Molecular detection of Coxiella burnetii from the formalin-fixed tissues of Q fever patients with acute hepatitis
Molecular detection of Coxiella burnetii from the formalin-fixed tissues of Q fever patients with acute hepatitis
Young-Rock Jang 0 1
Yong Shin 0
Choong Eun Jin 0
Bonhan Koo 0
Se Yoon Park 0 1
Min-Chul Kim 0 1
Taeeun Kim 0 1
Yong Pil Chong 0 1
Sang-Oh Lee 0 1
Sang-Ho Choi 0 1
Yang Soo Kim 0 1
Jun Hee Woo 0 1
Sung-Han Kim 0 1
Eunsil Yu 0
0 Editor: Xue-Jie Yu, University of Texas Medical Branch , UNITED STATES
1 Departments of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea, 2 Division of Infectious Disease, Department of Internal Medicine, Gil Medical Center, Gachon University College of Medicine, Incheon, Republic of Korea, 3 Department of Convergence Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea, 4 Division of Infectious Diseases, Department of Internal Medicine, Soonchunhyang University Seoul Hospital, Soonchunhyang University College of Medicine, Seoul, Republic of Korea, 5 Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine , Seoul , Republic of Korea
Data Availability Statement: All relevant data are
within the paper.
Funding: This study was supported by a grant
from the Korea Health Technology R&D Project
through the Korea Health Industry Development
Institute (KHIDI), funded by the Ministry of Health
& Welfare, Republic of Korea (grant no.
Competing interests: The authors have declared
that no competing interests exist.
A total of 11 patients with acute Q fever hepatitis were selected and analyzed. Of the 11
patients, 3 (27%) had exposure to zoonotic risk factors and 7 (63%) met the serologic
criteria. Granulomas with either circumferential or radiating fibrin deposition were observed in
10 cases on liver biopsy and in 1 case on bone marrow biopsy. 8 (73%) revealed positive
Coxiella burnetii PCR from their formalin-fixed liver tissues. In contrast, none of 10 patients
with alternative diagnosis who had hepatic granuloma revealed positive C. burnetii PCR
from their formalin-fixed liver tissues.
Q fever PCR from formalin-fixed liver tissues appears to be a useful adjunct for diagnosing
Q fever hepatitis.
Q fever occurs in humans when small-particle aerosols containing Coxiella burnetii are
inhaled. The most commonly identified sources of Q fever are farm animals such as cattle,
goats, and sheep. Recently, household cats and dogs have also been reported as potential
sources of urban outbreaks [
]. Clinical manifestations of Q fever are varied and non-specific:
in acute cases, Q fever often presents as pneumonia or hepatitis, and in chronic cases,
endocarditis is most often observed.
Serological test remains the method of choice for diagnosing C. burnetii infection as it is
easy to establish and widely applicable. However, antibodies are detected only after 2±3 weeks
from the onset of disease making a diagnosis of Q fever too slow in most clinical settings [
Another method of direct detection of C. burnetii is cell culture, but this method requires
biosafety level 3 (BSL-3) laboratories and has a varying degree of sensitivity [
polymerase chain reaction (PCR) techniques have been developed for Q fever testing and
successfully employed to detect DNA in both cell cultures and clinical samples [
there are limited data on Q fever PCR from formalin-fixed tissues, especially liver biopsy
tissues. We thus investigated the diagnostic utility of Q fever PCR from formalin-fixed liver
tissues in Q fever patients with acute hepatitis.
Materials and methods
We retrospectively reviewed the clinical and laboratory data of diagnosed acute Q fever
hepatitis patients who underwent liver biopsy in our institution from May 2000 to December
2016, and whose biopsied tissues were available. Acute Q fever hepatitis was diagnosed if 2
of the following clinical, serologic, or histopathologic criteria were met: (1) An infectious
hepatitis-like clinical feature such as fever ( 38ÊC) with elevated hepatic transaminase
levels, (2) Exhibition of a phase II immunoglobulin G (IgG) antibodies titer by IFA of 1:128
in single determination or a four-fold or greater rise between two separate samples obtained
two or more weeks apart, (3) histologic finding of biopsy tissue showing characteristic fibrin
ring granuloma. As controls, we tested biopsy liver tissues from the patients with confirmed
alternative diagnoses who underwent liver biopsy between 2013 and 2016 and in whom
their histologic findings of biopsy tissues showed granuloma. Controls were utilized to
determine the specificity of PCR assay and to detect any cross-reaction with samples from
patients with unrelated disease. Verbal and written consent were obtained from the study
participants. This study was approved by the Institutional Review Boards of Asan Medical
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(1) DNA extraction. To detect C. burnetii, DNA was extracted from the formalin-fixed
liver tissues of Q fever patients with acute hepatitis. For each paraffin block, five slices of
sections (5μm thick) were cut and placed in microtube. First, xylene was added to each tube and
centrifuged (12,000rpm, 5min), and the supernatant was discarded. This procedure was
carried out for three times. The specimens were then rehydrated through graded ethanol and
centrifuged in each washing step. Finally, the tubes were kept open for the remaining ethanol
evaporation. DNA extraction was performed using QIAamp DNA Mini Kit (Qiagen, Hilden,
Germany) according to the manufacturer's instruction with minor modifications. For tissue
digestion, AL buffer and proteinase K were added, and samples remained in a water bath for
18h. Washing steps with these buffers were done twice, and samples were eluted in 100 μl of
TAE buffer and stored at −20ÊC until use.
(2) PCR assay. End-point PCR was performed to detect the C. burnetii from the tissues.
The gene target was determined to be that derived from the transposase gene insertion element
IS1111a of C. burnetii isolate LBCE 13265 (NCBI Nr. KT 965031.1). The forward (5’-GAGCG
AACCATTGGTATCG-3’) and reverse (5’-TTTAACAGCGCTTGAACGT-3’) primers were
synthesized at the usual length of around 24 bp. The end-point PCR process consisted of an
initial denaturation step at 95ÊC for 15 min; 45 cycles of 95ÊC for 30s, 57ÊC for 30s, and 72ÊC
for 30s; and a final elongation step at 72ÊC for 7 min. 5 μL of DNA were amplified in a total
volume of 25 μL containing 10X PCR buffer (Qiagen), 2.5 mM MgCl2, 0.25 mM
deoxynucleotide triphosphate, 25 pmol of each primer, and 1 unit of Taq DNA polymerase (Qiagen). Gel
electrophoresis was used to separate PCR products on a 2% agarose gel containing ethidium
bromide (EtBr), and visualized using a GelDoc System (Clinx Science Instruments).
(3) Sequencing of PCR product and sequence analysis. For direct sequencing of DNA,
all DNA samples were amplified with primers for C. burnetii, and then purified by using Expin
PCR SV (GeneAll, Korea). The purified samples were directly sequenced using BigDyeTerminal
chemistry with the forward primer of Q-fever_IS111. We used Macrogen sequencing service
(Macrogen Inc. Korea), through which the DNA sequencing reactions were electrophoresed on
ABI's3730XL DNA Analyzers (Applied Biosystems, USA), which produces read lengths of 800±
A total of 11 patients with acute Q fever hepatitis were selected and analyzed. Baseline clinical
characteristics and laboratory findings are shown in Table 1. All cases were males, with median
patient age (range) of 50 years (29±70 years). All patients had acute onset fever, and none had
history of chronic liver disease. Three patients had exposure to zoonotic risk factors such as
cows and dogs. All patients revealed abnormal liver function tests. One patient (case 2) with
severe jaundice showed marked elevation of total and direct bilirubin levels up to 23-fold
higher than reference levels, whereas others revealed only slight increase in liver enzymes. No
patients showed bacterial growth on blood cultures. Antibodies for leptospirosis, brucellosis,
hepatitis A, B, and C, as well as autoantibodies including anti-nuclear antibody, anti-double
strand DNA antibody, and anti-neutrophilic cytoplasmic antibody were all negative.
Granulomas with either circumferential or radiating fibrin deposition (Fig 1A) were
observed in 10 cases on liver biopsy and 1 case on bone marrow biopsy (case 2). Of 11 patients,
8 (73%) revealed positive C. burnetii PCR results from their formalin-fixed liver biopsy
specimen (Table 1). So, the sensitivity of Coxiella burnetii PCR was 73% (95% CI 43±90). All eight
PCR products were sequenced; the corresponding data are summarized in Table 1, and an
agarose gel profile of the PCR products is presented in Fig 2. Of the 3 patients (case 9, 10, 11) in
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Fig 1. Representative photomicrographs of Q fever hepatitis (case no. 3 in case group, x200) and hepatic mucormycosis (case no. 6 in control
group, x100). (A) Characteristic fibrin ring granulomas consisting of a central fat globule or epitheloid cells with fibrin ring (arrow) (B) A suppurative
granuloma consists of multinucleated giant cells with fungal hyphae (arrow) and polymorphous lymphoid cell including eosinophils.
whom the stored blood samples were available, 2 (case 9, 10) revealed positive Coxiella burnetii
PCR from their blood samples. The time intervals from the onset of symptoms to blood
sampling were 14, 10, and 15 days in case 9, 10, and 11, respectively. None of the aforementioned 3
patients received doxycycline before blood sampling.
As for control liver biopsy tissues, 10 patients with confirmed diagnosis unrelated to Q
fever showing granuloma in liver biopsy tissue (Fig 1B) were evaluated to test if there were any
cross-reaction. Three patients with hepatic tuberculosis, 2 patients with chronic disseminated
candidiasis, 2 patients with primary biliary cirrhosis, 1 patient with hepatic mucormycosis, 1
patient with alcoholic hepatitis, and 1 patient with post-transplant lymphoproliferative disease.
The clinical diagnosis and PCR results for these 10 control patients are summarized in Table 2.
Of these 10 control patients, none revealed positive C. burnetii PCR from their live biopsy
tissues. The agarose gel profile of PCR products are shown in Fig 2.
Fig 2. Agarose gel electrophoresis of Coxiella burnetii IS1111a gene. DNA amplification with Q-fever-IS1111a primers for the detection of Coxiella
burnetii. Gel electrophoresis of 202 bp products by using end-point PCR. M: 50bp DNA size marker; 1±11: DNAs from the case patients with Q fever
hepatitis and N: negative control (left). M: 50bp DNA size marker; PC: C. burnetii DNA control (case number 1 of case patient group); 1±10: DNAs from the
control patients and N: negative control (right).
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Pathologic findings of liver biopsy
Chronic granulomatous inflammation with caseous necrosis
PCR result from liver tissues
In this study, we found that PCR targeting for C. burnetii IS1111 multicopy sequence from
formalin-fixed, paraffin-embedded liver tissues are useful for diagnosing patients with suspected
Q fever. A previous study showed that PCR has been successfully applied for the direct
detection of C. burnetii in 12 cardiovascular biopsy specimens [
]. It has been also reported that
PCR for Q fever can be also performed on cerebrospinal fluid, pleural fluid, bone marrow,
bone biopsies, liver biopsies, milk, placenta, and fetal tissue [5±7]. However, there are limited
data on whether PCR on formalin-fixed liver tissue can be used for diagnosing Q fever
All patients in the present study had fever and feature of acute hepatitis, and underwent
percutaneous or transjugular liver biopsy. Patients with fever of unknown origin (FUO)
occasionally undergo liver biopsy to establish a final diagnosis [
]; therefore, liver biopsy
specimens are frequently obtained in FUO patients with hepatitis, and it is important this sample is
available for direct diagnosis. Acute Q fever hepatitis is one of the most common
manifestations of C. burnetii infection [
]. In patients with FUO related to Q fever, liver biopsy
reveals a typical fibrin ring type granuloma [
], which has a central lipid vacuole
surrounded by a dense fibrin ring. These granulomas are highly suggestive of Q fever but not
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definitive, because they are also seen in cases of Hodgkin's disease, infectious mononucleosis,
typhoid fever, and allopurinol hypersensitivity [
]. After acute infection of C. burnetii, the
bacterial DNA remains for an extended period in several tissues, particularly in the bone
marrow and liver; in these tissues, DNA can be detected up to decades after infection, even in the
absence of serological evidence for C. burnetii infection [
]. In this context, our data
suggest that Q fever PCR from the liver tissue can be a useful adjunct for differential diagnosis in
febrile patients with fibrin ring type granulomas. One patient showed a discrepancy of PCR
results from blood sample (negative PCR result) and liver tissue (positive PCR result); such
discrepancy of PCR results between blood samples and the tissues has also been reported in a
previous study . One possible explanation for this observation is that whereas blood
coxiellemia persists in the acute phase (first 2 weeks from the symptom onset) but disappears 24±48
hours after antibiotic treatment [
], organ tissues are able to harbor DNA and antigen up
to decades after C. burnetii infection [
5, 7, 14
]. Further studies are needed on the comparison
of diagnostic usefulness according to the clinical specimens.
This study has several limitations. First, we did not exam the C. burnetii DNA by PCR from
fresh liver biopsy specimen. To diagnose acute Q fever hepatitis, the PCR from fresh liver
biopsy specimen would be more useful than that from formalin-fixed liver tissues. However,
our positive data on the C. burnetii DNA by PCR from the formalin-fixed liver tissues warrant
the further prospective study on the usefulness of the C. burnetii DNA by PCR from fresh liver
biopsy specimen in patients with suspicious acute Q fever hepatitis. Second, we included data
on patients who had similar pathology with Q fever hepatitis used as controls. Because some
control patients might have different clinical presentations from the patients with Q fever
hepatitis, they are not representative of the population to which the test will be applied in clinical
practice. However, this study provides the useful information in the setting of the pathologic
findings suggesting Q fever hepatitis.
In conclusion, Q fever PCR from formalin-fixed liver tissues appears to be a useful adjunct
for diagnosing Q fever hepatitis. This diagnostic method might prove helpful in determining
Q fever hepatitis in patients with FUO who have fibrin-ring type granuloma, especially in the
absence of serologic evidence in acute phase of Q fever and in cases in convalescent period,
during which a direct detection for microbiologic evidence using PCR or culture from blood
samples is impossible.
Conceptualization: YS SYP SHK EY.
Formal analysis: YRJ YS SHK EY.
Funding acquisition: YS SHK.
Investigation: YRJ YS SHK EY.
Methodology: YRJ YS SHK EY.
Project administration: YS SYP SHK EY.
Resources: YS SYP SHK EY.
Software: YS SYP SHK EY.
Supervision: YS SHK EY.
Data curation: YRJ YS CEJ BK SYP MCK TK YPC SOL SHC YSK JHW SHK EY.
7 / 8
Validation: YRJ YS SHK EY.
Visualization: YRJ YS SHK EY.
Writing ± original draft: YRJ YS SHK EY.
Writing ± review & editing: YRJ YS SHK EY.
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