Shinshu M ed J, 59 ⑷ :223〜228, 2011

An Assessment by In Situ Hybridization Method for Pathogens ofSevere Respiratory Infection
Mikako MATSUBARA ＊, Tomonobu KOIZUMI , Atsuhito USHIKI
Toshiki YOKOYAMA , Masanori YASUO , Kenji TSUSHIMA
Hiroshi YAMAMOTO , Keishi KUBO , Yoshitaka YAMAZAKI
and Takayuki HONDA
1） First Department of Internal Medicine, Shinshu University School of Medicine
2） Department of Laboratory Medicine, Shinshu University School of Medicine

It has been shown that the in situ hybridization (ISH)method for the detection of phagocyted bacteria inpolymorphonuclear neutrophils is more sensitive than conventional blood culture in patients with sepsis.The
present study was designed to further evaluate the clinical utility of the ISH method, using a commerciallyavailable kit (Hybrizep ), for detecting pathogens in patients with severe respiratory infections. Peripheral
blood was taken from patients with severe respiratory infections for both routine blood culture and the ISH
method,respectively.In addition,focal samples including sputum,bronchoalveolar lavage,central and thoracic
catheter, etc. were simultaneously examined for bacterial culture. A total number of 22 specimens was
examined.The positive cases by ISH were 50.0% in the respiratory infections,which were signiﬁcantlyhigher
than those in blood cultures (9.1%).However,identical pathogens in the ISH method were not isolated fromblood and/or other sources in respiratory sites.
These ﬁndings suggest a possibilityof high frequencyof bacteremia or multifactorious pathogens in severe
respiratory infections.The ISH method may provide additional information on serious respiratory infections
for the detection of bacteremia. However, the clinical utility by the ISH method for patients with severe
respiratory infection remains undetermined. Shinshu Med J 59 : 223 ―228, 2011
(Received for publication M arch 1, 2011;accepted in revised form April 7, 2011)
Key words:bacterial genes, neutrophil, hospital‑acquired pneumonia, blood culture

Introduction

speciﬁc for genomic DNA of pathogens.Subsequently,Shimada et al, reported that the ISH analysis of

Recently,several biological techniques have been

genes of pathogens surviving in neutrophil smears

developed for the detection of bacterial pathogens.

increased the accuracy of bacterial identiﬁcation

Neutrophils ingest bacteria at an early stage ofcompared with that by conventional blood cultures

bacteremia and retain some surviving bacteria in

in patients with sepsis.Several clinical studies using

the intracellular sites . M atsuhisa et al, devel-

ISH were conducted and showed that the sensitivity

oped a method of identifying causative pathogens inof ISH in the diagnosis of bacteremia was approxi-

neutrophil blood smear by in‑situ hybridization

mately four times higher than that of conventional(ISH) using biotinylated DNA probes which was

blood culture in sepsis .
The ISH probes can detect ﬁve microorganismsʼ

Corresponding author:Mikako Matsubara
First Department of Internal M edicine, Shinshu Univer-DNA,but do not include a speciﬁc probe for Strepto-

sity School of M edicine, 3‑1‑1 Asahi, M atsumoto‑city,

coccus pneumoniae. Streptococcus pneumoniae is a

Nagano‑prefecture 390‑8621, Japan

common pathogen in respiratory infection, espe-

No. 4, 2011223

M atsubara.Koizumi.Ushiki et al.

cially in community‑acquired pneumonia.Theo-

the analysis. Written informed consent was

retically,it is speculated that the ISH method is lessobtained from each patient prior to the study.

useful for etiologic diagnosis in respiratory infec-

Samples for ISH and blood cultures were taken

tions. However, numerous studies have emphasized

simultaneously in each patient. The blood samples

polymicrobial infections in the etiology of hospital‑

were obtained by arterial or venous puncture foracquired pneumonia or severe respiratory infec-

routine blood culture and ISH methods. The sam-tions

.It has been suggested that blood culture

ples for general culture were incubated in aerobic

is an optional tool to isolate an etiologic pathogen in

and anaerobic culture bottles, respectively,at 37 ℃

pulmonary infection, but, the positive rate forand then checked daily. The other samples fromblood culture is less than 20% in subjects withparts suspected of harboring infected foci including

respiratory infections in several studies

sputum, bronchial alveolar ﬂuid (BAL), pleural.

Our hypothesis was that bacteremia might beeﬀusion, catheter, etc. were collected on the same

more frequent than suggested by the clinical evi-day. Thoracocentesis for the sampling of pleuraldence in respiratory infection,especially in patients

ﬂuid was also performed when applicable.Its resultswith hospital‑acquired pneumonia. Thus, the study

were compared with each of the specimens.

was conducted to evaluate the performance of theIntracellular bacteria from blood samples were

ISH method in patients with respiratory infections

tested by the ISH method using a commerciallyand its substantial role in deﬁning the etiology.We

available kit (Hybrisep kit ,Fuso Pharmaceuticals

prospectively analyzed the results obtained with theIndustries, Osaka, Japan). The ISH method was

ISH method in patients with severe respiratory

performed based on the manufacturerʼs protocol,

infection and compared them with results con-which had been reported previously

comitantly obtained by the conventional techniques

heparinized blood samples (5 ml) were ﬁxed, per-including blood and other cultures from pulmonary

meabilized and incubated with digoxygenin (Dig)‑sites.

labeled DNA hybridization probes. The process
Method and Subjects. Brieﬂy,

involves the extraction of the DNAs of the targetedbacterium from the white blood corpuscles in the

The present study was performed from December

blood and ﬁxing them on slide glass. The process

2003 to M arch 2005 at Shinshu University Hospital.

was performed at Shinshu University Hospital.

Patients who required hospital admission because ofNext, the second treatment‑stage consists ofcommunity‑acquired pneumonia or who developed

hybridization with a speciﬁc DNA probe to target

pneumonia during hospitalization in the criticalthe bacterial genome, using color, and observation

care unit or respiratory center in Shinshu Univer-under the microscope.After washing,anti‑Dig anti-sity were eligible for this study. The diagnosis ofbody labeled with alkaline phosphatase (ALP) was

respiratory infection was based on the detection ofused to detect the Dig‑labelled hybridized signals.

a new, persistent pulmonary inﬁltrate and on the

To visualize the signals, color development waspresence of at least two of the following clinicalperformed using nitro blue tetrazolium(NBT)and 5‑

criteria :1)fever or hypothermia (temperature ＞38

bromo‑4‑chloro‑3‑indolyl phosphate (BCIP) as aor ＜35.5 ℃);2) leukopenia or leukocytosis (whitesubstrate for ALP.The identiﬁcation of color reac-

12,000/μl);or 3)purulenttion by intracellular signals was done by light

respiratory secretions. The clinical diagnosis of

microscopic examination (×1,000). The detection

hospital‑acquired pneumonia was made based on

and/or identiﬁcation of the bacterial genes werethe criteria of the Japanese Respiratory Society

carried out at Miroku Medical Laboratory. The

Guidelines . The diagnosis of pleuritis or pyothor-

ISH probes in Hybrisep were speciﬁc for micro-

ax was made bythe presence of pleural eﬀusion andorganisms such as Staphylococcus aureus, (SA) Sta-blood cells

224

4,000/μl or

Shinshu Med J Vol.59

ISH method for pathogen of respiratory infection

phylococcus epidermidis (SE), Pseudomonas aer-

blood culture, while half of the enrolled patientsuginosa (PA), Enterococcus faecalis (EF), and awere positive by the ISH method.Thus,the positive

group of colibacillus (EK) (Escherichia coli, Enter-rate by ISH was signiﬁcantly higher than that inobacter cloacae, Klebsiella pneumoniae).

blood cultures (9.1%). In addition,the positive rateby the ISH method was identical in patients bothpre‑treated and untreated with antibiotics, indicat-

Statistics
The continuous variables were compared using

ing independence of the use of antibiotics.

the Mann‑WhitneyU‑test and categorical variables

The corresponding ﬁndings based on ISH andwere contrasted by the Chi‑squared test or Fisherʼs

subculture tests for each case are shown in Table 2.

exact test. The level of signiﬁcance was set at lessCase numbers 1 to 7 were community‑acquired

than 5%.

pneumonia who required admission, and the others
were hospital‑acquired pneumonia.In cases of com-

Results

munity‑acquired pneumonia, possible causative

A total of 26 subjects were enrolled in the present

pathogens were obtained in sputum cultures in 5

study. Among them, 4 patients were excluded

cases,but were not detectable in the blood cultures

because of subsequent deﬁnite diagnosis of pneumo-of all patients. However, ISH revealed bacteremia

nia due to inﬂuenza viral infection and diﬀuse pul-in four cases of community‑acquired pneumonia

monary hemorrhage. The characteristics of theand the detectable pathogens were diﬀerent frompatients and the positive rate in blood culture andthose observed in sputum. In cases of hospital‑

ISH are summarized in Table 1. The mean age ofacquired pneumonia,there was a patient who repor-

the enrolled patients was 60.0 years and 8 patients

ted positive for both blood culture and ISH signal,

were pre‑treated with antibiotics at the study.

but the detectable pathogens were diﬀerent (case 8).

Mean peripheral blood white cell counts and C‑

In addition, among the cases positive for ISH sig-

reactive protein (CRP)in patients both pre‑treatednals,the same pathogens were not detected in otherand untreated with antibiotics are shown in Table 1.

sources including sputum,BAL,pleural eﬀusion,orThere were no signiﬁcant diﬀerences in these values

catheters. Thus, identical pathogens in the ISH

between the groups.Two patients were positive formethods were not isolated from blood and/or other

Table 1 Patient characteristics and positive rates of ISH and blood culture
Patients
Antibiotics(＋)n＝8(−)n＝14

Total n＝22

Mean Age yr (range)

55.9 (37−77)

67.1 (19 −87)

60.0 (19 −87)

17,200±9,800(6040−35,790)

8,940±4,200(3,000−20,470)

11,630±7,300(3,000−35,790)

CRP mg/dl
mean ± SD (range)

13.6±9.3(4.1−30.4)

10.3±7.9(2.2−24.9)

12.0±8.8(2.2−30.4)

Blood culture Positive
case (%)

1 (12.5%)

1 (7.1%)2 (9.1%)

ISH Positive
case (%)

4 (50.0%)

7 (50.0%)

11 (50.0%)

WBC μlmean ± SD(range)＊＜0.05 vs blood culture
Patient characteristics and positive rates in blood culture and the ISH method.
ISH ;In situ hybridization.
No. 4, 2011225

M atsubara.Koizumi.Ushiki et al.
Table 2 Positive pathogen results for each patient with respiratory infection
case

Underlying disease
orcomplications

In situ Hybridization

Blood

SputumSA SE PA EF EK culture

cultureOthers cultures

Antibiotics

−

＋

−

−

−

(−)

SA

N.D

(−)

−

−

−

−

＋

(−)

Streptococcus
pneumoniae

N.D

(−)

＋

−

−

−

−

(−)

(−)

N.D

(−)

−

−

−

−

＋

(−)

N.D

pleural eﬀusion (−)

(−)

5

−

−

−

−

−

(−)

KP

N.D

(−)

6

−

−

−

−

−

(−)

SA

N.D

(−)

7

−

−

−

−

−

(−)

HI

N.D

(−)

1
Bronchial asthma,
Heart failure

234

Pleurisy

8

Lung cancer

−

−

−

−

＋

SE

N.D

N.D

(＋)

9

ARDS

−

−

−

−

−

SE

(−)

bronchoalveolar lavage:(−)(−)

10 Hodgkinʼs disease, ARDS −

−

−

−

＋

(−)

SA,
N.D
Candida albicans

(＋)

11

MDS myeloﬁbrosis

−

−

−

−

＋

(−)

SE

N.D

(−)

12

COPD

−

−

−

−

＋

(−)

(−)

pharynx :Streptococcus Group G

(−)

13

Lung cancer

−

−

−

−

＋

(−)

N.D

14

Pneumothorax

−

−

−

−

＋

(−)

N.D

thoracostomy tube:M RSA,
pleural eﬀusion :SA

(−)

15

Overlap syndrome,interstitial pneumonia

−

−

−

−

＋

(−)

(−)

N.D

(＋)

16

Diﬀuse panbronchiolitis,−
Pulmonary hypertension

−

−

−

−

(−)

SA, PA

N.D

(＋)

17

Interstitial pneumonia,
Dermatomyositis

−

−

−

−

−

(−)

KP

bronchoalveolar lavage:(−)

(−)

18

COPD

−

−

−

−

−

(−)

M RSA

N.D

(−)

−

−

−

−

−

(−)

PA

N.D

(＋)

19 Diﬀuse panbronchiolitis

(＋)

20

Lung cancer,
Cancerous pleurisy

−

−

−

−

−

(−)

N.D

pleural eﬀusion (−)

(−)

21

Interstitial pneumonia

−

−

−

−

−

(−)

(−)

N.D

(＋)

22

Aspiration pneumonia,−
DIC, Acute renal failure

−

−

−

−

(−)

(−)

CV catheter (−)(＋)

Abbreviations;ARDS ;acute respiratory distress syndrome, DIC ;disseminated intravascular coagulation, SA ;staphylococcus
aureus, SE ; staphylococcus epidermidis, PA ; pseudomonas aeruginosa, EF ; entercoccus faecalis, EK ; a group of colibacillus(Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae (KP)). M RSA ; methicillin resistant Staphylococcus aureus, EA ;Enterobacter aerogenes, HI ;Haemophilus inﬂuenzae, CV catheter;central venous catheter, N.D ;not done.

sources in respiratory sites.
Discussion

Thus, the sensitivity of ISH in the diagnosis ofbacteremia was approximately four times higher
than that of conventional blood culture in these

Shimada et al, analyzed 292 cases with sepsis

studies. In the present study, we applied the ISH

and found positive ISH signals in 123 cases (42.0%),

method in patients with severe respiratory infec-

while only 32 cases (11%) were positive by bloodtions. We found an almost similar positive rate byculture.Subsequently,Kubo et al,reported a 41.7%

the ISH method as those previously reported inpositive ISH signal, compared with 11.7% in blood

sepsis .The present studythus suggests a frequentculture in 60 patients with the criteria for sepsis .

coexistence of bacteremia in patients with hospital‑

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Shinshu Med J Vol.59

ISH method for pathogen of respiratory infection

acquired pneumonia or community‑acquired pneu-

by blood culture in two patients. However, themonia requiring hospitalization. In addition, it has

isolation of S. epidermidis in blood culture may be a

been shown that the ISH method is unrelated to theresult of contamination, rather than infection .

prior use of antibiotics,which is consistent with theIndeed, the identical pathogen was not detected inpresent study. This is a remarkable advantage of

the ISH method and other sources in respiratory

the ISH method,compared with conventional bloodsites.Thus,whether the pathogen is responsible forculture.

bacteremia or respiratory infection remains un-

Blood culture is an optional tool to isolate an
etiologic pathogen in pulmonary infection

determined in these cases. Compared with conven-. In

tional culture methods, the ISH method is consid-

general, the positive rates of isolating bacteremia

ered less susceptible to contamination in the han-by conventional blood cultures were 8‑20% in hos-dling process

pital‑acquired pneumonia

method is a useful tool for detecting bacteremia.

and 6.6‑13% incommunity‑acquired pneumonia admitted to thehospital. In terms of sensitivity, the ISH

Although the present result is preliminary, con-. Because of these low positive rates,

comitant testing using the ISH method with sub-the clinical usefulness of blood cultures for isolating

cultured samples from pulmonary sites might indi-

the pathogen in both community‑ and hospital‑

cate the severity of the respiratory infection and/or

acquired pneumonia remains questionable.

alternative therapy. The sample size of the present

However, it has been suggested that positive blood

study was too small to evaluate the clinical utility

culture is a predictive factor for the prognosis andof the ISH method. Further case control studies

severity in pulmonary infection.Thus,our data

focusing on the clinical outcomes including alterna-

indicate that the ISH method, can at least detect ation of therapy are needed. In addition, bacterial

relatively higher incidence of bacteremia in patients

isolation by ISH can methodologically be obtainedwith severe respiratory infection than that reported

in 8 hours, which is another superior merit as

for conventional blood cultures.

opposed to the conventional culture technique.

However, it is still unclear whether the pathogen

The clinical usefulness in the time reduction to theidentiﬁed by the ISH method can be considered an

diagnosis was unable to be evaluated in the present

exact etiologic or diagnostic indicator in patients

study,because our results for ISH were obtained bywith respiratory infections. We found that neither

two separated steps. However, we believe that thethe identical pathogen in blood culture nor in otherrapid and simultaneous identiﬁcation of pathogenic

sites was detected among patients positive for ISH

bacteria in blood samples by the ISH method may

signals. Thus, the isolation of a microorganism

improve the diagnosis and clinical management inusing the ISH method does not always conﬁrm thecertain types of respiratory infection,such as venti-

microorganism as the pathogen causing the respira-lator‑associated pneumonia.

tory infection. In other words, the multiple path-

In summary,it can be stated that in‑situ hybridi-

ogens detected with the ISH method and other sites

zation (Hybrisep )is a highly reliable non‑culturalin patients with respiratory infections suggested themethod for the detection of bacteremia and that it

coexistence of polymicrobial pathogens in thecan provide an alternative to the conventional cul-enrolled subjects. On the whole, our data based onture techniques. However, the clinical utility of thethe ISH method indicate that bacteremia can be

ﬁndings obtained with the ISH method remains

detected more frequently than by the routine clini-

uncertain. Further clinical studies using the in‑situ

cal examinations and that multi‑factorial infection

hybridization method might be warranted for thecan exist in patients with severe respiratory infec-

identiﬁcation of etiologic pathogens and clinicaltions.

outcomes in pulmonary infections.

In the present study, S. epidermidis was isolated
No. 4, 2011227

M atsubara.Koizumi.Ushiki et al.

especially Kazue Tuge, Kiyoko Tamai, and Eiji

Acknowledgments

Yanagisawa, for assistance with the measurements

We thank Miroku M edical Laboratory Co., Ltd.,of ISH in the present study.

Saku‑city, Nagano‑prefecture, 384‑2201, Japan,
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