Genes Encoding Toxin of Clostridium difficile in Children with and without Diarrhea

The presence of gene 16S rRNA and genes encoding toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA/cdtB) of Clostridium difficile in stool samples from children with (110) and without (150) diarrhea was determined by using a TaqMan system. Fifty-seven (21.9%) out of 260 stool samples harbored the 16S rRNA gene. The genetic profile of tcdA+/tcdB− and cdtA+/cdtB+ was verified in one C. difficile-positive diarrhea sample and of tcdA+/tcdB+ in three C. difficile-positive nondiarrhea samples. The presence of tcdA+/tcdB+ in stools obtained from children without diarrhea, suggests that they were asymptomatic carriers of toxigenic strains.


Introduction
Clostridium difficile is the primary etiological agent of antibiotic-associated diarrhea and pseudomembranous colitis and is recognized as a cause of nosocomial diarrhea worldwide [1]. The pathogenic effects of C. difficile are mucosal damage to the colon that is caused by the production of toxin A (308 kDa) and/or toxin B (270 kDa) [2]. Some C. difficile strains also produce a binary toxin encoded by cdtA and cdtB genes, and both genes have been observed in strains producing C. difficile-associated diarrhea (CDAD) [3]. In Brazil, few studies have shown the presence of toxigenic C. difficile harboring tcdA and/or tcdB but not the binary toxin genes in stool samples from children with acute diarrhea [4,5].
The cytotoxic assays performed from stool samples or isolated strains for detecting toxigenic C. difficile are considered the "gold standard" [6]. Other methods such as culture and enzyme immunoassay (EIA) are not specific for the identification of these toxigenic strains, but EIA method is used in most laboratories worldwide [7].
In this study, a quantitative detection of genes encoding toxins A and B and binary toxin directly from C. difficilepositive stool samples of children with and without acute diarrhea was determined.

Sample Collection and DNA Extraction.
Two hundred sixty stool samples were obtained from 110 children with diarrhea and from 150 children without diarrhea, aged from 1 month to 8 years old. Stool samples were collected from March 2008 through November 2010. Children did not display other illnesses or comorbidities than diarrhea, and they were not under antibiotic treatment for at least three months prior to the sample collection. Diarrhea was defined as three or more unformed stools in the 24 h prior to enrollment. The Ethics Committee of the Biomedical Science Institute at the University of Sao Paulo (number 743/CEP) approved this study. Total bacterial genomic DNA was obtained from stools by using a QIAamp DNA stool minikit (Qiagen, Germany) according to the manufacturer's instructions.  F-TTG AGC GAT TTA CTT CGG TAA AGA  R-TGT ACT GGC TCA CCT TTG ATA TTC ND: not determined.
All toxigenic C. difficile-positive stool samples were evaluated for the presence of the toxins A/B with an enzyme immunoassay (EIA) by using a commercial kit Xpect C. difficile Toxin A/B Test (Remel, USA).

PCR Amplification.
Real-time PCR assays were carried out in duplicate and performed in a total volume of 25 L, containing 2X TaqMan Universal Master Mix (Applied Biosystems, USA), 100 M of each primer, 100 M of TaqMan probe, and 2 ng of DNA. Amplifications were performed in a thermal cycler programmed as follows: denaturation at 95 ∘ C for 10 min, followed by 45 cycles of two steps: denaturation at 95 ∘ C for 15 s and an annealing temperature at 60 ∘ C for 1 min, for detecting C. difficile 16S rRNA gene; and annealing temperatures for different toxin-encoding genes were as follows: at 51 ∘ C for detecting tcdA and cdtA genes and at 58 ∘ C for detecting tcdB and cdtB genes. The primer/probe sets used are shown in Table 1.
Bacterial DNA amplifications were adjusted using 2 values > 0.900. A sample was considered positive for a target gene when the detected fluorescence generates a curve above the background fluorescence, which was established by Rotor Gene 6000 analytical software (Corbett Life Science, Australia).

Statistical Analysis.
Mean values ± SD were calculated for each toxin gene using a Student's -test. A difference of < 0.05 was considered statistically significant (GraphPad Prism Software Inc.).

Results
By using a commercial kit Xpect, no toxin-positive stool sample was observed. Fifty-seven (21.9%) out of 260 stool samples analyzed harbored C. difficile (20 from children with diarrhea and 37 from children without diarrhea). Twenty C. difficile-positive diarrhea samples were obtained from children between six months and 7 years old, and, among them, a 1-year-old child harbored the tcdA gene and another 1-year-old child (tcdA−/tcdB−) harbored both binary toxin genes (cdtA and cdtB). Also, the 37 (24.6%) C. difficilepositive nondiarrhea samples were obtained from 1-to-5-year old children, and, of them, three children (a 1 year old and 2 years old) harbored both genes tcdA and tcdB ( Table 2). None of the C. difficile-positive nondiarrhea samples harbored the binary toxin genes.
The presence of the target genes and their respective copy numbers are shown in Table 2. The tcdA was observed in diarrhea sample with a value of log 10 2.1. Similarly, binary toxin genes were observed in log 10 2.7 (cdtA) and log 10 3.7 (cdtB). Moreover, in nondiarrhea samples tcdA was detected from log 10 1.3 to log 10 3.6 and tcdB from log 10 3.2 to log 10 5.3. The specific primer/probe sets here used showed an accurate standard curve over a 5-log-unit linear range that permitted the copy number determination from standard amplifications and detection to a limit of the 100 copies of the target DNA in unknown samples. In this study, C. difficile was detected in diarrhea (18.2%) and nondiarrhea (24.6%) stool samples.

Discussion
In Brazil, there are few epidemiological studies involving this pathogen as a cause of diarrhea in children [4,8]. Since a stool sample harbored the profile of toxigenicity tcdA+/tcdB− and another cdtA+/cdtB+, it suggested a low circulation of toxigenic C. difficile in Brazilian children, and the acute diarrhea in children could have been caused by other enteropathogens than C. difficile, like typical viral or bacterial pathogens, such as Norovirus, Rotavirus, Salmonella, or Campylobacter, and this needs to be investigated.
The presence of toxins A/B and binary toxin-producing C. difficile in diarrhea outbreak has been associated to severe CDAD processes [9]. On the other hand, it is possible that strains can harbor toxins A/B and/or binary toxin genes without any expression, and it could explain the failure to detect the presence of toxins when the EIA was used.
The quantitative PCR by using the TaqMan system provided a direct detection of tcdA/tcdB and cdtA/cdtB from diarrhea and nondiarrhea stool samples, and it could determine a rapid bacterial screening in patients with acute diarrhea, and, due to the high sensitivity to detecting C. difficile strains, this assay has been useful in surveillance of nosocomial diarrhea when required [10,11].
The presence of toxin genes (tcdA+/tcdB+) in stool samples obtained from three children without diarrhea suggests that they were asymptomatic carriers of toxigenic strains. In addition, an asymptomatic colonization by toxigenic C. difficile was recently described in neonates, suggesting that healthy children display some tolerance to toxigenic strains, and it could play a role in the development of the intestinal immune system [12][13][14][15].
Our results suggest that more studies are necessary to determine the real role of toxigenic C. difficile strains and their asymptomatic colonization in the human intestinal tract, mainly in children.