Clostridium difficile is the most common cause of Antibiotic-Associated Diarrhea and Colitis, occurring after exposure to antibiotics . Although there are many diagnostic methods ,the nucleic acid-based approach has been largely performed in several laboratories, due to its high sensitivity and specificity as well as rapid . This is the first study in Iraq for detection of toxins A&amp;B genes, and to focuses on diagnosis of C. difficile by PCR method to confirmed isolation were tested previously by culturing , Api20A and ELISA methods .Results show that all 75 positive isolation previously tested ,were having toxin A(tcdA) and toxinB(tcdB) genes . The results suggested that the combination of sample processing with the high-performance detection method would be applicable for routine diagnostic use in clinical setting. Keywords: Clostridium difficile , PCR, Api20A , ELISA  

AL-Mossawei, Mona Turkey

Mehdi, Luma  Yousif

English

International Institute for Science, Technology and Education (IISTE): E-Journals

Journal of Health, Medicine and Nursing                                                                                                                                          www.iiste.org ISSN 2422-8419     An International Peer-reviewed Journal Vol.15, 2015  58 PCR for Detection of Clostridium difficile toxin A (tcdA)and toxin B (tcdB)  genes  in  Iraq   Luma  Yousif Mehdi1 ⃰              Mona Turkey AL-Mossawei2 1, College of Health  and Medical Technology , Baghdad ,Iraq. 2, Biology Department , College of  Science for Women ,Baghdad University , Iraq.  Abstract Clostridium difficile is the most common cause of Antibiotic-Associated Diarrhea and Colitis, occurring after exposure to antibiotics . Although there are many diagnostic methods ,the nucleic acid-based approach has been largely performed in several laboratories, due to its high sensitivity and specificity as well as rapid . This is the first study in Iraq for detection of toxins A&B genes, and to focuses on diagnosis of C. difficile by PCR method to confirmed isolation were tested previously by culturing , Api20A and ELISA methods .Results show that all 75 positive isolation previously tested ,were having toxin A(tcdA) and toxinB(tcdB) genes . The results suggested that the combination of sample processing with the high-performance detection method would be applicable for routine diagnostic use in clinical setting. Keywords:  Clostridium difficile , PCR, Api20A , ELISA  .  INTRODUCTION Clostridium difficile is a motile, rod-shaped, Gram-positive bacterium, which is known to be a leading cause of antibiotic associated diarrhea, especially nosocomial infections [1]. Two large toxins, TcdA enterotoxin and TcdB cytotoxine(308 kDa and 270 kDa, respectively), are recognized as the main virulence factors of C. difficile disrupting tight junctions of the intestinal epithelial cells resulting in inflammation and increased permeability of the intestine [2]. The two genes are part of the PaLoc operon, which also contains tcdR, tcdE and tcdC, of which tcdC is a putative negative regulator of tcdA and tcdB [3]. C. difficile infection results in a wide range of symptoms including fever, abdominal pains, mild diarrhea, and pseudomembranous colitis ,the hypervirulent strains that are resistant to current therapy , are able to produce high titers of toxins poses a challenge to the treatment of infection worldwide [4]. C. difficile can be diagnosed by culturing stool samples on selective media, and toxigenic strains producing TcdA and ⁄ or TcdB may subsequently be identified by ELISA. Diagnostic strategies targeting nucleic acids, including PCR methods [5,6,7] and real-time PCR methods [8,9], have been developed for the detection of the genes encoding TcdA and ⁄ or TcdB. This is the first study in Iraq, that detection of toxin A and B by ELISA and PCR method.  AIM OF STUDY The study was designed to evaluate PCR method to confirmation diagnosis of C.difficile by previous methods .   MATERIALS AND METHODS Samples : Four hundred thirty ,the total stool samples were collected during the period of first of June 2013 , till the end of April 2014, from Iraqi patient ,children and adults suffering from antibiotic associated diarrhea and colitis ,and apparently healthy children and adults as showing in table (1) .  Primary identification Stool samples were streaked on selective media( CCFA ) (Sigma Aldrich, UK)+7%horse blood as[ 10] ,incubation in anaerobic conditions at 37C0 for 48hrs ,and isolates were presumptively identified (Gram stain , and Malachite green for spore ) ,definitive identification was performed by Api20A kit (BioMerieux ,USA), and detection of two toxins A& B in stool samples by ELISA Kit (Premier toxin A&B from Meridian Bioscience ,USA), according to the manufacturer’s recommendations. Genomic DNA of all 75 positive isolation were tested by previously methods, were extracted and analyzed with PCR. The aim of the present study was to use new molecular methods for conformation the detection of pathogenic C. difficile isolates, including PCR for detecting the genes encoding TcdA, TcdB. Journal of Health, Medicine and Nursing                                                                                                                                          www.iiste.org ISSN 2422-8419     An International Peer-reviewed Journal Vol.15, 2015  59 Table (1): Distribution of study samples . Age Total No. of patient samples Positive No. of patient isolates Total No. of healthy samples  Positive No. of healthy isolates Children (after birth -- 15 years) 240 51 80 12 Adults (16 to ≥ 61) 70 11 40 1 Total 310 62 120 13  DNA extraction: Bacterial genomic DNA was extracted from BHI broth samples by employment of Genomic DNA Purification kit (Promega, USA).  PCR amplification: The primers used were tcdA (forward, , 5'-GCA TGA TAA GGC AAC TTC AGT GGT A--3' and (reverse, 5'- AGT TCC TCC TGC TCC ATC AAA TG-3') primer pairs (Alpha DNA, Canada) which were described previously [11,12]. PCR amplifications were carried out in total reaction volumes of 25µl containing 1 µl of each primer, 5µl of Master mix (Accu power® PCR PreMix ,BioNeer ,Korea), 6µl DNA template and 13 µl of ultra-pure distilled water. The amplification thermo cycle parameters condition were 5min at 95C0,followed by 35 cycles of 30s at 95C0 ,1min at 53 C0 and 1min at 72C0,and final extention of 5 min at 72Co . Expected products of amplification are 625bp[11]. The primers used were tcdB (forward, 5'-CCA AAG TGG AGT GTT ACA AAC AGG TG-3') and (reverse, 5'- GCA TTT CTC CGT TTT CAG CAA AGT A-3') primer pairs (Alpha, Canada) which were described previously [11,12]. PCR amplifications were carried out in total reaction volumes of 25µl containing 1 µl of each primer, 5µl of Master mix (Accu power® PCR PreMix, BioNeer ,Korea), 6µl DNA template and13 µl of ultra-pure distilled water. The amplification thermo cycle parameters condition were 5min at 95C0,followed by 35 cycles of 30s at 95C0 ,1min at 56 C0 and 1min at 72C0,and final extention of 5 min at 72Co . Expected products of amplification are 410bp[11]. Samples were loaded 7 µl carefully into the individual wells of gel( 1% w\v agarose, Promega ,USA)stained with ethidium bromide (5 µl \100ml), and then electrical power was turned on at 70volt for 1 hrs .  Results and Discussion Clostridium difficile is the most common causative agent of primary and recurrent antibiotic-associated diarrhea and colitis in hospitalized patients[13]. The disease is caused mainly by two exotoxins, TcdA and TcdB, produced by the bacteria. The symptoms of C. difficile infection range from asymptomatic colonization to mild diarrhea and severe life threatening pseudomembranous colitis, a cause of antibiotic-associated diarrhea (AAD), is an inflammation of the colon. Therefore, a rapid and cost effective method to detect C. difficile directly from stool samples facilitates patient management to control infection.[14,15]. Seventy-five strains contained both tcdA (figure 1) and tcdB(figure2) genes .Pathogenic strains of C.difficile produce tcdA and tcdB ,and several strains isolated from outbreaks and severe infection have been shown harbour the genes encoding toxins.  Journal of Health, Medicine and Nursing                                                                                                                                          www.iiste.org ISSN 2422-8419     An International Peer-reviewed Journal Vol.15, 2015  60  Figure (1): Agarose gel electrophoresis of PCR amplification products of C.difficile Toxin A (tcdA )gene .Expected products of amplification are 625bp.   Figure (2): Agarose gel electrophoresis of PCR amplification products of C.difficile Toxin B (tcdB )gene .Expected products of amplification are 410bp. This is the first study in Iraq, that detection of toxin A and B genes by PCR . This rapid and cost-effective diagnostic assay provides an alternative approach for the detection of C. difficile, thereby improving the CDI management.  References [1] Carroll K. C. and Bartlett J. G., (2011). “Biology of Clostridium difficile: implications for epidemiology and diagnosis,” in Annual Review of Microbiology, vol. 65, pp. 501–521. [2] Oezguen N. , Power T. D. , Urvil P. et al.,( 2012). “Clostridial toxins: sensing a target in a hostile gut environment,” Gut Microbes, vol. 3, no. 1, pp. 35–41,. [3] Rupnik M, Dupuy B, Fairweather NF et al. (2005). Revised nomenclature of Clostridium difficile toxins and associated genes. J Med Microbiol; 54: 113–117. [4] Cartman S. T. ,. Heap J. T ,. Kuehne S. A , Cockayne A. , and Minton N. P. (2010). “The emergence of ’hypervirulence’ in Clostridium difficile,” International Journal of Medical Microbiology, vol. 300, no. 6, pp. 387–395. Journal of Health, Medicine and Nursing                                                                                                                                          www.iiste.org ISSN 2422-8419     An International Peer-reviewed Journal Vol.15, 2015  61 [5]. Lemee L, Dhalluin A, Testelin S et al. (2004). Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile. J Clin Microbiol; 42: 5710–5714. [6]. Kato H, Kato N, Watanabe K et al.( 1998). Identification of toxin A-negative, toxin B-positive Clostridium difficile by PCR J Clin Microbiol; 36: 2178–2182.. [7]. Gumerlock PH, Tang YJ, Weiss JB, Silva J Jr. (1993). Specific detection of toxigenic strains of Clostridium difficile in stool specimens. J Clin Microbiol; 31: 507–511. [8]. van den Berg RJ, Bruijnesteijn van Coppenraet LS, Gerritsen HJ, Endtz HP, van der Vorm ER, Kuijper EJ. (2005). Prospective multicenter evaluation of a new immunoassay and real-time PCR for rapid diagnosis of Clostridium difficile- associated diarrhea in hospitalized patients. J Clin Microbiol; 43: 5338–5340. [9]. Belanger SD, Boissinot M, Clairoux N, Picard FJ, Bergeron MG. (2003).Rapid detection of Clostridium difficile in feces by realtime PCR. J Clin Microbiol; 41: 730–734. [10]-George WL, SutterVL, CitronD, finegold SM,(1979).Selective and differential medium for isolation of C.difficile .J Clin Microbiol 9:214-219. [11]- Persson S., Torpdahl M. and Olsen K. E. P.(2008). New multiplex PCR method for the detection of Clostridium difficile toxin A (tcdA) and toxin B (tcdB) and the binary toxin (cdtA ⁄ cdtB) genes applied to a Danish strain collection. Clin Microbiol Infect; 14: 1057–1064. [12]-Soren P.;Joan NJ,and Katharina E.P.O.(2011). multiplex PCR method for the detection of tcdA , tcdB ,,cdtA, and cdtB and Internal In-Frame of tcdC. Journal Of Clinical Microbiology ,P.4299-4300. [13]-Abhishek D, Vinay P, Preethi P, Gati A and David D.K. R.(2011). Repeat Stool Testing to Diagnose Clostridium difficile Infection Using Enzyme Immunoassay Does not Increase Diagnostic Yield. CLINICAL GASTROENTEROLOGY AND HEPATOLOGY., 9:665–669. [14]-Marie Céline Z. T., Martine L. S., Philippe B., Jean L. F.(2014). Recurrent Clostridium difficile infections: The importance of the intestinal microbiota. World J Gastroenterol 2014 June 21; 20(23): 7416-7423. [15]-Kevin B., Kim V., David F., Andrew S., Nick D., (2015). Hospital Ward Antibiotic Prescribing and the Risks of Clostridium difficile Infection . JAMA Intern Med. Published online February 23.   The IISTE is a pioneer in the Open-Access hosting service and academic event management.  The aim of the firm is Accelerating Global Knowledge Sharing.  More information about the firm can be found on the homepage:  http://www.iiste.org  CALL FOR JOURNAL PAPERS There are more than 30 peer-reviewed academic journals hosted under the hosting platform.   Prospective authors of journals can find the submission instruction on the following page: http://www.iiste.org/journals/  All the journals articles are available online to the readers all over the world without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself.  Paper version of the journals is also available upon request of readers and authors.   MORE RESOURCES Book publication information: http://www.iiste.org/book/ Academic conference: http://www.iiste.org/conference/upcoming-conferences-call-for-paper/   IISTE Knowledge Sharing Partners EBSCO, Index Copernicus, Ulrich's Periodicals Directory, JournalTOCS, PKP Open Archives Harvester, Bielefeld Academic Search Engine, Elektronische Zeitschriftenbibliothek EZB, Open J-Gate, OCLC WorldCat, Universe Digtial Library , NewJour, Google Scholar   

PCR for Detection of Clostridium difficile toxin A (tcdA)and toxin B (tcdB)  genes  in  Iraq

https://www.iiste.org/Journals/index.php/JHMN/article/download/24453/25029

PCR for Detection of Clostridium difficile toxin A (tcdA)and toxin B (tcdB) genes in Iraq

Abstract

Similar works

Full text

Available Versions

International Institute for Science, Technology and Education (IISTE): E-Journals