By Chaudhry, Rama Joshy, Lovely; Kumar, Lalit; Dhawan, Benu
Background & objectives: Frequent use of broad spectrum antibiotics in hospitalized patients has increased the incidence of Clostridium difficile diarrhoea in recent years. In our tertiary care hospital in north India, C. difficile was responsible for 15 per cent of cases of nosocomial diarrhoea in 1999. A retrospective study was carried out to determine the frequency of C. difficile associated diarrhoea (CdAD) in our hospital, and to assess the effect of awareness among the hospital personnel and control measures taken to present C. difficile infection following the previous report. Methods: A retrospective chart review of all suspected cases of CdAD diagnosed at the hospital from January 2001 to December 2005 was done. Clinical specimens comprised 524 stool samples. All the samples were analyzed for C. difficile using culture and ELISA for toxin A and B. Attempts were made to type isolates using antibiogram, SDS-PAGE, gas liquid chromatography (GLC), PCR for toxin A and B gene fragments and restriction fragment length polymorphism (RFLP).
Results: A total of 37 (7.1%) specimens were positive for C. difficile toxin (11.2% in 2001, 9.4% in 2002, 8.6% in 2003, 5% in 2004 and 4% in 2005). The highest number of C. difficile toxin positive cases were from stool samples of patients hospitalized in the haematology/oncology ward (67.5% of all positive cases) followed by gastrointestinal surgery, neurology and nephrology wards. Of the C. difficile toxin positive samples, 15 (41%) were also positive for C. difficile culture. The isolates were grouped in to one, 3 and 5 groups using antibiogram, SDS-PAGE and PCR RFLP respectively. We observed an increase in the number of stool specimens tested for C. difficile infection but a decrease in C. difficile positives.
Interpretation & conclusions: A decrease in the number of C. difficile positive cases were noted during the 5 year study period though number of samples tested was increased. This may be due to stringent surveillance and an improved antibiotic policy followed in the hospital.
Key words C. difficile – culture – toxin A – toxin B – typing
(ProQuest: … denotes obscured text omitted.)
Diarrhoea is one of the most frequent side effects of antibiotic treatment. The symptoms may vary from slight abdominal discomfort to severe diarrhoea to colitis1. The aetiology of antibiotic associated diarrhoea (AAD) varies. The disruption of normal enteric flora caused by antibiotics may lead to overgrowth of pathogens and functional disturbances of the intestinal carbohydrate and bile acid metabolism, resulting in osmotic diarrhoea1. Allergic, toxic and pharmacological effects of antibiotics may also affect the intestinal mucosa and motility2. Cytotoxin producing Clostridium difficile has been reported to be the causative agent of approximately 20 per cent of AAD and of nearly all cases of pseudomembraneous colitis, the most severe manifestation of AAD1. Because of the frequent use of broad spectrum antibiotics, the incidence of C. difficile diarrhoea has risen dramatically in recent years3,4. Established guidelines should be followed to minimize exposure to the pathogen which include judicious use of antibiotics, rapid detection of C. difficile by immunoassays for toxin A and B, isolation of patients who had C. difficile associated diarrhoea (CdAD), proper disinfection of objects and education of staff members5. In our hospital which is a tertiary care hospital in north India, C. difficile was responsible for 15 per cent of cases of nosocomial diarrhoea in 1999(6). Standard control measures were implemented in our hospital to minimize the spread of this nosocomial pathogen after this report. This retrospective analysis was carried out in continuation of our earlier study6 to determine the effect of awareness and control measures taken to contain C. difficile infection in our hospital during the subsequent years.
Material & Methods
The study comprised retrospective analysis of faecal specimens from 524 patients suspected on clinical grounds to have CdAD2. The patients were hospitalized in All India Institute of Medical Sciences, New Delhi, India, over a period of 5 yr (January 1, 2001 – December 31, 2005). These included 80 patients in 2001, 96 in 2002, 92 in 2003, 106 in 2004 and 150 patients in 2005 respectively. Of these, 53 per cent were males and 82.4 per cent were in all age group > 12-60 yr (Table I).
Clinical information about the cause of diarrhoea underlying disease and antimicrobial therapy was obtained by reviewing the patient charts. A patient was considered to have CdAD if AAD was present and a stool specimen was positive in a toxin dependent C. difficile assay.
Sample collection and isolation of C. difficile: All the stool specimens were processed immediately for culture of C. difficile and stool aliquots were stored at -20[degrees]C for
The plates were incubated anaerobically at 37[degrees]C in an anaerobic jar for 48 h. After incubation, the plates were examined and colonies which resembled C. difficile were Gram stained and identified by biochemical reactions using standard methods7.
When culture plate were negative for C. difficile, subcultures were made from cooked meat broth onto CCFA and BHIA and incubated anaerobically at 37[degrees]C up to 5 days before being discarded as negative.
Enzyme immunoassay for toxin A and B: Detection of enterotoxin and cytotoxin (toxin A and toxin B) of C. difficile was performed on the stool specimens by a double sandwich enzyme-linked immunosorbent assay technique using a commercial kit (Premier toxins A & B; Meridian Diagnostics, Inc., Cincinnati, Ohio, USA). The assay was performed according to the manufacturer’s instructions.
Characterization of C. difficile isolates: All the C. difficile isolates were characterized phenotypically using antibiogram, SDS- PAGE6, gas liquid chromatography (GLC)7, and genotypically using PCR for toxin A gene and RFLP8,9.
Antibiogram typing – Antibiogram patterns were determined by disc diffusion method10. The antibiotics tested were chloramphenicol (30 [mu]g), penicillin G (10 units), clindamycin (2 [mu]g), vancomycin (5 [mu]g); metronidazole (5 [mu]g), tetracycline (30 [mu]g) and erythromycin (10 [mu]g). The results were expressed as susceptible or resistant.
Analysis of volatile fatty acids by gas liquid chromatography (GLC): All isolates were inoculated to cooked meat broth and incubated anaerobically for 48 h or more for GLC analysis to detect volatile fatty acids produced as metabolic end products. 1 ml of RCM broth was acidified with 0.2 ml of 50 per cent sulphuric acid and extracted with 1 ml of diethyl ether. The mixture was shaken vigorously and centrifuged at 176 g for 3 min; 1.5 [mu]l of the extracted ether layer was injected to the injection port of preconditioned GLC column with a 10 [mu]l Hamilton syringe. Chromatography was performed on a Nucon Series 5700, fitted with a flame ionization detector (FID)7. Operating conditions were as follows: carrier gas (oxygen free nitrogen): 60 ml/min oven temperature: detector 240[degrees]C, column 175[degrees]C, injector 240[degrees]C, attenuation 4X, sensitivity of the detector was set at 1000X. Fatty acids were identified by comparing the retention times of peaks in the test samples with those of known standard solutions which were examined each day7.
PCR assay for toxin gene fragments: The presence of toxin A gene in all isolates of C. difficile was determined by specific PCR using published primers8. PCR to detect the toxin B gene was performed in the C. difficile isolates using primers that had been developed and validated by Gumerlock et at9 to yield a 399-bp fragment for toxin B gene. PCR was performed in a 25 [mu]l reaction volume. Each reaction tube contained 1 X buffer (10 mm Tris HCl, pH 8.3, 50 mm KCl, 2.5 mm MgCl^sub 2^, 0.001% gelatin), each deoxynuclotide at a concentration of 100 mm (MBI, Fermentas, USA), each primer at a concentration of 20 pmol, 1.25 U Taq polymerase (MBI, Fermantas, USA) and 10 [mu]l of DNA. PCR was performed for 2 min at 95[degrees]C followed by 30 cycles of 1 min of denaturation at 95[degrees]C, 1 min of annealing at 52[degrees]C and 1 min of extension at 72[degrees]C. After the 30th cycle, extension was continued for an additional 10 min. 10 [mu]l of the amplified product was analyzed in 1 per cent agarose gel stained with ethidium bromide.
PCR-restriction fragment length polymorphism (RFLP) analysis: The amplified toxin A gene fragment was then digested with Alu I(10 units) restriction enzyme, under conditions recommended by the supplier (MBI-Fermentas). These digests were then subjected to electrophoresis on 2.5 per cent agarose gel at 60 V, along side a PCR size marker (100 bps, Sigma, USA).
Comparisons of patterns were performed visually. Strains with patterns differing alteast by one band were assigned to different types.
Results
A total of 524 stool specimens were analyzed for C. difficile from suspected cases of CdAD. The maximum number of C. difficile suspected cases were from oncology ward (378 cases, 72%), followed by other wards such as gastrointestinal surgery, neurology, nephrology and other medical wards. A total of 95 per cent of the analyzed group were on multiple antibiotics which included, 65 per cent on cephalosporins, 35 per cent on quinolones, 43 per cent on aminoglycosides, 12 per cent on macrolides, 69 per cent in vancomycin and metronidazole. Of the analyzed group, 37 (7.1%) patients were positive for C. difficile infection by the toxin dependent assay. Of these, 9 samples (11.2%) were positive in 2001, 9 (9.4%) in 2002, 8 (8.6%) in 2003, 5 (5%) in 2004 and 6 patients (4%) in 2005 (Fig. 1). Fifteen (41%) of the 37 toxin positive stool samples were also positive for C. difficile by culture. Eight of the 37 toxin positive cases expired, the cause of death was not directly related to C. difficile diarrhoea, although this might have been a contributory factor. Other pathogenic clostridia isolated from the patient group included C. perfringens (2.5%).
The highest number of C. difficile toxin positive cases were from stool samples of patients hospitalized in the haematology/oncology ward (25 samples, 67.5% of all positive cases), followed by gastrointestinal surgery, neurology and nephrology wards. Recovery rates of C. difficile in patient populations surveyed and summarized in Table II.
Of the 37 positive cases, 19 (51%) were males; 32 patients (86%) experienced diarrhoea during antibiotic treatment or within 15 days after the start of antibiotic treatment. The median time of occurrence of symptoms was 7 days (ranges 0-16 days) after start of antibiotic treatment and 8 days after admittance to hospital. All the patients were on multiple drugs and 50 per cent of the positive cases were on 3rd generation cephalosporins. None of the positive cases was on clindamycin. C. difficile positive cases were treated with metronidazole or vancomycin.
Antibiogram grouped all 15 isolates together as all were sensitive to erythromycin, chloramphenicol, penicillin, tetracyclin, clindamycin, vancomycin and metronidazole.
The identical fatty acid producers were grouped into 2 groups based on the production of isocaproic acid. Except one, all the isolates were producing isocaproic acid.
Based on the protein profiles observed on SDSPAGE, the isolates were placed into 3 groups; 12 isolates in group A, 2 in group B and 1 in group C.
PCR and RFLP analysis: All the …lates were positive for toxin A (1.2 kb fragment) … (399 bp) gene by PCR. Five different restriction profiles were obtained using Alu I endonucleases. The isolates were classified into five RFLP groups. The most frequent RFLP type was group I (6 isolates) group II, III, IV and V had 5, 2, 1 and 1 isolates respectively.
Discussion
C. difficile is considered as the most frequent aetiological agent of nosocomial diarrhoea occurring in hospitalized patients, spreading easily to the environment, the hands of health care workers and subsequently to other patients, particularly in large hospitals12. A trend of increasing prevalence of C. difficile has been reported in Europe and USA during the past 10 years13.
In our hospital C. difficile was found to be responsible for 15 per cent of the cases of nosocomial diarrhoea in our earlier study6. In this study, C. difficile was isolated mainly from patients in the haematology/ oncology wards. This points to the high risk areas for nosocomial spread of C. difficile isolates14. However, the percentage of infection showed a gradual decrease during the recent years.
Standard laboratory methods for diagnosing these infections include stool culture and identification of bacterial isolate, faecal toxin detection and C. difficile antigen detection. The culture lacks specificity due to the possible faecal carriage of non- toxigenic isolates, therefore many laboratories rely on toxin detection rather than culure for diagnosis of C. difficile infection15. A European survey of diagnostic methods for C. difficile, showed that culture of the organism is performed only in few countries. Mostly C. difficile toxin EIAs were used for diagnosis of CdAD16. In this study we used ELISA for toxin A, B and culture for diagnosing C. difficile infection. However, to the previous study6 we used C. difficile toxin A dependent ELISA for the analysis.
There was a gradual decrease in the percentage of … infection during 2001 and 2005. The fact … the 22 culture negative cases were on … or vancomycin at the time of sample … might be responsible for the decrease in isolation of organism as compared with the ELISA.
Older age, female gender and a prolonged hospital stay have been identified as risk factors in hospitalized CdAD patients17. In the current study, there was no gender prevalence among the positive cases and the median age of positive cases were 39 yr. However, highest percentage of culture positives was seen among patients >60 yr of age. Prolonged courses of antibiotic treatment have been related to an increased risk of AAD18,19. The median time for occurrence of symptoms was 7 days after the start of treatment in the present study, which was in accordance with other studies1,20. This suggests that disturbance of the normal colonic flora, eventually resulting in diarrhoea, takes place within about one week of antibiotic treatment. Prolonged duration of hospital stay has also been reported to be associated with AAD and CdAD19,21. In the present study, the median time of hospital stay was 8 days.
AAD was found to be frequently associated with cephalosporins, clindamycin and broad spectrum penicillins and quinolones22-25. In this study, about 50 per cent of our CdAD cases were on cephalosporins. However, since all the patients were on multiple antibiotics, the association with a particular group was not identified.
Discontinuation of antibiotic therapy withdraws the offending agents but is often not appropriate if the indication for such therapy was correct. An alternative is to change to antibiotics that do not belong to the high risk groups for induction of CdAD, such as quinolones, sulphonamides, parenteral aminoglycosides, cotrimoxazole, etc26. Metronidazole is suggested as the first line drug for the treatment of C. difficile infection2, and therefore the policy of the use of metronidazole in the treatment of suspected CdAD in our hospital is justified.
No nosocomial outbreak of C. difficile was reported during the study period. In this study we found antibiogram was least discriminatory of the typing strategies evaluated. The detection of short chain fatty acids by GLC is commonly utilized in bacteriological laboratories to identify anaerobes27. As all the isolates were positive for toxin A gene, we looked forward to analyze the variability of toxin A gene among C. difficile isolates by RFLP analysis. As the sequence analysis of the amplified 1.2 kbp toxin A gene fragment does not show any restriction sites for the previously reported restriction enzymes like Hinc II, Hind III, Ace I, EcoR I28, we decided to examine the amplified gene structure using restriction enzyme AIu I (5′ AG[arrow down]CT 3′, 3’TC[arrow up]GA 5′), which showed multiple restriction sites (8 sites) in the amplicon.
Although PCR-RFLP types 1 and II clustered some patient isolates, there was no epidemiological association between them. The locations where these patients were housed were different, and were admitted at different time periods. Better discriminatory methods such as pulsed field gel electrophoresis (PFGE) or ribotyping may be used to analyze the epidemiology of the pathogen.
In our recent prospective study, all the C. perfringens isolates were analyzed for the presence of enterotoxin by reverse passive later agglutination (RPLA), ELISA and by PCR assay for the presence of enterotoxin gene29. Of these, two were positive by PCR, RPLA and ELISA for C. perfringens enterotoxin. None of these samples had a co- infection with C. difficile.
Prevention of C. difficile infection is challenging. A change in antibiotic policy and implementation of standard infection control measures reduce the incidence of C. difficile symptomatic infections30,31. Combined approach, involving effective control measures, the use of rapid and sensitive techniques for laboratory diagnosis as well as prudent use of antibiotics, is necessary to reduce morbidity and mortality due to C. difficile associated infections in hospitalized patients.
In conclusion, we observed a decrease in the number of C. difficile toxin positive cases during the 5 yr of the study though there was an increase in the number of stool specimens tested per year for C. difficile. This possibly could be a result of stringent surveillance and antibiotic policy followed in our hospital especially in high risk areas such as haematology/oncology wards. Secondly, the use of clindamycin has been minimized in the hospital. Thirdly, antibiotics effective against C. difficile such as metronidazole have been included as the first line drugs in suspected CdAD cases. Isolation of the patients having C. difficile infection and regular awareness programmes conducted in the hospital might also have contributed.
Acknowledgment
Authors thank Ms. Sonam, Ms. Poornima and Shri Madho Prasad for technical assistance and acknowledge the Indian Council of Medical Research (ICMR), New Delhi, for financial suppport.
References
1. Wistrom J, Norrby SR, Myhre EB, Eriksson S, Granstron G, Legergrea, et al. Frequency of antibiotic-associated diarrhoea in 2462 antibiotic-treated hospitalized patients: a prospective study. J Antimicrob Chemother 2001; 47 : 43-50.
2. Hogenauer C, Hammer HF, Krejs GJ, Reisinger EC. Mechanisms and management of antibiotic-associated diarrhea. Clin Infect Dis 1998; 27 : 702-10.
3. Dallal RM, Harbrecht BG, Boujoukas AJ, Sirio CA, Farkas LM, Lee KK, et al. Fulminant Clostridium difficile: an underappreciated and increasing cause of death and complications. Ann Surg 2002; 235 : 363-72. 4. Kyne L, Hamel MB, Polavaram R, Kelly CP. Health care costs and mortality associated with nosocomial diarrhea due to Clostridium difficile. Clin Infect Dis 2002; 34 : 346-53.
5. Schroeder MS. Clostridium difficile-associated diarrhea. Am Fam Physician 2005; 71 : 921-8.
6. Dhawan B, Chaudhry R, Sharma N Incidence of Clostridium difficile infection: a prospective study in an Indian hospital. J Hosp Infect 1999; 43 : 275-80.
7. Sutter VL, Citron DM, Edelstein MAC, Finegold SM. Wadsworth Anaerobic Manual. Belmont, California: Star Publishers, 1985. p. 71- 5.
8. Kato N, Ou CY, Kato H, Bartley SL, Brown VK, Dowell VR Jr, et al. Identification of toxigenic Clostridium difficile by the polymerase chain reaction. J Clin Microbiol 1991; 29 : 33-7.
9. Gumerlock PH, Tang YJ, Weiss JB, Silva J Jr. Specific detection of toxigenic strains of Clostridium difficile in stool specimens. J Clin Microbiol 1993; 3 : 507-11.
10. Lambe DW Jr., Laslie WW. A standard disc diffusion method for antibiotic susceptability testing for anaerobes; four years antibiotic data. In: Lambe DW Jr, Gena RJ, Carson KJM, editors. Anaerobic bacteria-selected topics. New York: Plenum Press, 1980.
11. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 1970; 227 : 680-5.
12. Wroblewska MM, Swoboda-Kopec E, Rokosz A, Nurzynska G, Bednarska A, Luczak M. Detection of Clostridium difficile and its toxin A (TcdA) in stool specimens from hospitalised patients. Pol J Microbiol 2005; 54 : 111-5.
13. Wongwanich S, Pongpech P, Dhiraputra C, Huttayananont S, Sawanpanyalert P. Characteristics of Clostridium difficile strains isolated from asymptomatic individuals and from diarrheal patients. Clin Microbiol Infect 2001; 7: 438-41.
14. Blot E, Escande MC, Besson D, Barbut F, Granpeix C, Asselain B, et al. Outbreak of C. difficile related diarrhea in an adult oncology unit: risk factors and microbiological characteristics. J Hosp Infect 1993; 53: 187-92.
15. Wilcox MH, Fawley WN, Settle CD, Davidson A. Recurrence of symptoms in Clostridium difficile infection-relapse or reinfection? J Hosp Infect 1998; 38 : 93-100.
16. Barbut F, Delmee M, Brazier JS, Petit JC, Poxton IR, Rupnik M, et al. ESCMID Study Group on Clostridium difficile (ESGCD). A European survey of diagnostic methods and testing protocols for Clostridium difficile. Clin Microbiol Infect 2003; 9 : 989-96.
17. Al-Eidan FA, McElnay JC, Scott MG, Kearney MP. Clostridium difficile-associated diarrhoea in hospitalised patients. J Clin Pharm Ther 2000; 25 : 101-9.
18. Spencer R. The role of antimicrobial agents in the aetiology of Clostridium difficile-associated disease. J Antimicrob Chemother 1998; 41 : 21-7.
19. Bignardi GE. Risk factors for Clostridium difficile infection. J Hosp Infect 1998; 40 : 1-15.
20. Thamilikitkul V, Danpakdi K, Chokloikaew S. Incidence of diarrhea and Clostridium difficile toxin in stools from hospitalized patients receiving Clindamycin, beta lactams, and nonantibiotic medications. J Clin Gastroenterol 1996; 22 : 161-3.
21. Clabots CR, Johnson S, Olson MM, Peterson LR, Gerding DN. Acquisition of Clostridium difficile by hospitalized patients: evidence for colonized new admissions as a source of infection. J Infect Dis 1992; 166 : 561-7.
22. Impallomeni M, Galletly NP, Wort SU, Starr JM, Rogers TR. Increased risk of diarrhea caused by Clostridium difficile in elderly patients receiving cefotaxime. BMJ 1995; 311 : 1345-6.
23. Pear SM, Williamson TH, Bettin KM, Gerding DN, Galgiani JN. Decrease in nosocomial Clostridium difficile-associated diarrhea by restricting clindamycin use. Ann Intern Med 1994; 720 : 272-7.
24. Pepin J, Saheb N, Coulombe MA, Alary ME, Carrivean MP, Authier S, et al. Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in Quebec. Clin Infect Dis 2005; 41 : 1254- 60.
25. Muto CA, Pokrywka M, Shutt K, Mendelsohn AS, Nouri K, Posey K, et al. A large outbreak of Clostridium difficile-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use. Infect Control Hosp Epidemiol 2005; 26 : 273-80
26. Bartlett JG. Antibiotic associated diarrhea. Clin Infect Dis 1992; 15 : 573-81.
27. Pepersack F, Labbe M, Nonhoff C, Schoutens E. Use of gas- liquid chromatography as a screening test for toxigenic Clostridium difficile in diarrhoeal stools. J Clin Pathol 1983; 36 : 1233-6.
28. McFarland LV, Surawicz CM, Rubin M, Fekety R, Elmer GW, Greenberg RN. Recurrent Clostridium difficile disease: Epidemiology and clinical characteristics. Infect Control Hosp Epidemiol 1999; 20 : 43-50.
29. Joshy L, Chaudhry R, Dhawan B, Kumar L, Das BK. Incidence and characterization of Clostridium perfringens isolated from antibiotic- associated diarrhoeal patients: a prospective study in an Indian hospital. J Hosp Infect 2006; 63 : 323-9.
30. Khan R, Cheesbrough J. Impact of changes in antibiotic policy on Clostridium difficile-associated diarrhoea (CDAD) over a five- year period in a district general hospital. J Hosp infect 2003; 54 : 104-8.
31. Riley TV. Nosocomial diarrhoea due to Clostridium difficile. Curr Opin Infect Dis 2004; 17 : 323-7.
Rama Chaudhry, Lovely Joshy, Lalit Kumar* & Benu Dhawan
Departments of Microbiology, * Medical Oncology, Institute- Rotary Cancer Hospital
All India Institute of Medical Sciences, New Delhi, India
Received December 7, 2006
Reprint requests: Dr Rama Chaudhry, Professor, Department of Microbiology, All India Institute of Medical Sciences
New Delhi 110029, India
e-mail: [email protected], [email protected]
Copyright Indian Council of Medical Research Apr 2008
(c) 2008 Indian Journal of Medical Research. Provided by ProQuest LLC. All rights Reserved.
Comments