Fungi inhabit nearly all terrestrial environments. In
this regard, the interiors of human dwellings and
workspaces are no exception. The mycobiota of
human-inhabited indoor environments consists of
a distinctive group of organisms that collectively are
not normally encountered elsewhere. Dust forma-
tion occurs as a result of the ongoing elutriation of
airborne organic and inorganic particulate matter
that originates from a multiplicity of indoor and
outdoor sources (S co tt , 2001). House dust is a
fibrous material composed primarily of a matrix
of textile fibers, hairs, and shed epithelial debris
(Scott, 2001). Fungi commonly isolated from indoor
air include Alternaria, Aspergillus, Aureobasidium,
Cladosporium, and Penicillium species. Many of
these species may contaminate indoor air through
heating, ventilating, and air-conditioning systems
(S im on s et al., 1997). It is well known that venti-
lation systems, even those without water-containing
components, may act as a potential microbial source
in indoor air (Pa sa ne n et al., 1997). Filters are
porous soft insulation material very often used in
air-conditioning systems. Direct microscopic exam-
ination of air filters reveals pollen particles, cellulose
fibers, synthetic fibers, plant hairs, decayed leaves,
insect parts, dust mites, and many organic com-
pounds (Mo r a y and Wi l l i am s, 1990). Cellulose
and synthetic fibers probably come from indoor
sources, while the other components most likely
originate from outdoor sources. Insulation materi-
als also absorb moisture and volatile organics and
provide suitable substrates for fungal colonization
(S im on s et al., 1997). These organics represent
excellent nutrients for fungal growth, with the result
that air filters harbor an abundance of fungal hyphae
and spores (Yo u n g, 1996). Dust and microorgan-
isms may accumulate in supply air ducts during
installation or later from the outdoor air due to
leakages between the filter cassette and the assembly
frame, or from insufficient efficiency of the filter
(P a s an en et al., 1997).
Human infections caused by indoor fungi are
very seldom because of the highly efficient defense
mechanisms of human cells, such as the cell-medi-
ated response (P er de l i et al., 2006). However,
conidia and fragments of hyphae may sometimes
cause allergenic responses, and some metabolites
produced by fungi may be toxic or have immu-
nomodulating activity in humans (S i mo ns et
al., 1997). Also, reduction in the host organism’s
defensive ability, whether due to cancer, AIDS,
FUNGAL COLONIZATION OF AIR-CONDITIONING SYSTEMS
MILICA LJALJEVIĆGRBIĆ, JELENA VUKOJEVIĆ, and M. STUPAR
Institute of Botany and Jevremovac Botanical Garden, Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia
Abstract — Fungi have been implicated as quantitatively the most important bioaerosol component of indoor air associ-
ated with contaminated air-conditioning systems. Rarely, indoor fungi may cause human infections, but more commonly
allergenic responses ranging from pneumonitis to asthma-like symptoms. From all air conditioner filters analyzed, 16
fungal taxa were isolated and identified. Aspergillus fumigatus causes more lethal infections worldwide than any other
mold. Air-conditioning filters that adsorb moisture and volatile organics appear to provide suitable substrates for fungal
colonization. It is important to stress that fungal colonization of air-conditioning systems should not be ignored, espe-
cially in hospital environments.
Key words: Indoor air, air-conditioning filters, micromycetes, human health
Arch. Biol. Sci., Belgrade, 60 (2), 201-206, 2008 DOI:10.2298/ABS0802201L
M. LJALJEVIĆ-GRBIĆ ET AL.
organ transplantation, or any other medical rea-
son, may lead to the uncontrolled multiplication of
fungi and consequent onset of infection, sometimes
with fatal effects (P er de li et al., 2006). There are
reports that four patients died and 11 contracted
the respiratory disease aspergillosis at the Alcala de
Hanares Hospital, near Madrid (Spain). The disease
was caused by inhaling the spores of Aspergillus
fumigatus, which was later detected in the hospital’s
air-conditioning system (http://www.acr-news.com/
MATERIALS AND METHODS
Fifteen dust samples were collected from air-
conditioning filters from school classrooms and
offices. Also, five swab samples were collected from
an air conditioner in a hospital surgical ward. None
of the analyzed filters from classrooms and offices
had been removed or cleaned for two years (Fig. 1a),
whereas those from the hospital were cleaned once
a month on a regular basis.
The tape lift technique used for direct examina-
tion allows for immediate determination of the pres-
ence of fungal spores and identification of the types
of fungi present. Direct examinations should only be
used to sample visible mold growth in contaminated
air-conditioning filters. Samples were collected by
pulling the tape of the filter surface with slow steady
pressure, holding only the tape edges, after which
they were put on slides for light microscopy.
Determination of total spore concentrations in dust
Culturable fungal spore concentrations are pre-
sented in terms of colony-forming units (CFU)/g of
dust. Sub-samples (0.5 g) were taken from each dust
sample and suspended in distilled water (0.0425 g/l
KH2PO4, 0.25 g/l MgSO4, 0.008 g/l NaOH, 0.02%
Tween 80 detergent). Dilution series were prepared
and three succesive dilutions were plated in tripli-
cate on malt agar medium (MA) with the antibiotic
streptomycin, which was added during the prepara-
tion process in order to prevent bacterial growth
(P a s an en et al., 1997). The plates were incubated
on 22 ± 2ºC and read after 72 to 120 hours. Fungal
colonies formed on the medium were identified
on the basis of both macroscopic and microscopic
characteristics of each isolated colony using identifi-
cation keys (A i n s wo rt h et al., 1973; A r x, 1974;
E ll i s and E l l i s , 1997; P i t t , 1979; R a pe r, and
F e n ne l 1965).
RESULTS AND DISCUSSION
The direct examination method revealed the
presence of different fungal structures: conidia,
conidiophores, chlamidospores and mycelia (Fig. 1b-
f). From all dust samples analyzed from classrooms,
offices, and hospital air-conditionning systems, six
fungal genera with different numbers of species were
recorded: Cladosporium, Penicillium, Aspergillus,
Alternaria, Epiccocum, and one Ascomycotyna from
the order Sphaeriales (Table 1). The fungal colonies
isolated from different sources were characterized
not only by the presence of different species, but also
by their different abundance (Fig. 2a). The genus
Aspergillus with five species was the most frequent
(Tabel 1, Fig. 2b, e, f). The abundance of fungal
colonies was much higher in classrooms and offices
than in the hospital. Aspergillus and Penicillium
species were dominant. Alternaria alternata and
Cladosporium cladosporioides were the most frequent
dematiaceous fungi (Fig. 2c, d). Fungal growth was
quantified by the number of CFUs. Culturable spore
concentrations in 15 dust samples varied from 104 to
106 CFU/g. In central Finland, culturable and total
fungi in dust accumulated in air ducts in single-fam-
ily houses varied from 104 to 108 CFU/g (P a sa ne n
et al., 1997).
The results of this research confirmed previous
findings that air-conditioning systems are highly
linked with fungal pollution of indoor air. The diver-
sity and abundance of fungal species isolated from
different air-conditioning systems can be attrib-
uted to different ways of maintaining the systems
themselves. Greater numbers of fungal genera and
species with much higher colony abundance were
expected and found in samples isolated from the air-
conditioning systems from classrooms and offices
since these systems were not cleaned after installa-
FUNGI FROM AIR CONDITIONING SYSTEMS 203
Fig. 1. Fungal structures from air conditioner filter dust: a) two years uncleaned air conditioner
filter; b) the conidial chain of Aspergillus ochraceus; c) conidiophores with terminal conidium of
Cladosporium sphaeospermum; d) mycelia with chlamydospores inside filter fiber; e) chlamidospore;
f) conidia of Alternaria sp.
tion (Fig. 1a). Only two fungal species were found
in samples from the hospital units, with very low
colony abundance. This result is worrying because
one of the two isolated species was Aspergillus versi-
color (Fig. 2e). Aspergillus is a large genus of fungal
molds, of which only a few species cause human
infections, most commonly A. fumigatus. Spores
from these species are widespread in the environ-
ment, occurring in soil, in dust, and in outdoor and
indoor air. Known as aspergillosus, fungal infection
with Aspergillus ranges from the benign to the fatal.
Healthy individuals usually inhale Aspergillus spores
M. LJALJEVIĆ-GRBIĆ ET AL.
without any untoward effects, but sometimes con-
tract relatively benign infections of the lungs and
sinuses. In susceptible compromised patients, how-
ever, inhalation leads to multiplication of the fungus
in the lungs and subsequent invasive infection that
may spread to any organ of the body. Dissemination
to the brain, gastrointestinal tract, and other organs
occurs in up to 30% of cases. The disseminated infec-
tion is usually fatal, partly because early diagnosis is
difficult and treatment often ineffective (F re nc h ,
Fig. 2. Micromycetes isolated from air conditioner filter dust: a) fungal colonies on MA;
b) Aspergillus ochraceus; c) Alternaria alternata; d) Cladosporium cladosporioides; e) As-
pergillus versicolor; f) Aspergillus fumigatus.
FUNGI FROM AIR CONDITIONING SYSTEMS 205
2005). Carpet dust samples (n = 11) contained A.
versicolor in concentrations ranging from <2.5 × 101
to 3.6 × 105 (median, 3.1 × 104) CFU/g of dust, and
the median proportion of A. versicolor in total cul-
turable fungi was 18%. Based on thin-layer chroma-
tography detection of sterigmatocystin, 49 of 50 A.
versicolor isolates (98%) where found to be toxigenic
in vitro (En ge l h a r t et al., 2002). Many Aspergillus
species are well known as potential producers of
mycotoxins and other volatile harmful compounds,
and many of them can cause aspergillosis in humans.
Hospitals and other medical facilities are places were
patients with damaged immune systems are com-
monly found. These patients are very receptive to
fungal infections. In order to avoid unwanted fungal
infections in hospitals, the air-conditioning systems
must be subjected to regular maintenance in order
to reduce potential fungal pollution.
Three features of mold biochemistry are of spe-
cial interest from the standpoint of human health.
Molds contain glucan, a compound with inflamma-
tory properties. Spores and mycelial fragments con-
tain allergens (G or ny et al., 2002). The spores of
some species contain low-molecular-weight chemi-
cals that are cytotoxic or have other toxic proper-
ties. Some molds, such as A. fumigatus, can cause
opportunistic infection in immunocompromised
and healthy individuals and severe allergic diseases,
such as asthma or cystic fibrosis (Burge, 2000). In
our previous investigations, Aspergillus species were
recorded in different substrata. Fungal spores can
spread from different sources and contaminate air
conditioning filters (K at ar a n o v s k i et al., 2007;;
L j a lj e v i ć and Vu k o j e vi ć , 1997).
Acknowledgments - The present research was funded by the
Serbian Ministry of Science and Environmental Protection
through project No. 143041.
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КОЛОНИЗАЦИЈА КЛИМА УРЕЂАЈА МИКРOГЉИВАМА
МИЛИЦА ЉАЉЕВИЋ ГРБИЋ, ЈЕЛЕНА ВУКОЈЕВИЋ и М. СТУПАР
Институт за ботанику и Ботаничка башта «Јевремовац», Биолошки факултет, Универзитет у Београду,
11000 Београд, Србија
Микрогљиве, као квантитативно најзначајни-
је биоаеросолне компоненте ваздуха у затворе-
ним просторијама, су чести контаминанти клима
уређаја. Оне могу изазвати алергијске реакције
типа пнеумонитиса и симптоме сличне астми, а
ређе могу бити и изазивачи хуманих инфекција.
Из анализираних клима уређаја изоловано је и
идентификовано 16 таксона микрогљива. Изо-
ловани Aspergillus fumigatus је познат коо изази-
вач леталних инфекција широм света. Филтери
клима уређаја који апсорбују влагу и органске
материје су погодан супстрат за колонизацију
микрогљивама. Веома је значајно напоменути
да се колонизација клима уређаја микрогљивама
не сме игнорисати и то нарочито у болничким