International Journal of Clinical Medicine, 2013, 4, 472-478
http://dx.doi.org/10.4236/ijcm.2013.410083 Published Online October 2013 (http://www.scirp.org/journal/ijcm)
The Effect Analysis of Different Experimental Methods for
the Diagnosis of Invasive Pulmonary Aspergillosis in a Rat
Model*
Jiancong Lin, Wenming Xu, Ming Li, Yanli Xin, Yuanyuan Niu, Changran Zhang#, Zelong Guo
Department of Internal Medicine, Huang Pu Hospital of the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
Received August 28th, 2013; revised September 25th, 2013; accepted October 10th, 2013
Copyright © 2013 Jiancong Lin et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Background: Consensus on the most reliable assays to detect invasive aspergillosis from minimally or noninvasive
samples has not been reached. In this study, we compared the efficacy of an enzyme-linked immunosorbent assay
(ELISA) for galactomannan (GM) detection and quantitative real-time PCR assay (qRT-PCR) for the diagnosis of inva-
sive pulmonary aspergillosis in a rat model. Methods: Neutropenic, male Sprague-Dawley rats (specific pathogen free;
8 weeks old; weight, 200 ± 20 g) were immunosuppressed with cyclophosphamide and infected with Aspergillus fumi-
gatus intratracheally. Tissue and whole blood samples were harvested on days 1, 3, 5, and 7 post-infection and exam-
ined with GM ELISA and qRT-PCR. Results: On day 7, A. fumigatus DNA was amplified from 14 of 48 whole blood
samples from immunosuppressed infected rats: day 1 (0/12), day 3 (0/12), day 5 (6/12), day 7 (8/12) post infection. The
sensitivity and specificity of the qRT-PCR assay were 29.2% and 100%, respectively. Receiver operating characteristic
curve (ROC) analysis indicated a Ct cut-off value of 15.35, and the area under the curve (AUC) was 0.627. The GM
assay detected antigen in sera obtained on day 1 (5/12), day 3 (9/12), day 5 (12/12), and day 7 (12/12) post-infection,
and thus had a sensitivity of 79.2% and a specificity of 100%. The ROC of the GM assay indicated that the optimal
cut-off value was 1.40 (specificity, 100%; AUC, 0.919). Conclusions: The GM assay was more sensitive than qRT-
PCR assay in diagnosing invasive pulmonary aspergillosis in rats.
Keywords: Invasive Pulmonary Aspergillosis; Aspergillus; Galactomannan Antigen; Quantitative Real-Time PCR;
Receiver Operating Characteristic Curve; Rat Model
1. Introduction
The prevalence of invasive fungal infections and conse-
quent mortality has increased throughout the last 2 dec-
ades, and the reported mortality from an epidemiological
study covering the period from 2002 to 2006 is 28.6% [1].
According to the TRANSNET database, half of extensive
fungal infections are caused by Aspergillus sp. [2]. Most
fungal infections occur in immunodeficient individuals,
such as transplant recipients or those with leukemia re-
ceiving chemotherapy, and pathological exacerbations of
lung infections and the inability to diagnose fungal infec-
tions are the major causes of death in these patients [3,4].
Early diagnosis of invasive fungal infections is critical
for rapid treatment [3], but the lack of sensitive and spe-
cific clinical symptoms and radiological patterns for in-
vasive aspergillosis (IA) hinders early diagnosis. Tradi-
tional histopathological examination and fungal culture
relying on invasive procedures are relatively insensitive
and not commonly used in clinical diagnosis due to the
challenges of sampling pulmonary fluids or tissues from
critically ill patients. While examination of bronchial-
veolar lavage (BAL) fluid yields a higher detection rate
than examination of blood or serum [5-7], obtaining BAL
fluid is invasive, and many patients with IA have other
severe diseases which limit BAL collection. Consensus
on the most reliable assays to detect IA from minimally
or noninvasive samples has not been reached, and is a
major topic of current research [8,9].
Galactomannan (GM) is a cell wall component of As-
pergillus, and its production increases during Aspergillus
growth. Assays that detect fungal antigens such as GM
*Yanli Xin and Jiancong Lin equally contributed to this manuscript.
The authors declare that they have no competing interests.
#Corresponding author.

The Effect Analysis of Different Experimental Methods for the Diagnosis
of Invasive Pulmonary Aspergillosis in a Rat Model
473
by enzyme-linked immunosorbent assay (GM assay) or
Aspergillus DNA by polymerase chain reaction (PCR)
are emerging diagnostic methods; however, their speci-
ficity and sensitivity require additional characterization
and refinement. The sensitivity of the GM assay ranges
from 60% to 100% for infected Aspergillus samples, and
the specificity ranges from 80% to 100% [10-14]. The
cut-off value has a significant impact on diagnosis be-
cause of cross-reactivity [8,12,15-17]. Furthermore, treat-
ment with anti-fungal therapies decreases the fungal load
and reduces the GM concentration, which can fall below
the detectable limit of the GM assay [18]. Given the ad-
vantage of high sensitivity, a PCR assay coupled with an
ELISA assay (PCR-ELISA) has been used for detection
of Aspergillus [19,20]. However, poor specificity has
been noted due to several interference factors, including
environmental contamination [19]. In addition, the PCR-
ELISA assay is more cumbersome for clinical laborato-
ries [5].
Recently, a quantitative real-time PCR (qRT-PCR)
assay was developed that may overcome the shortcom-
ings of other methods [8,21]. qRT-PCR analysis of se-
rum samples of patients with hematological malignancies
at risk for IA was shown to have a sensitivity of 72.7%
[5]. A commercially available qRT-PCR for the detection
of Aspergillus DNA (MycAssay™ Aspergillus) has shown
a sensitivity of 60% - 70% and a specificity of 95% for
the detection of IA [22]. However, a study by Scotter et
al. [19] indicated that GM testing by ELISA and the
PCR-ELISA were more capable of early detection of
fungal infection than RT-PCR examination.
The aim of the study is to compare the sensitivity and
specificity of the GM assay to the Aspergillus-specific
nucleic acid qRT-PCR assay in a rat model of pulmonary
IA.
2. Methods
2.1. Aspergillus fumigatus Preparation
Lyophilized Aspergillus fumigatus (A. fumigatus) was
recovered in 0.5 mL sterile broth, cultured in Sabouraud
agar medium at 37˚C for 48 h, and subsequently at 30˚C
for 3 - 5 days. Spores were eluted from the agar surface
with 10 mL PBS containing 0.05% Tween-80. The sus-
pension was filtered through 8 layers of sterile gauze to
remove hyphae. The spore suspension was then trans-
ferred to a 15-mL tube and centrifuged at 10,000 ×g for
15 min. The supernatant was discarded, and the pellet
containing spores was resuspended in normal saline.
Spore count was determined on a blood count plate, and
the concentration was adjusted to 8 × 105 spores/L. Spore
viability was determined by culture of serial dilutions of
the spore suspension.
2.2. Induction of Pulmonary Aspergillosis in
Rats
Neutropenic rats were infected with Aspergillus as de-
scribed by Zhang et al. [23]. Briefly, male Sprague-
Dawley rats (specific pathogen free; 8 weeks old; weight,
200 ± 20 g) were housed with food and water ad libatum
according to animal care guidelines. Rats received one of
four treatments: immunosuppression and A. fumigatus
infection (n = 48), immunosuppression (no infection, n =
6), infection (no immunosuppression, n = 6) and vehicle
control (no immunosuppression, no infection, n = 12).
Persistent immunosuppression was induced in the indi-
cated groups by intraperitoneal injection of cyclophos-
phamide, as described by Leenders et al. [24]. The dos-
age of cyclophosphamide was determined by a prelimi-
nary experiment. Cyclophosphamide (50 mg/kg i.p.) was
injected 5 days prior to the Aspergillu s spore suspension
inoculation. A second cyclophosphamide injection (40
mg/kg) was performed 1 day prior to inoculation, and a
third injection (30 mg/kg) was performed on day 3 after
inoculation. Control groups were injected with an
equivalent volume of normal saline. After treatment with
cyclophosphamide, all rats were consistently kept in a
clean environment, and injected with levofloxacin, 10
mg/kg/d.
Immunosuppressed and normal rats of the indicated
groups were infected with A. fumigatus (8 × 105 spores)
after anesthesia (chloral hydrate (3.5 mL/kg i.p.) and
intubation. After connecting the endotracheal tube with a
syringe, 0.1 mL of the spore suspension was injected.
Rats were kept erect and rotated for 30 seconds, ensuring
that the inoculation entered the trachea and was distrib-
uted evenly in both lungs. Rats were sacrificed after 1 - 7
days and whole blood was collected by heart puncture.
Rats which received both immunosuppression and in-
fection (n = 48) were sacrificed on days 1, 3, 5, and 7
after inoculation, and were referred to as group 1, 2, 3,
and 4, respectively (n = 12/group). Four ml of whole
blood was collected and 3 mL blood was used to deter-
mine Aspergillus DNA using the qRT-PCR method, and
100 μL of serum was prepared to measure the GM con-
centrations using the ELISA method. Lung tissue was
collected for biopsy and tissue culture.
2.3. Isolation of A. fumigatus DNA from Blood
A.fumigatus DNA was isolated as described previously
[25]. We used the physical method of grinding, similar to
the bead beating, to break the cell walls of the fungi to
release fungal DNA. The efficiency of fungal DNA ex-
traction was about 96.8%. In brief, 3 mL of whole blood
were treated with 1 mL of EDTA anticoagulant, lysed in
1 mL of erythrocyte lysis buffer (0.0l mol/L Tris-HCl pH

The Effect Analysis of Different Experimental Methods for the Diagnosis
of Invasive Pulmonary Aspergillosis in a Rat Model
474
7.6, 0.01 mol/L NaCl, 0.005 mol/L MgCl2), mixed thor-
oughly, and further treated with 1 mL of lysis buffer
twice, allowing extensive erythrocyte lysis. After centri-
fuged at 8000 ×g for 10 min, the pellet was washed with
normal saline, treated with l00 μL proteinase K lysis
buffer (20 mM Tris-HC1, 2.0 mM EDTA, 1.0% Triton
X-l00, 2 mg/mL proteinase K), and subsequently 50 μL
lysostaphin at a concentration of 50 μg/mL. The lysate
was incubated at 60˚C for 60 min, treated with an equi-
valent volume of phenol, chloroform, and isoamyl alco-
hol. Thirty μL of A fumigatus DNA was precipitated us-
ing ethanol, centrifuged at 12000 rpm/min for 20 min,
and dissolved in Tris-EDTA buffer (10 mM Tris-Cl, pH
7.5 with 1 mM EDTA) for reserve.
2.4. Polymerase Chain Reaction and Product
Analysis
Specific primers were based on the target mRNA se-
quence in GeneBank, harboring the CDS region of the
mitochondrial translation optimization gene Mto1 of A.
fumigatus. Primers were designed using Primer Express
2.0 software: forward primer, 5’-tttctccacccaggaacgtt-3’;
reverse primer, 5’-cgaatccggagaggtgatacc-3’; probe, 5’-
FAM-cagttgtgatgacgacacgcccagt-TAMRA-3’. Primers were
synthesized using the ABI 3900 high-throughput DNA
synthesizer. To determine the specificity of the qRT-PCR
reaction, the aforementioned primers were assessed for
their ability to amplify DNA from A. niger, A. flavus, A.
terreus, Candida albicans, Cryptococcus neoformans,
Staphylococcus aureus, and Pseudomonas aeruginosa
[26].
The qRT-PCR reaction (50 μL) included 10 μL of 5 ×
reaction buffer (10 mM Tris-HCl pH 8.0, 50 mM KCl, 2
mM MgCl2), 10 pmol of each primer (10 pmol/uL), 2μL
dNTPs (10 mM), 3U Taq DNA polymerase, and 4 μL
cDNA or positive standard. Reaction was performed as
follows: 93˚C for 3 min, and 40 cycles of 93˚C 30 s,
55˚C 45 s. Real-time PCR was carried out in an auto-
mated fluorescent quantitative PCR cycler (ABI 7500),
and the amplification curve was analyzed based on the
exponential amplification and Ct value (cycle threshold).
The Ct was defined as the number of cycles required for
the fluorescent signal to cross the threshold (i.e., exceed
background level). The Ct value was dose-dependent on
the positive standard
The standard curve quantification method used the
target gene synthesized in Sangon (Shanghai). Ten-fold
serial dilutions of the positive standard were prepared to
produce the quantitative qRT-PCR gradients. Distilled
water was used as a negative control. The optical density
(OD) 260/280 of the purified DNA was >1.8, and thus
qualified. DNA concentration (copies/μL) was calculated
with OD 260 over the fragment length, namely the posi-
tive standard. After dilution of recombinant plasmid,
qRT-PCR amplification was performed following the
optimal procedures. The LOD was measured according
to amplification curve derived from qRT-PCR. The cor-
relation between Ct value and DNA copy was Ct =
−3.347424 × log copy number + 35.885406.
2.5. GM Antigen Detection (GM Assay)
The GM assay was performed using the Platelia Asper-
gillus kit (Bio-Rad Corporation, France), following the
manufacturer’s instructions for the preparation of sam-
ples. Optical densities (OD) at a primary wavelength of
450 nm and a secondary wavelength of 620 nm were
determined. In each experiment, positive serum control,
negative serum control, standardized control, and serum
samples were run in triplicate. The mean OD derived
from the standardized control was used as a standard
(GM OD Index = OD of sample/OD of standard serum),
and kept in the range of 0.3 to 0.8. All experiments were
internally controlled: The ratio of OD from the positive
control to OD from the standard was over 2, and the ratio
of OD from the negative control to the OD of the stan-
dard was below 0.4.
2.6. Histopathological Examination and Tissue
Culture
Rats that received both cyclophosphamide and infection
were sacrificed on 1, 3, 5, and 7 days after A. fumigatus
inoculation (n = 12 each group). Rats in the other groups
were sacrificed on day 7. Blood samples and right lung
tissue were used for fungal culture described above. The
left lung tissue was fixed in 10% formalin and embedded
in paraffin. The paraffin sections were primarily stained
with hematoxylin & eosin (HE), and further with peri-
odic acid-Schiff stain followed by histopathological ex-
amination.
2.7. Statistical Analysis
The values of the GM concentration for each group were
reported as mean ± standard deviation. Wilcoxon rank
sum test was used to analyze the difference between each
two groups. A receiver operating characteristic (ROC)
curve was created with the statistical software used for
all analyses (SPSS version 16.0). A value of p < 0.05 was
considered to indicate statistical significance.
3. Results
3.1. Histopathology of Pulmonary A. fumigatus
Infection
Immunosuppressed, infected rats showed progressive
accumulation of hyphae in the alveoli (Figure 1). Many

The Effect Analysis of Different Experimental Methods for the Diagnosis
of Invasive Pulmonary Aspergillosis in a Rat Model
475
(a) (b) (c)
(d) (e)
Figure 1. Histopathological analysis. Lung tissues were harvested from immunosuppressed, infected rats on day 1(a), 3(b),
5(c), and 7(d) (100× magnification), and from the control groups on day 7(e) (400× magnification). Lung tissue from healthy
controls.
Aspergillus spores and mild inflammation were found in
the lung tissue of the neutropenic rats on the 1st day post
infection (Figure 1(a)). On the 3rd day, the alveoli exhib-
ited accumulated spores, a small presence of hyphae,
hyperplasia of alveolar epithelial cells, and broadened
alveolar septa. Cellulose exudate and hemorrhage in the
alveolar spaces were also observed (Figure 1(b)). On the
5th day post-infection abnormal morphology of the al-
veolar structures was apparent, and the spores had
bloomed: hollow, colorless and acute angle-branching
hyphae and granuloma were present (Figure 1(c)). Clear
exudate was located in the alveolar spaces, and severe
hemorrhage was evident in the capillaries. The alveolar
septa had broadened, and part of the alveolar structure
was damaged. On the 7th day, the aforementioned cha-
racteristics had become more apparent and the alveolar
structure was no longer clear (Figure 1(d)). The alveoli
of the vehicle control group had normal morphology
(Figure 1(e)) No apparent inflammation was noted in
nonimmunosuppressed infected mice.
3.2. Detection of A. fumigatus DNA Isolated from
Immunosuppressed-Infected Rats
No A. fumigatus target sequence was amplified from the
uninfected control group (immunosuppression and no
infection, n = 6), and vehicle control group (no immuno-
suppression, no infection, n = 12). The target sequence
was successfully amplified in 14 out of 48 samples from
immunosuppressed-infected rats. The 14 positive sam-
ples were obtained on day 5 (6/12), and day 7 (8/12): no
positive samples were obtained on day 1 or day 3. No
DNA was identified in the no immunosuppression/infec-
tion control samples (0/6) or in the no infection/immu-
nosuppression control samples (0/6).
Amplification curve and quantitative analysis showed
that the DNA content ranged from 5 × 102 to 5.77 × 103
copies/μL of blood. The lower limit for qRT-PCR detec-
tion was 100 copies/μL of blood, and was determined
according to amplification curve derived from qRT-PCR.
Only samples from infected animals yielded positive
RT-PCR curves, and the PCR reactions spiked with DNA
from A. niger, A. fla vus, A. terreus, Ca n d ida albicans,
Cryptococcus neoformans, Staphylococcus aureus, and
Pseudomonas aeruginosa yielded negative results. Thus,
the primer set for A. fumigatus showed high specificity.
Receiver operating characteristic curve (ROC) analysis
indicated a Ct cut-off value of 15.35, and the area under
the curve (AUC) was 0.627.
3.3. Determination of GM by ELISA
A commercial kit was utilized for the determination of
GM, and the mean concentrations for each group are
shown in Table 1. As expected, the concentration of GM
ncreased from day 1 to day 7, in agreement with the i

The Effect Analysis of Different Experimental Methods for the Diagnosis
of Invasive Pulmonary Aspergillosis in a Rat Model
476
Table 1. qPCR detction of Mto1 gene copy number and serum GM levels.
Group Number Positive/Total Serum GM level (index) qPCR of blood (copies/μL)
Infection and immunosuppression
Day 1 (group 1) 5/12 1.236 ± 0.169 NA
Day 3 (group 2) 9/12 1.889 ± 0.247 NA
Day 5 (group 3) 12/12 2.548 ± 0.218 35.29 ± 31.25
Day 7 (group 4) 12/12 3.520 ± 0.215 1183.69 ± 1653.60
Control groups at Day 7
Immunosuppression/no infection 0/6 0.857 ± 0.103 NA
No immunosuppression/infection 0/6 0.683 ± 0.130 NA
No immunosuppression/no infection 0/12 0.600 ± 0.109 NA
NA = could not be detected. Wilcoxon rank sum test showed that the serum GM levels in the immunosuppressed infected animals at day 1, 3, 5, and 7 were
significantly different from the control groups (p < 0.01). A significant difference was also present between group 1 and group 4 (p < 0.01). No significant
difference was observed in the GM levels between the three control groups (all, p > 0.05).
infiltration observed in the histopathological specimens.
The GM assay detected antigen in only some of the in-
fected animals (group 1, 5/12; group 2, 9/12; group 3,
12/12; group 4, 12/12; infection control, 0/6). Since only
samples from infected animals tested positive in the GM
assay, the specificity for the GM assay was 100%, and
the sensitivity was 79.2%. The ROC of the GM assay
indicated that the optimal cut-off value was 1.40 (speci-
ficity, 100%; AUC, 0.919).
4. Discussion
In this study, the efficacies of GM assay and qRT-PCR
assays for the detection of Aspergillus infection were
evaluated in a well-established rat model of pulmonary
IA. The results showed that while both assays were
100% specific for the diagnosis of IA and GM assay ex-
hibited much greater sensitivity and allowed for earlier
detection.
Serum-based assays for diagnosis of Aspergillus infec-
tion have been sought for at least four decades, but in-
adequate sensitivity has restricted the general application
of many methods [8]. A commercial ELISA method
measures GM antigen at concentrations as low as 0.5 - 1
ng/mL, depending on the cutoff value [23,25]. Our cut-
off value of 1.4 provided a sensitivity of 79.2% for days
1 - 7 infected samples in this immunosuppressed rat
model. Using an A. fumigatus-infected guinea pig model,
McCulloch et al. [18] found that the GM assay was able
to detect A. fumigatus infection in samples from 0/3 ani-
mals on day 1, 1/3 on day 2, and 3/3 on days 3 - 5 for an
overall sensitivity of 67%. Lengerova et al. [6] observed
a 100% sensitivity in BAL fluids in an IA rat model, but
only a 26% sensitivity in serum samples (1/5 on day 3,
0/5 day 5, 3/5 day 7). Becker et al. [13] found that GM
assay detected GM in 8% of day 1 samples in an IA rat
model, 89% of day 3 samples, and reached 100% by day
7. In comparison, our results showed that GM was de-
tected in the sera of 5 of 12 samples on day 1, 9 of 12
samples on day 3, and all samples on day 5 and day 7,
which were consistent with those of reports [18]. The com-
mercially available MycAssay Aspergillus DNA assay
and an “in house” qRT-PCR assay have shown promise
in the clinical diagnosis of IA via testing BAL fluid sam-
ples [27]. Compared with conventional PCR, qRT-PCR
lowers the risk of cross-contamination by using a sealed
tube during amplification and detection. The fluores-
cence signal generation depends on both probe-template
recognition and amplification of template, ensuring the
high specificity of qRT-PCR method. Although Asper-
gillus sp. DNA was usually detected at a higher frequency
in BAL samples [27], Hadrich et al. [5] observed that
RT-PCR and PCR-ELISA assays exhibited higher sensi-
tivities in serum samples than in BAL samples, with sen-
sitivities ranging from 64% - 94%. In our rat model, the
sensitivity of the qRT-PCR was 25% with whole blood
samples harvested from infected, immunosuppressed rats
on days 1 - 7, which was similar to the 26% sensitivity
using serum samples from a guinea pig IA model (days 1
- 7) reported by Lengerova et al. [6]. Likewise, Becker et
al. [13] found that in an IA rat model PCR did not detect
Aspergillus DNA from day 1 or 2 serum samples, but the
sensitivity improved from 20% on day 3 to 40% on day 7,
and the authors concluded that the sensitivity may be
related to the methodology. One obvious difference be-
tween humans and animal models is that in most cases
humans will have sought testing due to the presence of
symptoms, suggesting a more fulminant Aspergillus in-

The Effect Analysis of Different Experimental Methods for the Diagnosis
of Invasive Pulmonary Aspergillosis in a Rat Model
477
fection at time of sampling than the early time points in
animal models. Presumably, the longer incubation period
would favor Asp ergillus replication, and may promote
alveolar damage and seepage of more Aspergillus spores
into the blood.
In this study, analysis of qRT-PCR results showed that
an AUC of 0.627 produced the maximum specificity, and
although highly consistent with the pathological changes
in lung tissue the qRT-PCR method was not sufficiently
sensitive (25.9%) to solely rely on for early diagnosis. In
contrast, the AUC of the GM assay was significantly
higher (0.919), and was able to detect Aspergillus in 79%
of day 1 - 7 samples. Interestingly, Torelli et al. [27] de-
scribed a cohort of patients with suspected IA, and >90%
of patients with BAL samples that were positive for As-
pergillus DNA by MycAssay and RT-PCR also had
GM-positive BAL samples. Together, it seems that GM
assay and qRT-PCR assays both are capable of early de-
tection of Aspergillus infection but further study for es-
tablishing a gold standard is warranted [28].
5. Acknowledgements
The research was supported by Science and Technology
Planning Project of Guangdong Province, China, No.
2011B090400118 , No. 2009B03081137 and Guangzhou
Public Health Bureau (Grant No. 2009-YB-181, 2009-
YB-183).
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