Vous êtes sur la page 1sur 6


Effects of Intrapartum Antibiotic Prophylaxis on Neonatal Acquisition of
Group B Streptococci
Meiwa Toyofuku, MD1,2, Miyuki Morozumi, PhD2, Mariko Hida, MD, PhD1, Yoshitake Satoh, MD, PhD3,
Hiroshi Sakata, MD, PhD4, Hiroyuki Shiro, MD, PhD1, Kimiko Ubukata, PhD2, Mitsuru Murata, MD, PhD5, and
Satoshi Iwata, MD, PhD2

Objectives To assess the incidence of colonization with group B streptococci (GBS) among neonates as influ-
enced by maternal GBS carriage and intrapartum antibiotic prophylaxis (IAP).
Study design Between October 2014 and May 2015, nasopharyngeal and rectal swab samples were collected
from 730 neonates at 1 week and 1 month after birth. GBS and capsular serotype were identified by real-time poly-
merase chain reaction and by culture. IAP at delivery was determined retrospectively from hospital records.
Results Sixty-four neonates (8.8%) were GBS-positive by real-time polymerase chain reaction and culture. Among
neonates born to mothers who were GBS carriers (n = 107), 94.4% (101/107) had maternal IAP; 19.6% nonethe-
less were GBS-positive, compared with 6.5% of neonates born to noncarrier mothers (P < .01). Among neonates
born to mothers receiving IAP, more were positive only at 1 month of age than at both 1 week and 1 month. The
frequency of GBS in neonates born to mothers receiving IAP was significantly lower than that in neonates born to
mothers not receiving IAP (P < .05). Capsular serotypes V (25%) and III (23.4%) were common, followed by Ib
(15.6%), Ia (14.1%), II (7.8%), IV (6.3%), nontypeable (4.7%), and VI and VIII (each 1.6%).
Conclusions Delayed colonization with GBS occurs in infants born to GBS carrier mothers receiving IAP. GBS
should be considered in all infants at 1 month after birth with signs of infection. (J Pediatr 2017;190:169-73).

roup B streptococcus (GBS) is a leading cause of severe infections in early infancy. These infections include early-
onset disease (EOD), occurring within 6 days and late-onset disease (LOD), occurring from 7 to 89 days. Among preg-
nant women, the GBS carrier rate was estimated to be near 13% (range, 12%-22%).1 In approximately one-half of
carriers, GBS is transmitted to the infant, with invasive GBS disease developing in 1%-2%.
To prevent EOD due to GBS, a guideline was introduced by the US Centers for Disease Control and Prevention and the Ameri-
can College of Obstetricians and Gynecologists in 1996. Universal screening for maternal GBS colonization and intrapartum
antibiotic prophylaxis (IAP) strategies implemented according to the guideline have reduced the incidence of EOD but had no
impact on LOD.2,3
In Japan, a revised guideline for obstetric practice published in 2014 recommended universal GBS screening by conven-
tional culture for all pregnant women at 33-37 weeks of gestation.4 IAP during labor or after premature rupture of mem-
branes was recommended for women with any of the following characteristics: GBS culture positivity, GBS infection during
infancy in previous offspring, or incidental detection of GBS in a urine culture during the current pregnancy. If GBS status in
the current pregnancy is unknown, IAP should be given to mothers with symptoms at delivery that might be attributable to
In this study, we investigated prevalence of GBS colonization in neonates examined at 1 week and 1 month after birth, in-
fluences of IAP and GBS carriage in the mother, and capsular serotype and sequence type (ST) of GBS isolates from neonates.

We conducted a prospective longitudinal cohort study at 4 hospitals, which were
located in Hokkaido, Gunma, and Kanagawa prefectures and in Tokyo. The study
was performed from October 2014 to May 2015. Nasopharyngeal and rectal swabs
From the 1Division of Pediatrics, Yokohama Rosai
Hospital, Yokohama; 2Department of Infectious Diseases,
Keio University School of Medicine, Tokyo; 3Division of
Pediatrics, Ota Memorial Hospital, Ota; 4Division of
Pediatrics, Asahikawa Kosei Hospital, Asahikawa; and
EOD Early-onset disease 5
Department of Laboratory Medicine, Keio University
GBS Group B streptococcus School of Medicine, Tokyo, Japan
IAP Intrapartum antibiotic prophylaxis M.T. received a grant from the Japanese Society for
LOD Late-onset disease Pediatric Infectious Diseases. The other authors declare
no conflicts of interest.
MLST Multilocus sequence typing
PCR Polymerase chain reaction 0022-3476/$ - see front matter. 2017 Elsevier Inc. All rights
ST Sequence type reserved.

THE JOURNAL OF PEDIATRICS www.jpeds.com Volume 190 November 2017

were collected from all eligible infants at 1 week and 1 month ing to previously described methods,6 with alleles and ST as-
after birth and transported immediately to our laboratory. All signments determined using the Streptococcus agalactiae MLST
samples were tested to identify GBS by amplification of the database.
dltS gene, which encodes a histidine kinase, and also to iden-
tify capsular types Ia, Ib, and III using real-time polymerase Statistical Analyses
chain reaction (PCR) as developed by Morozumi et al.5 Cor- Statistical analyses were performed with Ekuseru-Toukei 2012
relation between results of real-time PCR and bacterial culture software for statistics (Social Survey Research information,
also was examined. Tokyo, Japan). The Mann-Whitney U test, the c2 test, Fisher
As shown in Figure 1 (available at www.jpeds.com), samples exact test, the Wilcoxon rank sum test, or univariate logistic
were collected via cotton-tipped swabs (BBL CultureSwab Plus, regression analysis was used as appropriate. A P value < .05
Copan, Italy). Immediately after receipt, swab samples were sus- was considered to indicate statistical significance.
pended in 0.5 mL of Todd-Hewitt broth (Becton, Dickinson, Field workers explained the purpose of the study to mothers
Franklin Lakes, New Jersey) and centrifuged at 2000g for of eligible infants, and each mother signed an informed consent
5 minutes at 4C to collect bacterial cells. After the superna- form just before discharge or at 1 week after delivery. The study
tant was discarded, 55 L of pellet was stirred and used for was approved by the Yokohama Rosai Hospital Ethics Com-
direct DNA extraction (50 L) and for culture (5 L). mittee (approval number 26-51).
Each sample of 50 L was placed in 45 L of a lysis solu-
tion that included SimplePrep reagent for DNA (Takara Bio,
Shiga, Japan) and 2 U of mutanolysin (Sigma Aldrich, St. Louis, Results
Missouri). The lytic reaction was carried out for 10 minutes
at 37C, followed by 3 minutes at 95C. The lysate was added Figure 2 is a flowchart showing transmission of GBS among
to each of the tubes containing PCR mixtures for the follow- 730 neonates according to GBS status of their mothers as de-
ing gene identifications: the dltS gene, which encodes a histi- termined by prenatal screening. GBS screening via bacterial cul-
dine kinase specific to GBS; and capsular types Ia, Ib, and III. tures was performed at 33-37 weeks of gestation for 710
Total volume (30 L) of the PCR mixture included 20 pmol mothers (97.3%) receiving care at institutions participating in
of each primer, 25 pmol of each probe, 2 Multiplex Powermix the study, and 107 mothers (14.7%) proved to be GBS carri-
(Bio-Rad, Hercules, California), and DNase- and RNase-free ers. Twenty mothers (2.7%) did not undergo screening.
distilled water. DNA amplification was carried out for 45 cycles GBS-positive neonates numbered 64, including 46 posi-
as follows: 95C for 10 seconds, 50C for 30 seconds, and 72C tive by both real-time PCR and culture and 18 positive only
for 20 seconds. by real-time PCR. Twenty-one neonates born to the 107 GBS
The remaining 5 L of each sample was inoculated on sheep carriers were positive for GBS (19.6%); among neonates born
blood agar (Nippon Becton, Dickinson, Tokyo, Japan), which to the 603 noncarriers, 39 (6.5%) were positive for GBS. Fre-
then was incubated at 37C for 20 hours in an atmosphere con- quency of GBS positivity in neonates differed significantly
taining 5% CO2. Colonies of b-hemolytic streptococci grown between carrier and noncarrier mothers (P < .001). Four neo-
on the blood agar plates underwent identification of capsu- nates born to mothers without GBS screening were positive
lar type (Ia, Ib, II, III, IV, V, VI, VII, and VIII) and the dltS gene for GBS (20%). No infants developed invasive GBS infection
by real-time PCR. during the study period.
For multilocus sequence typing (MLST) analysis, primer sets Relationships between time points of GBS positivity in neo-
corresponding to 7 housekeeping genes (adhP, atr, glnA, glcK, nates and maternal IAP are shown in Table I.
pheS, sdhA, and tkt) used for MLST were constructed with ref- GBS-positive neonates were divided into 3 groups: GBS per-
erence to the MLST Web site (http://pubmlst.org/sagalactiae/). sistence (n = 16, 25%), indicating GBS positivity at both 1 week
MLST was applied to the sequences for these 7 genes accord- and 1 month after birth; GBS clearance (n = 7, 10.9%),

Mother & infant (n = 730)

Maternal GBS (+) Maternal GBS (-) Maternal GBS unknown

14.7% (n = 107) 82.6% (n = 603) 2.7% (n = 20)

Infant GBS (+) Infant GBS (-) Infant GBS (+) Infant GBS (-) Infant GBS (+) Infant GBS (-)
19.6% (n = 21) 80.4% (n = 86) 6.5% (n = 39) 93.5% (n = 564) 20.0% (n = 4) 80.0% (n = 16)

Figure 2. Flowchart showing presence or absence of GBS carriage in mothers and neonates.

170 Toyofuku et al

Table I. Association between GBS positivity in neonates and maternal GBS colonization
GBS(+) (n = 107) GBS() (n = 603) Screening () (n = 20)
Neonates n (%) Yes (n = 101) No (n = 6) Yes (n = 196) No (n = 407) Yes (n = 17) No (n = 3)
Persistence group, n (%) 16 (25.0) 2 (10.5) 1 1 10 (31.3) 1 1
Clearance group, n (%) 7 (10.9) 2 (10.5) 0 0 5 (15.6) 0 0
Delayed positivity group, n (%) 41 (64.1) 15 (78.9) 1 6 17 (53.1) 2 0
Total 64 19 2 7 32 3 1

*Among mothers with IAP, cefazolin was given to 189; ampicillin to 107; and other agents to 15.
One woman was not given IAP despite preterm delivery. Two women were not given IAP, in accordance with the guideline.
Persistence group, positive for GBS at 1 wk and 1 mo after birth; clearance group, positive only at 1 wk after birth; Delayed positivity group, negative for GBS at 1 wk, but positive at 1 mo after

indicating GBS positivity at 1 week but not at 1 month after Maternal and neonatal characteristics are analyzed for re-
birth; or delayed GBS positivity (n = 41, 64.1%), indicating GBS lationships to neonatal GBS positivity at 1 month after birth
negativity at 1 week but positivity at 1 month after birth. in Table III (available at www.jpeds.com). Only maternal
Of the 107 GBS carrier mothers, 101 (94.4%) received IAP, GBS carrier status showed a significant difference between
among whom ampicillin was administered to 84 (83.2%), GBS-positive neonates (n = 57) and GBS-negative neonates after
cefazolin to 14 (13.9%), and other agents to 3 (3%). Al- birth (n = 673) at 1 month (P < .001). Univariate logistic re-
though IAP was given, 19 neonates (18.8%) born to GBS carrier gression analysis of these characteristics identified maternal GBS
mothers were positive for GBS after birth. Notably, most of these colonization as a risk factor associated with neonatal GBS posi-
neonates, numbering 15 (78.9%), belonged to the delayed- tivity (OR 3.6; 95% CI 2.0-6.6).
positivity group. Capsular serotypes for all GBS isolates were identified by
Of 603 pregnant women found to be GBS noncarriers, 196 real-time PCR. As shown in Table IV, capsular serotypes V
(32.5%) received intravenous antibiotics during labor; among (n = 16, 25%), III (n = 15, 23.4%), Ib (n = 10, 15.6%), and Ia
their infants, 6 of the 7 GBS-positive neonates were in the (n = 9, 14.1%) were predominant, followed by II (n = 5),
delayed-positivity group. Among GBS-positive neonates whose IV (n = 4), nontypeable (n = 3), VI and VIII (n = 1, respec-
mothers received no antibiotic agents, 10 (31.3%) had GBS per- tively). In addition, the most common serotype in the
sistence, 5 (15.6%) had clearance, and 17 (53.1%) had delayed persistence group (n = 16), clearance group (n = 7) and delayed-
positivity. positivity group (n = 41) was Ib (31.3%), III (57.1%), and V
Generally, delayed positivity was significantly more preva- (29.3%), respectively.
lent in neonates born to mothers who received IAP (P = .04) Forty-five GBS strains isolated from 64 GBS-positive samples
than in neonates born to mothers not receiving IAP. In the per- by bacterial culture in neonates (70.3%) were analyzed for cap-
sistence group, a significant difference was not evident between sular serotype and MLST. Although 10 STs were found, ST1
infants of mothers with and without IAP (P = .08). (28.9%), ST335 (24.4%), and ST10 (15.6%) accounted for
Table II shows a correlation between threshold cycle values 68.9% of all 45 isolates. Most isolates with capsular serotype
derived from real-time PCR assays in neonates and imple- III were ST335, which is derived from ST19. ST17, linked with
mentation of IAP. Neonates born to mothers receiving IAP high virulence, was found in only 1 strain.
showed a significantly lower GBS bacterial load than neo-
nates born to mothers not receiving IAP (P = .04). Discussion
In this prospective cohort study of neonatal GBS coloniza-
tion following the 2014 introduction of the current guide-
Table II. Threshold cycle values derived from real- lines for obstetric practice in Japan, combining real-time PCR
time PCR in neonates according to IAP with culture methods, we found an overall prevalence of GBS
Mothers (n = 710) colonization within 1 month after birth of 8.8%. Notably, the
Threshold cycle and number IAP IAP positivity rate in neonates born to GBS carrier mothers, almost
of bacterial cells Yes (n = 297) No (n = 413) Total all of whom received IAP, was significantly greater (19.6%) than
<24 cycles 2 (18.2) 9 (81.8) 11 positivity in neonates whose mothers were not carriers (6.5%).
(>5 104 cells/sample) Positivity rates in our Japanese infants were considerably lower
25-28 cycles 8 (44.4) 10 (55.6) 18
(103 5104 cells/sample)
than those among infants in Denmark (11.3%),7 Gambia
>29 cycles 19 (54.3) 16 (45.7) 35 (24.8%),8 and Italy (21.9%).9 We previously estimated GBS car-
(<103 cells/sample) riers among pregnant women to be 15.7% by real-time PCR
Total 29 35 64
and 12.6% by bacterial culture.5 We believe that high compli-
P = .04. P values were calculated with the Wilcoxon rank sum test. ance with the obstetric guideline in Japan most likely was
Effects of Intrapartum Antibiotic Prophylaxis on Neonatal Acquisition of Group B Streptococci 171
THE JOURNAL OF PEDIATRICS www.jpeds.com Volume 190

Table IV. Relationships between capsular type and ST

Capsular type
STs* Total, n (%) Ia Ib II III IV V VI VIII NT
ST1 13 (20.3) 4 8 1
ST10 7 (10.9) 7
ST17 1 (1.6) 1
ST19 1 (1.6) 1
ST23 4 (6.3) 3 1
ST26 1 (1.6) 1
ST130 1 (1.6) 1
ST144 2 (3.1) 2
ST196 4 (6.3) 4
ST335 11 (17.2) 11
Unknown 19 (29.7) 4 3 1 1 6 1 3
Total 64 9 10 5 15 4 16 1 1 3
(%) (14.1) (15.6) (7.8) (23.4) (6.3) (25.0) (1.6) (1.6) (4.7)

*Only GBS isolates by culture were tested.

ST335 was derived from ST19.
Capsular type in 16 cases was identified by real-time PCR.

responsible for lower neonatal GBS positivity than in other rotype V has shown little involvement in invasive GBS disease
countries. during early infancy, whereas in the US serotype V is increas-
Neonatal GBS colonization of the pharynx, intestine, or um- ing in prevalence both in pregnancy and in invasive disease
bilical cord is well known to precede development of inva- during early infancy.16 Importantly, serotype patterns can shift.
sive GBS infection.10,11 Reported incidence of GBS disease was ST17 GBS, which has high virulence and belongs to capsular
0.57 per 1000 live births in Europe and 0.67 in the Americas.1 serotype III, accounted for 50% of invasive GBS isolates in
According to a previous report,12 incidence of invasive GBS Japan,13 similar to reports from the Netherlands.3 In our study,
disease in Japan was much lower, 0.18 per 1000 live births, only one ST17 GBS was identified.
which may be attributable to a low rate of GBS maternal trans- Our study has several limitations. First, US Centers for
mission to infants. Disease Control and Prevention guidelines indicate that IAP
Interestingly, delayed positivity was significantly more fre- administration beginning at least 4 hours before delivery has
quent than persistent positivity, especially in neonates born been found highly effective.2 Regrettably, detailed informa-
to mothers receiving IAP. The density of GBS colonization in tion about when IAP was administered relative to delivery was
neonates with delayed positivity was estimated to be rela- unavailable for our study. Second, bacterial culture of mater-
tively low by real-time PCR. In contrast, persistent positivity nal samples was carried out without use of a selective medium
was more frequent among neonates born to mothers without for GBS isolation at each institution. Therefore, false-negative
IAP compared with those born to mothers with IAP. We cases may have been included among noncarrier mothers.
suspect that after IAP was initiated, density of GBS coloniza- Application of real-time PCR or conventional PCR with high
tion of mothers decreased temporarily, but the organism was sensitivity and specificity is expected to enhance accuracy of
not eradicated. These small numbers of GBS, transmitted maternal testing. Finally, we could not identify capsular sero-
from mother to newborn, may then have multiplied gradu- types in GBS-colonized mothers, leaving open the possibility
ally in the respiratory or intestinal tract after escaping detection that instances of GBS colonization of infants may not all have
at 1 week after birth. Accordingly, IAP strategies could reduce resulted from vertical transmission at birth. Uniform testing
incidence of EOD but lack impact on incidence of LOD. that combines real-time PCR with bacterial culture for both
Pregnant women carrying GBS therefore remain an impor- pregnant women and neonates will be necessary to further
tant risk factor for neonatal invasive GBS infection. Neonatal address this question. However, such testing cannot distin-
GBS infection also could occur following transmission from guish transmission at delivery from subsequent transmission
the mother by a route unrelated to delivery, or by horizontal involving ordinary maternal contact.
transfer from another person such as a family member after The ability of IAP to prevent LOD due to GBS may be limited
discharge from the hospital. even with high-precision testing for both pregnant women and
In our study, the predominant capsular serotypes of GBS neonates. Further studies should determine whether surveil-
carried by neonates were types V and III. Serotype III is most lance microbiologic assessment at 1 month after birth and aug-
common among isolates from infants with invasive GBS.12-14 mented hygiene can reduce risk of LOD. GBS vaccination of
Relative capsular serotype prevalence is similar to that previ- pregnant women represents an alternative way to protect infants
ously reported worldwide.15 Among GBS isolates from Japa- from invasive GBS disease. This strategy is under develop-
nese maternal carriers, the predominant capsular serotype was ment, with the leading candidate for maternal vaccination being
type Ib, followed by types V and III5; other countries have dif- a trivalent GBS vaccine (CRM197-conjugated capsular poly-
fered in maternal carrier serotype prevalence.8,9 In Japan, se- saccharides of GBS serotype Ia, Ib, and III).17
172 Toyofuku et al

We thank Atsuo Sato, MD, Masaki Nakayama, MD, Miyuu Matsui, MD, 8. Le Doare K, Jarju S, Darboe S, Warburton F, Gorringe A, Heath PT, et al.
Tomoko Furutani, MD, and Kumiko Tago, MD for collecting data. Risk factors for Group B Streptococcus colonisation and disease in Gambian
women and their infants. J Infect 2016;72:283-94.
Submitted for publication Apr 22, 2017; last revision received Jun 22, 2017; 9. Berardi A, Rossi C, Creti R, Gherardi G, Venturelli C, Rumpianesi F, et al.
accepted Jul 19, 2017 Group B Streptococcal colonization in 160 mother-baby pairs: a pro-
spective cohort study. J Pediatr 2013;163:1099-104, e1.
10. Baker CJ, Barrett FF. Transmission of group B streptococci among
References parturient women and their neonates. J Pediatr 1973;83:919-
1. Kobayashi M, Vekemans J, Baker CJ, Ratner AJ, Le Doare K, Schrag SJ. 25.
Group B Streptococcus vaccine development: present status and future 11. Carl MA, Ndao IM, Springman AC, Manning SD, Johnson JR, Johnston
considerations, with emphasis on perspectives for low and middle income BD, et al. Sepsis from the gut: the enteric habitat of bacteria that cause
countries. F1000Res 2016;5:2355. late-onset neonatal bloodstream infections. Clin Infect Dis 2014;58:1211-
2. Verani JR, McGee L, Schrag SJ. Prevention of perinatal group B strepto- 8.
coccal diseaserevised guidelines from CDC, 2010. MMWR Recomm Rep 12. Matsubara K, Hoshina K, Suzuki Y. Early-onset and late-onset group B
2010;59:1-36. streptococcal disease in Japan: a nationwide surveillance study, 2004-
3. Van Dyke MK, Phares CR, Lynfield R, Thomas AR, Arnola KE, Craig AS, 2010. Int J Infect Dis 2013;17:e379-84.
et al. Evaluation of universal antenatal screening for Group B Strepto- 13. Morozumi M, Wajima T, Kuwata Y, Chiba N, Sunaoshi K, Sugita K, et al.
coccus. N Engl J Med 2009;360:2626-36. Associations between capsular serotype, multilocus sequence type,
4. Minakami H, Maeda T, Fujii T, Hamada H, Iitsuka Y, Itakura A, et al. and macrolide resistance in Streptococcus agalactiae isolates from
Guidelines for obstetrical practice in Japan: Japan Society of Obstetrics Japanese infants with invasive infections. Epidemiol Infect 2014;142:812-
and Gynecology (JSOG) and Japan Association of Obstetricians and Gy- 9.
necologists (JAOG) 2014 edition. J Obstet Gynaecol Res 2014;40:1469- 14. Chang B, Wada A, Hosoya M, Oishi T, Ishiwada N, Oda M. Character-
99. istics of group B streptococcus isolated from infants with invasive
5. Morozumi M, Chiba N, Igarashi Y, Mitsuhashi N, Wajima T, Iwata S, et al. infections: a population-based study in Japan. Jpn J Infect Dis 2014;356-
Direct identification of Streptococcus agalactiae and capsular type by real- 60.
time PCR in vaginal swabs from pregnant women. J Infect Chemother 15. Edmond KM, Kortsalioudaki C, Scott S, Schrag SJ, Zaidi AK, Cousens S,
2015;21:34-8. et al. Group B streptococcal disease in infants aged younger than 3 months:
6. Jones N, Bohnsack JF, Takahashi S, Oliver KA, Chan MS, Kunst F, et al. systematic review and meta-analysis. Lancet 2012;379:547-56.
Multilocus sequence typing system for group B streptococcus. J Clin 16. Puopolo KM, Madoff LC. Type IV neonatal early-onset Group B Strep-
Microbiol 2003;41:2530-6. tococcal disease in a United States hospital. J Clin Microbiol 2007;45:1360-
7. Hansen SM, Uldbjerg N, Kilian M, Sorensen UB. Dynamics of Strepto- 2.
coccus agalactiae colonization in women during and after pregnancy and 17. Heath PT. Status of vaccine research and development of vaccines for GBS.
in their infants. J Clin Microbiol 2004;42:83-9. Vaccine 2017;34:2876-9.

Effects of Intrapartum Antibiotic Prophylaxis on Neonatal Acquisition of Group B Streptococci 173

THE JOURNAL OF PEDIATRICS www.jpeds.com Volume 190

Figure 1. Protocol of real-time PCR and culture for identification of GBS in samples collected from neonates.

Table III. Maternal and neonatal characteristics with

respect to neonatal GBS positivity at 1 month after birth
GBS-positive GBS-negative P
Characteristics n = 57 (%) n = 673 (%) value
Boys, n 30 (52.6) 367 (54.5) .782
Born at full term, n (%) 51 (89.5) 614 (91.2) .654
Birth weight, mean SD, g 2852 565 2898 454 .638

Vaginal delivery, n (%) 46 (80.7) 504 (74.9) .328
Maternal age, mean SD, y 32.7 5.3 32.5 4.4 .518
Maternal GBS colonization, n (%) 19 (33.3) 88 (13.1) <.001
One or more prior live births, n (%) 25 (43.9) 316 (47.0) .653
Only breastfeeding, n (%) 18 (31.6) 245 (36.4) .481
Maternal mastitis, n (%) 1 (1.8) 5 (0.7) .323

173.e1 Toyofuku et al