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Previous Article | Next Article 
Journal of Clinical Microbiology, July 1998, p. 2063-2067, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Q Fever in the Greek Island of Crete: Detection,
Isolation, and Molecular Identification of Eight Strains of
Coxiella burnetii from Clinical Samples
Ioanna
Spyridaki,
Achilleas
Gikas,*
Diamantis
Kofteridis,
Anna
Psaroulaki, and
Yannis
Tselentis
Clinical Bacteriology, Parasitology, and
Geographical Medicine, University Hospital of Heraklion,
Voutes-Heraklion TK 71110 Crete, Greece
Received 13 October 1997/Returned for modification 10 November
1997/Accepted 26 March 1998
 |
ABSTRACT |
Over a period of 6 years (1989 to 1995), serum samples from 3,300 patients suspected to be infected by Coxiella burnetii were assayed for the presence of antibodies against antigen phase II of the
microorganism by the indirect immunofluorescence antibody technique
(IFAT). One hundred fifty-two cases were recorded, and blood samples
from 17 patients were cultured for the isolation of the pathogen. By a
centrifugation shell vial technique, eight strains were isolated from
patients suffering from acute Q fever. The microorganism was detected
in the cultures by IFAT, by Gimenez staining, and by the cytopathogenic
effect on Vero and human embryonic lung (HEL) cells. PCR followed by
restriction fragment length polymorphism analysis was used to confirm
the diagnosis and identify the Coxiella burnetii strains
within the cell cultures as well as to compare them with reference
strains. In order to avoid time-consuming cultures, to achieve direct
detection of Coxiella burnetii in clinical samples (blood,
buffy coat, etc.), and to increase the specificity and sensitivity of
the detection, nested PCR was performed. The first step of DNA
extraction was performed with the QIAamp blood kit 250. For the second
step of the PCR assays, the conditions of temperature and times of
recycling were properly modified, and the microorganism was detected
within 4 h. Our study demonstrates that Q fever is an endemic
disease in Crete and that the diagnosis of Coxiella
burnetii infection can be rapidly achieved by the detection of
the microorganism in buffy coat samples by nested PCR. Although the
presenting symptoms of the disease in this study differed from those in
other studies, the Cretan strains do not differ genotypically from the
reference strains (Nine Mile and Q212).
| |
INTRODUCTION |
Coxiella burnetii, an
obligate intracellular parasite with a worldwide distribution, is
the causative agent of Q fever in humans and animals (1,
25).
The bacterium exists as a strict intracellular parasite when infecting
its host but can also survive in the environment (1). Early
assessments of the epidemiology of Q fever suggested that the diseases
can be transmitted through contact with infected animals, blood
transfusions, inhalation of infectious aerosols, the digestive tract,
skin trauma, and sexual contact and, rarely, by a mother to the fetus
(21, 24).
In humans, the infection has two forms, acute and chronic. The acute
form is manifested by pneumonia, prolonged fever, granulomatous hepatitis, and, rarely, meningoencephalitis (23). The main
clinical manifestation of chronic Q fever is endocarditis (1, 23, 24, 34).
In Greece in 1946, Caminopetros detected the microorganism in sera of
German soldiers (2). Since 1950, only sporadic cases have
been reported, and the microorganism was never cultured.
In a previous study on the Greek island of Crete, Tselentis et al.
reported that the predominant clinical manifestations of the infection
were fever and respiratory disease, whereas hepatitis occurred in only
a minority of the infected patients (34). Reports from
Australia (4), Great Britain (3, 8), the United States (10), Spain (17), France (5),
and Canada (35) indicate that epidemiological and
clinical features of the disease may vary from one area of the world to
another. For example, in two Australian studies the prevailing clinical
presentation was fever of unknown origin (16); in two
studies from Nova Scotia (15) and Switzerland
(6), the prevailing clinical presentation was pneumonia,
while in a French study, hepatitis was the prevailing feature
(5).
Since the reason for this clinical diversity between acute cases of Q
fever is not known, the causative roles of strain differences cannot be
excluded. However, no firm conclusions can be drawn because of the
small number of C. burnetii strains isolated from patients suffering from acute Q fever (18-20, 36). Thus,
isolation of C. burnetii strains from different
geographic areas is needed.
Laboratory diagnosis of Q fever is mainly based on serological tests
(29). The isolation of C. burnetii in
cultures is time-consuming and hazardous and may give false-negative
results. To overcome these problems, PCR and nested PCR techniques were
developed (12, 29, 36). A number of C. burnetii strains originating from patients suffering from either
chronic or acute Q fever have been isolated by a shell vial culture
method. The method was successfully applied on valves, arterial
prostheses, bone, skin biopsies, bone marrow, and blood (11, 18,
20, 29, 30).
The purpose of this study was (i) the isolation and molecular
identification of clinical strains of C. burnetii in
Greece, (ii) the comparison of our isolates with the reference strains by PCR-restriction fragment length polymorphism (RFLP), and the improvement of the methodology of rapid detection of C. burnetii in patient samples.
In this study, we report the isolation of eight strains of
C. burnetii from Greek patients, the identification of
these strains by PCR-RFLP with material from cell cultures, and the
direct detection of the pathogen by nested PCR in buffy coat samples
within 4 h.
| |
MATERIALS AND METHODS |
Our laboratory is the National Reference Centre of Parasitology,
Zoonoses, and Geographical Medicine and a collaborating center of the
World Health Organization. Over a period of 6 years (1989 to 1995),
serum samples from 3,300 patients suspected to be infected by
C. burnetii were assayed for the presence of antibodies
against antigen phase II of the microorganism. Using the indirect
immunofluorescence antibody technique (IFAT), we considered titers of
immunoglobulin G (IgG) of 
1/960 or titers of IgM of 1/400 and/or a
fourfold increase of the titers between two assays as a strong
indication of acute infection. A fever of 38°C, respiratory disease
(dyspnea, expectoration, cough, and chest pain with associated X-ray
abnormalities), hepatitis (a higher-than-twofold increase in serum
glutamic oxalacetic transaminase and/or serum glutamic pyruvic
transaminase levels), central nervous system involvement (neurological
symptoms associated with normal or abnormal cerebrospinal fluid
findings), and skin rash were considered cardinal manifestations of Q
fever. The diagnosis was made according to clinical and serological
criteria of the disease. One hundred fifty-two cases of Q fever were
recorded.
Physicians were asked to provide buffy coat samples from patients who
had not received C. burnetii-specific antibiotics at admission.
Samples.
Samples from 17 patients were assayed for the
detection and identification of the microorganism. For blood cultures,
a 5-ml sample of heparinized blood was obtained, and after
sedimentation for 40 min, the supernatant monolayer was inoculated into
the shell vials.
Isolation of C. burnetii.
Human embryonic lung
(HEL) fibroblasts were grown in minimum essential medium with 10%
fetal calf serum and then 1% glutamine. Shell vials (3 and 7 ml;
Sterilin, Felthan, England) with 12-mm-diameter coverslips were seeded
with 1 ml of medium containing 50,000 cells and incubated in a 5%
CO2 incubator for 3 days to obtain a confluent monolayer. A
portion of the buffy coat fraction of each sample (0.5 ml) was diluted
with 1 volume of growth medium.
One milliliter of the mixture was placed in each shell vial. The shell
vials were centrifuged at 700 × g for 1 h at
22°C. The inoculum was then removed, and 1 ml of growth medium
was added to the cells. The shell vials were incubated in a 5%
CO2 incubator at 37°C. At least three shell vials
were inoculated per sample. The cytopathic effect of C. burnetii in HEL and Vero cells was also observed (20).
Immunofluorescence detection of C. burnetii.
The
cell monolayers in the shell vials were examined for C. burnetii by IFAT on day 6 and again on day 12 if the first test was negative. For detection of C. burnetii, human serum
samples collected by our laboratory (which display a high titer of
immunofluorescent antibody to C. burnetii of
>1/40,000), at a dilution of 1/100, and fluorescein-conjugated goat
antiserum to human IgG (dilution, 1/200) (Kallestad, Austin, Tex.) were
used. Specificity was evaluated by simultaneous staining of
uninoculated cell monolayers and inoculated cultures with human serum
negative for antibodies to C. burnetii.
Detection by PCR and nested PCR.
The DNA extraction from
buffy coat was performed with the QIAamp blood kit 250 (Qiagen, Hilden,
Germany) according to the instructions of the manufacturer. Infected
cells were used for PCR detection and RFLP identification of
C. burnetii. Two hundred microliters of the cell
suspension was incubated in the presence of 400 ng of proteinase K per
ml (stock solution, 20 mg/ml in H2O) overnight at
56°C. Subsequently, proteinase K was inactivated by boiling for
10 min, and the solution was centrifuged at 2,000 rpm for 5 min at
22°C in a Beckman GS-6R centrifuge. The supernatant was kept at
20°C.
DNA amplification.
In order to perform DNA amplification of
the Greek strains and compare them with the reference strains, Nine
Mile and Q212, three different genomic primers were used. Additionally,
in order to ascertain whether the Greek isolates contained plasmids,
two additional types of primers which have been described to indicate acute or chronic Q fever were used (Table
1) (36). Primers QpH11 and
QpH12 detect plasmids present during acute Q fever, whereas primers
QpRS01 and QpRS02 detect plasmids present in chronic infection due to
C. burnetii (36).
PCR was performed with 10 µl of supernatant from the
proteinase-K-treated cell suspension in a total volume of 100 µl. The PCR mixture contained 1 µM of each primer, 200 µM of each
deoxynucleoside triphosphate, 2.0 mM MgCl2, and 0.5 U of
Taq polymerase (GIBCO BRL Life Technologies, Gaithersburg,
Md.). The primers used, as well as the cycling conditions, are listed
in Table 1.
For the direct PCR detection of C. burnetii in the
buffy coat, a nested PCR assay was performed with primers Hfrag1 and
Hfrag2 in the first PCR and primers HF1 and HF2 in the nested PCR. The conditions used are described in Table 1. The PCR products were separated on a 2% agarose gel and visualized by UV illumination.
Restriction endonuclease digestion.
The specificity of the
amplification was evaluated by restriction analysis of the PCR
products. The CB1 and CB2 and Hfrag1 and Hfrag2 products from infected
cells were digested with the enzymes TaqI and
AluI (New England Biolabs) as previously described (29,
36). The restriction fragments were examined by electrophoresis on a 3% low-melting-point agarose gel (GIBCO BRL), stained with ethidium bromide, and viewed by UV illumination. The restriction fragments were compared with those obtained with the reference strains,
Nine Mile and Q212.
| |
RESULTS |
Isolation of C. burnetii.
Three cultures were
obtained from each patient prior to any administration of antibiotics.
We were able to isolate C. burnetii in blood cultures
from eight of 17 patients suffering from Q fever. All patients except
one presented with fever and pneumonia. The last patient had had fever
for 10 days upon admission. Clinical, serological, therapeutic, and
culture data from these eight patients are presented in Table
2.
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TABLE 2.
Clinical, serological, therapeutic, and culture data for
eight Q fever patients with blood cultures positive
for C. burnetii
|
|
The microorganism was detected between days 6 and 12 by IFAT, Gimenez
staining, and PCR (7). The subcultures derived from the
initial shell vial were considered heavily infected after 15 to 21 days
of incubation. At this point, the voluminous vacuolar formations in the
cell cytoplasm, due to the cytopathic effect of C. burnetii, were prominent (20).
PCR identification.
C. burnetii-specific sequences
were amplified by PCR from DNA derived from infected Vero or HEL cells.
For further characterization of the isolates, PCR was carried out with
infected cells with the genomic primers CB1 and CB2 and the plasmidic
primers QpH11 and QpH12, which were initially designed as primers
specific for acute infection (29, 36). Our results showed
clearly that the specific PCR product appeared in all our samples and
the Nine Mile strain but not in the Q212 strain (36). On the
other hand, the primers QpRS01 and QpRS02, which were referred to as
amplifying only chronic infection from specific sequences, did not give
any amplification with our samples and the Nine Mile strain but
produced a PCR-specific product with the Q212 strain (36).
The PCR methods were not sensitive enough to detect C. burnetii directly in the clinical samples (buffy coat,
blood, etc.).
The assay specificity has been previously evaluated by restricted
digestion of the PCR products (29). PCR products obtained (with primers CB1 and CB2) from our samples and the reference strains
were subsequently digested with AluI, resulting in the generation of fragments of 186, 68, and 3 bp (Fig.
1), whereas cleavage with TaqI
gave fragments of 118, 57, 43, and 39 bp (Fig. 2). The product of amplification with
primers Hfrag1 and Hfrag2 in infected cells was digested with
TaqI, and the sizes of the generated fragments were
identical to those of the fragments derived from the reference strains,
Nine Mile and Q212 (36).
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FIG. 1.
Restriction endonuclease profile analysis of the 257-bp
amplification products of eight C. burnetii isolates
digested with AluI. Lanes: A, strain Nine Mile; B, reagent
control; C to F, four C. burnetii isolates; G,
molecular size markers ( X174 cleaved with HinfI); H to K,
four C. burnetii isolates.
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FIG. 2.
Restriction endonuclease profile analysis of the 257-bp
amplification products of eight C. burnetii isolates
digested with TaqI. Lanes: A, strain Nine Mile; B, reagent
control; C, strain Q212; D to G, four C. burnetii
isolates; H, molecular size markers (X174 cleaved with
HinfI); I to L, four C. burnetii isolates.
|
|
Nested PCR detection.
Detection of C. burnetii
by nested PCR (Fig. 3 and
4) succeeded only with buffy coat samples
by the DNA extraction method with the QIAamp blood kit. When whole
blood was used, we were unable to detect a positive signal, even in the
samples from which C. burnetii had been previously
isolated. The nested PCR test for the detection of C. burnetii was modified from the one previously described
(36) in three ways. (i) Extraction of DNA was done by us
with the QIAamp blood kit instead of by the traditional boiling method
(36), (ii) the temperatures were changed, and (iii) the
times of recycling were modified (Table 1). Thus, the overall duration
of nested PCR was 4 h.
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FIG. 3.
Direct detection of C. burnetii in buffy
coat by nested PCR. Amplification products (183 bp in size) from four
buffy coat samples. An agarose gel electrophoretogram of amplified DNA
after 35 cycles of amplification. Lanes: A, reagent control; B to E,
four buffy coat samples; F, strain Nine Mile; G, molecular size markers
(X174 cleaved with HaeIII).
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FIG. 4.
Direct detection of C. burnetii in buffy
coat by nested PCR. Amplification products (183 bp in size) from four
buffy coat samples. An agarose gel electrophoretogram of amplified DNA
after 35 cycles of amplification. Lanes: A, strain Nine Mile; B,
reagent control; C, buffy coat negative control; D, strain Q212; E,
buffy coat sample; F, reagent control; G, molecular size markers
(X174/HinfI); H and I, buffy coat samples; J, buffy coat
negative control; K, buffy coat sample.
|
|
C. burnetii was also isolated from the same samples by
culture. The product obtained by the nested PCR had a size of 183 bp (Fig. 3).
| |
DISCUSSION |
C. burnetii is an obligate intracellular parasite
that was isolated initially in animals (guinea pigs) and
embryonated chicken eggs and later in cell cultures
(Vero and L929). These modes of isolation are time-consuming,
hazardous, and restricted to specialized laboratories. A less-hazardous
technique for the isolation of C. burnetii was proposed
by Raoult et al., who used a simplified shell vial culture system
(20). Although this rapid culture radically accelerates the
identification of C. burnetii, the process is still too
lengthy to be employed in everyday clinical practice. The shell vial
technique proved to be very efficient and able to yield large
quantities of C. burnetii for further studies of strain
identification and antibiotic susceptibility (22, 26). Although the genome of C. burnetii is still thought to
be highly conserved, previous studies have shown that C. burnetii isolates can be differentiated by RFLP (9, 32)
and/or plasmid DNA content (27, 36). The first C. burnetii plasmid, QpH1, was isolated and described by Samuel et
al. (28). This low-copy-number plasmid (13) was
obtained from tick isolate Nine Mile, the prototype strain of acute Q
fever. Another plasmid, QpRS, also described by Samuel et al.
(27), was obtained from a goat placenta C. burnetii isolate and found to be common to most of the chronic Q
fever isolates (14).
PCR-RFLP is useful for detection and identification of C. burnetii in early shell vial cultures, for diagnosis of both acute and chronic infections, and for detection of the bacteria in certain clinical specimens (heart valves) (29).
The classification of C. burnetii strains into acute
and chronic isolates by PCR is still preliminary. It is not yet known whether the virulence potential of C. burnetii is
encoded by plasmids or genomic sequences or dependent on host factors
as well (31, 33). We used both primers (genomic and
plasmidic) in order to confirm the above differentiation.
All our patients presented the clinical manifestations of acute Q fever
infection. Follow-up analysis of IgM and IgG showed a decline in
antibody titers. No other clinical manifestations (e.g., cardiac or
chronic hepatic involvement, etc.) were present during a 2- to 3-year
follow-up period.
Our strains were detected with the primers QpH11 and QpH12, derived
from plasmids of strains associated with acute Q fever, but not with
primers derived from strains associated with chronic Q fever.
Recently, a nested PCR approach was used for the highly sensitive and
specific direct detection of C. burnetii in clinical samples collected from animals and humans, with primers based on
conserved plasmid sequences (33, 36).
However, this technique also proved inconvenient, since the procedure
times are not yet appropriate for the everyday clinical practice and
inhibitors of the reaction can falsify the results. Our method, which
involved changing the DNA extraction procedures and optimizing the
temperature and time conditions, improved the time required for the PCR
procedure, making results available within 4 h.
This study presents the first successful attempt to isolate
C. burnetii in Greece from patients suffering from the
acute form of the infection.
In conclusion, we have successfully isolated C. burnetii in Greece from eight Greek patients by the shell vial
assay. Isolates were detected and identified by molecular biology
techniques. Optimization of nested PCR conditions allowed direct
detection of C. burnetii within 4 h. The strains
isolated did not differ from the standard reference strains.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Clinical
Bacteriology, Parasitology, and Geographical Medicine, University
Hospital of Heraklion, Voutes-Heraklion, TK 71110 Crete, Greece. Phone: 30-81-392 360. Fax: 30-81-392 847. E-mail:
achille{at}edu.uch.gr.
| |
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Journal of Clinical Microbiology, July 1998, p. 2063-2067, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
