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Development of TaqMan-based quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools.

Kubota H, Sakai T, Gawad A, Makino H, Akiyama T, Ishikawa E, Oishi K - PLoS ONE (2014)

Bottom Line: CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method.Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively.Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

View Article: PubMed Central - PubMed

Affiliation: Yakult Honsha European Research Center for Microbiology ESV, Gent-Zwijnaarde, Belgium; Yakult Central Institute, Tokyo, Japan.

ABSTRACT

Background: Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.

Methods: TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).

Results: The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA-TcdB+, and TcdA-TcdB- types), TcdB-producing strains (TcdA+TcdB+ and TcdA-TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA-TcdB- strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.

Conclusion: Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

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Related in: MedlinePlus

C. difficile carriage rates in four nursing home populations.On the basis of the qPCR counts for three genes (16S rRNA, tcdA, and tcdB), the toxigenic types (A+B+, A−B+, or A−B−) of C. difficile predominating in individual specimens were identified. The rates of carriage of each toxigenic type of C. difficile at three stool samplings (S1, S2, and S3) were calculated with respect to each nursing home (n = 11, 14, 24, and 33, respectively) and the total population (n = 82).
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pone-0111684-g002: C. difficile carriage rates in four nursing home populations.On the basis of the qPCR counts for three genes (16S rRNA, tcdA, and tcdB), the toxigenic types (A+B+, A−B+, or A−B−) of C. difficile predominating in individual specimens were identified. The rates of carriage of each toxigenic type of C. difficile at three stool samplings (S1, S2, and S3) were calculated with respect to each nursing home (n = 11, 14, 24, and 33, respectively) and the total population (n = 82).

Mentions: We obtained detection rates and qPCR counts of whole C. difficile, TcdB-producing strains, and TcdA-producing strains in elderly residents of different nursing homes at three samplings (S1∶82 specimens; S2∶79 specimens; and S3∶74 specimens) (See Table S2). Detection rates of whole C. difficile in S1, S2, and S3 specimens from the four nursing homes were 2/82 (2.4%), 5/79 (6.3%), and 5/74 (6.8%), respectively. The detection rates of TcdA-producing strains were 1/82 (1.2%), 3/79 (3.8%), and 2/72 (2.7%), respectively, and those of TcdB-producing strains were exactly the same. On the basis of the qPCR counts, we determined the toxigenic types of the predominating C. difficile in each specimen; the rates of carriage of the respective types by the 82 subjects are shown in Figure 2. Whereas site 01 had no C. difficile carrier throughout the 6-month period, the other sites had some carriers of either the A+B+ strain or the A−B− strain, or both. The overall carriage rate of C. difficile in the 82 subjects fluctuated below 10% during the 6-month test period, except in one case (S3 at site 02).


Development of TaqMan-based quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools.

Kubota H, Sakai T, Gawad A, Makino H, Akiyama T, Ishikawa E, Oishi K - PLoS ONE (2014)

C. difficile carriage rates in four nursing home populations.On the basis of the qPCR counts for three genes (16S rRNA, tcdA, and tcdB), the toxigenic types (A+B+, A−B+, or A−B−) of C. difficile predominating in individual specimens were identified. The rates of carriage of each toxigenic type of C. difficile at three stool samplings (S1, S2, and S3) were calculated with respect to each nursing home (n = 11, 14, 24, and 33, respectively) and the total population (n = 82).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216139&req=5

pone-0111684-g002: C. difficile carriage rates in four nursing home populations.On the basis of the qPCR counts for three genes (16S rRNA, tcdA, and tcdB), the toxigenic types (A+B+, A−B+, or A−B−) of C. difficile predominating in individual specimens were identified. The rates of carriage of each toxigenic type of C. difficile at three stool samplings (S1, S2, and S3) were calculated with respect to each nursing home (n = 11, 14, 24, and 33, respectively) and the total population (n = 82).
Mentions: We obtained detection rates and qPCR counts of whole C. difficile, TcdB-producing strains, and TcdA-producing strains in elderly residents of different nursing homes at three samplings (S1∶82 specimens; S2∶79 specimens; and S3∶74 specimens) (See Table S2). Detection rates of whole C. difficile in S1, S2, and S3 specimens from the four nursing homes were 2/82 (2.4%), 5/79 (6.3%), and 5/74 (6.8%), respectively. The detection rates of TcdA-producing strains were 1/82 (1.2%), 3/79 (3.8%), and 2/72 (2.7%), respectively, and those of TcdB-producing strains were exactly the same. On the basis of the qPCR counts, we determined the toxigenic types of the predominating C. difficile in each specimen; the rates of carriage of the respective types by the 82 subjects are shown in Figure 2. Whereas site 01 had no C. difficile carrier throughout the 6-month period, the other sites had some carriers of either the A+B+ strain or the A−B− strain, or both. The overall carriage rate of C. difficile in the 82 subjects fluctuated below 10% during the 6-month test period, except in one case (S3 at site 02).

Bottom Line: CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method.Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively.Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

View Article: PubMed Central - PubMed

Affiliation: Yakult Honsha European Research Center for Microbiology ESV, Gent-Zwijnaarde, Belgium; Yakult Central Institute, Tokyo, Japan.

ABSTRACT

Background: Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.

Methods: TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).

Results: The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA+TcdB+, TcdA-TcdB+, and TcdA-TcdB- types), TcdB-producing strains (TcdA+TcdB+ and TcdA-TcdB+ types), and TcdA-producing strains (TcdA+TcdB+ type), respectively, with a lower detection limit of 103 cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA+TcdB+ strain in six specimens and TcdA-TcdB- strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 104.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.

Conclusion: Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.

Show MeSH
Related in: MedlinePlus