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Abstract
Background
Causative bacterial agents of infectious diarrheal disease were traditionally diagnosed by stool cultures. Stool culture, however, has a problem because of relatively low sensitivity and long turnaround time. In this study, we evaluated multiplex PCR applied on stool specimens directly to diagnose enteropathogenic bacteria.
Methods
From June to September 2009, 173 diarrheal stools submitted for stool cultures were tested by SeeplexⓇ Diarrhea ACE Detection kit (Seegene, Korea) to detect 10 enteropathogenic bacteria. Specimens were cultured for Salmonella, Shigella, Vibrio, and Yersinia. Late 50 specimens were also cultured for Campylobacter. The specimens positive for verotoxin-producing Escherichia coli (VTEC) were further subcultured for detecting enterohaemorrhagic Escherichia coli O157:H7. Electronic medical records were reviewed for clinical and laboratory findings.
Results
Of 173 specimens, multiplex PCR and cultures identified enteropathogens in 36 (20.8%) and 8 specimens (4.6%), respectively. While multiplex PCR detected 5 Salmonella, 15 Campylobacter, 1 Vibrio, 4 Clostridium difficiles toxin B, 5 Clostridium perfringens, 1 Yersinia enterocolitica, 5 Aeromonas, and 2 VTEC, cultures detected 5 Salmonella, 1 Vibrio, 1 Y. enterocolitica, 1 Aeromonas, and 2 E. coli O157:H7.
Conclusion
Multiplex PCR would be useful to detect Campylobacter, VTEC and C. perfringens, as well as have equivalent sensitivity to conventional culture for ordinary enteropathogens such as Salmonella, Shigella, Vibrio, Y. enterocolitica. Direct application of multiplex PCR combined with conventional cultures on stool warrants remarkable improvement of sensitivity to diagnose enteropathogenic bacteria.
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Table 1.
Target genes and their product size of multiplex PCR
Table 2.
Distribution of diarrhea pathogens confirmed by multiplex PCR and culture results
| Pathogenic organism | No. (%) of positive specimens | |
|---|---|---|
| Multiplex PCR | Conventional culture | |
| Salmonella | 5 (13.2) | 5, Salmonella B, C, D, E |
| Campylobacter | 15 (39.5) | 0∗ |
| Vibrio | 1 (2.6) | 1, V. parahemolyticus |
| CDB | 4 (10.5) | Not tested |
| Clostridium perfr | ringens 5 (13.2) | Not tested |
| Yersinia enteroco | olitica 1 (2.6) | 1 |
| Aeromonas | 5 (13.2)† | 1, A. sobria |
| E. coli O157:H7 | , VTEC 2 (5.2) | Not tested |
| Total | 36 (100)† | 8 |
Table 3.
Clinical characteristics of PCR-positive patients
| Pathogenic organism | No. of patients | Age | Sex | No. of NI | Fever | Abdominal pain | No. positive/No. tested | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Stool | Peripheral blood | ||||||||||
| ≤15 | ＞15 | M: F | OB | WBC | ↑WBC∗ | ↑CRP† | |||||
| Campylobacter | 15 | 5 | 10 | 2.8: 1 | 0 | 12 | 13 | 5/13 | 6/6 | 5/15 | 13/13 |
| Salmonella | 5 | 3 | 2 | 2:: 3 | 0 | 5 | 5 | 0/5 | 0/5 | 1/5 | 5/5 |
| Aeromonas | 5‡ | 0 | 5 | 4: 1 | 0 | 4 | 4 | 1/2 | 1/5 | 2/5 | 4/5 |
| VTEC | 2 | 1 | 1 | 1: 1 | 0 | 2 | 2 | 1/2 | 1/2 | 1/2 | 2/2 |
| CDB | 4 | 2 | 2 | 1: 1 | 3 | 2 | 2 | 1/4 | 0/3 | 0/4 | 2/4 |
| C. perfringens | 5 | 4 | 1 | 1: 4 | 0 | 0 | 5 | 0/4 | 0/4 | 0/5 | 0/5 |
| Vibro | 1 | 0 | 1 | 0: 1 | 0 | 0 | 1 | 0/1 | 0/1 | 1/1 | 1/1 |
| Y. enterocolitica | 1 | 1 | 0 | 0: 1 | 0 | 1 | 1 | 0/1 | 0/1 | 0/1 | 0/1 |
| Total | 36‡ | 16 | 22 | 1.2: 1 | 3 | 25 | 33 | 8/32 | 8/27 | 11/38 | 29/38 |
‡ No. of patient was corrected, since Aeromonas was detected together in each one patient of Campylobacter and Vibrio, total 2 patients. Abbreviations: NI, nosocomial infection; OB, occult blood; WBC, white blood cell; CRP, C-reactive protein; VTEC, verotoxin-producing E. coli; CDB, C. difficile Toxin B.
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