, including a Verticillium (a

filamentous taxon with no valid pathogenic record inhuman dermatological mycology) from a nail biopsy

positive only for yeast elements, and a Candida as well

as a ‘Madurella ’ (a purely tropical group of nonspor-

ulating soil fungi not known to cause onychomycosis

and only technically recognizable either by sequencing

or by demonstrating black fungal grains in a case of

mycetoma) from two nails biopsy positive for derma-

tophyte-like, penetrating filaments. Lawry et al. [25], inattempting to confirm etiology for NDF growing in

initial culture from specimens histopathologically po-

sitive for fungal filaments, sought to elicit consistent

NDF outgrowth in a second culture trial done with

residual portions of the original specimen. This in

effect amounted to a single-specimen inoculum-count-

ing method with an even lower standard for acceptance

of etiology (two appearances of the fungus in culture)than the proven unreliable standard [21] of five culture-

positive inocula out of 20 planted as originally

proposed by Walshe and English [11]. Gupta et al.

[21] have shown that many NDF, especially members of

the heavily sporulating genera Aspergillus and Scopu-

lariopsis, can give high growth rates from single nail

specimens in which they are found purely as insignif-

icant contaminants deriving from the specimen (i.e. notderiving from transport materials or from process

contamination in laboratory workup). Splitting the

initial specimen into two portions and culturing the



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second portion later is highly unlikely to alter this effect

due to transitory, but potentially heavy, body surfacecontamination. Needless to say, such practices are not

conducive to accurate comparative analyses of diag-

nostic methodologies.

Preliminary studies have suggested, again without

reference to gold-standard data, that PCR can improve

the rate of detection of dermatophytes from nail

specimens [37]. Unfortunately, because contaminating

molds can sometimes be present in large quantities onnails, direct PCR amplification with universal fungal

primers has no potential advantage in the investigation

of NDF infections: any attempt to make this applica-

tion would simply reinvent liberal simple-association.

Indeed, modern PCR techniques are so sensitive that

even one or a few contaminating spores of an NDF on

a nail could lead to a positive signal; the confusion

potentially caused by this amplification of contaminat-ing fungi has already been recognized in PCR studies of

suspected deep mycotic lesions and of fungi occurring

on nasal sinus membrane surfaces [38,39]. PCR results

for common environmental fungi detected on normally

unsterile body surfaces, including lesional surfaces, can

only be interpreted in the context of direct microscopy,

culture and other corroborating studies such as follow-

up specimens.The flow cytometry and differential immunohisto-

chemistry techniques investigated by Pierard et al . [20],

if they could be made practicable for routine or even

most reference laboratories, would have clear advan-

tages in sensitivity and first-specimen specificity over

existing routine methods. On the other hand, main-

taining immunohistochemical reagent stocks for all the

possible agents of NDF infection would be arduous,and it is clearly easier to initially detect which species is

involved in onychomycosis by culturing than by screen-

ing a panel of up to 35 or more histopathologic sections

with specific reagents for each possible etiologic fungus.

Of course, specific immunohistochemicals could be

used as a follow-up to culture to determine accurately

if filaments corresponding to a cultured NDF are

present in nail tissue. However, such a system wouldby no means solve all the problems intrinsic to correct

diagnosis of NDF infections: confusion could still be

caused by chance filament-negative samples from true

infections (in histopathologically studied nail dystrophy

patients who grew a dermatophyte in culture, ‘false’-

negative biopsies were found by Suarez et al. [40] in five

of 74 (6.8%) of cases and by chance-negative cultures as

well as wrong mold isolations as defined above. In flowcytometry, defining parameters for all possible agents

of infection would be a challenge. New agents of well-

verified onychomycosis are still being described [41]

and users of techniques such as immunohistochemistry

and flow cytometry must be aware that their judgmentcriteria should allow for the possibility of unknown

fungi, including some that might confound established

differentiating criteria (e.g. via antigenic cross-reac-

tion).

With regard to the dermatophyte components of

onychomycosis, it has been shown that most if not all

untreated cases of T. rubrum infection will show

demonstrable levels of the characteristic mycotoxinxanthomegnin in the nail tissue [42]. This technique

may warrant further investigation and extension to

additional dermatophyte species known to produce

xanthomegnin or closely related metabolites in vitro

[42].

The one modification of the classic system intro-

duced in the present study, namely, the use of Note 2,

increased the sensitivity of the method, allowing sevenmore NDF cases to be correctly recognized than would

have been confirmed otherwise. On the other hand, the

cultured NDF triggering use of this note turned out to

be insignificant in slightly more than half of the cases in

which the note would have been issued. This is in

contrast to Note 1, which in our study population

would have directly rewarded the labor of taking a

second patient sample 67.2% of the time (41 confirmedNDF cases/61 issuances). What must be borne in mind

in conjunction with these numbers is that because in

both our data and those of Gentles [7], around 15% of

causal dermatophytes are shown to be first grown in

the second specimen, and because adding in a second

specimen raises the accuracy of dermatophyte culture

alone from around 75 to around 90%, soliciting a

second specimen in some cases where an NDF grows,even in cases where the NDF turns out later to have

been insignificant, is a practice that in the aggregate

will improve the overall chances of correctly under-

standing the cases investigated. In the present study, the

issuance of Note 1 as per routine practice would have

resulted in the elucidation of four pure dermatophy-

toses that would not have been elucidated otherwise,

as well as the dermatophyte component of one mixedinfection. Use of Note 2 would also have yielded the

dermatophyte component of one mixed infection.

For practical purposes at the moment, it is clear that

despite its imperfections there is no known substitute

for classic methodology, inclusive of its handling of

NDF cases via soliciting of additional specimens

(though possibly direct microscopy based on use of

hydroxide with or without fluorescent fungal wallstains could be supplanted by histopathology). As

pointed out by Gupta et al. [21], the classic methodol-

ogy can also be enhanced with minimal extra trouble


Onychomycosis 57

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and cost by the use of inoculum counting, not in the

manner originally recommended by Walshe and Eng-

lish [11], but rather as a supplementary study to place

probabilities on the likelihood that NDF isolated in

initial specimens will be confirmed as etiologic in

follow-up studies. A chart correlating positive inocu-

lum numbers with probabilities of etiologic significance

for NDF in general is available [21]. Although low

inoculum counts can never be used to rule out an NDF

infection, and though the Walshe and English 5/20 rule

(as detailed above) is highly unreliable [21], high counts

(e.g. 15 inocula positive for the same NDF out of 15

planted) in conjunction with an initial microscopic-

positive specimen can indicate a probability of up to

90% that the isolated NDF is etiologic. In such a case

involving high initial NDF counts, if the suspected

NDF agent were to be missed due to chance sampling

effects alone in a second, confirmatory sample, the

physician would know that a third sample should be

taken or, alternatively, given the original 90% chance of

an NDF infection vis-a-vis the approximately 11%

chance (as shown by the present study and Gentles

[7]) of a dermatophyte growing only from a third or

later specimen, could decide that the patient could

reasonably be treated prospectively as having an NDF

onychomycosis.

However, even when culturing efforts are vigorously

made, it should be recognized that physicians diagnos-

ing cases of onychomycosis are currently obliged to

work to some extent in the dark, knowing that

additional details about at least a few of the cases

they are dealing with could only be elucidated by

taking a number of samples that is not practical.

Therefore the breadth of the activity spectrum of drugs

used in therapy may be important to consider even in

seemingly straightforward cases, provided that this

consideration does not conflict with efficient elimina-

tion of the most prevalent etiologic agents, the derma-

tophytes. The prevailing drugs used in onychomycosis

change over time, as do perceptions of their activity

spectra, and the foregoing statement is not made in

reference to any particular drugs in current use.

Acknowledgements

The authors thank the staff of the Ontario Ministry of

Health mycology laboratory and of Mediprobe La-

boratories who participated in analyzing or collecting

samples in this study, and also thank the patients who

volunteered.

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Onychomycosis: a critical study of techniques and criteria for confirming the etiologic significance of nondermatophytes