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Journal of Clinical Microbiology, August 1999, p. 2434-2438, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Specific Detection of Fusarium Species
in Blood and Tissues by a PCR Technique
Francois-Xavier
Hue,1,*
Michel
Huerre,2
Marie Ange
Rouffault,1 and
Claude
de Bievre1
Laboratoire de Mycologie
Médicale1 and Laboratoire
d'Histopathologie,2 Institut Pasteur, 75724 Paris cedex 15, France
Received 14 October 1998/Returned for modification 1 February
1999/Accepted 30 April 1999
 |
ABSTRACT |
Fusarium species are opportunistic nosocomial pathogens
that often cause fatal invasive mycoses. We designed a primer pair that
amplifies by PCR a fragment of a gene coding for the rRNA of
Fusarium species. The DNAs of the main Fusarium
species and Neocosmospora vasinfecta but not the DNAs from
11 medically important fungi were amplified by these primers. The lower
limit of detection of the PCR system was 10 fg of Fusarium
solani DNA by ethidium bromide staining. To test the ability of
this PCR system to detect Fusarium DNA in tissues, we
developed a mouse model of disseminated fusariosis. Using the PCR, we
detected Fusarium DNA in mouse tissues and in spiked human
blood. Furthermore, F. solani, Fusarium
moniliforme, and Fusarium oxysporum were testing by
random amplified polymorphic DNA (RAPD) analysis. The bands produced by
RAPD analysis were purified, cloned, and sequenced. The information was
used to design primer pairs that selectively amplified one or several
Fusarium species. The method developed may be useful for
the rapid detection and identification of Fusarium species
both from culture and from clinical samples.
| |
INTRODUCTION |
Disseminated fungal infections
constitute one of the most difficult challenges for clinicians caring
for immunocompromised patients (3). Candidiasis and
aspergillosis remain the most common mycoses in neutropenic patients.
However, other life-threatening infections caused by new opportunistic
pathogens also occur. One of the most frequently occurring of these
pathogens is Fusarium (15, 19).
Members of the genus Fusarium are ubiquitous fungi commonly
found in soils and plants (22). Fusarium species
have long been recognized as a cause of localized infections
(11). Because of bone marrow grafts and immunosuppressive
therapy, invasive Fusarium infections have increased during
the last decade. The immunologic status of the host and the extent of
the infection are the most important factors for the clinical outcome
of Fusarium infections (13).
Because an invasive Fusarium infection may mimic
aspergillosis, patients are usually treated with amphotericin B, an
antifungal agent with poor activity against fusariosis (9).
Hence, early identification is an important factor for a successful
outcome. Furthermore, diagnosis requires the demonstration of hyphae in pathological samples; however, hyphae of Aspergillus,
Scedosporium, and Fusarium are difficult to
discriminate. Positive culture is thus needed for the identification of
a Fusarium sp. Currently, the identification of members of
the genus Fusarium is based on the characteristic colony
morphology and the microscopic characters, which include the production
of multiseptated sickle-shaped conidia called macroconidia; however,
recognition may be difficult when the macroconidia are not produced in
culture (11). This usually happens with strains isolated
from clinical samples which have been developed in unfavorable
conditions. In this case, the isolates can be confused with other
genera such as Acremonium and Verticilium. Furthermore, precise determination of Fusarium species
remains a prerequisite for studying the spread, host infections, and treatment.
The PCR technique is extremely sensitive and has been used successfully
for the specific detection of several fungi (20). We report
here on the use of a competitive PCR technique for the detection of
Fusarium spp. in blood and tissues and PCRs for the identification of the Fusarium species. In order to test the
PCR system, we also developed a mouse model of fusariosis.
| |
MATERIALS AND METHODS |
Culture conditions.
DNA was isolated from several
Fusarium species and a range of medically important fungi.
The collection used is listed in Table 1.
The Fusarium isolates were maintained on potato dextrose agar at 25°C. The other fungi were maintained on
Sabouraud-chloramphenicol (SC) at 30°C. Malassezia furfur
was cultured on Dixon agar at 37°C.
DNA preparation from fungal cells.
The total cell DNA was
extracted from mycelium or yeast grown in YPG liquid medium by the
method described by Dellaporta et al. (8). Briefly, the
mycelium and the yeast were mechanically disrupted within liquid
nitrogen and were mixed with 15 ml of fungal extraction buffer
(Tris-HCl, 10 mM; EDTA, 50 mM; NaCl, 500 mM), 1 ml of sodium dodecyl
sulfate (10%) was added, and the mixture was incubated for 20 min at
65°C. Then, 5 ml of potassium acetate (3 M) was added, and the
homogenate was incubated for 30 min on ice. After centrifugation, the
DNA was precipitated with 0.7 volume of isopropanol, and the samples
were centrifuged at 9,000 × g for 20 min at 4°C. The
pellet was dissolved in 700 µl of Tris (Tris-HCl, 10 mM [pH 8]) and
incubated at 65°C, and the DNA was precipitated with 0.7 volume of
isopropanol and 0.1 volume of sodium acetate (3 M). The DNA was washed
with 70% ethanol, dried, and resuspended in 200 µl of Tris (10 mM;
pH 8).
Oligonucleotide design, internal control, PCR amplification, and
detection of PCR products.
The design of oligonucleotides P28SL
and P58SL was based on comparison of the sequences of the ribosomal
genes (rDNAs) from a large number of isolates belonging to the genus
Fusarium found in the EMBL/GenBank database (Table
2). The sequences were analyzed with the
PILEUP program of the Genetics Computer Group software package as
reported earlier (9, 10). Primers P28SL and P58SL (Oligoexpress, Paris, France) amplified a fragment of 329 bp containing ITS2 and a portion of 5.8S and 28S rDNA.
To avoid false-negative results, a positive internal control was made
from a part of
phage DNA (
4). We designed two primers,
primers C1 and C2 (Table
3), whose 3'
ends correspond to the
DNA and whose 5' ends correspond to the
primers used in the
PCR amplification. The first PCR generated a
fragment of 517 bp
which contains the
Fusarium sequence at
the ends. This fragment
was amplified with the
Fusarium
primers and was purified with
the Qiaquick PCR amplification kit
(Qiagen, Courtaboeuf, France).
After dilution of the fragments, PCRs
were performed. The highest
dilution that gave a positive result after
electrophoresis was
chosen as the internal control. One microliter of
internal control
was added to each PCR mixture.
The PCRs were performed according to the manufacturer's instructions
(Ready to go kit; Pharmacia Biotech, Uppsala, Sweden),
with the
following temperatures cycles: initial denaturation at
94°C for 5 min, followed by 40 cycles of denaturation at 94°C
for 1 min,
annealing at 68°C for 1 min, and extension at 72°C
for 1 min. The
thermal cycles were terminated by a final extension
of 10 min at
72°C. One nanogram of template DNA was used per reaction
mixture.
To ensure that no contaminating DNA could give a positive result, one
sample without DNA was included in each series of reactions.
To examine
the specificity of the P58SL and P28SL primer pair,
the samples of
genomic DNA extracted from fungi and from human
blood were tested to
verify whether the primer pair selectively
amplified the DNA from
Fusarium (Table
1). To determine the lower
limit of
detection, PCRs were performed with water samples containing
1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, and 0.1 fg of
genomic
Fusarium solani DNA.
Aliquots (10 µl) of the PCR products were analyzed on a 2% agarose
gel after electrophoresis in Tris-acetate-EDTA buffer.
A 100-bp DNA
Ladder (Boehringer Mannheim GmbH, Mannheim, Germany)
was used as a
molecular size marker. The gels were stained with
ethidium bromide (1 µg/ml), visualized under UV light, and photographed
with Polaroid 667
film.
Preparation of PCR template from F. solani-containing
blood.
Human blood (obtained from healthy volunteers after they
provided appropriate written consent) was collected in tubes containing EDTA as an anticoagulant and was inoculated with 10-fold dilutions of
Fusarium mycelium in sterile saline. The cells in the
Fusarium-spiked blood samples were lysed (GFX genomic blood
DNA purification kit; Pharmacia Biotech) and mixed with 200 µl of
enzyme buffer (sorbitol, 0.9 M; Tris, 0.1 M; EDTA, 0.1 M) and 20 µl
of Lyticase (Sigma, St. Louis, Mo.). Following incubation at 37°C for
90 min, proteinase K (10 µl, 20 mg/ml; Sigma) was added, and each
sample was incubated at 55°C for 30 min, followed by the addition of
5 µl of RNase (Boehringer Mannheim GmbH). After incubation at 37°C
for 30 min, the DNA was extracted by the use of the GFX genomic blood
DNA purification kit, according to the manufacturer's instructions. PCR was performed by using these extracts as templates (2 µl). Aliquots (10 µl) of the PCR products were analyzed by electrophoresis on a 2% agarose gel as described above.
Mouse model of invasive fusariosis.
Four male BALB/c mice
(Charles River, Saint Aubin Les Elbeuf, France) weighing between
20 g and 25 g were immunosuppressed with three
intraperitoneal injections of triamcinolone (Kenacort; Bristol-Myers
Squibb, Paris, France) at a dose of 2 mg/kg of body weight on days 
2,
0, and +3. On day 0, they were also inoculated in the lateral tail
veins with 106 conidia of F. solani IP 1684.87 in a 200-µl volume of sterile saline. One control mouse was treated
in the same way and received 200 µl of sterile saline on day 0. When
the mice died, portions of organs and blood were removed. A total of 50 µl of each blood sample was cultured on SC at 25°C. Various organs
(spleen, lung, heart, kidney, and liver) were cultured on SC at 25°C.
A small piece of each organ was retained for histology. These sections were stained with hematoxylin-eosin-safran and Gomori methenamine silver stains. The remainder of each organ was ground in a 1.5-ml microcentrifuge tube and was stored at 20°C. DNA from both blood and tissues was extracted by the protocol described above.
Differentiation of Fusarium species.
For random
amplified polymorphic DNA (RAPD) analysis, DNA fragments were amplified
with primers F 24 and A8 (12), as follows: one cycle of 5 min at 92°C, followed by 36 cycles of denaturation at 92°C for
30 s, annealing at 36°C for 1 min, and extension at 72°C for 2 min, with a final cycle of 2 min at 72°C. The DNAs of the three main
Fusarium species (F. solani, Fusarium
moniliforme, and Fusarium oxysporum) were tested. Three
high-intensity bands produced by RAPD analysis for each species were
purified from agarose and were cloned into the vector pUC 18. The ends
of the cloned fragments were sequenced according to the manufacturer's instructions (Thermosequenase Cycle Sequencing; Amersham Life Science,
Buckinghamshire, United Kingdom), and the sequences obtained were used
to design the primer pairs. The PCR amplifications and the analysis of
the DNA were performed as described above.
| |
RESULTS |
Oligonucleotide design.
After alignment and visual assessment
of the Fusarium and non-Fusarium sequences,
primers pair P58SL and P28SL, which amplified a 329-bp fragment, was
selected (Table 3).
Specificity and sensitivity studies.
On the basis of the
sequences of the 5.8S and 28S regions of the rDNA complex, a product of
329 bp was amplified by PCR with primer pair P58SL and P28SL from all
27 strains of 11 medically important Fusarium species and
from Neocosmospora vasinfecta but not from any of the other
fungi tested (Fig. 1). The specificity of
the 329-bp fragment was verified with the restriction enzymes HincII and XbaI (Gibco Life Technologies,
Cergy-Pontoise, France); as expected, the 329-bp fragment was not
digested by XbaI and gave two fragments of 200 and 129 bp
upon HincII digestion. No amplification was observed with
human DNA.
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FIG. 1.
Specificity of PCR with primer pair P58SL and P28SL. An
ethidium bromide-stained agarose gel of PCR products amplified from 1 ng of genomic DNA templates from various fungi is shown. (A) Lanes: 1, F. moniliforme IP 1579.85; 2, F. moniliforme IP
233.95; 3, F. solani IP 2330.95; 4, F. solani IP
2447.97; 5, F. solani IP 2451.98; 6, F. oxysporum
9582; 7, F. oxysporum IP 625.72; 8, F. oxysporum
9026; 9, F. proliferatum 68G; 10, F. dimerum IP
1516.83; 11, F. semitectum 2239; 12, F. subglutinans 2241; 13, F. nivale 2238. (B) Lanes: 1, Candida albicans; 2, Cryptococcus neoformans; 3, Penicillium purpurogenum; 4, Aspergillus
fumigatus; 5, Acremonium strictum; 6, Trischoporon cutaneum; 7, Malassezia furfur; 8, Exophiala jeanselmei; 9, Trichophyton rubrum; 10, Alternaria alternata; 11, Aspergillus flavus; 12, Neocosmospora vasinfecta; 13, F. solani IP
1681.87; lane T, negative control. Lanes M, 100-bp DNA ladder.
|
|
The sensitivity allowed detection of as little as 10 fg of
F. solani DNA by visualization under UV light. There was no amplified
product from the negative control (sample without
Fusarium DNA).
PCR template from Fusarium-containing blood.
As
shown in Fig. 2, with primer pair P58SL
and P28SL the PCR system was able to amplify the Fusarium
DNA from blood artificially inoculated with mycelium. Amplified
products were not detected from control samples that were devoid of
Fusarium DNA.
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FIG. 2.
Ethidium bromide-stained agarose gel of products from
F. solani IP 1684.87-spiked blood samples in serial 10-fold
dilutions. The fragment of 517 bp corresponds to the internal control.
The fragment of 329 bp corresponds to amplified F. solani
DNA. Lane M, 100-bp DNA ladder.
|
|
Mouse model.
A summary of mouse survival and the culture,
histology, and PCR results is given in Table
4. Fifteen samples had positive results
by culture. Seven culture-positive samples were also positive by PCR
(with primer pair P58SL and P28SL), and 3 of 13 culture-negative samples also had positive PCR results. The result for sample 24 was
nonconclusive: the fragment corresponding to the internal control was
not observed (Fig. 3). Despite the use of
proteinase K and a column extraction, amplification inhibitor factors
may not have been eliminated. All PCR products led to the amplification of a single fragment. The result of PCR, culture, and histology were
all negative for uninfected mice.
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FIG. 3.
Three possible results expected by competitive PCR with
primers P28SL and P58SL. The results of gel electrophoresis of PCR
products amplified from genomic DNA extracted from mouse organs are
shown. The fragment of 517 bp corresponds to the internal control. The
fragment of 329 bp corresponds to amplified F. solani DNA.
Lane 1, inconclusive PCR result; lanes 2, 3, 4, 5, and 6, PCR-negative
results; lanes 7, 8, and 9, PCR-positive results; lane T, negative
control.
|
|
Differentiation of the Fusarium species.
Table
5 shows the primer pairs that were
selected and their characteristics. These primer pairs were used to
perform new PCR assays. The use in the same PCR mixture of the two
primer pairs OX 31-OX 32 and OX 41-OX 42 allowed the amplification of the F. oxysporum DNAs tested (Fig.
4). Although primer pair SOL 31-SOL 32 was specific for F. solani, only four of five strains tested
were amplified (Table 5).
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FIG. 4.
Specificity of the primer pairs OX 31-OX 32 and OX
41-OX 42. An ethidium bromide-stained agarose gel of PCR products
amplified from 1 ng of genomic templates from various fungi is shown.
Lane 1, F. oxysporum IP 625.72; lane 2, F. oxysporum 9582; lane 3, F. oxysporum 9026; lane 4, F. moniliforme IP 1579.85; lane 5, F. solani IP
2330.95; lane 6, F. proliferatum IP 2240.94; lane 7, F. dimerum IP 1516.83; lane 8, F. semitectum
2239; lane 9, F. subglutinans 2241; lane 10, F. chlamydosporum IP 1542.84; lane 11, Penicillium
purpurogenum 9701; lane 12, Aspergillus fumigatus; lane
13, Acremonium strictum IP 2234.96; lane 14, Candida
albicans IP 48.72; lane T, negative control. Lane M, 100-bp DNA
ladder. pb, base pairs.
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| |
DISCUSSION |
The results obtained in the present study demonstrate that the
rDNA-based PCR method has high degrees of sensitivity and specificity for the detection of a wide range of medically important
Fusarium species from spiked blood and from tissue from an
animal model.
Several previous studies have used PCR technology to detect fungi
(20). The primers used target three kinds of sequences: (i)
sequences coding for an antifungal target, (ii) fungus-specific regions
of conserved proteins such as actin, and (iii) repeated sequences such
as those of rDNA and mitochondrial DNA. For diagnostic purposes, it is
essential that repeated sequences be used as targets to ensure good
sensitivity, which justifies our choice of primers. We reported that
the binding sites for primer pair P58SL and P28SL, within the 5.8S and
the 28S regions, were conserved among Fusarium species. They
amplified DNAs from all 11 different Fusarium species tested
and also from N. vasinfecta, which is a sexual form of an
Acremonium sp. whose species name is not yet defined. Only two cases of infection with the latter species have been reported in
humans (7).
One of the limitations of the molecular methods of diagnosis of
microbial infections, especially PCR amplification, are the false
positivity and the false negativity of the test results. These issues
are being addressed in several ways: standardization of the technical
procedure (one-step procedure) and the use of an internal control
(2). Impurities in nucleic acid preparations (e.g., ethanol
and isopropanol) or in biological samples (e.g., hemoglobin, EDTA, and
heparin) can inhibit or reduce the sensitivity of PCR amplification
(16). Thus, the internal control is necessary both for
clinical diagnosis and for the development of a DNA extraction procedure.
Although many studies regarding the PCR detection of yeast (5, 6,
17, 18, 21, 24) and Aspergillus fumigatus (25) have been reported, few PCR systems for the detection
of other fungi have been developed (1, 26). Moreover, the
PCRs described for the latter case have not been tested with blood or
tissues. For this reason, we thought it desirable to test the PCR with
primers P28SL and P58SL for the detection of Fusarium both
in spiked blood and in tissues and blood from experimentally infected
mice. In the mouse model, the correlation between PCR results and
culture results was not high (46%); however, 23% of the samples with
culture-negative results were positive by PCR amplification. One
explanation is the poor efficiency of the extraction protocol. Another
explanation is the existence of a localized necrotic abscess. Indeed,
before extraction histology and culture, each sample was divided into
three portions, but not all portions may have been infected.
Different molecular methods are used to differentiate species of fungi:
restriction fragment length polymorphism (RFLP) analysis (14,
16) or RAPD analysis (23). RFLP analysis is long and laborious and often requires the use of radioactive probes. RAPD analysis is poorly reproducible, and results are difficult to interpret. Furthermore, RAPD and RFLP analyses require axenic cultures
of the fungus, whereas PCR can be performed with tissue samples. PCR
should be considered as complementary method for the identification of
Fusarium species when microscopic characterization cannot
distinguish between two species.
Further experiments will be necessary to determine whether specificity
and sensitivity similar to those obtained in vitro and with spiked
blood samples can be obtained with clinical samples. Larger
prospectives studies that focus on immunocompromised patients at high
risk of invasive fusariosis should evaluate both the utility and the
sensitivity of this PCR assay in a routine setup in comparison to those
of other available methods (culture and histology).
| |
ACKNOWLEDGMENTS |
We are grateful to Nalin Rastogi and Christophe Sola for helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Present address: Institut
Pasteur, Morne Jolivière, BP 484, 97165 Pointe à Pitre
cedex, Guadeloupe, France. Phone: 590.896940. Fax: 590896941. E-mail:
cneyret{at}ipagua.gp.
| |
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Journal of Clinical Microbiology, August 1999, p. 2434-2438, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
