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.
REFERENCES
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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 |
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]
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) |
Table 4.
Pattern of co-infecting diarrheagenic pathogens in 32 stool specimens detected by multiplex PCR assay
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 |