Journal of Clinical Microbiology, December 1999, p. 3865-3871, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Specific Detection of Aspergillus Species in Blood and
Bronchoalveolar Lavage Samples of Immunocompromised Patients by
Two-Step PCR
Heyko
Skladny,1
Dieter
Buchheidt,1,*
Corinna
Baust,1
Frank
Krieg-Schneider,1,
Wolfgang
Seifarth,1
Christine
Leib-Mösch,1,2 and
Rüdiger
Hehlmann1
III. Medizinische Klink, Klinikum Mannheim,
University of Heidelberg, D-68305 Mannheim,1
and Institute of Molecular Virology, GSF-National Research
Center for Environment and Health, D-85764
Neuherberg,2 Germany
Received 22 February 1999/Returned for modification 12 July
1999/Accepted 9 August 1999
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ABSTRACT |
The increasing incidence of aspergillosis, a life-threatening
infection in immunocompromised patients, emphasizes the need to improve
the currently limited diagnostic tools. We developed a two-step PCR
assay that specifically amplifies a region of the 18S rRNA gene that is
highly conserved in Aspergillus species. A number of
primers with the least homology to equivalent human or
Candida gene sequences were screened for the pairs that
gave the highest sensitivity and specificity. No cross-reaction with the wide range of fungal and bacterial pathogens so far tested was
observed. This assay allows direct and rapid detection of down to 10 fg
of Aspergillus DNA corresponding to 1 to 5 CFU per ml of
blood. A total of 315 blood and bronchoalveolar lavage samples from 140 subjects, including 93 patients at risk for invasive fungal disease,
were screened. The result was a 100% correlation between positive
histology, culture, or high-resolution computed tomography findings and
PCR results. The test specificity was 89%. Our data point to the
considerable potential clinical value of this simple, specific, rapid,
and inexpensive PCR assay for improving the means of early diagnosis of
systemic aspergillosis in high-risk patients.
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INTRODUCTION |
The incidence of life-threatening
systemic fungal infections has been increasing in recent years, and the
increasing incidence has been correlated with increasing numbers of
immunocompromised patients (1). Patients at the greatest
risk are those with prolonged periods of neutropenia after intensive
immunosuppressive chemotherapy, for example, during treatment for acute
leukemia or after bone marrow transplantation (31, 39).
Particularly on the increase are invasive infections with
Aspergillus species, resulting in high mortality rates or,
if the patient survives, causing high levels of morbidity that often
limit further antileukemic therapies. Antifungal prophylaxis,
especially prophylaxis against molds, is controversial and is not
generally practiced (3, 10). Moreover, amphotericin B, at
present the "gold standard" of antifungal therapy, is toxic.
In contrast to other infections, only limited conventional diagnostic
tools with poor sensitivity and reliability are available for early
detection of invasive aspergillosis (13, 21, 50), with the
systemic infection frequently being diagnosed late or confirmed only at
autopsy (7, 12, 20, 35, 36). The tests commonly used in the
initial period of infection during neutropenia are often insufficient
not only for accurate early diagnosis but also for monitoring of the
subsequent course of invasive aspergillosis (2, 6, 11, 21-23,
25-27, 32, 34, 42, 43, 46-49). In view of the low specificity
and sensitivity rates of these methods, the diagnosis of an invasive
aspergillosis can be proven conclusively only by positive histology or
culture results. However, establishing cultures from blood and
bronchoalveolar lavage (BAL) samples is often unsuccessful due to the
low yields of CFU, and in the case of immunocompromised high-risk
patients who are febrile, neutropenic, thrombocytopenic, and often
seriously ill, tissue biopsy specimens, in general, are not available
(15, 37, 53).
The diagnosis of invasive aspergillosis by molecular methods such as
Southern blot analysis has been performed successfully with lung and
liver material from animal models (41). The method showed a
high degree of sensitivity but is limited by the necessity of
performing invasive tissue biopsy. When applied to clinical samples
which were obtained noninvasively, such as blood and BAL samples,
hybridization methods were unsuccessful (41). As
successfully accomplished for other pathogenic organisms that are
difficult to detect (e.g., human immunodeficiency virus,
cytomegalovirus, Borrelia burgdorferi, and Toxoplasma
gondii) (9, 16, 24, 33, 40), more sensitive and rapid
detection assays have been established by use of the PCR method,
particularly following the identification and sequencing of multicopy
gene templates in a range of fungi and other organisms. PCR assays for
the detection of fungal nucleic acids may be the optimal diagnostic
approach because they are potentially more sensitive than current
culture-based methods and may be designed to encompass the desired
range of genera and specimen types. Previous studies evaluating
PCR-mediated detection of Aspergillus species showed
significantly improved sensitivity but involved assays with different
methods and objectives, partly to optimize culture assays (9,
28) and partly for typing in epidemiological studies (4, 5,
18). Therefore, the results of different groups are not
consistent or comparable. By using PCR primers specific for the
multicopy 28S rRNA gene, very small amounts (down to 1 pg) of genomic
DNA from Aspergillus fumigatus have been detected, the
sensitivity being increased to 100 fg by a subsequent Southern blot
assay (41). Studies with clinical samples, e.g., blood or
BAL samples, were mostly done retrospectively and with small numbers of
patients (8, 16, 29, 30, 41, 44, 45, 51, 52).
Melchers et al. (30) first described a PCR assay for the
detection of DNA from an Aspergillus sp. in
immunocompromised patients with primers based on the coding sequence of
the 18S rRNA gene which is highly conserved and which is amplified some
hundredfold in the Aspergillus genome. PCR products were
obtained from BAL samples of immunocompromised and neutropenic
patients, while no amplicons were obtained from immunocompetent
individuals. However, another report pointed to the high risk of BAL
specimen contamination by Aspergillus conidia (8)
resulting in approximately 25% false-positive results. Yamakami et al.
(52) first described the use of a two-step PCR to detect
Aspergillus spp. in blood with increased specificity and
sensitivity, but that study was performed with only a small number of
patients. Einsele et al. (17) described a PCR assay with
subsequent Southern blot analysis which allowed the detection of fungal
pathogens (including Aspergillus spp.) in blood samples. A
recent publication described a panfungal PCR assay for amplification of
a variety of fungal pathogens in human blood specimens, with the
specific fungal species or genera being identified by subsequent Southern blot hybridization (45).
In order to achieve an improved, specific, and rapid means of detection
of Aspergillus spp. in clinical specimens, we developed a
two-step PCR assay for peripheral blood and BAL samples. Two optimal
pairs of oligonucleotide primers derived from sequences of the 18S rRNA
gene which are specific for Aspergillus spp. were selected
from among a number of different candidates. The assay was evaluated
for its sensitivity and specificity in vitro and was used to analyze
clinical samples of immunocompromised patients. The results of the
assay were also compared with the results of conventional diagnostic methods.
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MATERIALS AND METHODS |
Strains and growth conditions.
Prior to extraction of DNA,
cultures of A. fumigatus were grown in Sabouraud agar for
72 h at 30°C. The cell density of the fungal suspensions
(conidia) was determined microscopically by counting the cell number in
a Neubauer cell chamber. Different titers of Aspergillus
cell suspensions were used to spike EDTA-anticoagulated blood of
healthy donors, and DNA was extracted by the method described below.
These dilution experiments were done in triplicate. Extraction of DNA
from the bacteria cultures was performed by ultrasonication of the
pelleted bacteria and subsequent phenol-chloroform extraction as
described by Sambrook et al. (38). Total DNA was used to determine the sensitivity of the PCR assay.
Strains tested for species specificity of PCR assay.
The
following fungal strains were used in the study: A. fumigatus DSM 819, A. fumigatus CS, Aspergillus
flavus DSM 1959, Aspergillus terreus DSM 1958, Aspergillus niger DSM 63263, A. niger CS,
Aspergillus versicolor DSM 1943, Aspergillus
clavatus DSM 3410, Candida albicans DSM 1386, C. albicans ATCC 44808, Candida tropicalis DSM 1346, Candida tropicalis DSM 5991, Candida krusei DSM
70079, Candida glabrata DSM 70614, Candida
parapsilosis DSM 70126, Emericella nidulans DSM 820, Penicillium chrysogenum DSM 844, Penicillium expansum DSM 1282, Penicillium funiculosum DSM 1944, Aureobasidium pullulans DSM 2404, Paecilomyces
variotii DSM 1961, Rhizopus oryzae DSM 854, Fusarium proliferatum DSM 848, Botrytis cinerea
DSM 877, Scopulariopsis brevicaulis DSM 1218, and
Neurospora crassa DSM 1257.
The following bacterial strains were used in the study:
Streptococcus sanguis DSM 20068, Streptococcus
mitis DSM 20568, Streptococcus pneumoniae DSM 20566, Staphylococcus aureus DSM 799, S. aureus ATCC
29213, S. aureus ATCC 25923, Staphylococcus
epidermidis DSM 709, Enterococcus faecalis DSM 2570, Enterococcus faecium DSM 2146, Escherichia coli
DSM 787, E. coli DSM 5923, E. coli ATCC 25921, Pseudomonas aeruginosa ATCC 27853, Klebsiella
pneumoniae DSM 681, Enterobacter cloacae DSM 6234, Serratia marcescens DSM 1636, Proteus mirabilis
DSM 788, and Haemophilus influenzae DSM 4690.
The fungal and bacterial test strains were obtained from the Deutsche
Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Braunschweig,
Germany, from the American Type Culture Collection (ATCC), Manassas,
Va., or from the Institute for Medical Microbiology and Hygiene,
Klinikum Mannheim, University of Heidelberg, Heidelberg, Germany.
Clinical samples and DNA preparation.
The primary diseases
of the immunocompromised patients group were hematological
malignancies: acute myeloid or lymphoblastic leukemias, chronic
myelogenous leukemia blast crisis, advanced myelodysplastic syndrome
(refractory anemia-excess of blasts, refractory anemia-excess of
blasts, transformation) while under antileukemic therapy, advanced or
relapsed high- or intermediate-grade malignant lymphoma, advanced or
relapsed Hodgkin's disease, or advanced chronic lymphocytic leukemia.
Human immunodeficiency virus-positive patients were not included.
Criteria for inclusion of immunocompromised patients were neutropenia
(granulocyte count < 1.0 × 109/liter), fever (body
temperature > 38.3°C), unresponsiveness to the first-line
antibacterial treatment, and/or newly arisen nonspecific pulmonary
infiltrates proven by conventional chest radiography.
Nonimmunocompromised patients had fever and lung infiltrates caused by
complicated community-acquired pneumonia and/or suspect tumor findings
by conventional chest radiography.
High-resolution computed tomography (HR-CT) scans of the lung were
performed by standardized techniques by the Department of Clinical and
Diagnostic Radiology, Klinikuum Mannheim, University of Heidelberg;
small angiotropic round infiltrates, halo signs around infiltrates, or
wedge-shaped small peripheral lung infarctions were judged as typical
early pulmonary aspergillosis findings, the "air crescent sign" was
considered a sign of a later disease stage. The HR-CT scans were
performed, and the findings were analyzed by a panel of radiologically
experienced staff.
Blood samples were obtained under sterile conditions by venipuncture,
usually simultaneously with blood samples for culture for
microbiological examination, in a sterile vessel containing potassium
EDTA to a final concentration of 1.6 mg of EDTA per ml of blood. The
sample volume was 5 to 7 ml.
A total of 3 to 5 ml of peripheral blood was mixed with 5 volumes of
erythrocyte lysis buffer (0.155 M NH4Cl, 0.01 M
NH4HCO3, 0.1 mM EDTA [pH 7.4]), and the
mixture was incubated for 10 min at 4°C. After lysis of erythrocytes,
the sample was centrifuged at 300 × g for 10 min. The
supernatant was discarded, and the leukocytes were washed once with 1×
phosphate-buffered saline (10× phosphate-buffered saline is 1.4 M
NaCl, 50 mM KCl, 90 mM Na2PO4 · 2H2O, and 20 mM KH2PO4 [pH 7.4])
and recentrifuged.
Bronchoscopy was performed according to the guidelines of the Deutsche
Gesellschaft für Pneumonologie (14), and BAL samples were obtained in a sterile vessel without conservation medium. The mean
sample volume was 10 ml; the total volume of BAL samples taken from one
patient at a time (ca. 100 ml) was aliquoted into 10-ml volumes, and
these were placed in appropriate sterile vessels.
BAL samples were transferred to 1.5-ml tubes, the tubes were
centrifuged at 13,000 rpm for 5 min (bench minifuge; Heraeus, and the
supernatant was removed. Sedimented cell material from both blood and
BAL specimens was processed as follows: the leukocyte pellet was
resuspended in 300 µl of 1× phosphate-buffered saline and the
mixture was incubated with 100 to 125 U of lyticase (50,000 U; Sigma)
for 30 min at 37°C to achieve degradation of fungal cells. Residual
human and fungal cell material was treated with 500 to 1,000 µg of
proteinase K (Boehringer Mannheim, Mannheim, Germany) and 0.5% sodium
dodecyl sulfate (Sigma) at 55°C for 1 h. Residual cell material
was then lysed by incubation with an additional 100 µl of 2×
Aspergillus extraction buffer (400 mM Tris-Cl, 1 M NaCl, 20 mM EDTA, 2% sodium dodecyl sulfate) for 30 min at 65°C. The
purification of fungal and human DNA was performed by conventional
phenol-chloroform extraction (38). The DNA was precipitated
by the addition of 0.7 volume of isopropanol, pelleted, and washed once
with 70% ethanol and air dried. The DNA concentration was assessed by
spectrophotometry at 260 and 280 nm.
Oligonucleotide primers, primer sequences, and PCR assay.
The alignment of the three DNA sequences was performed with the program
Geneworks (Intelligenetics, Inc.) by using standard algorithms. Primers
were designed to have sequences homologous to those of various
Aspergillus spp. but not to include the human 18S rRNA gene
or the 16S rRNA genes of Candida spp. or other pathogenic microorganisms. Therefore, selection of the primer sequences was based
on a close check for sequences with matching homologies in current DNA
databases (GenBank, release June 1998) with a DNA alignment program (Blast).
By using a nested, two-step PCR technique, 15 different primers (Table
1) were tested, and the optimum two pairs (primers AFU5S and AFU5AS and
primers AFU7S and AFU7AS) were chosen for all subsequent PCR assays. As
an internal control, a 138-bp PCR fragment encoded by the human
glucose-6-phosphate dehydrogenase gene (GenBank accession no. X55448)
was amplified with primers G6PD1S and 1AS (Table
1) in each clinical sample.
PCR.
Per 25-µl PCR mixture, approximately 50 to 150 ng of
total DNA was used as the template. The standard PCR mixture contained 0.5 U of Taq DNA polymerase, 6.25 nmol of the
deoxynucleoside triphosphates, 10 pmol of primer (first step, primer
AFU7S-AFU7AS; second step, primer AFU5S-AFU5AS). In preliminary studies
the optimum reaction conditions were established by testing different DNA, primer, enzyme, and deoxynucleoside triphosphate concentrations as
well as a range of cycling conditions. PCR was performed in a thermal
cycler (Perkin-Elmer Cetus), as follows: for the first PCR, 2 min at
94°C and then 23 cycles of 40 s at 94°C, 1 min at 65°C, and
1 min at 72°C with a terminal step of 5 min at 72°C and then the
mixture was held at 4°C; for the second PCR, 2 min at 94°C and then
35 cycles of 40 s at 94°C, 1 min at 65°C, and 1 min at 72°C,
with a terminal step of 5 min at 72°C, and then the mixture was held
at 4°C. For the second PCR, approximately 1 to 2 µl of the
first-round PCR product was used. The PCR products were separated by
2.5% agarose gel electrophoresis, stained with ethidium bromide, and
visualized with UV light. Control samples included all the constituents
in the reaction mixture except genomic DNA. As negative and positive
PCR controls, DNA from the human cell line T47D or dilute samples of
A. fumigatus, respectively, were used as templates.
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RESULTS |
Strategy for primer selection.
The alignment of the 18S rRNA
genes of Aspergillus spp., humans, and Candida
spp. revealed several regions of significant divergence which were the
basis for selection of the primers. In order to establish a PCR assay
specific for several Aspergillus species of clinical
importance the 18S rRNA gene (rDNA) sequence of A. fumigatus
(GenBank accession no. AB008401) was aligned with the human 18S rDNA
sequence (GenBank accession no. M10098) and the 16S rDNA sequence of
C. albicans (GenBank accession no. X53497), another
ubiquitious microorganism of major clinical importance. Fifteen
different primers, comprising 7 sequences upstream and 8 sequences
downstream from various divergent regions of the Aspergillus
species gene which showed the least homology with the human or
Candida genes, were selected for the evaluation and the
optimization of the PCR assay.
Three different strategies were used. (i) A nested PCR with
Aspergillus-specific primers in both PCR steps was used.
Most of these primer combinations were relatively insensitive,
detecting 1 pg of Aspergillus DNA. The pairs described
below, however, were more sensitive. (ii) A nested PCR with one primer
(universal primer) that had broad specificity and that matched most of
the 18S rDNA sequences analyzed in combination with a primer specific
for Aspergillus in the first step and a primer pair specific
for only Aspergillus in the second step was used. None of
these primer combinations reached a sensitivity sufficient to amplify a
detectable DNA fragment from less than 1 pg of Aspergillus
DNA, or they proved to be unspecific, amplifying human and
Candida DNA in the second step. (iii) The intergenic region
of the 18s rRNA gene of A. fumigatus was amplified with the
primer pair AFU4S-AFU4AS to generate specific intergenic regions. This
assay did not amplify any detectable PCR products, probably because the
fragment length was too long for productive amplification.
The two primer pairs that produced PCR products with the highest
sensitivity and species specificity were the
Aspergillus-specific primers AFU7S and AFU7AS, which
amplified a fragment of 405 bp, followed by AFU5S and AFU5AS, which
produced an internal fragment of 236 bp (Fig.
1 and 2;
primer sequences in Table 1). These primer-binding sites are located in
the 3' part of the 18S rRNA gene and in variable region V7-V9 (AFU7AS)
or V8-V9 (AFU5AS), with no sequence overlap between the primers used in
the first and second PCRs to reduce contamination problems (Fig. 2).
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FIG. 1.
Locations of primer pairs AFU5S-AFU5AS and AFU7S-AFU7AS
used in the two-step PCR to detect Aspergillus DNA. The
primers are derived from the 18S rRNA gene of Aspergillus
spp. The first PCR step (with AFU7S-AFU7AS) results in amplification of
a 405-bp fragment, and the second step (with AFU5S-AFU5AS) amplifies an
internal fragment of 236 bp.
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FIG. 2.
Alignment of DNAs of 18S rRNA genes of A. fumigatus (GenBank accession no. AB008401) and humans (GenBank
accession no. M10098) and the corresponding 16S rRNA gene of C. albicans (GenBank accession no. X53497). The locations of primer
pairs AFU5S-AFU5AS and AFU7S-AFU7AS used in the two-step PCR to detect
Aspergillus DNA are indicated by arrows. Homologous regions
are boxed.
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Sensitivity of two-step PCR.
The sensitivity of the PCR assay
with the optimal two pairs of nested oligonucleotides AFU7S-AFU7AS and
AFU5S-AFU5AS was determined by dilution of A. fumigatus
template in human leukocyte DNA. Signals derived from as little as 10 fg of A. fumigatus DNA could still be clearly detected by
ethidium bromide staining of agarose gels (Fig.
3), whereas PCR with human or C. albicans DNA alone did not produce any detectable amplified DNA.
The detection limit of this assay corresponds to 1 to 5 CFU/ml of
blood, as additionally confirmed by spiking blood samples from healthy
donors with serial dilutions of fungal conidia and performing similar
two-step PCR amplification assays (Fig.
4).
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FIG. 3.
Determination of the sensitivity of the two-step PCR
assay with purified A. fumigatus DNA diluted in human DNA
(50 ng). The signal derived from 10 fg of Aspergillus
template DNA was clearly detectable by ethidium bromide staining of an
agarose gel. As a positive control, only DNA extracted from A. fumigatus (10 pg) was used in a single PCR amplification of the
236-bp fragment with the second primer pair (lane +). A negative
reagent control amplification without addition of DNA (lane  ) as well
as purified DNA from a human cell line (T47D) resulted in no bands. The
123-bp ladder (Gibco BRL) was used as molecular size marker (lane M).
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FIG. 4.
Peripheral blood samples from healthy donors were spiked
with defined numbers of conidia from A. fumigatus. The
signal derived from 101 to 100 CFU per ml of
blood were still detectable by ethidium bromide staining of an agarose
gel. As a positive control, DNA extracted from A. fumigatus
(10 pg) was used for single-step PCR amplification of the 236-bp
fragment (lane +). Negative reagent control amplification without
conidia (0) resulted in no bands. The 123-bp ladder (Gibco BRL) was
used as a molecular size marker (M).
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Genus and species specificity of PCR assay.
The products
obtained after the second PCR from an A. fumigatus test
strain were cloned and sequenced, confirming that the DNA sequences
exactly matched the corresponding sequence of the A. fumigatus 18S rRNA gene from a database (GenBank). To further test
the PCR specificity, template DNA extracted from a wide range of fungal
and bacterial pathogens, listed in Materials and Methods, was
investigated with the two primer pairs. PCR products could be obtained
only from DNAs of cultures of Aspergillus spp. (A. fumigatus, A. flavus, A. niger, A. terreus, A. clavatus, A. versicolor, and the
Aspergillus-like species E. nidulans), whereas
the PCRs with the other fungal and bacterial test strains and DNA from a human cell line were negative. In the cases in which the PCR products
were obtained from clinical samples (see below), three blood samples
were chosen for cloning and sequencing of the amplified fragment. These
samples had results identical to those for the control PCR product
clones from the A. fumigatus test strain: the DNA sequences
matched the sequence of the A. fumigatus 18S rRNA gene from
the database for all samples (data not shown). These results indicate
the high species specificity and sequence stringency of this two-step
PCR assay.
Detection of Aspergillus DNA in clinical samples.
A total of 315 samples (250 blood samples and 65 BAL samples) from 140 subjects including 47 healthy individuals or nonimmunocompromised patients and 93 febrile neutropenic patients with malignant hematologic diseases (predominantly patients with acute leukemias) at high risk for
invasive fungal disease were screened by the two-step PCR assay.
Several specimens from all patients were tested (average, 3.0 specimens). Typical PCR results for clinical samples (peripheral blood)
showed either no amplification product or a single defined band of 236 bp, corresponding exactly to the results for the Aspergillus controls.
A positive DNA sample containing 100 fg of Aspergillus DNA
mixed with 50 ng of human DNA was used in PCRs with samples from each
set of patients to check for proper amplification. If no signal was
obtained for this positive control, the PCR was repeated.
Consecutive blood samples from 22 neutropenic leukemia patients had
positive PCR signals, and aspergillosis was proven in 7 of these
patients by the results of histology and/or culture. Histologic tissue
samples were obtained from two patients by lung biopsy and from two
subjects at autopsy. The three positive culture results that were
obtained were exclusively for BAL specimens, thus highlighting the
difficulties associated with obtaining positive culture results,
particularly for blood samples.
No convincing clinical evidence of an Aspergillus infection
could be found for only one of the leukemic patients (patient 13; Table
2), and only a single positive PCR result
was obtained for one blood sample from that patient. By monitoring the
clinical course of this febrile neutropenic patient, in whom a small
lung infiltration was detected by chest X ray, we could neither prove nor exclude invasive aspergillosis. Antimycotic and antibacterial therapy and a prompt rise in granulocyte counts resulted in
defervescence within a few days; no relapse of the pneumonia occurred
during the next subsequent neutropenic phase. Subsequent positive PCR results were not obtained until 8 months later, following bone marrow
transplantation, and invasive aspergillosis was also proven at this
time by a positive result for a tracheal lavage specimen culture.
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TABLE 2.
Positive PCR results for febrile neutropenic patients
with lung infiltrates and proven (by histology and/or by culture)
or probable (by HR-CT findings)
invasive aspergillosisa
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The remaining 14 (of the 22) patients had characteristic HR-CT findings
that suggested invasive aspergillosis. (For almost all patients, blood
and BAL specimens were collected before HR-CT scans were performed.)
Therefore, the correlation between the positive histopathology results,
positive culture results, or positive HR-CT findings and the PCR
results was 100%. None of our patients had positive HR-CT findings and
a negative PCR result. Among the samples from nonimmunocompromised
patients and healthy volunteers, the PCR assay gave one single positive
result with a blood sample (2.1%) and four positive results with BAL
samples (8.5%) from different individuals.
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DISCUSSION |
PCR has been shown to be a highly sensitive diagnostic tool for
the detection of infectious fungi in diverse specimens, but current PCR
assays applied in clinical diagnostics have considerable drawbacks due
to possible contamination with ubiquitious fungal conidia,
coamplification of human DNA, or the necessity for additional time-consuming hybridization steps. Our aim was to establish a new,
highly sensitive, and specific PCR assay for the rapid detection of the
full range of human pathogenic Aspergillus species in blood and BAL samples. This would result in a test with increased sensitivity and increased predictive value.
Pioneering work in this direction (41) was initiated after
multicopy gene targets, such as the 28S rRNA gene, had been identified and sequenced. The first PCR assay for the detection of
Aspergillus DNA in immunocompromised patients to be
described (30) used primers derived from the multicopy 18S
rRNA gene, a gene of numerous microorganisms that has now been
sequenced. More recent reports described PCR techniques with primers
designed to target conserved 18S rRNA sequences common to a variety of
fungal pathogens (17, 45). However, additional Southern blot
analyses with longer probes were also necessary to achieve the
detection sensitivity (17) for the identification of the
specific fungal species or genera present (45).
Nested PCR assays improved the detection sensitivity. In the first
description of such a two-step PCR for the detection of Aspergillus spp. in serum samples of patients, Yamakami et
al. (52) reported that a PCR with two sets of 18S rRNA
primers had considerably improved sensitivity compared to that of a PCR
assay with a single set of primers. This could be further improved by subsequent Southern blot hybridization. These data also highlighted for
the first time the use of PCR for the detection of
Aspergillus DNA in blood samples, which can be obtained by a
less noninvasive procedure that is associated with fewer risks than the
procedure used to collect BAL specimens, which cannot be performed
repeatedly with immunocompromised patients.
We also used a two-step PCR procedure with carefully selected primers
to increase the sensitivity and specificity for Aspergillus detection, thereby eliminating a subsequent hybridization step otherwise obligatory for sensitive detection or verification of the
amplified PCR products. Use of a two-step PCR increases the probability
of contamination. Therefore, negative controls (human DNA and water)
were used in each set of PCRs. If these controls gave a positive
result, the set of PCRs was repeated. The multicopy 18S rRNA gene is
highly conserved in all species but includes variable regions that are
conserved among most Aspergillus species. On the basis of
comparisons with sequences in a data bank, we chose oligonucleotides
from the variable regions that specifically matched only the 18S rRNA
gene from various Aspergillus species to avoid problems
arising from coamplification of human or bacterial DNA or contamination
with other fungus-derived DNA. A total of 15 different primers were
screened for their sensitivities and specificities, and the optimal two
pairs of nested primers were chosen for use in all subsequent assays.
The negative test results for a wide range of fungal pathogens,
including several Penicillium spp., and bacterial pathogens
or a human cell line indicated the high species and genus specificity
of this PCR assay. PCR products were obtained only from DNA from
Aspergillus cultures (A. fumigatus, A. flavus, A. niger, A. terreus, A. clavatus, A. versicolor) and the
Aspergillus-like species E. nidulans. In order to
exclude possible contamination with Aspergillus conidia, a
problem associated with BAL specimens (8), and also because
of the clinically relevant ease of with which repeated blood samples
can be obtained, peripheral blood from volunteer donors and patients
was preferentially analyzed.
The detection threshold of the PCR assay described here was about 10 fg
of template DNA, or between 1 and 5 CFU per ml of blood (Fig. 3 and 4).
This demonstrates a sensitivity that may allow this PCR assay to be
useful for the detection of small amounts of pathogen and demonstrates
that this PCR assay may be a valid diagnostic tool. In addition,
elimination of a subsequent hybridization step greatly simplifies and
speeds up the diagnostic assay. Contamination with ubiquituous fungal
spores may be a disadvantage of this PCR assay. The conditions and the
origins of contamination are various and cannot be methodically
excluded completely. The contamination rate that occurs during
processing of DNA and PCRs was low under our optimized conditions. To
assess the clinical implications and applicability of the assay, we
screened clinical samples from 47 healthy individuals or
nonimmunocompromised patients and 93 febrile neutropenic patients with
malignant hematologic diseases, predominantly acute leukemias, at high
risk for invasive fungal disease. No convincing evidence of an
Aspergillus infection could be found in one leukemic patient
who provided one blood sample that had a positive PCR result.
Conversely, all 21 patients with clinically proven invasive
aspergillosis (positive histopathology results, positive culture
results, or positive HR-CT scans) had positive PCR results,
demonstrating a test sensitivity of 100%.
Interestingly, only three positive culture results were obtained, and
all of these were with BAL specimens, highlighting the difficulties
associated with obtaining positive culture results, particularly with
blood samples. In contrast, PCR results for all blood samples were
consistent with those for BAL samples or positive clinical test
results, demonstrating the sensitivity and potential clinical value of
this PCR assay. For the group of nonimmunocompromised patients, the
results indicate a test specificity of 89% due to a low contamination
rate of one blood sample and four BAL samples. Preliminary data
(unpublished data) from follow-up PCR assays correlated with the
clinical course of the infected patients, indicating that this assay
may provide a better means of monitoring antimycotic therapy. It has
already been reported that the persistence of PCR signals reveals a
trend toward a worse, possibly fatal outcome (17).
Due to the low incidence of proven invasive aspergillosis in
immunocompromised patients, the prospective clinical validation of this
assay requires large numbers of patients at high risk for fungal
infection. The clinical validation of the assay in a prospective
multicenter study to define the predictive value of the assay and its
experimental validation with a histologically defined animal model of
aspergillosis are in progress.
In summary, a specific two-step PCR assay for the rapid detection of
the full range of human pathogenic Aspergillus species in
both BAL and blood samples was established, facilitating improved early
diagnosis and better monitoring of systemic aspergillosis during
therapy in high-risk patients.
A patent of the assay is pending (23a).
| |
ACKNOWLEDGMENTS |
We thank H. Hof and M. Kretschmar, Institute for Medical
Microbiology and Hygiene, Klinikum Mannheim, University of Heidelberg, Germany, for helpful support, especially with bacterial and fungal test
strains, and H. Niachos for technical assistance. We thank A. Arthur-Goettig for critically reading the manuscript.
This work was supported in part by a grant from the Forschungsfonds der
Fakultät für Klinische Medizin Mannheim der
Universität Heidelberg (project nos. 62 and 2062).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: III.
Medizinische Klinik, Klinikum Mannheim der Universität
Heidelberg, Wiesbadenerstraße 7-11, D-68305 Mannheim, Germany. Phone:
49-621-383-4115. Fax: 49-621-383-4201. E-mail:
dieter.buchheidt{at}urz.uni-heidelberg.de.
Present address: QIAGEN GmbH, Hilden, Germany.
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Journal of Clinical Microbiology, December 1999, p. 3865-3871, Vol. 37, No. 12
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Abstract
Introduction
