Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 1, 2011, pp. 56-59

Indian Journal of Medical Microbiology, Vol. 29, No. 1, January-March, 2011, pp. 56-59

Original Article

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

C Vaishnavi, G Singh, K Singh

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

Correspondence Address: C Vaishnavi, Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh-160 012, India, cvaishnavi@rediffmail.com

Date of Submission: 01-Mar-2010
Date of Acceptance: 18-Jan-2011

Code Number: mb11013

PMID: 21304197

DOI: 10.4103/0255-0857.76526

Abstract

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

Introduction

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

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

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.

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].

Discussion

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

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

References

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