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Lee, Park, Lee, Kim, Kim, Lee, and Yoo: Detection of 13 Enteric Bacteria and 5 Viruses Causing Acute Infectious Diarrhea Using Multiplex PCR from Direct Stool Specimens
Original Article

Ann Clin Microbiol 2013;16(1):33-38.

Published online: 5 March 2013

DOI: https://doi.org/10.5145/ACM.2013.16.1.33

Detection of 13 Enteric Bacteria and 5 Viruses Causing Acute Infectious Diarrhea Using Multiplex PCR from Direct Stool Specimens

Seungok Lee1, Yeon-Joon Park2, Hae Kyung Lee3, Soo-Young Kim4, Ja-Young Kim5, So-Young Lee6, Jin-Kyung Yoo2

1Department of Laboratory Medicine, Incehon St. Mary’ s Hospital, The Catholic University School of Medicine, Incheon, Korea

2Department of Laboratory Medicine, Seoul St. Mary’ s Hospital, The Catholic University School of Medicine, Seoul, Korea

3Department of Laboratory Medicine, Uijeongbu St. Mary’ s Hospital, The Catholic University School of Medicine, Uijeongbu, Korea

4Department of Laboratory Medicine, St. Vincent’ s Hospital, The Catholic University School of Medicine, Suwon, Korea

5Department of Laboratory Medicine, Daejeon St. Mary’ s Hospital, The Catholic University School of Medicine, Daejeon, Korea

6Department of Laboratory Medicine, Yeouido St. Mary’ s Hospital, The Catholic University School of Medicine, Seoul, Korea

Correspondence: Yeon-Joon Park, Department of Laboratory Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea, 505, Banpo-dong, Seocho-gu, Seoul 137-040, Korea. (Tel) 82-2-2258-1640, (Fax) 82-2-2258-1719, (E-mail) yjpk@catholic.ac.kr

Received 1 August 201223 October 2012       Accepted 23 October 2012

Copyright © 2013 The Korean Society of Clinical Microbiology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

초록

Background

We investigated the prevalence of various pathogens (13 enteric bacteria and 5 viruses) which cause diarrhea using multiplex PCR of stool specimens and compared two multiplex PCR methods for detecting diarrheagenic Escherichia coli.

Methods

A total of 405 stool specimens submitted between November 2010 to February 2011 for routine culture of enteric pathogens were included and screened for five viruses (astrovirus, Group A rotavirus, enteric adenovirus, norovirus G1/G2) and eight bacteria (Salmonella spp., Shigella spp., Campylobacter spp., Vibrio spp., C. difficile Toxin B, C. perfringens, Y. enterolytica, Aeromonas spp.) using the SeeplexⓇ Diarrhea ACE detection kit (Seegene). In addition, virulence-associated genes of enteropathogenic E. coli, (EPEC), enterohemorrhagic E. coli (EHEC), enteroin-vasive E. coli, (EIEC), enterotoxigenic E. coli (ETEC), and enteroaggressive E. coli (EAEC) were detected using 16-plex PCR and a commercial diarrheagenicE. coli detection (DEC) PCR kit (SSI Diagnostica).

Results

Overall, 138 (34.1%) of 405 samples was positive for pathogen. The positive rate for virus was 18.5%. norovirus G2, Group A rotavirus, enteric adenovirus, astrovirus and norovirus G1 were detected in 40, 23, 8, 3 and 1 samples, respectively. The positive rate for bacteria was 24.4% (99/405). C. difficile toxin B was the most frequently detected, followed by C. perfringens, EPEC, and EAEC. The agreements of the two multiplex PCR methods for detecting EPEC and EHEC were 99.3% and 100%, respectively.

Conclusion

The detection rate was high (34.1%) in-cluding various diarrheagenic E. coli (6.2%) and C. perfringens (5.2%). Multiplex PCR is thus useful for detecting various pathogens.

Keywords: Agreement, Diarrheagenic Escherichia coli, Norovirus, Rotavirus

REFERENCES

1.Korea Centers for Disease Control and Prevention. The prevalence and characteristics of bacteria causing acute diarrhea in Korea, 2008. Public Health Weekly Report.http://www.cdc.go.kr/CDC/notice/CdcKrInfo0301.jsp?menuIds=HOME001-MNU0004-MNU0036-MNU0037&cid=12298/[Online. (last visited on 25 July 2012).
2.Antikainen J., Tarkka E., Haukka K., Siitonen A., Vaara M., Kirveskari J. New 16-plex PCR method for rapid detection of diarrheagenic Escherichia coli directly from stool samples. Eur J Clin Microbiol Infect Dis. 2009. 28:899–908.
3.Nataro JP., Bopp CA, et al. Escherichia, Shigella, and Salmonella. Murray PR, Baron EJ, editors. Manual of Clinical Microbiology. 9th ed. Washington: American Society for Microbiology;2007. p. 670–87.
4.Higgins RR., Beniprashad M., Cardona M., Masney S., Low DE., Gubbay JB. Evaluation and verification of the Seeplex Diarrhea-V ACE assay for simultaneous detection of adenovirus, rotavirus, and norovirus genogroups I and II in clinical stool specimens. J Clin Microbiol. 2011. 49:3154–62.
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6.Persson S., Olsen KE., Scheutz F., Krogfelt KA., Gerner-Smidt P. A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory. Clin Microbiol Infect. 2007. 13:516–24.
7.Fujioka M., Kasai K., Miura T., Sato T., Otomo Y. Rapid diagnostic method for the detection of diarrheagenic Escherichia coli by multiplex PCR. Jpn J Infect Dis. 2009. 62:476–80.
8.Huang DB., Mohanty A., DuPont HL., Okhuysen PC., Chiang T. A review of an emerging enteric pathogen: enteroaggregative Escherichia coli. J Med Microbiol. 2006. 55:1303–11.
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Table 1.
Primer sets and target genes for the detection of 5 viral and 8 enteric bacterial pathogens in the SeeplexⓇ Diarrhea ACE detection kits [4,5]
Primer set Pathogen Target gene
Diarrhea-V ACE Astrovirus ORF1a
Detection Group A rotavirus VP4
Enteric adenovirus Hexon
Norovirus-G1 ORF2
Norovirus-G2 ORF2
Diarrhea-B1 ACE Vibrio spp. hly, tlh, wh
Detection Clostridium difficile toxin B tcdB
Salmonella spp. sopB
Shigella spp. vif, ipaH
Campylobacter spp. hip, asp
Diarrhea-B2 ACE Clostridium perfringens cpa
Detection∗ Yersinia enterocolitica inv
Aeromonas spp. hly, ela

∗ Evaluation for E. coli O157, E. coli H7 and VTEC in the Diarrhea-B2 ACE Detection kit was excluded in this study.

Table 2.
Comp etween 16-ple parison of the target g ex PCR [2] and DEC P enes of diarrheagenic E. coli PCR kit [6]
Pathotype 16-plex PCR DEC PCR kit
EPEC [eaeA, escV, ent]*, bfp eae
EHEC hly, stx1, stx2 vtx1 (stx1), vtx2 (stx2)
EIEC ipaH, invE ipaH
ETEC elt, est1a, est1b eltA, estA-human, estA-porcine
EAEC astA, aggR, pic Not available
Internal control uidA 16sDNA

∗ eaeA, escV and ent can be found in both EPEC and EHEC.

Table 3.
Characteristics of 174 acute diarrheagenic pathogens detected by multiplex PCR assay from 405 stool specimens in six university hospitals between October 2010 and February 2011
Classification Pathogens No. (%) positive isolates Age of patients Laboratory tests∗ No. (%) coinfection
0-5 6-18 19-64 ＞65 Stool OB† Stool WBC
Viruses Norovirus-G2 40 (9.9) 29 4 4 3 3/39 (7.7) 1/38 (2.6) 11/40 (27.5)
Group A rotavirus 23 (5.7) 19 2 0 2 2/22 (9.1) 1/22 (4.5) 9/23 (39.1)
Enteric adenovirus 8 (2.0) 6 0 1 1 2/8 (25.0) 1/8 (1.3) 4/8 (50.0)
Astrovirus 3 (0.7) 1 1 1 0 1/3 (33.3) 0/3 (0) 3/3 (100)
Norovirus-G1 1 (0.2) 1 0 0 0 0/1 (0) 0/1 (0) 1/1 (100)
Bacteria C. difficile toxin B 33 (8.1) 9 6 4 14 13/32 (40.1) 8/29 (27.6) 10/33 (41.7)
C. perfringens 21 (5.2) 6 5 3 7 6/20 (30.0) 1/20 (0.5) 11/21 (52.4)
Aeromonas spp. 10 (2.5) 3 2 1 4 0/10 (0) 1/9 (11.1) 5/10 (50.0)
Campylobacter spp. 5 (1.2) 2 0 2 1 3/5 (60.0) 2/5 (40.0) 2/5 (40.0)
Salmonella spp. 3 (0.7) 1 1 1 0 2/3 (66.6) 2/3 (66.6) 0/3 (0)
Vibrio spp. 1 (0.2) 0 1 0 0 1/1 (100) 1/1 (100) 0/1 (0)
Y. enterocolitica 1 (0.2) 1 0 0 0 0/1 (0) 0/1 (0) 0/1 (0)
Pathogenic E. coli EPEC 11 (2.7) 5 1 4 1 5/9 (55.5) 0/11 (0) 6/11 (54.5)
EAEC 11 (2.7) 4 1 3 3 3/11 (27.3) 0/10 (0) 5/11 (45.5)
EHEC 1 (0.2) 0 0 1 0 0/1 (0) 0/1 (0) 0/0 (0)
Mixed‡ 2 (0.5) 0 0 1 1 1/2 (50.0) 0/2 (0) 0/0 (0)

∗ No. positive/no. tested (%);

† Stool occult blood;

‡ The isolate showed mixed type of EPEC and EAEC.

Table 4.
Pattern of co-infecting diarrheagenic pathogens in 32 stool specimens detected by multiplex PCR assay
Classification Co-infecting pathogens (n)
Viral (n=6) Norovirus-G2 & Astrovirus (1)
Norovirus-G2 & Enteric adenovirus (1)
Norovirus & Group A rotavirus (1)
Norovirus-G2 & Norovirus-G1 (1)
Group A rotavirus & Enteric adenovirus (1)
Enteric adenovirus & Astrovirus (1)
Bacterial (n=10) C. difficile toxin B & Aeromonas spp. (1)
C. difficile toxin B & C. perfringens (1)
C. difficile toxin B & EAEC (1)
C. difficile toxin B & EPEC (1)
C. perfringens & Aeromonas spp. (1)
C. perfringens, Aeromonas spp. & Campylo-
bacter spp. (1)
C. perfringens, Aeromonas spp. & EAEC (1)
C. perfringens & Campylobacter spp. (1)
C. perfringens & EAEC (1)
C. perfringens & EPEC (1)
Viral and bacterial (n=16) Norovirus-G2 & C. difficile toxin B (3)
Norovirus-G2 & C. perfringens (1)
Norovirus-G2, C. perfringens & C. difficile toxin B (1)
Norovirus-G2, C. perfringens & EAEC (1)
Norovirus-G2 & EPEC (1)
Rotavirus & Aeromonas app. (2)
Rotavirus & C. difficile toxin B (1)
Rotavirus & C. perfringens (1)
Rotavirus & EAEC (1)
Rotavirus & EPEC (2)
Adenovirus & C. difficile toxin B (1)
Astrovirus & EPEC (1)
Table 5.
Detection of virulence genes of diarrheagenic E. coli by 16-plex PCR [6] and DEC PCR kit [7] from 405 stool specimens in six university hospitals between October 2010 and February 2011
Pathotyp 16-plex PCR DEC PCR kit Agreement (%)
Positive gene(s) No. isolates Positive gene(s) No. isolates
EPEC bfpB, escV 1 - - 99.3%
eaeA 2 - -  
escV, eaeA 4 eae 4  
escV, eaeA, astA 3 eae 3  
escV, eaeA, astA, hly 1 eae 1  
Mixed∗ ent, escV/aggR, astA 2 eae 2  
EHEC escV, hly, stx2 1 vtx2 1 100%
EAEC pic, aggR 5 - - Not available
aggR 2 - -
astA, aggR 1 - -  
pic, astA 1 - -  
pic, aggR, astA 2 - -  
astA† astA 32 - - Not available

∗ Mixed pathotype of EPEC and EAEC;

† astA gene-positive E. coli.

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