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 ~  Abstract
 ~  Introduction
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Acknowledgments
 ~  References
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2011  |  Volume : 29  |  Issue : 1  |  Page : 56-59
 

Cost-effective screening of pooled faecal specimens from patients with nosocomial diarrhoea for Clostridium perfringens enterotoxin


Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh-160 012, India

Date of Submission01-Mar-2010
Date of Acceptance18-Jan-2011
Date of Web Publication7-Feb-2011

Correspondence Address:
C Vaishnavi
Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh-160 012
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.76526

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 ~ Abstract 

Purpose: Clostridium perfringens is a significant cause of nosocomial AAD. The prevalence of C. perfringens enterotoxin (CPE)-positive stool specimens in hospitalised patients is very low in the Indian setting making the diagnostics very expensive. Therefore, a cost-effective diagnostic approach to screen faecal specimens for CPE was devised. Materials and Methods: Faecal specimens from 540 hospitalised patients with various ailments and from 340 healthy subjects were investigated for CPE. An aliquot of pooled faecal supernatants was made by mixing 100 μl each of 10 specimens to be tested. Each aliquot was investigated for the presence of CPE by an enzyme immunoassay. A repetition of the assay was done with individual specimens of the pooled aliquots from each positive well as seen visually by colour development. Results: Of the 540 patient specimens tested, 405 (75%) patients were on antibiotics, the predominant ones being cephalosporins, penicillin, quinolones, aminoglycosides, etc. During the time of sampling, diarrhoea was present in 481 (89%), abdomen pain in 203 (37.6%) and fever in 242 (44.8%) patients. C. perfringens enterotoxin was positive in nine wells of the 540 pooled test specimens whereas all of the pooled 340 control samples were negative. Repeat of individual specimens comprising the nine wells with positive samples helped to identify the individual patients positive for CPE. Conclusion: Only two CPE kits were needed for a total of 880 faecal specimens tested. The cost-effective diagnostic approach to screen faecal specimens for CPE, as described herein will help to save institutional resources.


Keywords: C. perfringens enterotoxin, cost-effective method, nosocomial diarrhoea


How to cite this article:
Vaishnavi C, Singh G, Singh K. Cost-effective screening of pooled faecal specimens from patients with nosocomial diarrhoea for Clostridium perfringens enterotoxin. Indian J Med Microbiol 2011;29:56-9

How to cite this URL:
Vaishnavi C, Singh G, Singh K. Cost-effective screening of pooled faecal specimens from patients with nosocomial diarrhoea for Clostridium perfringens enterotoxin. Indian J Med Microbiol [serial online] 2011 [cited 2019 Sep 22];29:56-9. Available from: http://www.ijmm.org/text.asp?2011/29/1/56/76526



 ~ Introduction Top


Clostridium perfringens Type A (CPA) is a common intestinal inhabitant of human beings and CPA isolates are associated with pathogenic outcomes. Vaishnavi et al., [1] indicated the prevalence of multiple serotype A of C. perfringens in patients who developed diarrhoea after hospitalisation and/or antibiotic usage. It has been reported that 2-5% of all CPA isolates produce enterotoxin. [2] CPA isolates may be responsible for some of the cases of antibiotic-associated diarrhoea (AAD) even though C. difficile is implicated in the most severe cases. Carman [3] reported that C. perfringens might be responsible for 5-20% of all cases of AAD. Moreover C. difficile is much less often found in diarrhoea or colitis where pseudomembranes are absent and interestingly some C. difficile-negative cases of antibiotic associated diarrhoea (AAD) present with bloody diarrhoea. Even though C. perfringens is believed to be a significant cause of nosocomial AAD, its exact role in AAD remains to be elucidated. It is not known whether antibiotics permit infection by enterotoxigenic C. perfringens or allow overgrowth of small numbers of the organisms that may normally be resident in the gut of these patients. Therefore it is necessary to study the prevalence of C. perfringens enterotoxin (CPE)-positive stool specimens from patients with nosocomial diarrhoea so that optimum control and treatment measures can be defined in the future. As the prevalence of CPE-positive samples in AAD is very little in the Indian setting, [4] the diagnostics become very expensive. Thus it is not possible to routinely investigate each specimen for CPE. Therefore, a cost-effective diagnostic approach to screen faecal specimens of patients with nosocomial diarrhoea for CPE was devised.


 ~ Materials and Methods Top


Study population and sampling

The Institute Research Committee cleared the proposal. A total of 540 consecutive faecal specimens submitted for C. difficile investigation irrespective of antibiotic intake, were received between April 2007 and October 2009. The specimens belonged to hospitalised patients suffering from various ailments such as sepsis, meningitis, renal disease, liver diseases, pancreatitis, ulcerative colitis and other inflammatory bowel disease and having diarrhoea. Patients with infective diarrhoea caused by enteric pathogens other than C. difficile or C. perfringens were excluded from the study. Faecal specimens from 340 age- and sex-matched healthy subjects who did not receive any antibiotic for at least six weeks prior to testing and who did not have diarrhoea served as controls. The faecal specimens were diluted 1 in 5 and supernatants prepared as described earlier. [5] The faecal supernatants were stored at -20°C till the time of assay.

Demographic and clinical profiles of the patients were recorded in preprinted proforma and included age, gender, presence of diarrhoea, fever and abdominal pain, the intake of antibiotics and C. difficile toxin (CDT) status.

C. perfringens ELISA

At the time of assay, an aliquot of pooled faecal supernatants were made by mixing 100 μl each of 10 samples to be tested. Each aliquot was investigated for the presence of CPE by the RIDASCREEN enzyme immunoassay (OSB Agencies Pvt. Ltd. 14/147, Main Road, Geeta Colony, Delhi-110031, India) in a microtitre plate as instructed by the manufacturer. Briefly, microtitre wells precoated with polyclonal antibodies directed against epitopes of CPE served as a solid phase . Aliquots of pooled faecal specimens were added to the wells along with an anti-C. perfringens enterotoxin monoclonal antibody conjugated to horse radish peroxidase. After 60-min incubation the wells were washed thrice with washing buffer. Next the substrate was added and the plate was incubated at 25 0 C in the dark for 20 min for colour development. Further reaction was stopped by the addition of stop solution.

A repetition of the ELISA was done with individual specimens of the pooled aliquots from each positive well as seen visually by colour development. Final dilution of each positive sample was made 1:11 before repeating the ELISA, as was required for the assay. The intensity of the colour developed was measured at 450 nm in an ELISA reader (Bio Rad Model 550, K.K. 2-2-24 Higashi Shinagawa Shinagawa-ku, Tokyo 140-0002, Japan). The cut-off value was calculated by extinction for the negative control +0.150 and was 0.192. Samples were considered positive if the absorbance value was higher than 10% over the determined cut-off value as mentioned by the manufacturers. Thus samples above 10% of 0.192 (i.e.> 0.201) were considered positive.


 ~ Results Top


The demographic and clinical profiles of the patients have been grouped in [Table 1]. Of the 540 patient specimens tested 349 belonged to males and 191 to females. The age of the patients ranged from 26 days to 98 years. Four hundred and five (75%) patients were on antibiotics, predominant ones being cephalosporins, penicillin, quinolones, aminoglycosides etc. During the time of sampling, diarrhoea was present in 481 (89%), abdomen pain in 203 (37.6%) and fever in 242 (44.8%) patients.
Table 1 :Demographic and clinical profile of test patients

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C. perfringens enterotoxin was positive in nine wells of the 540 pooled test specimens whereas all of the pooled 340 control samples were negative. Repeat of individual specimens comprising the nine wells with positive samples helped to identify the individual patients positive for CPE. The details of the patients with CPE in their faecal specimens are given in [Table 2].
Table 2 :Individual patient details in CPE-positive cases

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 ~ Discussion Top


Clostridium difficile has been in the medical limelight due to its role in AAD resulting in increased morbidity and mortality. Even though C. perfringens is the second most frequent pathogen for AAD, due to its low prevalence it is considered less important for clinicodiagnosis. Several years ago Borriello et al.,[6] reported the prevalence of C. perfringens enterotoxin in 1.6% and C. difficile toxin in 14.4% with 2/11 of patients excreting both the organisms. Soon other reports on the association of C. perfringens with AAD followed. Samuel et al.,[7] reported 25/721 (3.5%) diarrhoeal specimens to be positive for enterotoxigenic C. perfringens. Residents of old people's homes and hospitalised patients receiving antibiotic therapy have a raised susceptibility for colonisation with pathogenic strains of C. perfringens. Satomura et al.,[8] reported CPE producing C. perfringens in the stool culture of a 40-year-old man who was administered with prophylactic antibiotics as he was undergoing haematopoietic stem cell transplantation.

The causal role of C. perfringens in non-food-borne diarrhoeal disease is proved by the presence of enterotoxin in the faeces of AAD patients or sporadic diarrhoea, in contrast to asymptomatic individuals or those suffering from infectious intestinal disease caused by other enteropathogens. [9] The samples investigated in the present study were exclusive of those caused by other infectious enteropathogens. It is unclear if antibiotic exposure primarily permits the proliferation of small numbers of resident C. perfringens strains or allows their acquisition. In spite of this C. perfringens AAD is recognised as a distinct entity.

C. perfringens forms both chromosomally as well as plasmid-coded enterotoxin. All strains capable of causing food poisoning excrete without exception chromosomally formed CPE during the sporulation phase, whereas strain isolates gained from patients with AAD or sporadic diarrhoea form only plasmid-coded enterotoxin. Strains with this episomally formed enterotoxin [9] are, however, pathogenetically far more effective than the disorders caused by food poisoning. These more severe and more protracted courses are presumably connected with the general state of health of the carriers of such C. perfringens strains.

Sparks et al.,[10] provided important evidence that plasmid clostridium perfringes entertoxin (cpe) isolates cause most C. perfringens-associated AAD and chromosomal cpe isolates cause most C. perfringens Type A food poisoning cases. Asha et al.,[11] in a prospective study of 4,659 hospitalised patients reported an incidence of 12.7% for C. difficile toxins A and B and 3.3% for C. perfringens enterotoxin amongst those suffering from AAD.

C. difficile may account for about 20-30% of all cases of AAD [12] and may rise even higher with the advent of the hypervirulent strain. [13] But some proportion of AAD cases are definitely due to C. perfringens.[14] Asha and Wilcox [15] reported up to 15% cases of AAD to be due to C. perfringens. Vaishnavi et al.,[4] detected CPE in diarrhoeal samples of 2% patients with antibiotic association. Pituch et al.,[16] reported 75% of 52 specimens positive for C. difficile toxin A and B, 40% for CPE and 31% positive for both. They recommend that CPE detection should be included for routine diagnosis of patients with presumed AAD.

Less than 5% of global C. perfringens isolates are estimated to carry cpe plasmid. [17] Specific ecologic niches may have different genotypes of CPE subpopulation and reservoirs and transmission routes remain unidentified due to conjugative transfer of cpe plasmid. [18] The CPE infecting dose of CPE causing AAD is considerably smaller than the infecting dose for C. perfringens Type A food poisoning. [3] This is true because a small number of plasmid cpe isolates might be able to establish human AAD by conjugative transfer to cpe-negative C. perfringens isolates found in the human intestine.

Symptoms of C. perfringens involve watery or bloody diarrhoea. Sixty seven percent of our CPE-positive patients had watery diarrhoea, 11% bloody diarrhoea, whereas 22% had semi-solid stools. Risk factors for nosocomial diarrhoea due to either C. difficile or C. perfringens are the same and include the use of proton pump inhibitors (PPI) and older age group. Seventy-seven percent of our CPE-positive patients were on PPI and 33% belonged to the older age group. Sporadic diarrhoea occurs when patients develop disease without prior exposure to antibiotics. Twenty-five percent of the 540 hospitalised patients were not on antibiotics.

It should be noted that 55% of our CPE-positive patients were on an antibiotics. However, as most of our patients were elderly and hospitalised, this association with antibiotics could be coincidental. It is however probable that in the majority of cases antibiotics disrupted the normal gut flora to allow small number of CPE strains to proliferate. Forward et al., [19] reported that age over 60 years was a factor associated with CPE detection and in the present study 33% of our CPE-positive patients were more than 60 years of age.

The increased use of antibiotics, PPI, immuno-suppressive and other drugs have resulted in an increase in the incidence of C. difficile-related diseases. [20] The same factors may also be involved in C. perfringens-related diseases as it is next in line as the aetiological agent for AAD. As detection of C. perfringens is not part of the routine laboratory investigation because of resource implications, one tends to miss out on the diagnosis of AAD due to this organism.

However, there is a limitation to the pooling of samples and performing the test as it may not be possible in a clinical set up unless there is an outbreak. It may not be relevant to an individual patient as batching of samples will delay the report. But because the prevalence of CPE-positive samples in AAD is small, pooling of samples will be useful to screen faecal specimens of patients with nosocomial diarrhoea and help to establish the local prevalence at any given place in a cost-effective manner. Pooling of samples will help to eliminate a large number of negative ones.

The criteria for initiating investigations have to be established to assess the true burden of C. perfringens by screening stool samples of AAD for C. perfringens enterotoxin. Even though the kit for CPE testing has been available for several years, testing for CPE in a routine diagnostic laboratory has not been advocated because of the low prevalence of AAD due to C. perfringens. In the present investigation only two CPE kits were needed for a total of 880 faecal specimens tested. The cost-effective diagnostic approach to screen faecal specimens for CPE, as described by us, will go a long way in saving institutional resources.


 ~ Acknowledgments Top


The authors thank Ms Monica Sharma for the technical help provided.

 
 ~ References Top

1.Vaishnavi C, Kaur S, Singh K. Clostridium perfringens type A and antibiotic associated diarrhoea. Indian J Med Res 2005;122:52-6.  Back to cited text no. 1
    
2.Kokai-Kun JF, Songer JG, Czeczulin JR, Chen F, McClane BA. Comparison of Western immunoblots and gene detection assays for identification of potentially enterotoxigenic isolates of Clostridium perfringens. J Clin Microbiol 1994;32:2533-9.   Back to cited text no. 2
    
3.Carman RJ. Clostridium perfringens in spontaneous and antibiotic associated diarrhoea of man and other animals. Rev Med Microbiol 1997;8:S43-5.  Back to cited text no. 3
    
4.Vaishnavi C, Kaur S. Clostridium perfringens enterotoxin in antibiotic-associated diarrhea. Indian J Pathol Microbiol 2008;51:198-9.  Back to cited text no. 4
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5.Vaishnavi C, Kochhar R, Bhasin DK, Thapa BR, Singh K. Detection of Clostridium difficile toxin by an indigenously developed latex agglutination assay. Trop Gastroenterol 1999;20:33-5.  Back to cited text no. 5
    
6.Borriello SP, Larson HE, Welch AR, Barday F, Stringer MF, Bartholomew BA. Enterotoxigenic Clostridium perfringens: A possible cause of antibiotic associated diarrhoea. Lancet 1984;305-7.  Back to cited text no. 6
    
7.Samuel SC, Hancock P, Leigh DA. An investigation into Clostridium perfringens enterotoxin associated diarrhoea. J Hosp Infect 1991;18:219-30.  Back to cited text no. 7
    
8.Satomura H, Odaka I, Sakai C, Kato H Antibiotic-associated diarrhea due to Clostridium perfringens. Kansenshogaku Zasshi 2009;83:549-52.  Back to cited text no. 8
    
9.Collie RE, McClane BA. Evidence that the enterotoxin gene can be episomal in Clostridium perfringens isolates associated with non food borne human gastrointestinal diseases. J Clin Microbiol 1998;36:30-6.  Back to cited text no. 9
    
10.Sparks SG, Carman RJ, Sarker MR, McClane BA. Genotyping of enterotoxigenic Clostridium perfringens faecal isolates associated with antibiotic-associated diarrhoea and food poisoning in North America. J Clin Microbiol 2001;39:883-8.  Back to cited text no. 10
    
11.Asha NJ, Tompkins D, Wilcox MH. Comparative analysis of prevalence, risk factors, and molecular epidemiology of antibiotic-associated diarrhea due to Clostridium difficile, Clostridium perfringens, and Staphylococcus aureus. J Clin Microbiol 2006;44:2785-91.  Back to cited text no. 11
    
12.Vaishnavi C. Bhasin DK, Kochhar R, Singh K. Clostridium difficile toxin and faecal lactoferrin assays in adult patients. Microbes Infect 2000;2:1827-30.  Back to cited text no. 12
    
13.Razavi B, Apisamthanarak A, Mundy LM. Clostridium difficile: Emergence of hypervirulence and fluoroquinolone resistance. Infection 2007;35:300-7.  Back to cited text no. 13
    
14.Hancock, P Antibiotic-associated diarrhoea: Clostridium difficile or Clostridium perfringens. Rev Med Microbiol 1997;8:66-7.  Back to cited text no. 14
    
15.Asha NJ, Wilcox MH. Laboratory diagnosis of Clostridium perfringens antibiotic associated diarrhoea. J Med Microbiol 2002;51:891-4.  Back to cited text no. 15
    
16.Pituch H, Obuch-Woszczatyñski P, Wultañska D, van Belkum A, Meisel-Mikolajczyk F, Luczak M. Laboratory diagnosis of antibiotic-associated diarrhea: A Polish pilot study into the clinical relevance of Clostridium difficile and Clostridium perfringens toxins. Diagn Microb Infect Dis 2007;58:71-5.  Back to cited text no. 16
    
17.Smedley JG, McClane, BA. Fine mapping of the N-terminal cytotoxicity region of Clostridium perfringens enterotoxin by site directed mutagenesis. Infect Immun 2004;72:6914-23.  Back to cited text no. 17
    
18.Brynestad S, Sarker MR, McClane BA, Granum PE, Rood JI. Enterotoxin plasmid from Clostridium perfringens is conjugative. Infect Immun 2001;69:3483-7.  Back to cited text no. 18
    
19.Forward LJ, Tompkins DS, Brett MM. Detection of Clostridium difficile cytotoxin and Clostridium perfringens enterotoxin in cases of diarrhoea in the community. J Med Microbiol 2003;52:753-7.  Back to cited text no. 19
    
20.Vaishnavi C. Established and potential risk factors for Clostridium difficile infection Indian J Med Microbiol 2009;27:287-98.  Back to cited text no. 20
    



 
 
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