Microbiol Immunol 2014; 58: 615–620 doi: 10.1111/1348-0421.12189
ORIGINAL ARTICLE
Reverse transcription polymerase chain reaction-based method for selectively detecting vegetative cells of toxigenic Clostridium difficile Mitsutoshi Senoh1, Haru Kato1, Tomoko Murase2, Hideharu Hagiya3, Yasuaki Tagashira4, Tadashi Fukuda1, Masaaki Iwaki1, Akihiko Yamamoto1 and Keigo Shibayama1 1
National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan, 2Tsuyama Chuo Hospital, Tsuyama, Okayama, Japan, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Okayama, Japan and 4Tokyo Metropolitan Tama Medical Center, Fuchu, Tokyo, Japan
3
ABSTRACT The laboratory diagnostic methods for Clostridium difficile infection (CDI) include toxigenic culture, enzyme immunoassays (EIAs) to detect the toxins of C. difficile, and nucleic acid amplification tests (NAATs) to detect C. difficile toxin genes, but each of these methods has disadvantages; toxigenic cultures require a long time to produce results, EIAs have low sensitivity, and NAATs that target DNA cannot distinguish vegetative cells from spores and dead cells. Here we report a new detection method that uses reverse transcription polymerase chain reaction to target the toxin-gene transcripts. This method was able to specifically detect the vegetative cells of toxigenic C. difficile in fecal samples in spike tests, with a minimum detection limit of 5 102 colony-forming units per 100 mg of stool specimen. The performance of this method was also demonstrated in a pilot scale evaluation using clinical fecal specimens, which showed that this method may be more sensitive than EIA and requires a shorter time than toxigenic culture. This method could potentially be applied in the clinical laboratory to detect C. difficile in fecal specimens. The ability of this method to discriminate the presence of vegetative cells from spores and dead cells could help to further the understanding of CDI. Key words
Clostridium difficile, Detection method, RT-PCR, Vegetative cells.
Clostridium difficile is the primary cause of healthcareand antibiotic-associated infections in developed countries. Both the incidence and severity of C. difficile infection (CDI) have increased in recent years (1, 2). Toxins A and B, which are encoded by tcdA and tcdB, are the major C. difficile virulence factors (3, 4), and the detection of these proteins or the genes that encode them are common targets in the laboratory diagnosis of CDI. Some C. difficile isolates are also capable of producing another toxin, i.e., binary toxin (5), but this toxin is generally not considered as a target for CDI diagnosis.
The reference methods for the laboratory diagnosis of CDI are the cell cytotoxicity assay, which detects neutralizable toxins in fecal specimens, and toxigenic culture, which establishes whether cultured C. difficile isolates can produce toxins in vitro. However, these methods are associated with a long turnaround time. Enzyme immunoassays (EIAs) that can detect toxin A, toxin B (6, 7), and glutamate dehydrogenase (GDH) (8) by C. difficile are more rapid compared with the reference methods, but EIAs for toxins A and B are less sensitive compared with the reference methods, and EIA for GDH is not able to distinguish between toxigenic and
Correspondence Mitsutoshi Senoh, Department of Bacteriology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan. Tel: 81-42-848-7097; Fax: 81-42-561-7173. email: [email protected] Received 24 June 2014; revised 1 August 2014; accepted 15 August 2014. List of Abbreviations: CCMA, cycloserine cefoxitin mannitol agar; CDI, Clostridium difficile infection; EIA, enzyme immunoassay; GDH, glutamate dehydrogenase; NAAT, nucleic acid amplification test.
© 2014 The Societies and Wiley Publishing Asia Pty Ltd
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nontoxigenic C. difficile. Nucleic acid amplification tests (NAATs) that detect the genes encoding C. difficile toxins, such as tcdA and tcdB, have been utilized in CDI diagnosis (7, 9, 10). These methods have been reported to be more sensitive than both EIAs and toxigenic culture (11, 12), but NAATs detect target DNA derived from all forms of C. difficile including vegetative cells, spores, and dead cells; since the DNA is present not only in vegetative cells but also in the spores and dead cells of C. difficile. In contrast, RNA is less stable and it is present in vegetative cells in large quantities; therefore, novel NAATs that target RNA could enable the detection of vegetative cells without detecting spores and dead cells. In this study, we established a reverse transcription (RT) PCR-based method that only detects the vegetative cells of toxigenic C. difficile in fecal specimens. The symptoms of CDI are due to toxins of C. difficile. It is considered that spores and dead cells of C. difficile do not cause CDI because spores and dead cells are unlikely to produce toxins. Therefore, to distinguish vegetative cells from spores and dead cells of C. difficile might provide useful information in clinical settings.
MATERIALS AND METHODS Bacterial strains and culture C. difficile clinical strains GAI 97660 (toxin A-positive, toxin B-positive, binary toxin-negative), GAI 95601 (toxin A-negative, toxin B-positive, binary toxin-negative), and OG45 (toxin A-positive, toxin B-positive, binary toxin-positive) were used in this study. These strains were anaerobically cultured on Brucella HK agar (Kyokuto Pharmaceutical Industrial Co. Ltd, Tokyo, Japan) plates at 35°C for 48 h. A colony was inoculated into brain–heart infusion (BHI) broth (Becton Dickinson, Sparks, MD) and anaerobically incubated at 35°C for 24 h. Preparation of dead cells and spores of C. difficile The BHI culture of C. difficile (108 CFU/ml) was autoclaved at 121°C for 15 min to prepare dead cells. C. difficile spores were prepared by alcohol treatment as follows. After the C. difficile culture in BHI was aerobically maintained for 2 months at room temperature, C. difficile was collected by centrifugation at 20,000 g for 10 min and soaked in 70% ethanol for 1 h. After another round of centrifugation at 20,000 g for 10 min, the spores were collected, washed twice with phosphatebuffered saline (PBS: Nissui Pharmaceutical Co. Ltd, Tokyo, Japan), and resuspended in 1 ml of PBS. The colony-forming units (CFU) of C. difficile spores were 616
counted using prereduced cycloserine cefoxitin mannitol agar (CCMA) EX plates (Nissui Pharmaceutical Co. Ltd). Gram staining was performed to check for contamination by C. difficile vegetative cells. Spike tests Vegetative cells, spores, and dead cells of the C. difficile strains (105 CFU or equivalent for dead cells) were separately prepared and added to stool specimens (100 mg), which had previously been confirmed as C. difficile-negative. To examine the minimum detection limit of RT-PCR, serial dilutions of vegetative cells of the C. difficile strain GAI 97660 were prepared and added to stool specimens (100 mg), which had previously been confirmed as C. difficile-negative. Stool specimens The study under protocol number 407 was approved by the institutional ethical committee “The Medical Research Ethics Committee of the National Institute of Infectious Diseases for the use of Human Subjects,” and written informed consent was obtained from the patients prior to sample collection as per the committee’s recommendation. Forty-four unformed stool specimens, i.e., one per patient, were collected from patients who had symptoms that were suggestive of CDI, such as diarrhea, and who had been admitted to the two hospitals included in this study. The stool specimens for toxigenic culture, EIA, and PCR were stored at 20°C until testing. Each stool specimen (100 mg) was suspended in 1.0 ml of RNAlater (Ambion, Carlsbad, CA) and stored at 20°C for RT-PCR. The storage steps were performed within 30 min after the stool specimens were collected. Toxigenic culture Cultivation from stool specimens and identification of C. difficile were performed according to the method by Kato et al. (13), with some modifications. The stool specimen (200 mg) was homogenized with 0.2 ml of 99.5% ethanol. The homogenate (100 mL) was cultured on prereduced CCMA EX plates and anaerobically incubated at 35 °C for 48 h. C. difficile-like colonies (yellow colonies) were picked and anaerobically cultured on Brucella HK agar plates at 35°C for 48 h, and these colonies were then transferred to BHI broth and anaerobically cultivated at 35°C for 24 h. The bacterial cultures prepared in this manner were analyzed to determine their toxigenic nature using EIA targeting toxins A and B (C. DIFF QUIK CHEK COMPLETE; Techlab, Blacksburg, VA). Bacterial culture (25 mL) was © 2014 The Societies and Wiley Publishing Asia Pty Ltd
Detecting C. difficile vegetative cells
subjected to EIA. EIA was performed according to the manufacturer’s instructions. EIA-based detection of GDH and toxins A/B in stool specimens The stool specimens were subjected to EIA that targeted the GDH of C. difficile and toxins A and B (C. DIFF QUIK CHEK COMPLETE). The assay was performed according to the manufacturer’s instructions. RNA and DNA extraction Isogen reagent (Nippon Gene Co. Ltd, Tokyo, Japan) was used to extract RNA and DNA from the stool specimens. A stool specimen (100 mg) was suspended in 1.0 ml of Isogen with 300 mg of 0.1 mm diameter glass beads (Yasui Kikai, Osaka, Japan). The suspension was disrupted by shaking at 2,500 rpm for 5 min at 4°C using a Multi-Beads Shocker (Yasui Kikai) and centrifuged at 20,000 g for 5 min at 4°C. After 0.2 ml of chloroform was added to the supernatant, the mixture was shaken vigorously and centrifuged at 20,000 g for 15 min at 4°C. Procedures from this step were performed according to the manufacturer’s instructions including DNase treatment for RNA extraction. One-tenth of total volume of RNA and DNA extracted was used for RT-PCR and PCR. PCR PCR to detect the nonrepeating sequence region of tcdA was performed using Platinum Taq DNA polymerase (Life Technologies, Carlsbad, CA). The primer set, i.e., NK3-NK2 (13, 14), can detect toxin A-positive toxin Bpositive strains as well as toxin A-negative toxin Bpositive strains such as toxinotype VIII because part of tcdA that includes the NK3-NK2 region is not deleted in toxin A-negative toxin B-positive strains (15). The size of the amplicon was 252 bp. After denaturation at 94°C for 2 min, 35 cycles of amplification were performed, each comprised the following: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. The other conditions followed the manufacturer’s instructions.
composed of Cd-lsu-F and Cd-lsu-R was used to detect 23S rRNA sequence region (16). The size of the amplicon was 301 bp. After reverse transcription at 45°C for 30 min and denaturation at 94°C for 2 min, 35 cycles of amplification were performed, each comprised the following: denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The other conditions followed the manufacturer’s instructions. Gel electrophoresis To visualize the RT-PCR and PCR amplicons, gel electrophoresis was performed using a 2% agarose (Nippon Gene Co. Ltd.) gel at a constant voltage of 100 V for 30 min.
RESULTS Spike tests The RT-PCR results were evaluated using spike tests. RNA and DNA were extracted from the stool samples spiked with 105 CFU or equivalent for dead cells and used for RT-PCR and PCR. RT-PCR to target tcdA produced an amplicon only when the stool sample was spiked with vegetative cells of C. difficile strain GAI 97660 (Fig. 1a). In contrast, PCR produced an amplicon from all the stool samples, when all the C. difficile forms were used to spike the sample (Fig. 1b). An amplicon of RT-PCR to target 23S rRNA was produced when the stool sample was spiked with vegetative cells and spores (Fig. 1c). Similar RT-PCR and PCR results were obtained from the stool samples spiked with C. difficile strains GAI 95601 and OG45 (data not shown). When the sample was spiked with dead cells of C. difficle prepared by soaking sodium hypochlorite (available chlorine ¼ 0.5%) for 10 min, RTPCR and PCR results were the same as the cases that the sample was spiked with dead cells of C. difficle prepared by autoclaving (data not shown). PCR was performed using RNA samples to check for contamination with DNA, and no amplicons were observed (data not shown). In addition, the minimum detection limit of RT-PCR for toxin gene was examined. The amplicon was detectable if 100 mg of the stool specimen contained more than 5 102 CFU of vegetative C. difficile (Fig. 2).
RT-PCR RT-PCR to target the same sequence region in tcdA transcript as the PCR was performed using a SuperScript III One-Step RT-PCR kit (Life Technologies) and primer set NK3-NK2. After reverse transcription at 45°C for 30 min and denaturation at 94°C for 2 min, the amplification reactions were performed using the same conditions as those described for PCR. Primer set © 2014 The Societies and Wiley Publishing Asia Pty Ltd
Comparison of RT-PCR for toxin gene with PCR, toxigenic culture, and EIA in clinical stool specimens RT-PCR for toxin gene, PCR, toxigenic culture, and EIA results are shown in Table 1. Among the 44 stool specimens tested, 12 were positive according to all tests and 22 specimens were negative in all tests. Three 617
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Fig. 2. Minimum detection limit of RT-PCR for toxin gene. The CFU of the vegetative cells of C. difficile strain GAI 97660 used to spike each stool specimen were 5 104 (lane 1), 5 103 (lane 2), 5 102 (lane 3), and 5 101 (lane 4). The numbers on the left represent the DNA sizes in base pairs.
and toxin-negative by EIA. The remaining four specimens were classified as negative by RT-PCR for toxin gene, positive by PCR, positive by toxigenic culture, and GDH-positive and toxin-negative by EIA.
DISCUSSION
Fig. 1. Results of the spike tests. (a) RT-PCR targeting tcdA was performed using RNA from Clostridium difficile-negative stool specimens spiked with the vegetative cells (lane 1), spores (lane 2), or dead cells (lane 3) of C. difficile strain GAI 97660. RNA from C. difficile-negative stool specimen was used as negative control (lane 4). (b) PCR targeting tcdA was performed using DNA from C. difficilenegative stool specimens spiked with the vegetative cells (lane 1), spores (lane 2), or dead cells (lane 3) of C. difficile strain GAI 97660. DNA from C. difficile-negative stool specimen was used as negative control (lane 4). (c) RT-PCR targeting 23S rRNA was performed using RNA from Clostridium difficile-negative stool specimens spiked with the vegetative cells (lane 1), spores (lane 2), or dead cells (lane 3) of C. difficile strain GAI 97660. RNA from C. difficile-negative stool specimen was used as negative control (lane 4). The numbers on the left represent the DNA sizes in base pairs.
specimens were negative by RT-PCR for toxin gene and PCR, negative by toxigenic culture, and GDH-positive and toxin-negative by EIA. The analysis of C. difficilelike colonies (yellow colonies) that appeared on CCMA EX from the three specimens showed that they were nontoxigenic C. difficile. The other three specimens were positive by RT-PCR for toxin gene and PCR, positive by toxigenic culture, and GDH-positive or GDH-negative 618
Laboratory methods, such as toxigenic culture, EIAs, and NAATs, are essential tests for the diagnosis of CDI. Toxigenic culture, which employs the cytotoxicity assays developed by Taylor et al. (17), is associated with a long waiting time before the results are known. Although EIAs developed by Yolken et al. (18) obtain results more rapidly, they have low sensitivity. NAATs that target DNA, such as PCR, which were first reported by Gumerlock et al. (19), are gradually replacing more conventional methods for CDI diagnosis. These methods are not only faster but also more sensitive than toxigenic culture and EIAs (20, 21). However, PCR cannot distinguish the vegetative cells from spores and dead cells of C. difficile. Thus, we developed an RT-PCR method based on one of the NAATs as a new method for detecting toxigenic C. difficile in fecal specimens. Spike tests demonstrated that RT-PCR for toxin gene only detected toxigenic C. difficile vegetative cells, but not spores and dead cells; whereas PCR detected all of these. The presence of RNA from spores of C. difficile was confirmed by RT-PCR for 23S rRNA. Comparisons of the RT-PCR for toxin gene, PCR, toxigenic culture, and EIA results showed that 34 out of 44 specimens yielded identical results for all four tests (all positive or negative). These specimens were either toxigenic C. difficile-positive or -negative. The RTPCR results for toxin gene of the specimens, which were positive by PCR, toxigenic culture, and EIA, were also positive. These results suggest that RNA is detectable from clinical specimens collected in hospitals using our method, although RNA is fragile. The results were also © 2014 The Societies and Wiley Publishing Asia Pty Ltd
Detecting C. difficile vegetative cells
Table 1. Comparison of the results of RT-PCR for toxin gene, PCR, toxigenic culture, and EIA EIA detection for Number of stool specimens 12 2 1 4 3† 22
RT-PCR for toxin gene
PCR
Toxigenic culture
GDH
Toxin A/B
þ þ þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
þ
†
Non-toxigenic C. difficile was isolated from these three specimens.
very clear for three specimens classified as positive for GDH detection by EIA and that contained nontoxigenic C. difficile. This suggests that our method could be used to distinguish between toxigenic and nontoxigenic C. difficile. Only the results of EIA to detect toxins A/B in the other three specimens were negative, which was probably because of the low sensitivity of EIA for toxins. Four specimens were negative for RT-PCR for toxin gene and EIA for toxins but positive for PCR, toxigenic culture, and EIA for GDH. These results suggest that the majority of the C. difficile cells in these four specimens were probably spores because of the absence of tcdA RNA. Our RT-PCR method for toxin gene may be more sensitive than EIA, and it is completed within 5 hours in a typical laboratory settings. Furthermore, this method only detected the vegetative cells of C. difficile in fecal specimens, and did not detect spores and dead cells. Distinguishing the presence of vegetative cells from spores and dead cells could lead to further insights into CDI, such as the different symptoms with each form of C. difficile.
ACKNOWLEDGMENTS This study was supported by a grant from the Ministry of Health, Labor, and Welfare, Japan (H23-Shinko-Shitei020).
DISCLOSURE There are no conflicts of interest to declare.
REFERENCES 1. Centers for Disease Control and Prevention (CDC). (2012) Vital signs: preventing Clostridium difficile infections. Morb Mortal Wkly Rep 61: 157–62.
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2. Lucado J., Gould C., Elixhauser A. (2012) Clostridium difficile Infections (CDI) in Hospital Stays, 2009. In: Healthcare Cost and Utilization Project Statistical Briefs. Statistical Brief #124. 3. Kuehne S.A., Cartman S.T., Heap J.T., Kelly M.L., Cockayne A., Minton N.P. (2010) The role of toxin A and toxin B in Clostridium difficile infection. Nature 467: 711–3. 4. Lyras D., O'Connor J.R., Howarth P.M., Sambol S.P., Carter G.P., Phumoonna T., Poon R., Adams V., Vedantam G., Johnson S., Gerding D.N., Rood J.I. (2009) Toxin B is essential for virulence of Clostridium difficile. Nature 458: 1176–9. 5. Gerding D.N., Johnson S., Rupnik M., Aktories K. (2014) Clostridium difficile binary toxin CDT: Mechanism, epidemiology, and potential clinical importance. Gut Microbes 5: 15–27. 6. Alcalá L., Sánchez-Cambronero L., Catalán M.P., SánchezSomolinos M., Peláez M.T., Marín M., Bouza E. (2008) Comparison of three commercial methods for rapid detection of Clostridium difficile toxins A and B from fecal specimens. J Clin Microbiol 46: 3833–5. 7. Sloan L.M., Duresko B.J., Gustafson D.R., Rosenblatt J.E. (2008) Comparison of real-time PCR for detection of the tcdC gene with four toxin immunoassays and culture in diagnosis of Clostridium difficile infection. J Clin Microbiol 46: 1996–2001. 8. Sharp S.E., Ruden L.O., Pohl J.C., Hatcher P.A., Jayne L.M., Ivie W.M. (2010) Evaluation of the C. Diff Quik Chek Complete Assay, a new glutamate dehydrogenase and A/B toxin combination lateral flow assay for use in rapid, simple diagnosis of Clostridium difficile disease. J Clin Microbiol 48: 2082–6. 9. Kim H., Jeong S.H., Kim M., Lee Y., Lee K. (2012) Detection of Clostridium difficile toxin A/B genes by multiplex real-time PCR for the diagnosis of C. difficile infection. J Med Microbiol 61: 274–7. 10. Pallis A., Jazayeri J., Ward P., Dimovski K., Svobodova S. (2013) Rapid detection of Clostridium difficile toxins from stool samples using real-time multiplex PCR. J Med Microbiol 62: 1350–6. 11. Dubberke E.R., Han Z., Bobo L., Hink T., Lawrence B., Copper S., Hoppe-Bauer J., Burnham C.A., Dunne W.M., Jr. (2011) Impact of clinical symptoms on interpretation of diagnostic assays for Clostridium difficile infections. J Clin Microbiol 49: 2887–93. 12. Eastwood K., Else P., Charlett A., Wilcox M. (2009) Comparison of nine commercially available Clostridium difficile toxin detection assays, a real-time PCR assay for C. difficile tcdB, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods. J Clin Microbiol 47: 3211–7. 13. Kato H., Kato N., Watanabe K., Iwai N., Nakamura H., Yamamoto T., Suzuki K., Kim S.M., Chong Y., Wasito E.B. (1998) Identification of toxin A-negative, toxin B-positive Clostridium difficile by PCR. J Clin Microbiol 36: 2178–82. 14. Kato N., Ou C.Y., Kato H., Bartley S.L., Brown V.K., Dowell V.R., Jr., Ueno K. (1991) Identification of toxigenic Clostridium difficile by the polymerase chain reaction. J Clin Microbiol 29: 33–7. 15. Rupnik M., Kato N., Grabnar M., Kato H. (2003) New types of toxin A-negative, toxin B-positive strains among Clostridium difficile isolates from Asia. J Clin Microbiol 41: 1118–25. 16. Matsuda K., Tsuji H., Asahara T., Takahashi T., Kubota H., Nagata S., Yamashiro Y., Nomoto K. (2012) Sensitive quantification of Clostridium difficile cells by reverse transcription-quantitative PCR targeting rRNA molecules. Appl Environ Microbiol 78: 5111–8.
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17. Taylor N.S., Bartlett J.G. (1979) Partial purification and characterization of a cytotoxin from Clostridium difficile. Rev Infect Dis 1: 379–85. 18. Yolken R.H., Whitcomb L.S., Marien G., Bartlett J.D., Libby J., Ehrich M., Wilkins T. (1981) Enzyme immunoassay for the detection of Clostridium difficile antigen. J Infect Dis 144: 378. 19. Gumerlock P.H., Tang Y.J., Meyers F.J., Silva J., Jr. (1991) Use of the polymerase chain reaction for the specific and direct
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detection of Clostridium difficile in human feces. Rev Infect Dis 13: 1053–60. 20. Humphries R.M., Uslan D.Z., Rubin Z. (2013) Performance of Clostridium difficile toxin enzyme immunoassay and nucleic acid amplification tests stratified by patient disease severity. J Clin Microbiol 51: 869–73. 21. Chapin K.C., Dickenson R.A., Wu F., Andrea S.B. (2011) Comparison of five assays for detection of Clostridium difficile toxin. J Mol Diagn 13: 395–400.
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ORIGINAL ARTICLE
Reverse transcription polymerase chain reaction-based method for selectively detecting vegetative cells of toxigenic Clostridium difficile Mitsutoshi Senoh1, Haru Kato1, Tomoko Murase2, Hideharu Hagiya3, Yasuaki Tagashira4, Tadashi Fukuda1, Masaaki Iwaki1, Akihiko Yamamoto1 and Keigo Shibayama1 1
National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan, 2Tsuyama Chuo Hospital, Tsuyama, Okayama, Japan, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Okayama, Japan and 4Tokyo Metropolitan Tama Medical Center, Fuchu, Tokyo, Japan
3
ABSTRACT The laboratory diagnostic methods for Clostridium difficile infection (CDI) include toxigenic culture, enzyme immunoassays (EIAs) to detect the toxins of C. difficile, and nucleic acid amplification tests (NAATs) to detect C. difficile toxin genes, but each of these methods has disadvantages; toxigenic cultures require a long time to produce results, EIAs have low sensitivity, and NAATs that target DNA cannot distinguish vegetative cells from spores and dead cells. Here we report a new detection method that uses reverse transcription polymerase chain reaction to target the toxin-gene transcripts. This method was able to specifically detect the vegetative cells of toxigenic C. difficile in fecal samples in spike tests, with a minimum detection limit of 5 102 colony-forming units per 100 mg of stool specimen. The performance of this method was also demonstrated in a pilot scale evaluation using clinical fecal specimens, which showed that this method may be more sensitive than EIA and requires a shorter time than toxigenic culture. This method could potentially be applied in the clinical laboratory to detect C. difficile in fecal specimens. The ability of this method to discriminate the presence of vegetative cells from spores and dead cells could help to further the understanding of CDI. Key words
Clostridium difficile, Detection method, RT-PCR, Vegetative cells.
Clostridium difficile is the primary cause of healthcareand antibiotic-associated infections in developed countries. Both the incidence and severity of C. difficile infection (CDI) have increased in recent years (1, 2). Toxins A and B, which are encoded by tcdA and tcdB, are the major C. difficile virulence factors (3, 4), and the detection of these proteins or the genes that encode them are common targets in the laboratory diagnosis of CDI. Some C. difficile isolates are also capable of producing another toxin, i.e., binary toxin (5), but this toxin is generally not considered as a target for CDI diagnosis.
The reference methods for the laboratory diagnosis of CDI are the cell cytotoxicity assay, which detects neutralizable toxins in fecal specimens, and toxigenic culture, which establishes whether cultured C. difficile isolates can produce toxins in vitro. However, these methods are associated with a long turnaround time. Enzyme immunoassays (EIAs) that can detect toxin A, toxin B (6, 7), and glutamate dehydrogenase (GDH) (8) by C. difficile are more rapid compared with the reference methods, but EIAs for toxins A and B are less sensitive compared with the reference methods, and EIA for GDH is not able to distinguish between toxigenic and
Correspondence Mitsutoshi Senoh, Department of Bacteriology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan. Tel: 81-42-848-7097; Fax: 81-42-561-7173. email: [email protected] Received 24 June 2014; revised 1 August 2014; accepted 15 August 2014. List of Abbreviations: CCMA, cycloserine cefoxitin mannitol agar; CDI, Clostridium difficile infection; EIA, enzyme immunoassay; GDH, glutamate dehydrogenase; NAAT, nucleic acid amplification test.
© 2014 The Societies and Wiley Publishing Asia Pty Ltd
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nontoxigenic C. difficile. Nucleic acid amplification tests (NAATs) that detect the genes encoding C. difficile toxins, such as tcdA and tcdB, have been utilized in CDI diagnosis (7, 9, 10). These methods have been reported to be more sensitive than both EIAs and toxigenic culture (11, 12), but NAATs detect target DNA derived from all forms of C. difficile including vegetative cells, spores, and dead cells; since the DNA is present not only in vegetative cells but also in the spores and dead cells of C. difficile. In contrast, RNA is less stable and it is present in vegetative cells in large quantities; therefore, novel NAATs that target RNA could enable the detection of vegetative cells without detecting spores and dead cells. In this study, we established a reverse transcription (RT) PCR-based method that only detects the vegetative cells of toxigenic C. difficile in fecal specimens. The symptoms of CDI are due to toxins of C. difficile. It is considered that spores and dead cells of C. difficile do not cause CDI because spores and dead cells are unlikely to produce toxins. Therefore, to distinguish vegetative cells from spores and dead cells of C. difficile might provide useful information in clinical settings.
MATERIALS AND METHODS Bacterial strains and culture C. difficile clinical strains GAI 97660 (toxin A-positive, toxin B-positive, binary toxin-negative), GAI 95601 (toxin A-negative, toxin B-positive, binary toxin-negative), and OG45 (toxin A-positive, toxin B-positive, binary toxin-positive) were used in this study. These strains were anaerobically cultured on Brucella HK agar (Kyokuto Pharmaceutical Industrial Co. Ltd, Tokyo, Japan) plates at 35°C for 48 h. A colony was inoculated into brain–heart infusion (BHI) broth (Becton Dickinson, Sparks, MD) and anaerobically incubated at 35°C for 24 h. Preparation of dead cells and spores of C. difficile The BHI culture of C. difficile (108 CFU/ml) was autoclaved at 121°C for 15 min to prepare dead cells. C. difficile spores were prepared by alcohol treatment as follows. After the C. difficile culture in BHI was aerobically maintained for 2 months at room temperature, C. difficile was collected by centrifugation at 20,000 g for 10 min and soaked in 70% ethanol for 1 h. After another round of centrifugation at 20,000 g for 10 min, the spores were collected, washed twice with phosphatebuffered saline (PBS: Nissui Pharmaceutical Co. Ltd, Tokyo, Japan), and resuspended in 1 ml of PBS. The colony-forming units (CFU) of C. difficile spores were 616
counted using prereduced cycloserine cefoxitin mannitol agar (CCMA) EX plates (Nissui Pharmaceutical Co. Ltd). Gram staining was performed to check for contamination by C. difficile vegetative cells. Spike tests Vegetative cells, spores, and dead cells of the C. difficile strains (105 CFU or equivalent for dead cells) were separately prepared and added to stool specimens (100 mg), which had previously been confirmed as C. difficile-negative. To examine the minimum detection limit of RT-PCR, serial dilutions of vegetative cells of the C. difficile strain GAI 97660 were prepared and added to stool specimens (100 mg), which had previously been confirmed as C. difficile-negative. Stool specimens The study under protocol number 407 was approved by the institutional ethical committee “The Medical Research Ethics Committee of the National Institute of Infectious Diseases for the use of Human Subjects,” and written informed consent was obtained from the patients prior to sample collection as per the committee’s recommendation. Forty-four unformed stool specimens, i.e., one per patient, were collected from patients who had symptoms that were suggestive of CDI, such as diarrhea, and who had been admitted to the two hospitals included in this study. The stool specimens for toxigenic culture, EIA, and PCR were stored at 20°C until testing. Each stool specimen (100 mg) was suspended in 1.0 ml of RNAlater (Ambion, Carlsbad, CA) and stored at 20°C for RT-PCR. The storage steps were performed within 30 min after the stool specimens were collected. Toxigenic culture Cultivation from stool specimens and identification of C. difficile were performed according to the method by Kato et al. (13), with some modifications. The stool specimen (200 mg) was homogenized with 0.2 ml of 99.5% ethanol. The homogenate (100 mL) was cultured on prereduced CCMA EX plates and anaerobically incubated at 35 °C for 48 h. C. difficile-like colonies (yellow colonies) were picked and anaerobically cultured on Brucella HK agar plates at 35°C for 48 h, and these colonies were then transferred to BHI broth and anaerobically cultivated at 35°C for 24 h. The bacterial cultures prepared in this manner were analyzed to determine their toxigenic nature using EIA targeting toxins A and B (C. DIFF QUIK CHEK COMPLETE; Techlab, Blacksburg, VA). Bacterial culture (25 mL) was © 2014 The Societies and Wiley Publishing Asia Pty Ltd
Detecting C. difficile vegetative cells
subjected to EIA. EIA was performed according to the manufacturer’s instructions. EIA-based detection of GDH and toxins A/B in stool specimens The stool specimens were subjected to EIA that targeted the GDH of C. difficile and toxins A and B (C. DIFF QUIK CHEK COMPLETE). The assay was performed according to the manufacturer’s instructions. RNA and DNA extraction Isogen reagent (Nippon Gene Co. Ltd, Tokyo, Japan) was used to extract RNA and DNA from the stool specimens. A stool specimen (100 mg) was suspended in 1.0 ml of Isogen with 300 mg of 0.1 mm diameter glass beads (Yasui Kikai, Osaka, Japan). The suspension was disrupted by shaking at 2,500 rpm for 5 min at 4°C using a Multi-Beads Shocker (Yasui Kikai) and centrifuged at 20,000 g for 5 min at 4°C. After 0.2 ml of chloroform was added to the supernatant, the mixture was shaken vigorously and centrifuged at 20,000 g for 15 min at 4°C. Procedures from this step were performed according to the manufacturer’s instructions including DNase treatment for RNA extraction. One-tenth of total volume of RNA and DNA extracted was used for RT-PCR and PCR. PCR PCR to detect the nonrepeating sequence region of tcdA was performed using Platinum Taq DNA polymerase (Life Technologies, Carlsbad, CA). The primer set, i.e., NK3-NK2 (13, 14), can detect toxin A-positive toxin Bpositive strains as well as toxin A-negative toxin Bpositive strains such as toxinotype VIII because part of tcdA that includes the NK3-NK2 region is not deleted in toxin A-negative toxin B-positive strains (15). The size of the amplicon was 252 bp. After denaturation at 94°C for 2 min, 35 cycles of amplification were performed, each comprised the following: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. The other conditions followed the manufacturer’s instructions.
composed of Cd-lsu-F and Cd-lsu-R was used to detect 23S rRNA sequence region (16). The size of the amplicon was 301 bp. After reverse transcription at 45°C for 30 min and denaturation at 94°C for 2 min, 35 cycles of amplification were performed, each comprised the following: denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The other conditions followed the manufacturer’s instructions. Gel electrophoresis To visualize the RT-PCR and PCR amplicons, gel electrophoresis was performed using a 2% agarose (Nippon Gene Co. Ltd.) gel at a constant voltage of 100 V for 30 min.
RESULTS Spike tests The RT-PCR results were evaluated using spike tests. RNA and DNA were extracted from the stool samples spiked with 105 CFU or equivalent for dead cells and used for RT-PCR and PCR. RT-PCR to target tcdA produced an amplicon only when the stool sample was spiked with vegetative cells of C. difficile strain GAI 97660 (Fig. 1a). In contrast, PCR produced an amplicon from all the stool samples, when all the C. difficile forms were used to spike the sample (Fig. 1b). An amplicon of RT-PCR to target 23S rRNA was produced when the stool sample was spiked with vegetative cells and spores (Fig. 1c). Similar RT-PCR and PCR results were obtained from the stool samples spiked with C. difficile strains GAI 95601 and OG45 (data not shown). When the sample was spiked with dead cells of C. difficle prepared by soaking sodium hypochlorite (available chlorine ¼ 0.5%) for 10 min, RTPCR and PCR results were the same as the cases that the sample was spiked with dead cells of C. difficle prepared by autoclaving (data not shown). PCR was performed using RNA samples to check for contamination with DNA, and no amplicons were observed (data not shown). In addition, the minimum detection limit of RT-PCR for toxin gene was examined. The amplicon was detectable if 100 mg of the stool specimen contained more than 5 102 CFU of vegetative C. difficile (Fig. 2).
RT-PCR RT-PCR to target the same sequence region in tcdA transcript as the PCR was performed using a SuperScript III One-Step RT-PCR kit (Life Technologies) and primer set NK3-NK2. After reverse transcription at 45°C for 30 min and denaturation at 94°C for 2 min, the amplification reactions were performed using the same conditions as those described for PCR. Primer set © 2014 The Societies and Wiley Publishing Asia Pty Ltd
Comparison of RT-PCR for toxin gene with PCR, toxigenic culture, and EIA in clinical stool specimens RT-PCR for toxin gene, PCR, toxigenic culture, and EIA results are shown in Table 1. Among the 44 stool specimens tested, 12 were positive according to all tests and 22 specimens were negative in all tests. Three 617
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Fig. 2. Minimum detection limit of RT-PCR for toxin gene. The CFU of the vegetative cells of C. difficile strain GAI 97660 used to spike each stool specimen were 5 104 (lane 1), 5 103 (lane 2), 5 102 (lane 3), and 5 101 (lane 4). The numbers on the left represent the DNA sizes in base pairs.
and toxin-negative by EIA. The remaining four specimens were classified as negative by RT-PCR for toxin gene, positive by PCR, positive by toxigenic culture, and GDH-positive and toxin-negative by EIA.
DISCUSSION
Fig. 1. Results of the spike tests. (a) RT-PCR targeting tcdA was performed using RNA from Clostridium difficile-negative stool specimens spiked with the vegetative cells (lane 1), spores (lane 2), or dead cells (lane 3) of C. difficile strain GAI 97660. RNA from C. difficile-negative stool specimen was used as negative control (lane 4). (b) PCR targeting tcdA was performed using DNA from C. difficilenegative stool specimens spiked with the vegetative cells (lane 1), spores (lane 2), or dead cells (lane 3) of C. difficile strain GAI 97660. DNA from C. difficile-negative stool specimen was used as negative control (lane 4). (c) RT-PCR targeting 23S rRNA was performed using RNA from Clostridium difficile-negative stool specimens spiked with the vegetative cells (lane 1), spores (lane 2), or dead cells (lane 3) of C. difficile strain GAI 97660. RNA from C. difficile-negative stool specimen was used as negative control (lane 4). The numbers on the left represent the DNA sizes in base pairs.
specimens were negative by RT-PCR for toxin gene and PCR, negative by toxigenic culture, and GDH-positive and toxin-negative by EIA. The analysis of C. difficilelike colonies (yellow colonies) that appeared on CCMA EX from the three specimens showed that they were nontoxigenic C. difficile. The other three specimens were positive by RT-PCR for toxin gene and PCR, positive by toxigenic culture, and GDH-positive or GDH-negative 618
Laboratory methods, such as toxigenic culture, EIAs, and NAATs, are essential tests for the diagnosis of CDI. Toxigenic culture, which employs the cytotoxicity assays developed by Taylor et al. (17), is associated with a long waiting time before the results are known. Although EIAs developed by Yolken et al. (18) obtain results more rapidly, they have low sensitivity. NAATs that target DNA, such as PCR, which were first reported by Gumerlock et al. (19), are gradually replacing more conventional methods for CDI diagnosis. These methods are not only faster but also more sensitive than toxigenic culture and EIAs (20, 21). However, PCR cannot distinguish the vegetative cells from spores and dead cells of C. difficile. Thus, we developed an RT-PCR method based on one of the NAATs as a new method for detecting toxigenic C. difficile in fecal specimens. Spike tests demonstrated that RT-PCR for toxin gene only detected toxigenic C. difficile vegetative cells, but not spores and dead cells; whereas PCR detected all of these. The presence of RNA from spores of C. difficile was confirmed by RT-PCR for 23S rRNA. Comparisons of the RT-PCR for toxin gene, PCR, toxigenic culture, and EIA results showed that 34 out of 44 specimens yielded identical results for all four tests (all positive or negative). These specimens were either toxigenic C. difficile-positive or -negative. The RTPCR results for toxin gene of the specimens, which were positive by PCR, toxigenic culture, and EIA, were also positive. These results suggest that RNA is detectable from clinical specimens collected in hospitals using our method, although RNA is fragile. The results were also © 2014 The Societies and Wiley Publishing Asia Pty Ltd
Detecting C. difficile vegetative cells
Table 1. Comparison of the results of RT-PCR for toxin gene, PCR, toxigenic culture, and EIA EIA detection for Number of stool specimens 12 2 1 4 3† 22
RT-PCR for toxin gene
PCR
Toxigenic culture
GDH
Toxin A/B
þ þ þ
þ þ þ þ
þ þ þ þ
þ þ þ þ
þ
†
Non-toxigenic C. difficile was isolated from these three specimens.
very clear for three specimens classified as positive for GDH detection by EIA and that contained nontoxigenic C. difficile. This suggests that our method could be used to distinguish between toxigenic and nontoxigenic C. difficile. Only the results of EIA to detect toxins A/B in the other three specimens were negative, which was probably because of the low sensitivity of EIA for toxins. Four specimens were negative for RT-PCR for toxin gene and EIA for toxins but positive for PCR, toxigenic culture, and EIA for GDH. These results suggest that the majority of the C. difficile cells in these four specimens were probably spores because of the absence of tcdA RNA. Our RT-PCR method for toxin gene may be more sensitive than EIA, and it is completed within 5 hours in a typical laboratory settings. Furthermore, this method only detected the vegetative cells of C. difficile in fecal specimens, and did not detect spores and dead cells. Distinguishing the presence of vegetative cells from spores and dead cells could lead to further insights into CDI, such as the different symptoms with each form of C. difficile.
ACKNOWLEDGMENTS This study was supported by a grant from the Ministry of Health, Labor, and Welfare, Japan (H23-Shinko-Shitei020).
DISCLOSURE There are no conflicts of interest to declare.
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