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Infectious diseases

ISSN: (Print) (Online) Journal homepage:https://www.tandfonline.com/loi/infd20

Diagnosis of pleural empyema/parapneumonic

effusion by next-generation sequencing

Yoshiki Shiraishi, Kirill Kryukov, Katsuyoshi Tomomatsu, Fumio Sakamaki,

Shigeaki Inoue, So Nakagawa, Tadashi Imanishi & Koichiro Asano

To cite this article:Yoshiki Shiraishi, Kirill Kryukov, Katsuyoshi Tomomatsu, Fumio Sakamaki,
Shigeaki Inoue, So Nakagawa, Tadashi Imanishi & Koichiro Asano (2021) Diagnosis of pleural
empyema/parapneumonic effusion by next-generation sequencing, Infectious Diseases, 53:6,
450-459, DOIs:10.1080/23744235.2021.1892178

To link to this article:https://doi.org/10.1080/23744235.2021.1892178

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INFECTIOUS DISEASES, https://doi.org/10.1080/23744235.2021.1892178
2021; VOL.
No. 6, 450–459

ORIGINAL ARTICLE

Diagnosis of pleural empyema/parapneumonic effusion by

next-generation sequencing

Yoshiki Shiraishia , Kirill Kryukovb,c , Katsuyoshi Tomomatsua, Fumio Sakamakid, Shigeaki Inouee,
So Nakagawab , Tadashi Imanishib and Koichiro Asanoa
aDivision of Pulmonary Medicine, Department of Medicine, Tokai University School of Medicine, Isehara, Japan;bDepartment of

Molecular Life Science, Tokai University School of Medicine, Isehara, Japan;cDepartment of Genomics and Evolutionary Biology, National
Institute of Genetics, Mishima, Japan;dDivision of Respiratory Disease, Department of Medicine, Tokai University Hachioji Hospital,
Tokyo, Japan;eDepartment of Emergency and Critical Care Medicine, Tokai University School of Medicine, Isehara, Japan

ABSTRACT
Background:Although a microbiological diagnosis of pleural infection is clinically important, it is often complicated by prior
antibiotic treatment and/or difficulties with culturing some bacterial species. Therefore, we aim to identify probable
causative bacteria in pleural empyema/parapneumonic effusions by combining 16S ribosomal RNA ( rRNA) gene
amplification and next-generation sequencing (NGS).

Methods:Pleural fluids were collected from 19 patients with infectious effusions and nine patients with non-infectious
malignant effusions. gene. Infectious and noninfectious effusions were distinguished by semi-quantitative PCR of the
16S rRNA gene.

Results:Only 8 (42%) effusions were culture-positive, however, NGS of the 16S rRNA gene amplicon identified 14 anerobes
and 7 aerobes/facultative anaerobes in all patients, includingStreptococcussp.n¼6),Fusobacteriumsp.n¼Five),
Porphyromonassp.n¼5), andPrevotellasp.n¼4), accounting for >10% of the total genomes. The culture and NGS results were
discordant for 3 out of 8 patients, all of whom had previously been treated with antibiotics. Total (2D.CTvalue in semi-
quantitative PCR of the 16S rRNA gene) and specific (total bacterial load multiplied by the proportion of primary bacteria in
NGS) bacterial loads could efficiently distinguish empyema/parapneumonic effusion from non-infectious effusion.

Conclusion:Combining NGS with semi-quantitative PCR can facilitate the diagnosis of pleural empyema/parapneumonic
effusion and its causal bacteria.

Abbreviations:A ACP: American College of Chest Physicians; GSTK: Genome Search Toolkit; NGS: next-generation sequencing; ROC:
receiver operating characteristic; rRNA: 16S ribosomal RNA;

KEYWORDS ARTICLE HISTORY CONTACT

Pleural empyema Received 27 July 2020 Revised Koichiro Asano
parapneumonic effusion 31 January 2021 Accepted 10 ko-asano@tokai-u.jp
next-generation sequencing February 2021 Division of Pulmonary Medicine, Department of
malignant pleural effusion Medicine, Tokai University School of Medicine, 143
Shimokasuya, Isehara, 259-1193,
Kanagawa, Japan

Supplemental data for this article can be accessedhere.

- 2021 Society for Scandinavian Journal of Infectious Diseases
 INFECTIOUS DISEASES 451

Background PCR with NGS to identify pathogenic bacteria in pleural

empyema/parapneumonic effusions.
A parapneumonic effusion is the accumulation of
pleural fluid caused by pneumonia. When
microorganisms infect the pleural space, a massive Methods
influx of neutrophils causes the accumulation of Patients
purulent effusion, resulting in empyema.
Patients with infectious effusions were prospectively
parapneumonic effusion and 6%–10% develop pleural
recruited from two tertiary referral university hospitals
empyema [1,2]. The aspiration of oral indigenous
between August 2015 and September 2019. Medical history,
bacteria in patients with poor dental hygiene is a major
laboratory, microbiological, and radiological data were
risk factor for empyema, which can be lethal, especially
obtained from their medical records. Patients were
among the elderly [3].
categorized into four risk categories for poor outcomes
Identification of the pathogenic bacteria causing pleural
according to the American College of Chest Physicians (AACP)
empyema is clinically important for determining suitable
consensus statement [twenty one] as 1 (very low), 2 (low), 3
antimicrobial therapy. Facultative anaerobic Grampositive
(moderate), and 4 (high), based on the volume, localisation,
cocci, includingStreptococcus pneumoniaeand Streptococcus
appearance, pH, and Gram staining/bacterial culture data of
angiosusgroup, are predominant in culture-positive patients,
the pleural effusions (Categories 1– 3, parapneumonic
whereas Gram-negative rods such asEscherichia coli,
effusion; Category 4, empyema). Patients with malignant
Klebsiella pneumoniae, Haemophilus influenzae,
effusion were recruited as non-infectious controls. The
Pseudomonas aeruginosa,and anaerobes including
institutional review board of Tokai University Hospital
Peptostreptococci, Fusobacterium nucleatum, andPrevotella
approved this study (#14 R-220), and all patients provided
sp., etc. are rare [3–6]. In one study, Gram-positive cocci were
written informed consent to participate.
isolated from 76% of patients, anaerobes from 17%, and
Gram-negative bacilli from 5% [2], whereas in another study,
aerobic, anaerobic, and both aerobic and anaerobic bacteria DNA extraction from pleural fluids
were isolated from 64%, 13%, and 23% of patients,
The pleural fluid samples (obtained by thoracentesis)
respectively [Four].
were fractionated into cellular components and spinal
Low sensitivity and specificity is a recurring problem for
by centrifugation at 50-gat 4-C for 15min. The
identifying pathogenic microorganisms by conventional
supernatants were divided into 1mL aliquots and
means. Up to 40% of the samples can be culturenegative [
stored at 80-C. Ice-cold aliquots of the liquids (1mL)
Five,7–11] because some bacterial species are
were centrifuged at 20,000-gat 4-C for 15min, and
uncultivable or require specific culture conditions DNA was extracted from the pellets using the DNeasy [3–6,12],
anerobes are more difficult to culture than aerobic bacteria [13,14],
Blood and most
& Tissue patients
Kit (Qiagen are treated
GmbH, Hilden, Germany).m
L of buffer (10mM
with antimicrobials before the pleural fluid is Tris-HCl, 0.1M NaCl, 1mM EDTA, 5% (v/v) Triton X-100, sampled [15
]. pH 8.0) with 20mg/mL lysozyme and 4mL of RNase
To overcome these problems, Kawanami and col- (100 ng/mL) and incubation at 37-C for 30min.
leagues applied clonal microflora analysis in which the Proteinase K (25mL, 20mg/mL) was added and incubated
16S ribosomal RNA (rRNA) gene was amplified, and 100 at 56-C from 30min to overnight. Following alcohol
randomly selected clones were sequenced using the precipitation, samples were dissolved in 20mL of
Sanger method [16]. This strategy identified anerobes nuclease-free water. DNA was quantified using a more
efficiently and comprehensively thanin vitroculture, but itQubit
is time and labor-intensive
TMsfluorometer and HS
with a dsDNA thus, impractical
assay for
kit (Thermo
clinical applications. Other researchers have applied next-generation sequencing
Fisher Scientific, (NGS)
Waltham, MA, technology for the
USA).
diagnosis of infectious respiratory diseases such as pneumonia and pleural empyema [17–20], however, it is
often difficult to differentiate pathogenic bacteria from contaminated species with NGS analysis alone.
16s rRNA gene amplification

For some experiments, DNA extracted from the pleural

effusion was PCR amplified before NGS. Variable regions
of the bacterial 16S rRNA gene were amplified using the
16S Primer Sets V2–4–8 that amplify variable regions
452 Y.SHIRAISHI ET AL.

V2, V4, and V8, and 16S Primer Sets V3–6,7–9 that amplify Enterococcus faecalis, Lactobacillus fermentum, Listeria
variable regions V3, V6-7, and V9 from the 16STMs monocytogenes,andStaphylococcus aureus)that are
Metagenomics Kit (Thermo Fisher Scientific). The difficult to lyse.mL of the mock microbial community
primer set V2-4-8 is designed to amplify variable standard was added to 1mL of malignant pleural fluid,
regions V2, V4, and V8 and the primer set V3-6,7-9 is which was then analyzed as above.
designed to amplify variable regions V3, V6-7 , and V9.
Fragments of DNA were amplified by PCR using the
Semi-quantitative PCR
ProFlexTMs3-32-well PCR System (Thermo Fisher
Scientific) according to the manufacturer's instructions. We estimated the bacterial loads of the pleural effusions,
Technologies, Santa Clara, CA, USA). by semi-quantitative real-time PCR of the V2 region of the
16S rRNA gene, using the primers, 50-AGNGGCG
NACGGGTGAGTAAC-30(forward), and 50-CTGCCTCCCGTA
GGAGTCTG-30(reverse). Reactions contained 5mL of Fast
SYBR Green master mix (Thermo Fisher Scientific), 0.3mM
of each primer, 1mL of -10 diluted genomic DNA
NGS and analysis
(equivalent to 2mL of pleural effusion solution), and
For the NGS, barcode adapters were attached using the ultrapure water (to 10mL final volume).
Ion XpressTMsBarcode Adapters 1–16 and Ion Plus StepOnePlus Real-Time PCR System (Thermo Fisher Fragment
Library Kit (Thermo Fisher Scientific), each bar- Scientific) was performed as follows: initial denaturation
Coded sample diluted to 26 pM and combined as at 95-C for 5min, 40 amplification cycles at 95-C for 20 sec,
described by the manufacturer. Thereafter, emulsion PCR and 60-C for 30 sec, and then the samples were held at 4-
was performed using the Ion OneTouchTMs2 System C. The reference cycle threshold (C.T.) value determined by
loaded onto an Ion 316TMsChip. The NGS-based 16S rRNA PCR using nuclease-free water was used to calculate the
gene amplicon was sequenced using the Ion PGMTMswith D.C.T.values. We defined the total bacterial load as 2D.CT
HiQ chemistry (Thermo Fisher Scientific). The Ion Reporter and the specific bacterial load as the total bacterial load
System (Thermo Fisher Scientific) generated FASTQ files multiplied by the proportion of specific bacterial species
and the statistics were evaluated using a SeqKit with a or genera as determined by NGS.
'stats' option [twenty two].
The sequence reads were taxonomically classified by a
Statistical analysis
BLASTN homology search using the Genome Search Toolkit
(GSTK;http://kirill-kryukov.com/study/tools/gstk/) against the All values are presented as medians and interquartile
reference database, the GenomeSync (http:// ranges (IQR). Total or specific bacterial loads were
genomesync.org); the GenomeSync contains genomes from compared between infectious and non-infectious
both GenBank [twenty three] and RefSeq [twenty four] and pleural fluids using Mann-Whitney U tests and receiver
taxonomy annotation from the NCBI Taxonomy Database [ operating characteristics (ROC) curves.p< .05 were
twenty five]. The taxonomic assignment was based on the considered significantly different. All data were
best-matching genomes between the query sequence of the statistically analyzed using GraphPad Prism 8 version
NGS results and the reference sequence in the database. We 9.0.0 (GraphPad Software, La Jolla, CA, USA).
selected the species with the highest alignment score
computed by BLASTN as the species of interest in the Results
sample. Visual reports were made using KronaTools [26].
Demographic data of the patients
To verify the ability of the 16S rRNA gene amplicon
sequencing to accurately detect bacterial species, we Pleural effusion samples were obtained from 19
used the microbial mixture, ZymoBIOMICS Microbial patients (male,n¼16;n¼3; median age: 69 years) with
Community Standard D6300 (Zymo Research, Irvine CA, infectious effusion, and nine patients with non-
USA), that contains eight bacterial strains (theoretical infectious, malignant pleural effusion (male,n¼6; n¼3;
proportion of each genomic DNA : 12%), including three median age, 73 years).Table 1shows the demo-
Gram-negative bacteria (Escherichia coli, Salmonellagraphic data. Seven patients with pleural empyema/par- enterica,
apneumonic effusion
andPseudomonas aeruginosa)that are easy to lyse and five Gram-positive had cancer,
bacteria (Bacillus five had underlying
subtilis,
respiratory diseases such as interstitial pneumonia,
 INFECTIOUS DISEASES 453

chronic obstructive pulmonary disease, or bronchiectasis, antibiotic therapy, 3 (60%) were culture-positive for
two had neuromuscular diseases, and four had a history anaerobes, whereas 5 (36%) of the 14 patients under
of stroke. One patient had an indwelling chest tube to antibiotic therapy were culture-positive, including two
treat malignant effusion at the onset of pleural empyema. with anaerobes. Sputum cultures were positive in eight
One patient was being treated with systemic patients and did not match the bacteria isolated from the
corticosteroids for eosinophilic pleurisy At the time of pleural effusions in any of the patients. Blood cultures
thoracocentesis, 14 patients had already been treated were negative in all patients.
with systemic antibiotics for 1–18 days. According to the
AACP consensus statement regarding the risk for poor
Verification of NGS protocol for
outcomes in patients with parapneumonic effusions [
parapneumonic effusion
twenty one], one, five, and 13 patients were placed in
categories 2, 3, and 4, respectively. Additional file 3:Table S3summarizes the details of the
Tables 2and3and Additional file 1:Table S1show the statistics of our NGS data. We conducted NGS of the
bacterial culture results of pleural fluid, sputum, and first seven samples (Additional file 3:Table S2), both
blood. with and without 16S rRNA gene amplification.
The pleural fluid was culture-positive in eight absence of PCR, bacterial reads accounted for only patients.
Among the five patients without prior 0.03% (IQR: 0.02% 0.04%) of the total reads.

Table 1.Clinical and laboratory features of 19 infectious and 9 non-infectious effusions.

Infectious effusions

Patient characteristics Non-infectious effusions AACP Categories 1–3 (Parapneumonic) AACP Category 4 (Empyema)
Male/Female 6/3 4/2 12/1
Age (y) 73 (70–75) 71 (66–76) 65 (49–76)
Pleural fluid analysis
Total cell count (cell number/mL) 1023 (539–1582) 3,508 (2,239–4,893) 4,000 (1,034–27,215)
Neutrophil count (cell number/mL) 5 (2–12) 2,500 (1,686–4,122) 2,080 (807–1,7908)
pH 7.44 (7.42–7.46) 7.44 (7.24–7.68) 7.20 (6.98–7.30)
Glucose (mg/dL) 112 (108–135) 120 (90–131) 28 (8–62)
Lactate dehydrogenase (U/mL) 297 (283–686) 557 (475–777) 2,111 (1,312–5,024)
Symptoms/Conditions
Fever 0 6 11
Chest pain 0 6 8
Prior antibiotic therapy 1 Four 8
Thoracostomy tube 0 0 1
Immunosuppressive drugs 1 0 1
Underlying diseases
Poor dental hygiene 0 1 9
Cancer 8 Four 2
Cardiac failure 2 1 Four
Respiratory diseases† Five 2 3
Diabetes mellitus Four 2 2
History of stroke 0 0 Four
Renal failure 2 1 1
Neurological disease 0 0 2
Values shown are medians with interquartile ranges.
†Respiratory
diseases included chronic obstructive pulmonary disease, interstitial pneumonia, and bronchiectasis.

Table 2.Major bacterial species identified by 16S rRNA metagenomics in non-infectious effusions.
Antibiotics Pleural fluid
Treatment NGS culture

Prior antibiotics Proportion of specific Specific Average nucleotide Bacterial

treatment D.C.T. Bacterial Species bacteria (%) bacterial load Identity (%) species
No 0.32 Leaf89 Candidatus 13.5 0.11 98.72 ND
No 0.96 Burkholderia crenata ND 11.8 0.23 96.84 ND
No 0.28 – – – ND
No 0.36 ND – – – ND
No 0.56 Cutibacterium acnes 21.2 0.31 98.72 ND
No 0.08 ND – – – ND
No 0.2 ND – – – ND
No 1.3 ND – – – ND
No 0.8 ND – – – ND
ND: not detected.
454 Y.SHIRAISHI ET AL.

Table 3.Major bacterial species and their proportions identified by 16S rRNA metagenomics in infectious effusions.
Antibiotic treatments NGS Pleural fluid culture

Specific Average
AACP Prior antibiotics period Proportion of bacteria nucleotide
Category treatment (Days) D.C.T. Bacterial Species specific bacteria (%) load Identity (%) Bacterial species
2 Yes Five 6.6 Streptococcus intermedius 81.1 78.73 99.30 ND
3 No – 7.5 Streptococcussp. 72.7 130.75 – ND
S. mitis 26.9 48.35 98.69
S. infantis 16.1 29.98 98.64
S. pneumoniae 29.7 53.42 98.54
3 No – 4.2 Lawsonella clevelandensis 14.4 2.71 98.96 ND
Cutibacterium acnes 13.7 2.57 98.09
3 Yes 3 7.7 Cutibacterium acnes 11.9 25.20 98.84 ND
3 Yes 3 6.8 Fusobacterium nucleatum 89.3 101.68 98.69 Pseudomonas aeruginosa
3 Yes 3 Porphyromonassp.
0.5 12.7 0.09 – ND
P. endodontalis 8.0 0.06 98.71
P. gingivalis 4.7 0.03 98.60
Four No – 11.8 Enterococcussp. 76.4 2724.46 – Enterococcus faecium
E. faecium 38.1 1358.47 98.79
E. phoeniculicola 19.6 699.05 98.71
E. durans 18.7 666.88 98.81
Four No – 1.6 Fusobacterium nucleatum 55.7 1.72 98.77 Fusobacteriumsp.
Prevotella buccae 12.8 0.39 96.79
Four No – 5.7 Fusobacterium nucleatum 62.5 31.68 99.32 Fusobacterium nucleatum
Four Yes 18 6.1 Porphyromonas endodontalis 24.6 17.17 98.61 Peptostreptococcussp.
Fusobacterium nucleatum 21.2 14.77 98.61 Campylobacter curvus
Prevotella oris 16.2 11.28 98.28
Four Yes 14 13.4 Prevotella oris 76.6 8561.83 98.07 Streptococcus angiosus
Four Yes 6 5.1 Streptococcus intermedius 16.4 5.53 98.78 ND
Four Yes Four 11.5 Porphyromonas gingivalis 84.1 2483.26 98.00 Porphyromonassp.
Prevotellasp.
Four Yes 2 1.0 Staphylococcisp. 63.1 1.26 – Staphylococcus aureus
S. aureus 39.1 0.78 98.95
S. simiae 24.0 0.48 98.90
Four Yes 9 0.6 Streptococcus intermedius 34.5 0.51 99.20 ND
Filifactor alocis 18.9 0.28 99.00
Four Yes 6 1.0 Streptococcus intermedius 53.1 1.08 99.11 ND
Four Yes Four 0.1 Streptococcus intermedius 37.9 0.39 99.25 ND
Four Yes 6 4.9 Porphyromonas gingivalis 54.4 16.26 98.62 ND
Fusobacteriumsp. 23.4 6.99 –
F. periodonticum 12.2 3.66 98.65
F. nucleatum 11.2 3.36 98.85
Four Yes Four 4.7 Enterococcussp. 56.9 14.57 – ND
E. durans 29.6 7.56 99.47
E. faecium 16.1 4.13 99.35
E. phoeniculicola 11.2 2.87 99.12
Prevotella oris 29.6 7.56 98.71
Porphyromonas gingivalis 11.5 2.94 98.66
ND: not detected.

contrast, >95% of the total reads from post-PCR 10.1%,Lactobacillus fermentum18.4%,Listeria

samples were annotated as bacterial DNA, regardless monocytogenes14.1%,Pseudomonas aeruginosa4.2%,
of the pleural effusion category (Additional file 2:Table Salmonella enterica10.4%, andStaphylococcus aureus15.5%.
S2). Comparing NGS with 16S rRNA PCR and NGS The measured proportion of each bacterial species [mean
without 16S rRNA PCR, only three out of seven cases (standard deviation)] were:Bacillus subtilis12.6 (2.5)%,
showed identical bacterial species. Enterococcus faecalis9.1 (1.6)%,Escherichia coli14.4
16S rRNA gene by PCR in the subsequent samples (3.6)%,Lactobacillus fermentum11.4 (1.5)%,Listeria before
NGS. monocytogenes11.5 (0.4)%,Pseudomonas aeruginosa
Additional file 4:Figure S1shows the results of the mock 3.8 (1.1)%,Salmonella enterica23.1 (5.4)%, and
standard verification. All bacterial species were detectable Staphylococcus aureus16.9 (3.0)%.
in the three samples of the malignant pleural effusion
spiked with the mock standard. The theoretical
NGS of non-infectious and infectious effusions
proportion of 16S rRNA genes based on the genome size
and copy number of 16S rRNA gene are:Bacillus subtilis We assessed malignant pleural effusions from nine
17.4%,Enterococcus faecalis9.9%,Escherichia coli patients with no signs of active intrapleural infection.
 INFECTIOUS DISEASES 455

Bacterial species accounting for more than 10% of the Discrimination between infectious and non-infectious
total bacterial genomes by 16S rRNA gene sequence pleural effusion

analysis were identified in three patients (33%); NGS can only determine the proportions of bacterial
Cutibacterium acnes, Methylobacteriumsp.Leaf89,genomes in a given sample.
Candidatus burkholderia crenatain one patient each ( infectious and non-infectious pleural effusions, we
Tables 2and3). analyzed the largest proportions of the bacterial species,
Major bacterial species accounting for >10% of the the bacterial genome load by real-time PCR of the V2
total genomes including fourteen anaerobes and five region of the 16S rRNA gene, and the specific bacterial
aerobes/facultative anaerobes were identified in all the load, which is the total bacterial load multiplied by the
pleural empyema/parapneumonic effusions. proportion of specific bacteria determined by NGS
Streptococcussp.n¼6),Fusobacteriumsp.n¼Five), (Table 2,Figure 2). Porphyromonassp.n¼Five),Prevotellasp.n¼4),
portions of bacterial species from infectious effusions Cutibacterium acnes (n¼2), andEnterococcussp.n¼2) were
identified in two or more effusions. Detection rates and bacterial
was 0.56species did0.75),
(IQR, 0.30 not differ
whichsubstantially based
was significantly on
higher
the AACP risk category or prior antibiotic treatment (Figure 1). that
than The observed
predominant bacteria comprising
in non-infectious effusions>50%
(0.07;total
IQR,
bacterial genomes identified in 12 samples were: Streptococcussp.S.
0.05 0.12,p<intermediusorS. mitis/S.
.0001). The area under theinfantis/S.
ROC curve (AUC)
pneumoniae)andF. nucleatumin three effusions each, was 0.96 and the cut-off value to discriminate between
infectious and non-infectious samples was 0.125 with a
Youden index of 0.73 (Figure 2 (A, B)). The median total
bacterial load in infectious pleural effusions was 33.7
(IQR, 5.8–146.9), which was also signifi-
Enterococcussp.E. durans/E. faecium/E. phoeniculicola)cantly higher than that observed non-infectious and
malignant
Porphyromonas gingivalisin two effusions each, and Prevotella effusions (1.3; IQR,aureus/S.
oris,andStaphylococcisp.S. 1.1–1.7;p< .01). The AUC
simiae/S.
of the
haemolyticus)in one effusion each. Four effusions were probably total bacterial
infected with twoload was bacteria,
or more 0.88 andthree
the cut-off
had to
multiple anaerobes, one hadEnterococcussp. and anaerobes,discriminate
and two hadbetween infectious
mixed infections and non-infectious
(Figure 1(A)).
samples was 2.73 with a Youden index of 0.79 (Figure 2
(C, D)). The median specific bacterial loads were 16.3
(IQR, 2.2–90.2) and 0.08 (IQR, 0.07–0.13) for infectious
and non-infectious effusions, respectively (p< .001).
The AUC of the specific bacterial load was 0.98 and the
cutoff to discriminate infectious from non-infectious
samples was 0.35 with a Youden index of 0.95 (Figure 2
Comparison of bacterial species identified by culture (E, F)).
and NGS

The predominant bacteria identified by NGS matched Discussion

the culture results in five effusions (Table 2). In
contrast, the predominant bacteria identified in three
effusions by NGS did not match those identified by
culture.Peptostreptococcussp. Campylobacter curvusin
culture, these bacteria represented only 0.01% and
1.6% of the total bacterial genomes, respectively,
whereas NGS identified the anaerobes,Porphyromonas
endodontalis (twenty five%),Fusobacterium nucleatum
(21%), andPrevotella oris (16%).Streptococcus angiosus
andPseudomonas aeruginosa,accounted for only 0.9%
and 0.2% of the total bacterial genomes, respectively.
Prevotella oris (77%) andF. nucleatum (89%) were the
predominant bacteria according to NGS in these
samples.
We show here that combining 16S rRNA gene amplicon
sequencing with semi-quantitative PCR can reliably detect
bacterial infection and identify the causative
microorganism(s) of pleural empyema and
parapneumonic effusions more efficiently and accurately
than conventional culture methods, even when patients
have previously been treated with antibiotics.
Prokaryote-specific 16S rRNA gene amplicon sequencing
has been widely applied to microbiome analyses, but few
studies have highlighted the value of NGS for diagnosing
infectious diseases such as pneumonia and pleural empyema
[17–20]. One reason could be the difficulty of identifying
pathogenic species due to the similarity of the 16S rRNA
gene among species. We transcended this issue using
GenomeSync and GSTK, our in-house database and
456 Y.SHIRAISHI ET AL.

(A) 1.0
, Cutibacterium

Proportion of Major
0.8 Enterococcus

Bacterial Genus
Fusobacterium
0.6 Porphyromonas
0.4 Prevotella
Pseudomonas
0.2 Staphylococci
Streptococcus
0
Other genus of Bacteria
(B) TenFour

Ten3
Specific bacteria
Genus Load

Ten2
Ten
1
Ten-1

Ten-2
AACP Category 23333344444444444444
Antibiotic treatments+ + + +++ ++++++++++
Non-infectious Infectious

Figure 1.Distribution of bacterial genera in non-infectious and infectious pleural effusions. (n¼9) and infectious (n¼19) samples are
presented with the AACP risk category and prior antibiotic treatment in the order shown in Table 2(A) Proportion of major bacterial
genera in each sample. (B) Specific bacterial load defined as total bacterial load (2D.CTin PCR of the 16S rRNA gene V2 region)
multiplied by the proportion of the most dominant bacterial genus determined by NGS.

Our sequence search comprised all-to-all non-infectious patients, suggesting that this bacterium, sequence
similarity searches without clustering in GSTK which is also found in the oral cavity, might have caused [27–31].
Besides, the GenomeSync database is continuously the pyogenic pleural infections as previously updated by
peer-to-peer networking with the NCBI reported [34–37].
GenBank [twenty three] and RefSeq [twenty four] databases and is now Several technical limitations remain in our
one of the largest genome databases worldwide. methodology. The efficiency of DNA extraction and PCR
Possible causative bacteria were identified by NGS in amplification is inconsistent among bacterial species. We
almost all effusion samples, whereas the culture-positive examined this variability using a mock standard that
rate was only 42% as previously reported [Five,7–11]. We contained eight bacterial species with theoretical found that
over 50% of our patients had empyema/para- Amounts of 16S rRNA genes (4.2–18.4%). The proportion
pneumonic effusions caused by mono- or poly- determined by 16S rRNA gene amplicon sequencing showed
infection with anaerobes, a result compatible with the some deviation from the theoretical proportion, ranging
NGS findings of Dyrhovden et al.17], who also found between 3.8% and 23.1%. Direct sequencing without 16S
discordant results with bacterial cultures in 63% of rRNA gene amplification would avoid variability during PCR
their samples, similar to our results (73%). amplification. , but the results were not reliable in 4 (57%) of
Streptococcus pneumoniae, S. anginosus group,and our samples due to a low bacterial load (Additional file 2:
other aerobes/facultative anaerobes are more likely to Table S2). Another problem was that multiple species in the
be detected in culture than anaerobic microbes [Four, same genus such asS. pneumoniae/mitis/infantis, E. faecium/
14,32]. However, one study found that anaerobes such phoeniculicola/durans,or
as Gram-positive anaerobic cocci,Fusobacterium, S. aureus/simiaewere annotated in some samples. The
microaerophilicStreptococci, Prevotella,andBacteroides alignment scores among these species were so close
species could be identified in 74% of empyema cases that we could not determine whether the results were
under culture conditions more suitable for anaerobes [ due to mixed infections or mis-annotation. Sequencing
33].Cutibacterium acnes, identified in two of our other targets, such as the atpA, groEL, RecN, or kat
patients with AACP category 3 effusions, might have genes, groES locus, or 16S-23S rRNA gene internal
originated from the skin during thoracentesis. transcribed spacer regions might help overcome these
problems [38–45], although it is necessary to design
primers that target specific groups of bacteria and
C. acneswas higher in these samples than in those from perform another round of NGS analysis.
 INFECTIOUS DISEASES 457

(A) (B)
1.0
periodontopathic microorganisms are more prone to
1.0
phagocytosis by neutrophils than periodontopathic
Largest Proportion of

0.8 0.8
Bacterial Species

pathogens such asPorphyromonasandPrevotellasp.47].

Sensitivity
0.6 0.6
This might explain why bacterial loads were smaller in
0.4 0.4
patients withS. intermedius-induced pleural empyema (
0.2 0.2
Table 3). Further analysis of more patients is needed to
0.0 0.0
0.0 0.5 1.0 establish appropriate cut-off values for specific genome
us
us

io
io

1-specificity
loads to discriminate infectious from non-infectious
ct
ct

fe
fe

In
- in

pleural effusions.
on
N

(C) (D)

Conclusions
NGS analysis combined with semi-quantitative PCR of the
16S rRNA gene can identify causative micro-organisms
more accurately than conventional culture methods, and
thus serve as a powerful tool that could facilitate
appropriate and timely treatment of pleural empyema
and parapneumonic effusions.
(E) (F)

Acknowledgments
We acknowledge the valuable technical support and comments
regarding the NGS analysis provided by Keiko Yokoyama,
Takuma Araki, Masayuki Tanaka, Tadayuki, Wang Ting, Tadayuki
Satou, Hideki Hayashi, and Nobuo Watanabe. , Tomoe Takeuchi,
Keito Enokida, and Shohei Obayashi for recruiting participants
and providing clinical support.

Figure 2.Comparison of non–infectious and infectious pleural

effusions. Proportions of the largest bacterial species (A), total (C),
and specific (E) bacterial loads of non-infectious and infectious Ethics approval and consent to participate
pleural effusions are shown as medians with IQR. ROC curve of The Institutional Review Board for Clinical Research at Tokai
proportions of bacterial genome (B), total (D), and specific (F)
University approved the study (Approval No: 14 R-220), which was
bacterial loads were analyzed to discriminate non-infectious from
conducted according to the Declaration of Helsinki (2013
parapneumonic samples.
amendment). The study was initiated after patients received written
explanations of the study and Its protocols. After ensuring all the
atpA and 16S rRNA gene amplification to identify a
details were fully understood, the patients provided written
bacterium that was annotated as a subspecies of
informed consent to participate.
Campylobacter curvus [27]. Alternatively, sequencing
longer fragments of 16S rRNA gene such as V1-V2 region
might improve annotation rates [46]. Author contributions
The NGS data provided information about the YS and KA contributed to the conceptualisation and the design of the
bacterial proportion, but not the number of specific study and data interpretation.
bacteria. Therefore, NGS alone cannot differentiate KK analyzed the NGS data. SN and TI supervised the
infectious from non-infectious pleural effusions. The bioinformatic analysis; KT, FS, and SI acquired the clinical data
and the pleural effusion samples. YS drafted the manuscript and
AUC indicated that the specific bacterial loads are
conducted statistical analyses. Approved its submission for
more suitable for identifying infectious pleural
publication.
effusions. Three of our patients with AACP category 4
effusions caused by Streptococcus intermediushad
specific bacterial loads close to the cut-off value.
Streptococciand other non- Disclosure statement
The authors declare that they have no competing interests.
458 Y.SHIRAISHI ET AL.
Funding
This study was supported by the Japan Initiative for Global Research
Network on Infectious Diseases (J-GRID) of Japan Agency for Medical
Research and Development (AMED) under Grant Number
JP17fm0108023, and the Takeda Science Foundation.
ORCID
Yoshiki Shiraishi http://orcid.org/0000-0001-5275-6318
Kirill Kryukov http://orcid.org/0000-0002-0286-0288
So Nakagawa http://orcid.org/0000-0003-1760-3839
Tadashi Imanishi http://orcid.org/0000-0002-1182-9127
Koichiro Asano http://orcid.org/0000-0002-9044-3061
Data availability statement
The dataset used and/or analyzed during the current study is
available from the corresponding author on reasonable request.
References
[1] Light RW. Parapneumonic effusions and empyema. Proc Am
Thorac Soc. 2006;3(1):75–80.
[2] Falguera M, Carratala J, Bielsa S, et al. Predictive factors,
microbiology and outcome of patients with parapneumonic
effusion. Eur Respir J. 2011;38(5):1173–1179.
[3] Davies CW, Kearney SE, Gleeson FV, et al. Predictors of outcome
and long-term survival in patients with pleural infection. Am J
Respir Crit Care Med. 1999;160(5):1682–1687.
[4] Brook I, Frazier EH. Aerobic and anaerobic microbiology of
empyema. A retrospective review in two military hospitals.
Chest. 1993;103(5):1502–1507.
[5] Barnes TW, Olson EJ, Morgenthaler TI, et al. Low yield of
microbiologic studies on pleural fluid specimens. Chest.
2005;127(3):916–921.
[6] Ozol D, Oktem S, Erdinc E. Complicated parapneumonic effusion
and empyema thoracis: microbiologic and therapeutic
aspects. Respir Med. 2006;100(2):286–291.
[7] Byington CL, Spencer LY, Johnson TA, et al. An epidemiological
investigation of a sustained high rate of pediatric
parapneumonic empyema: risk factors and microbiological
associations. Clin Infect Dis. 2002;34(4):434–440.
[8] Jimenez D, Diaz G, Garcia-Rull S, et al. Routine use of pleural
fluid cultures. Are they indicated? Limited yield, minimal
impact on treatment decisions. Respir Med. 2006;100(11):
2048–2052 .
[9] Labelle AJ, Arnold H, Reichley RM, et al. A comparison of culture-
positive and culture-negative health-care-associated
pneumonia. Chest. 2010;137(5):1130–1137.
[10] Medford AR, Medford A, Maskell N. Pleural effusion.
Postgrad Med J. 2005;81(961):702–710.
[11] Thomson AH, Hull J, Kumar MR, et al. Randomized trial of
intrapleural urokinase in the treatment of childhood
empyema. Thorax. 2002;57(4):343–347.
[12] Maskell NA, Davies CWH, Nunn AJ, et al. UK Controlled trial
of intrapleural streptokinase for pleural infection.
Engl J Med. 2005;352(9):865–874.
[13] Bartlett JG. Anaerobic bacterial infections of the lung. Chest.
1987;91(6):901–909.
[14] Menzies SM, Rahman NM, Wrightson JM, et al. Blood culture
bottle culture of pleural fluid in pleural infection. Thorax.
2011;66(8):658–662.
[15] Vincent JL, De Backer D. Circulatory shock. N Engl J Med.
2013;369(18):1726–1734.
[16] Kawanami T, Fukuda K, Yatera K, et al. A higher significance of
anaerobes: the clone library analysis of bacterial pleurisy.
Chest. 2011;139(3):600–608.
[17] Dyrhovden R, Nygaard RM, Patel R, et al. The bacterial
aetiology of pleural empyema. A descriptive and
comparative metagenomic study. Clin Microbiol Infect.
[18] Sung JY, Hwang Y, Shin MH, et al. Utility of conventional culture
and MALDI-TOF MS for identification of microbial
communities in bronchoalveolar lavage fluid in comparison
with the GS junior next generation sequencing system.
Ann Lab Med. 2018;38(2):110–118.
[19] Kitsios GD, Fitch A, Manatakis DV, et al. Respiratory
microbiome profiling for etiologic diagnosis of
pneumonia in mechanically ventilated patients. Front
Microbiol. 2018;9: 1413.
[20] Yang L, Haidar G, Zia H, et al. Metagenomic identification of
severe pneumonia pathogens in mechanically-ventilated
patients: a feasibility and clinical validity study. Respir Res.
2019;20(1):265.
[21] Colice GL, Curtis A, Deslauriers J, et al. Medical and surgical
treatment of parapneumonic effusions: an evidence-based
guideline. Chest. 2000;118(4):1158–1171.
[22] Shen W, Le S, Li Y, et al. SeqKit: a cross-platform and ultrafast
toolkit for FASTA/Q file manipulation. PLoS One. 2016;
11(10):e0163962.
[23] Benson DA, Cavanaugh M, Clark K, et al. GenBank. Nucleic Acids
Res. 2017;45(D1):D37–D42.
[24] O'Leary NA, Wright MW, Brister JR, et al. Reference sequence
(RefSeq) database at NCBI: current status, taxonomic
expansion, and functional annotation. Nucleic Acids
Res. 2016;44:D733–45.
[25] Federhen S. Type material in the NCBI Taxonomy Database.
Nucleic Acids Res. 2015;43(Database issue):D1086–D1098.
[26] Ondov BD, Bergman NH, Phillippy AM. Interactive metagenomic
visualization in a Web browser. BMC Bioinformatics.
2011;12:385.
[27] Horio Y, Shiraishi Y, Watanabe N, et al. Empyema associated
with Campylobacter curvus infection.
Rep. 2017;5(4):e00234.
[28] Kryukov K, Ueda MT, Imanishi T, et al. Systematic survey of non-
retroviral virus-like elements in eukaryotic genomes.
Virus Res. 2019;262:30–36.
[29] Mitsuhashi S, Kryukov K, Nakagawa S, et al. A portable system
for rapid bacterial composition analysis using a nanopore-
based sequencer and laptop computer. Sci Rep. 2017;
7(1):5657.

[30] Watanabe N, Kryukov K, Nakagawa S, et al. Detection of
pathogenic bacteria in the blood from sepsis patients using
16S rRNA gene amplicon sequencing analysis. PLoS One.
2018;13(8):e0202049.
[31] Nakagawa S, Inoue S, Kryukov K, et al. Rapid
sequencingbased diagnosis of infectious bacterial species
from meningitis patients in Zambia. Clin Transl
Immunology. 2019; 8(11):e01087.
[32] Ferguson AD, Prescott RJ, Selkon JB, et al. The clinical course
and management of thoracic empyema. QJM. 1996;
89(4):285–289.
[33] Boyanova L, Vladimir D, Gergova G, et al. Anaerobic
microbiology in 198 cases of pleural empyema: a
Bulgarian study. Anaerobe. 2004;10:261–267.
[34] Pan SC, Wang JT, Hsueh PR, et al. Propionibacterium acnes:
an easily ignored pathogen. J Infect. 2005;51(4):e229–
e231.
[35] Odunukan OW, Masannat F, Baka JJ.Propionibacterium
acnesbrain abscess in an immunocompetent man in the
absence of prior neurosurgery. SD Med. 2016;69:71–73.
[36] Veitch D, Abioye A, Morris-Jones S, et al.Propionibacterium
acnesBMJ Case Rep. 2015;2015:bcr2015212431.
[37] Claeys G, Verschraegen G, De Potter C, et al.
Propionibacterium acnes. European journal of clinical
microbiology & infectious. Eur J Clin Microbiol Infect Dis.
1994;13(9):747–749.
[38] Ward NL, Rainey FA, Hedlund BP, et al. Comparative
phylogenetic analyzes of members of the order
Planctomycetales and the division Verrucomicrobia: 23S
rRNA gene sequence analysis supports the 16S rRNA gene
sequence-derived phylogeny. Int J Syst Evol Microbiol.
2000;50 Pt 6:1965–1972.
[39] Wang Y, Qian PY. Conservative fragments in bacterial 16S
rRNA genes and primer design for 16S ribosomal DNA
amplicons in metagenomic studies. PLoS One. 2009;4(10):
e7401.
INFECTIOUS DISEASES 459
[40] Miller WG, Yee E, Jolley KA, et al. Use of an improved atpA
amplification and sequencing method to identify members of
the Campylobacteraceae and Helicobacteraceae.
Appl Microbiol. 2014;58(6):582–590.
[41] Tang Y, Underwood A, Gielbert A, et al. Metaproteomics analysis
reveals the adaptation process for the chicken gut
microbiota. Appl Environ Microbiol. 2014;80(2):478–485.
[42] Glazunova OO, Raoult D, Roux V. Partial recN gene
sequencing: a new tool for identification and phylogeny
within the genus Streptococcus. Int J Syst Evol Microbiol.
2010;60(Pt 9):2140–2148.
[43] Kim W, Park HK, Hwang WJ, et al.Streptococcus
pneumoniae, S. mitis,andS. oralisby a novel multiplex PCR
assay targeting the gyrB gene. J Clin Microbiol.
2013;51(3):835–840.
[44] Blaiotta G, Fusco V, Ercolini D, et al. Diversity of
Staphylococcus species strains based on partial kat
(catalase) gene sequences and design of a PCR-restriction
fragment length polymorphism assay for identification
and differentiation of coagulase-positive species (S.
aureus, S. delphini, S. hyicus, S. intermedius, S.
pseudintermedius,andS. schleiferisubsp. coagulans. J Clin
Microbiol. 2010;48(1): 192–201.
[45] Conrads G, Citron DM, Tyrrell KL, et al. 16S-23S rRNA gene
internal transcribed spacer sequences for analysis of the
phylogenetic relationships among species of the genus
Porphyromonas. Int J Syst Evol Microbiol. 2005;55(Pt 2) :
607–613.
[46] Watts GS, Youens-Clark K, Slepian MJ, et al. 16S rRNA gene
sequencing on a benchtop sequencer: accuracy for
identification of clinically important bacteria. J Appl Microbiol.
2017;123(6):1584–1596.
[47] Ji S, Hyun J, Park E, et al. Susceptibility of various oral bacteria to
antimicrobial peptides and to phagocytosis by neutrophils. J
Periodontal Res. 2007;42(5):410–419.