Diagnosis of Coccidioidomycosis

by Culture
Safety Considerations, Traditional Methods,
and Susceptibility Testing
DEANNA A. SUTTON
Fungus Testing Laboratory, Department of Pathology, University of Texas
Health Science Center at San Antonio, San Antonio, Texas 78229, USA

ABSTRACT: The recovery of Coccidioides spp. by culture and confirma-

tion utilizing the AccuProbe nucleic acid hybridization method by Gen-
Probe remain the definitive diagnostic method. Biosafety considerations
from specimen collection through culture confirmation in the mycology
laboratory are critical, as acquisition of coccidioidomycosis by labora-
tory workers is well documented. The designation of Coccidioides spp. as
select agents of potential bioterrorism has mandated strict regulation of
their transport and inventory. The genus appears generally susceptible,
in vitro, although no defined breakpoints exist. Susceptibility testing may
assist in documenting treatment failures.

KEYWORDS: Coccidioides; culture; susceptibility; select agent

INTRODUCTION

The three mainstays of laboratory diagnosis of coccidioidomycosis in-

clude culture, histopathology, and serology.1 Because a thorough review of
histopathological findings and immunologic methods may be found elsewhere
in these proceedings, the objective of this brief article is to review traditional
culture methods available to routine mycology laboratories. Although recent
PCR procedures that amplify coccidioidal DNA from clinical specimens show
diagnostic promise,2,3 the recovery of Coccidioides spp. by culture continues
to be the most definitive diagnostic method.4 Molecular characterization of the
genus has now identified two species, Coccidioides immitis and Coccidioides
posadasii; however, the morphology of the two species, the clinical manifes-
tations of the disease, and the therapeutic options are essentially identical.5
Address for correspondence: Deanna A. Sutton, Ph.D., M.T., S.M. (A.S.C.P.), R.M., S.M. (N.R.M.),
Department of Pathology—MSC 7750, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. Voice:
210-567-4032; fax: 210-567-4076.
[email protected]

Ann. N.Y. Acad. Sci. 1111: 315–325 (2007). 

C 2007 New York Academy of Sciences.

doi: 10.1196/annals.1406.005
315
316 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

Coccidioides immitis is indigenous to California and C. posadasii to other

areas, although animal isolates appear to delineate this distinction more ac-
curately than do patient cases.6 With the advent of Coccidioides spp. being
included in the select agent list of potential bioterrorism agents, the additional
safeguards and requirements regarding biosafety, transport, storage, and report-
ing are in effect. While the clinical utility of antifungal susceptibility testing
is not universally embraced, numerous clinical isolates have been tested and
continue to be submitted for in vitro evaluation.

SPECIMEN COLLECTION

The collection, transport, and processing of appropriate clinical specimens

is an important consideration in the recovery of Coccidioides spp., and several
specific guidelines apply to specimens collected for the recovery of dimorphic
pathogens. Common respiratory sites include sputum, bronchoalveolar lavage,
transtracheal aspirates, and lung biopsies. Sputum is typically contaminated
with bacteria, yeasts from the oral cavity, and inhaled conidia from a variety
of saprobic moulds, and should be transported and processed as quickly as
possible to curtail overgrowth by these more rapidly growing bacteria and
endogenous flora. Recovery from blood, bone marrow, cerebrospinal fluid, and
other body fluids, as well as cutaneous and/or soft tissue biopsy specimens is
common when there is dissemination to extrapulmonary sites. Aspirates or pus
should be submitted in syringes, when possible. Syringes must have the needle
removed and be recapped prior to transport.7,8 Several sources provide a more
detailed guide to specimen collection.7,9 All specimens should be considered
potentially hazardous, and universal precautions should be observed during
collection and processing.7 Specimens should be accompanied by a properly
completed requisition, and alerting the laboratory regarding potential cases of
coccidioidomycosis is always advised.
Although it is recommended that all direct specimens be plated within 24 h,
laboratories that must ship these samples to other sites for culture may do so by
adhering to the Category B guidelines for biological substances as defined by
the U.S. hazardous materials regulations and packaged according to the Depart-
ment of Transportation guidelines for Transportation of Hazardous Materials.
Shipment of filamentous cultures of suspected or known Coccidioides spp. is
addressed later.

THE MYCOLOGY LABORATORY

Hyperendemic areas for coccidioidomycosis in the U.S. include Kern County

in the San Joaquin Valley of California, and Maricopa (Phoenix) and Pima
(Tucson) counties in Arizona. The risk of acquisition in these areas increases
SUTTON 317

with events such as dust storms, earthquakes, and droughts that disrupt the soil
and thereby disperse high concentrations of arthroconidia into the atmosphere,
facilitating airborne transmission of the fungus.10–15 Handling of clinical spec-
imens with the subsequent growth and recovery of Coccidioides spp. in labo-
ratory cultures provides a scenario in which arthroconidia could potentially be
present several orders of magnitude higher than in the air of endemic areas, and
thus the concern for laboratory-acquired infections among healthcare workers.
This method of acquisition is well documented.16–28 Laboratory workers are
also at risk of accidental percutaneous inoculation of the spherule form of the
fungus while they are processing direct specimens and handling unfixed his-
tology specimens.29,30 Some reports stating that 90% of laboratory-acquired
infections result in clinical disease31 further support the need for clinical lab-
oratories to adhere to the safety standards promulgated by the Centers for
Disease Control Office of Health and Safety in their 5th edition of Biosafety
in Microbiological and Biomedical Laboratories (BMBL).32 This document
is available online at http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm
and summarizes the four different recommended biosafety levels for infectious
agents, and addresses which types of biological safety cabinets are appropriate
for each biosafety level. Biosafety Level 2 practices and facilities are recom-
mended for handling and processing clinical specimens and identifying isolates
of Coccidioides spp. Biosafety Level 2 practices differ from Biosafety Level
1 practices in that (1) laboratory personnel have specific training in handling
pathogenic agents and are directed by qualified scientists; (2) access to the lab-
oratory is limited when work is being conducted; (3) extreme precautions are
taken with contaminated sharp items; and (4) procedures in which infectious
aerosols or splashes may be created are conducted in Class II biological safety
cabinets. Class II biological safety cabinets have vertical laminar airflow with
HEPA (high-efficiency particulate air) filtered supply and exhaust air. They
protect the worker, the product, and the environment.32

DIRECT MICROSCOPY

Clinical specimens such as fresh wet sputa, centrifuged bronchoalveolar

lavage fluids, cerebrospinal fluid, aspirates, exudates, tissue biopsies, skin
scrapings, and other similar specimens should also be examined microscop-
ically. Direct microscopy often provides a rapid, presumptive diagnosis of
coccidioidomycosis when intact or partially intact spherules are observed, and
this procedure carries much less risk to laboratory workers than do cultural
methods. Mature spherules are thick-walled, may measure up to 80 m in
diameter, and are filled with endospores. These spherules rupture, releasing
small endospores approximately 2–4 m in diameter that in turn undergo
progressive maturation to variously sized spherules (FIG. 1). A preliminary
diagnosis is more problematic when only small spherules (10–20 m) lacking
318 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

FIGURE 1. Hematoxylin and eosin (H&E) stain of spherules of Coccidioides immitis

seen in various stages of maturation. Original magnification approximately 960×.

endospores or a few endospores of varying sizes are seen. These may often
be mistaken for phagocytic cells, artifacts, or other fungi, particularly atypical
forms of Blastomyces dermatitidis and yeasts such as Histoplasma capsu-
latum, Candida glabrata, and Cryptococcus species. A simple procedure to
rule out artifacts such as pollen and other spherical fungi is to place a small
amount of the primary specimen suspected of containing endospores or small
spherules in a drop of sterile saline on a glass slide and then seal the speci-
men with a petroleum-edged coverslip. This provides a microculture in which
spherules put out tubular structures and become hyphae within 2 to 3 days at
room temperature.33 Hyphal forms have also been observed in chronic cavi-
tary and granulomatous lesions and in cerebrospinal fluid.34–36 Several direct
microscopy methods may be employed such as the Gram stain, a KOH prepara-
tion, and the calcofluor white stain.37 The cytopathological Papanicolaou stain
is also particularly useful for the detection of spherules.38

CULTURE SETUP AND MEDIA CONSIDERATIONS

All direct inoculation procedures for specimens suspected of containing

Coccidioides spp. should be performed only within a biological safety cabi-
net and be cultured onto tubes rather than plates. Select bloody, purulent, or
caseous portions from respiratory specimens, and mince tissue biopsies with
a scalpel; do not grind. Coccidioides spp. are not particularly fastidious, and
SUTTON 319

a variety of primary isolation media may be used. Common mycologic media

include brain–heart infusion agar, Sabouraud dextrose agar, potato dextrose
agar, and Sabouraud dextrose agar with cycloheximide. For non-sterile sites,
prevention of overgrowth by bacteria and more rapidly growing saprobic
fungi is necessary. Antibiotics commonly added to media to suppress bacte-
rial growth include chloramphenicol, gentamicin, streptomycin, and penicillin.
The addition of cycloheximide inhibits some saprobic fungi and is useful as
a selective medium as it supports the growth of all dimorphic fungi. Media
without cycloheximide should also be included in the battery so as not to in-
hibit other opportunistic pathogens.8 Cultures are typically incubated at 30◦ C;
however, room temperature is also suitable. Coccidioides spp. are also com-
monly recovered on a variety of bacterial media. Unopened tubes should be
examined daily for the first 10 days and weekly thereafter for 3 weeks. It is
seldom necessary to hold cultures for longer than 3 weeks to recover spp.
of Coccidioides unless patients are receiving antifungal agents at the time of
specimen collection.

CULTURAL FEATURES

Colonies develop readily, usually within 3–5 days, and are initially white
to cream glistening, glabrous and tenacious, adhering to the medium. As they
mature, discrete concentric rings develop and filamentous areas containing
arthroconidia become visible. While species of Coccidoidies are generally
white to off-white, various other colors have been observed, ranging from tan
to yellow, rose to lavender, and pale brown to gray-brown. Tease-mounts for
observation of microscopic morphology should be made in lactophenol cotton
blue and the coverslips should be sealed with a mounting medium and all ma-
nipulations of cultures must be performed within a Class II biological safety
cabinet. Hyphae are thin, hyaline, and septate. Thicker side branches give
rise to unicellular, barrel-shaped arthroconidia (3–4 × 3–6 m) that alternate
with thin-walled, empty disjunctor cells (FIG. 2). At maturity the arthroconi-
dia are released, and remnants of the disjunctor cells are seen as “annular
frills” on the ends of individual arthroconidia.8,39 The arthroconidia of Cocci-
doides species must be differentiated from some “look-alike” genera with al-
ternating arthroconidia such as Malbranchea, Chrysosporium, and Geomyces,
and from Sporotrichum pruinosum. Isolates morphologically consistent with
Coccidioides spp. are commonly confirmed in clinical laboratories using the
AccuProbe nucleic acid hybridization method by GenProbe, San Diego, Cali-
fornia.40–42 This method is based on the ability of complementary nucleic acid
strands to specifically align and associate to form stable double-stranded com-
plexes. The method uses a chemiluminescent labeled, single-stranded DNA
probe complementary to rRNA of the target organisms to form a DNA:RNA
hybrid that is measured in a luminometer. A selection reagent differentiates
320 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

FIGURE 2. Lactophenol cotton blue tease-mount of Coccidioides immitis showing

chains of arthroconidia separated by disjunctor cells. Original magnification approximately
960×.

the non-hybridized from the hybridized probe. The cutoff for a positive test is
50,000 RLU (relative light units). Repeat testing should be performed for iso-
lates with RLU between 40,000 and 49,999. False negative results may occur
with formaldehyde-killed cultures.43 Exoantigen testing may also be used for
confirmation. The method detects the presence of cell-free antigens, known
as exoantigens. Mature slants are overlaid with merthiolate 1:5,000 overnight
and extracts sterilized through a membrane filter.44 Exoantigens are demon-
strated by immunodiffusion of antigens forming precipitin lines of identity with
reference antisera. This method has mostly been replaced by the AccuProbe
method. Conversion of arthroconidia to spherules in Converse medium is not
performed in routine clinical laboratories.45,46

BIOTERRORISM AND REQUIREMENTS FOR REPORTING

Coccidioides immitis and C. posadasii are now classified as select agents
of potential bioterrorism and their recovery from clinical samples must be
documented and reported. Clinical or diagnostic laboratories that have iden-
tified Coccidioides species must report their findings to the Centers for
Disease Control within seven calendar days on CDC Form 4 and either
SUTTON 321

destroy cultures by a recognized method, or forward these isolates to a se-

lect agent laboratory registered with CDC select agent programs. Shipment
of these isolates previously identified as Coccidioides immitis or C. posadasii
must be through approved hazardous materials channels for infectious agents
and may be shipped as Category A biological substances. Select agent lab-
oratories may keep culture collections, but must follow stringent documen-
tation and inventory control of cultures and adhere to the guidelines for
handling select agents. Clinical laboratories sending isolates suspected of
being Coccidioides spp. to select agent laboratories for purposes of identi-
fication and/or susceptibility testing may ship these isolates as Category B
biological substances. Complete handling and transfer regulations for select
agents are found in the Federal Register 42 CFR Part 1003, Possession, Use,
and Transfer of Select Agents and Toxins; Final Rule, March 18, 2005 at
http://www.cdc.gov/od/sap/pdfs/42 cfr 73 final rule.pdf

SUSCEPTIBILITY TESTING

In vitro antifungal susceptibility testing for Coccidioides spp. remains un-

standardized outside specialized laboratories experienced in this testing. How-
ever, modifications of the Clinical Laboratory Standards Institute M38-A
method for filamentous fungi have been used for testing the arthroconidial
form of the fungus.47 All testing must be performed within a biological safety
cabinet. The Fungus Testing Laboratory utilizes an enclosed macrobroth mod-
ification of the M38-A method with spectrophotometric standardization of the
inoculum to 95% T at 530 nm (1 × 104 to 5 × 104 CFU/mL), incubation at 35◦ C,
and visual endpoint readings. Because no defined breakpoints exist, “suscepti-
bility” may be viewed as minimum inhibitory concentrations, minimum lethal
concentrations, or minimal effective concentrations of the agent being tested
that are within safely achievable levels in the patient. In vitro MICs/MECs be-
yond levels attainable would suggest potential resistance. Coccidioides spp.,
in vitro, are generally “susceptible” to amphotericin B, fluconazole, itracona-
zole, voriconazole, posazonazole48 as well as the echinocandins.49 Fluconazole
treatment failures have been reported, however,50,51 and may, in some cases,
be partially attributed to resistant organisms displaying MICs > 64 g/mL.
It should be noted, however, that there may be a lack of correlation between
in vitro activity and clinical efficacy for coccidioidomycosis,52 and that fac-
tors such as the pharmacokinetics of the drug, general host factors, and site of
infection all influence clinical outcomes.53

SUMMARY

The recovery of Coccidioides immitis or Coccidioides posadasii by culture

remains the “gold standard” for a definitive diagnosis. Appropriate specimens
322 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

must be submitted, and those from non-sterile sites should be plated on media
that will support growth as well as inhibit bacteria and rapidly growing saprobic
fungi. Direct microscopy should always be included in the culture process, as
a preliminary diagnosis may be made with the observance of spherules and/or
endospores. Although colonies of Coccidioides spp. are generally white to
cream to buff, many other colors have been reported and should be expected
occasionally. Confirmation of suspected Coccidioides isolates is commonly
made using the AccuProbe DNA probe. While in vitro susceptibility testing
remains non-standardized, it may be useful in detecting potential resistance
and documenting treatment failures.

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