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An Unusual
Aspergillus Species at a Major Cancer Center: Course
Number: DL-977 © California Association
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An Unusual
Aspergillus Species at a Major Cancer Center:
Implications for the Clinical Laboratory
After completing this course the participant will be able to:
- define and illustrate terms essential to the discussion of Aspergillus species and aspergillosis.
- name seven Aspergillus spp. that have been documented to cause invasive aspergillosis.
- discriminate among the seven Aspergillus spp. using morphologic characteristics.
- outline at least three ways to optimize the recovery and rapid identification of aspergilli in the clinical laboratory.
NOTICE ABOUT THIS COURSE
This web version is a more recent version than the published Newsline
article
When taking the On-line Quiz, it may be useful to print the pdf version of the
course and
Open Table I and the Essential Vocabulary page in seperate windows to have this
material available during the Quiz
The On-line Quiz will require reference to Table I and the Case above questions
9 and 10 for completion.
INTRODUCTION:
During the past 30 years, opportunistic fungi, both yeasts and
moulds, have emerged as life-threatening pathogens among patients with AIDS
and hematologic malignancies, among recipients of hematopoietic stem cell transplants
(HSCT), and among other immunocompromised patients. Aspergillus spp.,
a group of saprobic fungi abundant in the environment worldwide, cause the majority
of opportunistic mould infections. Among the approximately 180 species of Aspergillus,
34 have been associated with disease (1). Aspergillus fumigatus remains
the most commonly isolated and clinically relevant Aspergillus spp. isolated in hospital laboratories. In addition to Aspergillus spp. ,
Fusarium spp. , Scedosporium spp. , the Zygomycetes, and other
saprobic fungi, cause fewer, though similar, opportunistic infections.
Within the past decade, aspergillosis caused by non-fumigatus
Aspergillus has increased. Documented infections have been attributed
to Aspergillus flavus, Aspergillus niger, Aspergillus
terreus, Aspergillus ustus, Aspergillus versicolor and
Emericella (Aspergillus) nidulans among others. The increase
in numbers of infections, the detection of resistance to amphotericin B among
isolates of A. fumigatus and A. terreus, and the
continuing high morbidity and mortality associated with invasive aspergillosis
(IA) have intensified the need for rapid, non-invasive, highly sensitive and
specific diagnostic tests. To meet this need, many promising serological
and molecular diagnostic tools have been developed. Culture and microscopy,
however, remain the traditional, commonly used laboratory methods for diagnosing
aspergillosis. In addition, the two methods can be inexpensive and accurate
in the hands of trained professionals. On the other hand, culture and
microscopy often become positive too late during the course of IA for successful
treatment. Too often the diagnosis of IA is made at autopsy. Given
this situation, Clinical Laboratory Scientists (CLS) are challenged not only
to optimize their skills using culture and microscopy but also to minimize
the turnaround time of the results.
CASE: OUTBREAK OF AN UNUSUAL ASPERGILLUS AT A MAJOR CANCER
CENTER (adapted from reference 2)
A retrospective search of records between 1993 and 2003 at a large
tertiary care center revealed that six hematopoietic stem cell transplant (HSCT)
recipients suffered invasive aspergillosis caused by a rarely isolated species,
Aspergillus ustus. The organism was identified using standard laboratory
methods. Three patients were infected in 2001 and three more in 2003. DNA relatedness
of the six previously frozen patient isolates was performed using randomly amplified
polymorphic DNA (RAPD) analysis. Results of RAPD showed that most patient isolates
were genetically similar, suggesting a common environmental source for one cluster,
or perhaps, both clusters of the moulds isolated in 2001 and 2003. The study
is limited by the lack of environmental isolates of A. ustus for genetic
comparison. Interestingly, a large hospital construction project began in 2001
and ended in 2003; all six cases occurred between the beginning and end of the
project. Antifungal susceptibility testing, using techniques described in document
M-38-A published by the National Committee for Clinical Laboratory Standards
(now Clinical and Laboratory Standards Institute), revealed relatively high
MICs, i.e. , increased resistance, to all antifungal drugs tested. Of further
interest is that three of the four patients treated with both voriconazole and
caspofungin were cured of A. ustus infection. According to the authors,
the six cases represent the first outbreak of A. ustus recorded in
the medical literature. In addition, the cases are instructive for Clinical
Laboratory Scientists interested in mycoses and fungal identification.
Detection of this outbreak would have been improbable without
the ability of the laboratory to identify the organism beyond the genus level. If the laboratory had issued a final report of simply Aspergillus,
not fumigatus, instead of Aspergillus ustus, these six isolates
would have been lumped together with all other species of non-fumigatus
Aspergillus isolated during the 10-year period of the study. Likewise,
the evidence that this particular species may be more drug resistant than some
other species of Aspergillus may have been missed. This case also demonstrates
the value of molecular strain identification for analysis of patient isolates
and its implied potential for preventing future outbreaks.
DISCUSSION
Clinical relevance of Aspergillus spp.
The spectrum of human diseases caused by Aspergillus spp. is extremely broad; it includes toxicoses, allergic reactions, as well
as local and systemic infections in both immunocompetent and immunocompromised
hosts. The clinical laboratory provides diagnostic tests for many of these
diseases, most commonly those involving the respiratory system. Among
respiratory diseases caused by aspergilli, allergies cause significant illness
although the risk of systemic invasion is low. Allergic bronchopulmonary
aspergillosis (ABPA), a steroid-dependent, asthma-like disease, can lead to
permanent lung damage. Another syndrome, recurrent sinusitis, may result
from an allergic response to local Aspergillus infection. Besides
allergic disease, inhalation of Aspergillus conidia may result in aspergillomas
(fungus balls) that occur in the lungs of patients with tuberculosis or other
underlying lung diseases. The fungal balls may expand and multiply causing
chronic pulmonary aspergillosis, which may progress to fibrosis of the lung.
Invasive disease signals that the immune system has failed to control
local infection. The mortality rate of these invasive infections is very
high among immunocompromised hosts. In several recent studies, in fact,
the mortality rate of invasive aspergillosis (IA) among HSCT recipients ranged
between 55% and 80% (1).
In addition to diseases of the respiratory system, aspergilli
also infect the eye (e.g. endophthalmitis and keratitis), ear (otomycosis),
skin, nails (onychomycosis), and, upon dissemination, the central nervous system
and other deep organs. Extensive review of the disease spectrum of Aspergillus
spp. and its relation to immunosuppression is beyond the scope of this
article. The following discussion focuses instead on the laboratory diagnosis
of IA.
Laboratory Diagnosis of Aspergillosis
Detection of Aspergillus with Microscopy
A physician who suspects IA may request that tissue samples be
stained with periodic acid-Schiff (PAS) and Gomori methenamine silver (GMS),
two stains used in the histopathology laboratory to detect fungi. A pathologist
reviews the slides for evidence of fungal structures. A CLS with extensive
experience identifying fungi can supply valuable input to the pathologist if
evidence of fungi is found.
In slides of stained tissue, opportunistic moulds, including Aspergillus
spp., Scedosporium spp., Paecilomyces spp., and Fusarium spp., normally appear as septate, acute-angle,
dichotomously branching hyphae. When such hyphae are seen in tissue slides
in the absence of more specific structures, Aspergillus spp. cannot
be distinguished from the other opportunistic fungi. The slide result
must be followed by isolation in culture of a mould with hyphal characteristics
consistent with those seen on the slide.
In the clinical laboratory, the CLS may detect hyphae and/or conidia
of fungi on a Gram stained slide. An ocular micrometer is a helpful tool
for distinguishing Rhizopus spp., Mucor spp.,
and other Zygomycetes (aseptate hyphae, diameter 10-30µm) from Aspergillus
spp. and similar opportunistic moulds (septate hyphae, diameter 2.5-8
µm). The potassium hydroxide (KOH) mount, made by mixing 10-20%
KOH in water, is the wet prep typically ordered when a fungal infection is suspected.
Commercially available fluorescent dyes, including Calcofluor, Blankophor,
and Uvitex 2B, can be added to KOH to improve detection of fungi with fluorescence
microscopy. Like histopathology stains and Gram stains, KOH mounts that
reveal only hyphal elements are not specific for Aspergillus spp. Culture
is necessary to isolate the causative fungus (3, 4).
Detection of Aspergillus with Culture
Common species of Aspergillus typically grow rapidly
in culture and mature within a few days of inoculation. They grow on a
wide variety of media and at a wide range of temperatures. Identification
is based on 1) the macroscopic morphology of a mature colony growing on a standard
fungal medium such as potato dextrose agar, 2) the microscopic morphology of
the mature conidial head and other distinguishing features, if present, and
occasionally 3) physiologic tests such as temperature tolerance at 35-37°C
and 48°C. An Aspergillus colony is “mature”
when reproductive bodies (conidia, and occasionally ascospores) have developed
sufficiently to distinguish one species from another, using published identification
schemes such as those found in references 5, 6, and 7.
The nomenclature used for the isolate is influenced by the type(s)
of reproductive structures observed in the mature colony. Conidia are
the product of mitosis, or asexual reproduction. Ascospores are the product
of meiosis, or sexual reproduction. Most medically important aspergilli
reproduce in culture only by mitosis. A few, for example, Emericella
nidulans, reproduce by both mechanisms. When both types of reproductive
bodies are observed in culture, use of the meiotic stage name (in this example,
Emericella), is considered taxonomically correct rather than the mitotic
stage name (in this example, Aspergillus). To avoid confusion,
some authors use both names, for example, Emericella (Aspergillus) nidulans.
Conidia of many Aspergillus spp. are intensely
pigmented, giving the surface of the colonies a distinctive color. The
hyphae of most species are hyaline (colorless), leaving the reverse side of
the colonies whitish or with variable, usually muted, colors. Some species
also produce bright pigments that diffuse into the agar. The production
of a distinct surface color is a signal that the colony is probably mature,
i.e. , that reproductive structures have been produced. The Scotch-tape
mount and/or tease mount are used to examine small bits of the colony for distinctive
microscopic features. Growth is removed from the pigmented part of the
colony surface and mounted in lacto-phenol or in lacto-phenol with an aniline
blue, or fuchsin, dye. Dyes may help highlight structural details but
may obscure pigments that are inherent to fungal structures, e.g. , the
pale brown pigment in conidiophores of Emericella (Aspergillus) nidulans.
Table I outlines some major macroscopic and microscopic
features of the seven species of Aspergillus listed in the Introduction.
Refer to the Essential Vocabulary and to Figures
1-15 as guides for interpretation of Table I. The Figures include
images of selected aspergilli isolated from patient specimens at the Clinical
Microbiology Virology Laboratory at Stanford Hospital and Clinics, Stanford,
CA and photographed by the author. Additional image sources are noted.
The print references listed in References and Bibliography supply further
identification aids, as do the following excellent websites:
• http://www.aspergillus.org.uk/indexhome.htm (For images of species,
click on “Image Bank,” option “Species Images. ”
Registration at the site may be required to view images. )
• http://www.cbs.knaw.nl/ (For images, drawings and other information
on species, click on “Databases,” option “Filamentous Fungi
Database,” option “Species Name. ”)
• http:// www.doctorfungus.org/ (For the image bank, go to www.doctorfungus.org/imageban/index.htm)
• http://www.mycology.Adelaide.edu.au/ (Click on “Kaminski’s
Digital Images Library. ”)
Attending a hands-on workshop is an effective method for learning
traditional fungal identification. When that option is unavailable, reference
fungal strains can be ordered from commercial sources including the American
Type Culture Collection (ATCC) and the Centraalbureau voor Schimmelcultures
(available at www.cbs.knaw.nl. ). These isolates can be rehydrated
and planted on available fungal media for study. It is important to note
that the physical characteristics of moulds actively growing on media may vary
somewhat from the images and descriptions in print and on websites. For
example, Klich (7) illustrates and describes Aspergillus spp. growing
on malt extract and Czapek agars, media traditionally used by reference laboratories
to describe aspergilli. In contrast, St. Germain and Summerbell
(6) use photographs from potato glucose and Sabouraud glucose media, agars that
are more commonly used in clinical laboratories. In summary, repeatedly
studying known pathogenic species in your laboratory on your media
with the assistance of a knowledgeable mycologist may be the most beneficial
learning experience.
Promise of non-culture methods
The search for improved tools to diagnose aspergillosis has produced
several immunologic and molecular tests. For example, the galactomannan
(GM) antigen, a cell wall polysaccharide found in most Aspergillus spp., can be detected by a FDA-approved ELISA assay from BioRad. The
GM test may detect IA significantly earlier than is possible by culture alone.
There is continuing debate, however, about the optimal frequency of the
test and the cut-off value for positive results. In addition, it has been
documented that there are false positive galactomannan assays when a patient
is receiving pipercillin-tazobactam. Other assays in development include
quantitative polymerase chain reaction that promises detection of aspergilli
directly from specimens. Conventional PCR, using ribosomal targets followed
by gene sequencing, offers molecular identification of culture isolates. Lack
of availability, cost, lack of standardization, technical problems, and lack
of consensus among the medical and research communities about how best to use
them are among the factors that prevent the widespread use of these non-culture
diagnostic tests. For an extensive discussion of the galactomannan test
and other immunodiagnostic, metabolic and molecular methods, the reader is referred
to references 3, 5, and 8.
Beyond Identification: Optimizing Culture Techniques and Turnaround Time
Successful detection and identification of Aspergillus spp., indeed, of all agents of infectious diseases, rests on proper specimen
collection and processing. In general, the same procedures used for recovery
of bacteria are appropriate for recovery of fungi. However, because the
number of infective units may be relatively low, maximizing the amount of specimen
used for culture is especially important for the diagnosis of fungal disease.
On the other hand, collecting blood cultures, while useful for diagnosing
many bacterial infections and a few fungal infections such as histoplasmosis,
is not an effective method for diagnosing aspergillosis (5, 9).
The selection of appropriate isolation media for fungal cultures,
the inhibitory antibiotics added to that media, and the temperature of incubation
may vary depending on the type of fungal pathogens most commonly recovered from
the patient population of a hospital or clinic. Optimally, a variety of
media are selected to ensure detection of all potential pathogens. Fortunately,
Aspergillus spp. grow quickly on a wide range of media and at
a wide range of temperatures. In fact, Aspergillus colonies may be initially
detected on the sheep blood and/or chocolate agars used for bacterial culture.
Media developed specifically for detection of fungi should be
fresh and poured into containers that provide a large surface area. To
reduce dehydration, using a large amount of media, e.g. 30-40 ml of media
per plate or flask, is preferable to using smaller amounts. For recovery
of Aspergillus spp., where the need for rapid detection may
be especially acute, the ideal medium will induce rapid growth and conidiation
and inhibit bacteria that might suppress fungal growth. Potato dextrose
or potato flake agars with antibiotics are among the media providing best results.
Many antibiotics, such as chloramphenicol and gentamicin, may be added
to inhibit bacteria present in mixed-flora specimens such as sputum. On
the other hand, cycloheximide, an antifungal agent used for the selective isolation
of dermatophytes, should be avoided when recovery of Aspergillus spp. and other opportunistic moulds is desired.
Besides selecting the most appropriate media, there are several
other options for improving the turnaround time of fungal detection in the clinical
laboratory. For example, reviewing fungus cultures daily for the first
seven days after inoculation may optimize detection of rapid growers such as
Aspergillus spp. Fungi isolated on fungal media, as well as on
media referred from routine bacteriologic cultures, should be studied as soon
as possible. A quick tape mount of a mature colony, followed by documentation
of the macroscopic and microscopic characteristics from a fungal medium such
as potato dextrose agar, is often adequate for a final identification of common
aspergilli. For atypical and/or less common isolates, a slide culture,
temperature tolerance, and/or prolonged incubation may be necessary. Identification
of clinically significant isolates which fail to produce conidia and/or which
are extremely atypical may be referred to a mycology reference laboratory. The
patient population, the particular strengths and limitations of the clinical
laboratory, and other factors will influence which of the techniques described
above are desirable and feasible. The CLS, however, must be aware that
diagnosing IA as quickly as possible is a critical laboratory service.
Because aspergilli are common and abundant in the environment,
a challenging task for the CLS is to determine whether an Aspergillus
isolate is a plate contaminant or not. A possible scenario might involve
one colony of A. fumigatus growing on one plate of a sputum culture;
the colony is located within the inoculated area of the plate. The patient
has been diagnosed with leukemia. In this case, the isolate may signal
invasive disease and should be identified and reported as quickly as possible.
If the single colony is growing outside the inoculated area and/or diagnostic
information is not available, the report should be more carefully considered.
It is important for the laboratory, in conjunction with physicians, to
develop clear and detailed reporting criteria for the isolation of all moulds,
including Aspergillus spp. In addition to the location of the
isolate on the inoculated area of culture media, the following factors suggest
that an isolate is clinically significant: 1) the presence of hyphal elements
in direct mounts of the specimen; 2) the presence of more than one colony on
one or more of the culture media; and 3) isolation of the same mould from several
specimen (5).
SUMMARY
The clinical laboratory offers important tests for diagnosing
invasive aspergillosis and for studying its epidemiology. The CLS has
a very important role in this process, including specimen collection, selection
of appropriate media, astute reading of stained specimens, timely detection
of colonies, accurate identification of isolates, and reporting of lab results
to the physician. Awareness and appropriate use of emerging immunologic
and molecular tests will ensure that patients, especially the immunocompromised,
receive the most rapid diagnosis possible. Given the numerous, reliable
resources online and in print, continual learning about Aspergillus spp. and aspergillosis is easily available. By increasing skill in fungal
identification and improving the effectiveness of laboratory procedures, the
CLS can provide valuable services for patients threatened by aspergillosis.
ACKNOWLEDGMENTS
I gratefully acknowledge Ellen Jo Baron, Ph.D. , Director of the Clinical Microbiology
Laboratory (CML), Stanford University Medical Center, Stanford, CA, for supporting
my photography sessions at Stanford University’s Pathology Photography
Laboratory. Thanks also go to the staff of the CML for saving isolates and for
ongoing support of laboratory education.
REFERENCES
- Barnes PD, Marr KA. Aspergillosis: Spectrum of disease, diagnosis, and treatment. Infect Dis Clin North Am. 2006; 20: 545-561.
- Panackal AA, Imhof A, Hanley EW, Marr KA. Aspergillus ustus infections among transplant recipients. Emerg Infect Dis. 2006; 12: 403-408.
- Hope WW, Walsh TJ, Denning DW. Laboratory diagnosis of invasive aspergillosis. Lancet Infect Dis. 2005; 5: 609-622.
- McClenny N. Laboratory detection and identification of Aspergillus species by microscopic observation and culture: the traditional approach. Med Mycol. Supplement 1 2005; 43: S125-S128.
- Sigler L, Verweij PE. Aspergillus, Fusarium, and Other Opportunistic Moniliaceous Fungi. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, eds. Manual of Clinical Microbiology. 8th ed. Washington, DC: ASM Press; 2003: 1726-1760.
- St-Germain G, Summerbell R. Identifying Filamentous Fungi: A Clinical Laboratory Handbook. Belmont, CA: Star Publishing Company; 1996.
- Klich MA. Identification of Common Aspergillus Species. Utrecht, The Netherlands: Centraalbureau voor Schimmelcultures; 2002.
- Hinrikson HP, Hurst SF, De Aguirre L, Morrison CJ. Molecular methods for the identification of Aspergillus species. Med Mycol. Supplement 1 2005; 43: S129-S137.
- Sutton, DA. Specimen Collection, Transport, and Processing: Mycology. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, eds. Manual of Clinical Microbiology, 8th ed. Washington, DC: ASM Press; 2003: 1659-1667.
BIBLIOGRAPHY
- De Hoog GS, Guarro J, Gene J, Figueras MJ. Atlas of Clinical Fungi. Baarn, The Netherlands: Centraalbureau voor Schimmelcultures; 2000.
- Larone DH. Medically Important Fungi. 4th ed. Washington, DC: ASM Press; 2002.
- NCCLS (now CLSI). Document M38-A: Reference method for broth dilution antifungal susceptibility testing of filamentous fungi: approved standard. Wayne, PA: NCCLS; 2002.
- Sutton DA, Fothergill AW, Rinaldi MG. A Guide to Clinically Significant Fungi. Baltimore, MD: Williams&Wilkins; 1998.
Course #DL-977
Choose the one best answer
- Which answer BEST completes the following statement: Aspergillus spp. are
a. a group of yeast-like fungi that cause opportunistic infections.
b. a group of mould-like fungi that cause infections primarily in immunocompetent patients.
c. saprobic moulds that cause opportunistic infections in humans.
d. a group of mushroom-like fungi. - Aspergilloma, or fungus balls, may result from
a. the inhalation of the conidia of Aspergillus spp.
b. skin trauma during which conidia of Aspergillus spp. enter the bloodstream.
c. ingestion of Aspergillus conidia.
d. penetration of mucous membranes by conidia of aspergilli. - The mortality rate of invasive aspergillosis among hematopoietic stem cell
transplant recipients, documented in recent studies, is
a. 10-25%
b. 80-95%
c. <10%
d. 55-80% - In a conidial head of Aspergillus spp., which structures produce
the conidia?
a. vesicles
b. metulae
c. conidiophores
d. phialides - While reading a KOH mount, a CLS detects acute-angle, dichotomously branching
septate hyphae in sinus tissue from a patient with leukemia. The hyphae measure
about 4µm in diameter. A mould grows from culture of the tissue specimen
and is quickly identified by standard laboratory methods. Among the following
fungi, the LEAST likely identification is:
a. Aspergillus spp.
b. Scedosporium spp.
c. Fusarium spp.
d. Rhizopus spp.
- Traditional microscopy and culture for the identification of opportunistic
fungi rely predominantly on which pair of characteristics:
a. macroscopic morphology and physiologic tests
b. microscopic and macroscopic morphology
c. macroscopic morphology and RAPD
d. microscopic morphology and RAPD - A non-invasive, FDA-approved test that may detect aspergillosis sooner than
is possible with microscopy and culture alone is:
a. RAPD
b. galactomannan ELISA
c. Calcofluor fluorescence
d. Caspofungin MIC - All the methods below will likely improve turnaround time for laboratory
diagnosis of invasive aspergillosis EXCEPT
a. maximizing the amount of tissue used for fungus culture
b. inspecting fungus culture media daily for the first few days after inoculation
c. ordering blood cultures
d. studying the microscopic morphology of an isolate as soon as pigment develops - The Aspergillus produced a green-colored colony. Which one of the
following species can be ELIMINATED as the pathogen based on that single characteristic?
a. Aspergillus fumigatus
b. Emericella (Aspergillus) nidulans
c. Aspergillus terreus
d. Aspergillus flavus
- The microscopic mount of the mature colony revealed a conidial head with
both uni- and bi-serrations. The conidiophores were distinctly echinulated. Small, dark, peppercorn-like bodies developed after the colony aged. The fungus
was identified as:
a. Aspergillus flavus
b. Emericella (Aspergillus) nidulans
c. Aspergillus versicolor
d. Aspergillus ustus
Case for Questions 9 and 10: In 1989-1990 three cases of fungal endophthalmitis were reported in Louisville, Kentucky. All of the patients were intravenous drug users and seronegative for HIV. The same species of Aspergillus grew from culture of the vitreous fluid of all 3 patients. Despite treatment with amphotericin B and flucytosine following vitrectomy, all the patients suffered some permanent vision loss. Epidemiologists were unable to uncover the exact source of infection. Given the restricted time frame and geographic area of the 3 cases, however, a contaminated drug supply was suspected as the likely source. The Aspergillus spp. that grew from all 3 patients is among the seven species described in Table 1 of this course. Use that table to answer the following questions.