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Clostridium difficile toxin and faecal lactoferrin assays in adult patients
Microbes and Infection, 2, 2000, 1827−1830 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1286457900013435/FLA
Clostridium difficile toxin and faecal lactoferrin assays in adult patients Chetana Vaishnavi*, Deepak Bhasin, Rakesh Kochhar, Kartar Singh Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India (Received 22 May 2000; accepted 20 September 2000)
ABSTRACT – Clostridium difficile is the primary aetiological agent of antibiotic-associated diarrhoea. The faecal lactoferrin (FL) assay is a simple in vitro test which is highly sensitive to the presence of a marker of polymorphonuclear cells. We evaluated the use of the FL assay in conjunction with the C. difficile toxin assay in faecal samples obtained from 231 adult patients. The relationship between C. difficile toxin and FL in both negative and positive status was highly significant statistically (P < 0.001). Therefore, the FL assay performed simultaneously with the C. difficile toxin assay can help rule out asymptomatic carriage of C. difficile. © 2000 Éditions scientifiques et médicales Elsevier SAS Clostridium difficile toxin / faecal lactoferrin / adjunct usage / asymptomatic carriage
1. Introduction Clostridium difficile is the primary aetiological agent of antibiotic-associated diarrhoea and pseudomembraneous colitis [1]. The disease results from overgrowth of C. difficile already present endogenously or of newly acquired exogenous organisms after suppression of competing gut flora by antibiotics. The organism is widespread in the environment and can be isolated from the stools of numerous animals as well as from the soil. It is commonly present in the stools of 5% of healthy human adults (usually in low numbers). It is unclear if this represents transient colonisation or is a component of stable flora. The incidence of C. difficile-associated diarrhoea is uncertain, even though both sporadic cases and outbreaks have been reported worldwide. Therefore, carriage of the organisms is as important as infection with them. The faecal lactoferrin (FL) assay is a simple in vitro test for a leucocyte marker that is highly sensitive to the presence of polymorphonuclear neutrophils [2]. The test was originally developed and described by Guerrant et al. [3]. We developed our own FL latex agglutination (FLLA) assay and have standardised it in our laboratory [4]. We have been using it as an adjunct to diagnose C. difficileassociated intestinal disease in faecal samples obtained from adult patients.
* Correspondence and reprints. E-mail address: [email protected] (C. Vaishnavi). Microbes and Infection 2000, 1827-1830
2. Materials and methods 2.1. Faecal samples
Faecal samples (231) sent to the microbiology section of the Department of Gastroenterology, Postgraduate Institute of Medical Education and Research (Chandigarh, India), form the basis of the present study. The samples were received between January 1998 and May 1999 with a specific request for C. difficile toxin assay. The patients, whose ages ranged from 18 to 95 years and whose samples we received, were admitted to the institute for treatment of various ailments. Stool samples received from all diarrhoeic patients are routinely investigated for salmonella and shigella. Other pathogens are looked for whenever requested. During analysis of the results, the samples were broadly divided into two main groups based on the information received on a pro forma document: i) samples from patients receiving single or multiple antibiotics for various ailments, and ii) samples from patients who had not received any antibiotic for at least 6 weeks prior to testing; they served as controls to the previous group. 2.2. C. difficile toxin assay
This was performed as described earlier [5]. In brief, 50 µL of a 1 in 5 diluted faecal supernatant was taken on a clean glass slide to which ready-to-use Clostridium sordelli antitoxin-coated latex beads were added. The slide was gently rocked manually and checked for macroscopic agglutination. A sample was considered C. difficile toxin (CDT)-positive when agglutination occurred within 2 min. Controls for both positive and negative assays were included. A known positive faecal sample obtained from a 1827
Original article
Vaishnavi et al.
Table I. The relationship between different parameters studied. Group 1. Antibiotics received (n = 191) diarrhoea present diarrhoea absent 2. CDT positive (n = 41) antibiotic received antibiotic not received diarrhoea present diarrhoea absent 3. FL positive (n = 100) antibiotic received antibiotic not received diarrhoea present diarrhoea absent
Patient samples
P value
179/191 (93.7%) 12/191 (6.3%)
< 0.05
37/191 (19.4%) 4/41 (10.0%) 40/41 (97.6%) 1/41 (2.4%)
> 0.05
84/191 (44%) 16/40 (40%) 97/100 (97.0%) 3/100 (3.0%)
> 0.05
patient with antibiotic-associated diarrhoea was the positive control. The two negative controls consisted of: (i) an unreactive faecal sample from a healthy volunteer who had no antibiotic exposure for 6 weeks prior to testing, and (ii) uncoated latex beads plus diluted test sample. All positive samples were subjected to further titration. Doubling dilutions from the faecal samples ranging from 1 in 10 to 1 in 640 were prepared, and agglutination tests were repeated with them. The titre of the toxin was recorded as the highest dilution of the faecal supernatant which gave a positive agglutination reaction using C. sordelli antitoxincoated latex beads. The advantages of using C. sordellicoated beads for CDT assay have been described in detail earlier [5]. 2.3. FLLA assay
The FLLA assay was also carried out as described earlier [4] on the same faecal samples. Based on our previous experience, latex beads were coated with anti-lactoferrin serum [6]. Briefly, anti-human lactoferrin (Sigma Chemical Company, USA) was coated onto 0.81 µm latex beads (Difco Labs, USA) after pre-titration of the optimum strength of anti-lactoferrin in phosphate-buffered saline, pH 7.2. The FLLA was carried out similarly to the C. difficile toxin assay, using anti-lactoferrin-coated beads thus prepared. The positive control consisted of a known positive faecal sample from a breast-fed infant. Uncoated latex beads plus the diluted faecal supernatant constituted the negative control. All positive faecal samples for lactoferrin were further titrated, and results were recorded as for the CDT assay. The cut-off value for intestinal inflammation was an FL titre of 1 in 40 or more. 2.4. Statistical analysis
The data were expressed by descriptive statistics. The statistical analysis for comparison of the CDT and FL assays between the group receiving antibiotics and the group not receiving antibiotics was carried out by the chi-square test for qualitative data. The quantitative data were analysed by an unpaired Student’s t-test. The relationship between CDT status and FL status was analysed by the McNemar’s test. P < 0.05 was considered a statistically significant difference. 1828
< 0.01
< 0.01
3. Results Of the 231 patients whose faecal samples we analysed, there were 129 males (55.8%) and 102 females (44.2%); 211 (91.3%) of the 231 patients had diarrhoea, while 191 (82.7%) had received antibiotics. Forty-one (17.7%) of the 231 samples were C. difficile toxin positive, and 100 (43.3%) were FLLA positive. No statistically significant difference (P > 0.05) in any of the above parameters was seen according to sex. Of the 191 patients receiving antibiotics, 179 (93.7%) had diarrhoea, while 12 (6.3%) did not. There was a statistically significant difference (P < 0.05) between antibiotic usage and occurrence of diarrhoea. However, CDT positivity was not influenced by prior antibiotic usage, as 37 (19.4%) patients receiving antibiotics were CDT positive compared with four (10.0%) of those who had not received antibiotics (P > 0.05), although diarrhoea was present in 40/41 CDT-positive patients (97.6%). This relationship was statistically significant (P < 0.01) (table I). When we analysed FLLA positivity, 97% of these patients had diarrhoea, while only 3% did not. This difference was highly significant statistically (P < 0.01). However, there was no statistical difference (P > 0.05) in FLLA positivity between patients who had received antibiotics (44%) and those who had not (40%) (table I). 3.1. Relationship between CDT status and FL status
In 123 patients (53.2%) both FL and CDT were negative, and in 33 patients (14.3%) both the assays were positive; but in six patients the FL titres were below cut-off value (< 1 in 40) and therefore not significant. However in 67 patients (29%) FL was positive but CDT was negative, whereas in eight patients (3.5%) FL was negative and CDT was positive (table II). This relationship in both negative and positive status is highly significant statistically (P < 0.001). Other pathogens like salmonella, shigella and campylobacter were not found in any of these patients.
4. Discussion Like virtually all bacterial pathogens, C. difficile causes a spectrum of conditions ranging from asymptomatic carMicrobes and Infection 2000, 1827-1830
Clostridium difficile toxin and faecal lactoferrin
Original article
Table II. The relationship between CDT status and FL status. CDT status CDT positive CDT negative Total
FL positive
FL negative
Total
≥ 1 in 40 < 1 in 40 significant insignificant 27 6 21 46 48 52
8 123 131
41 190
riage to full-blown clinical disease. Many people may have mild to profuse diarrhoea that may last for several weeks or months after administration of the implicated drugs is discontinued. In a recent report it was found that FLLA is a good predictor of symptomatic C. difficile diarrhoea [7]. Therefore, FLLA can be used as an adjunct for diagnosing C. difficile-induced enteric disease in adults in the absence of idiopathic inflammatory colitis. There are numerous reports of cases of C. difficileinduced enteric disease in nursing homes and hospitals [8, 9]. The rate of asymptomatic carriage is 20–30% in patients receiving antibiotics and in hospitalised patients [8, 10]. All of our patients were hospitalised and a majority of them (82.7%) received one or several antibiotics, and of these 93.7% developed diarrhoea. In our study, CDT was present in only 37 patients (19.4%) who received antibiotics. Diarrhoea was present in 97.6% of CDT-positive patients. At times, however, CDT may be present in an individual without the presence of clinical manifestations [11], especially after treatment, as seen in some of our patients. The quantity of lethal toxin produced by different C. difficile strains may vary and might reflect the degree of colitis involved [5], as seen in patients whose CDT titre ranged from 1 in 5 to 1 in 320. There was a highly significant correlation between CDT positivity and FL positivity. Twenty-seven samples were both CDT positive and had significant levels of FL. These probably represented true symptomatic infection, as diarrhoea was present in 97.6% of these patients. Only three patients without prior antibiotic treatment had significant FL titres (i.e. ≥ 1 in 40) with low titres of CD toxin (1 in 10 (n = 2); 1 in 20 (n = 1)). These patients had clinically diagnosed diabetes mellitus with hypothyroiditis, relapsed ulcerative colitis and cirrhosis, respectively, and were over 30 years of age. Diarrhoea was present in all three cases. Here it may be noted that occasionally we may come across C. difficile colitis that cannot be ascribed to antibiotic exposure [12] even though antibiotic therapy accounts for 98% of all cases of C. difficile-associated diarrhoea [13]. The six CDT-positive patients with insignificant levels of FL had rheumatic heart disease, pancreatitis, AIDS, pneumonitis, cirrhosis or encephalitis. It should be remembered that C. difficile may also be present without actually causing harm to the individual. In contrast, nine CDT-negative patients with significant FL levels were ulcerative colitis patients, where FL levels are expected to rise due to intestinal inflammation. In the Microbes and Infection 2000, 1827-1830
same group, nine other patients had septicaemia, where cross-gut border infection can give rise to increased FL titres. One tuberculous meningitis patient also had a high FL titre, probably because of an intestinal focus. Another had portal hypertension, where oedema of the gut wall is expected. It is difficult to say whether this was responsible for the significant FL titre in this case. We are also unable to explain one more case of a patient with herpes simplex viral encephalitis who had significant FL titre. The incidence of C. difficile infection increases with age and is particularly high in the elderly. This could be due to increased use of antibiotics in older patients and also due to the presence of predisposing factors such as gastrointestinal malignancy and major surgical procedures [14, 15]. In our study 51% of the CDT-positive patients were over 45 years of age. C. difficile pseudomembraneous colitis may result from nosocomial infection, since clustering of cases is observed, and contacts of infected patients become carriers of the organism [16, 17]. Studies that examine only colonisation with C. difficile may not be identifying the critical virulence factors associated with diarrhoea [18]. Therefore assays of in vitro CDT production would be more beneficial, along with FL assay. However, interpretation cannot be made using FL titres in neutropenic patients. We believe that FLLA, which is a very simple in vitro test for intestinal inflammation, should be used as an adjunct for diagnosing C. difficile-associated enteritis. It can help rule out asymptomatic carriage. It would be more appropriate to evaluate paired samples for CDT and FL simultaneously, once at the time of admission of the patient and the other one after the development of diarrhoea, for final interpretation of the diagnosis.
Acknowledgments The authors express their gratitude to the World Health Organisation, Geneva and Statens Seruminstitut, Denmark for the gift of C. sordelli antitoxin. We are also thankful to Dr K. Thennarasu for his help in statistical evaluation of this study.
References [1] Bartlett J.G., Chang T.W., Gurwith M., Gorbach S.L., Onderdonk A.B., Antibiotic associated pseudomembraneous colitis due to toxin producing clostridia, N. Engl. J. Med. 298 (1978) 531–534. [2] Choi S.W., Park C.H., Silva T.M.J., Zaenker E.I., Guerrant R.L., To culture or not to culture: Fecal lactoferrin screening for inflammatory bacterial diarrhea, J. Clin. Microbiol. 34 (1996) 928–932. [3] Guerrant R.L., Araujo V., Soares E., Kotloff K., Lima A.A.M., Cooper W.H., Lee A.G., Measurement of fecal lactoferrin as a marker of fecal leucocytes, J. Clin. Microbiol. 30 (1992) 1238–1242. 1829
Original article
[4] Vaishnavi C., Kochhar R., Bhasin D.K., Thapa B.R., Singh K., Faecal lactoferrin latex agglutination assay for Clostridium difficile associated intestinal diseases, Indian J. Med. Microbiol. 16 (1998) 81–83. [5] Vaishnavi C., Kochhar R., Bhasin D.K., Thapa B.R., Singh K., Detection of Clostridium difficile toxin by an indigenously developed latex agglutination assay, Trop. Gastroenterology 20 (1999) 33–35. [6] Vaishnavi C., Agnihotri N., Kumar B., Kaur S., Ganguly N.K., Field utility of phenolic glycolipid coated latex agglutination test for rapid detection of bacilliferous leprosy cases, J. Hyg. Epid. Microbiol Immunol. 36 (1992) 169–174. [7] Manabe Y.C., Vinetz J.M., Moore R.D., Merz C., Charache P., Bartlett J.G., Clostridium difficile colitis; an efficient clinical approach to diagnosis, Ann. Intern. Med. 123 (1995) 835–840. [8] McFarland L.V., Mulligan M.E., Kwok R.Y., Stamm W.E., Nosocomial acquisition of Clostridium difficile infection, N. Engl. J. Med. 320 (1989) 204–210. [9] Silva J. Jr, Lessi C., Clostridium difficile as a nosocomial pathogen, J. Hosp. Infect. 11 (suppl. A) (1988) 378–385. [10] Viscidi R., Willey S., Bartlett J.G., Isolation rates and toxigenic potential of Clostridium difficile is a glutamate dehydrogenase (letter), J. Clin. Microbiol. 30 (1992) 1363–1364. [11] Lima A.A., Lyerly D.M., Wilkins T.D., Innes D.J., Guerrant R.I., Effect of Clostridium difficile toxins A and B in rabbit small and large intestine in vivo and on cultured cells in vitro, Infect. Immun. 56 (1998) 582–588.
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[12] Keighley M.R.B., Burdon D.W., Arabi Y., Alexander-Williams J., Thompson H., Youngs D., Johnson M., Bentely S., George R.H., Mozz G.A.G., Randomised controlled trial of vancomycin for pseudomembraneous colitis and postoperative diarrhoea, Br. Med. J. 2 (1978) 1667–1669. [13] Dhawan B., Chaudhry R., An update on Clostridium difficile infection, Trop. Gastroenterology 18 (1997) 149–152. [14] Kim K.H., Clostridium difficile colitis associated with cancer chemotherapy, Arch. Intern. Med. 142 (1982) 333–335. [15] Panichi G., Pantosti A., Gentile G., Testore G.P., Venditti M., Martions P., Serra P., Clostridium difficile colitis in leukemia patients, Eur. J. Cancer Clin. Oncol. 21 (1985) 1159–1163. [16] Walters B.A.J., Roberts R., Stafford R., Seneviratne E., Relapse of antibiotic associated colitis. Endogenous persistence of Clostridium difficile during vancomycin therapy, Med. J. Australia 24 (1983) 206–212. [17] Heard S.R., O’Farrell S., Holland D., Crook S., Barnett M.J., Tabaqchali S., The epidemiology of Clostridium difficile with use of typing scheme: nosocomial acquisition and cross-infection among immunocompromised patients, J. Infect. Dis. 153 (1986) 159–162. [18] Kotloff K.L., Wade J.C., Morris J.G., Lack of association between Clostridium difficile toxin and diarrhea in infants, Pediatr. Infect. Dis. J. 7 (1988) 662–663.
Microbes and Infection 2000, 1827-1830
Clostridium difficile toxin and faecal lactoferrin assays in adult patients Chetana Vaishnavi*, Deepak Bhasin, Rakesh Kochhar, Kartar Singh Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India (Received 22 May 2000; accepted 20 September 2000)
ABSTRACT – Clostridium difficile is the primary aetiological agent of antibiotic-associated diarrhoea. The faecal lactoferrin (FL) assay is a simple in vitro test which is highly sensitive to the presence of a marker of polymorphonuclear cells. We evaluated the use of the FL assay in conjunction with the C. difficile toxin assay in faecal samples obtained from 231 adult patients. The relationship between C. difficile toxin and FL in both negative and positive status was highly significant statistically (P < 0.001). Therefore, the FL assay performed simultaneously with the C. difficile toxin assay can help rule out asymptomatic carriage of C. difficile. © 2000 Éditions scientifiques et médicales Elsevier SAS Clostridium difficile toxin / faecal lactoferrin / adjunct usage / asymptomatic carriage
1. Introduction Clostridium difficile is the primary aetiological agent of antibiotic-associated diarrhoea and pseudomembraneous colitis [1]. The disease results from overgrowth of C. difficile already present endogenously or of newly acquired exogenous organisms after suppression of competing gut flora by antibiotics. The organism is widespread in the environment and can be isolated from the stools of numerous animals as well as from the soil. It is commonly present in the stools of 5% of healthy human adults (usually in low numbers). It is unclear if this represents transient colonisation or is a component of stable flora. The incidence of C. difficile-associated diarrhoea is uncertain, even though both sporadic cases and outbreaks have been reported worldwide. Therefore, carriage of the organisms is as important as infection with them. The faecal lactoferrin (FL) assay is a simple in vitro test for a leucocyte marker that is highly sensitive to the presence of polymorphonuclear neutrophils [2]. The test was originally developed and described by Guerrant et al. [3]. We developed our own FL latex agglutination (FLLA) assay and have standardised it in our laboratory [4]. We have been using it as an adjunct to diagnose C. difficileassociated intestinal disease in faecal samples obtained from adult patients.
* Correspondence and reprints. E-mail address: [email protected] (C. Vaishnavi). Microbes and Infection 2000, 1827-1830
2. Materials and methods 2.1. Faecal samples
Faecal samples (231) sent to the microbiology section of the Department of Gastroenterology, Postgraduate Institute of Medical Education and Research (Chandigarh, India), form the basis of the present study. The samples were received between January 1998 and May 1999 with a specific request for C. difficile toxin assay. The patients, whose ages ranged from 18 to 95 years and whose samples we received, were admitted to the institute for treatment of various ailments. Stool samples received from all diarrhoeic patients are routinely investigated for salmonella and shigella. Other pathogens are looked for whenever requested. During analysis of the results, the samples were broadly divided into two main groups based on the information received on a pro forma document: i) samples from patients receiving single or multiple antibiotics for various ailments, and ii) samples from patients who had not received any antibiotic for at least 6 weeks prior to testing; they served as controls to the previous group. 2.2. C. difficile toxin assay
This was performed as described earlier [5]. In brief, 50 µL of a 1 in 5 diluted faecal supernatant was taken on a clean glass slide to which ready-to-use Clostridium sordelli antitoxin-coated latex beads were added. The slide was gently rocked manually and checked for macroscopic agglutination. A sample was considered C. difficile toxin (CDT)-positive when agglutination occurred within 2 min. Controls for both positive and negative assays were included. A known positive faecal sample obtained from a 1827
Original article
Vaishnavi et al.
Table I. The relationship between different parameters studied. Group 1. Antibiotics received (n = 191) diarrhoea present diarrhoea absent 2. CDT positive (n = 41) antibiotic received antibiotic not received diarrhoea present diarrhoea absent 3. FL positive (n = 100) antibiotic received antibiotic not received diarrhoea present diarrhoea absent
Patient samples
P value
179/191 (93.7%) 12/191 (6.3%)
< 0.05
37/191 (19.4%) 4/41 (10.0%) 40/41 (97.6%) 1/41 (2.4%)
> 0.05
84/191 (44%) 16/40 (40%) 97/100 (97.0%) 3/100 (3.0%)
> 0.05
patient with antibiotic-associated diarrhoea was the positive control. The two negative controls consisted of: (i) an unreactive faecal sample from a healthy volunteer who had no antibiotic exposure for 6 weeks prior to testing, and (ii) uncoated latex beads plus diluted test sample. All positive samples were subjected to further titration. Doubling dilutions from the faecal samples ranging from 1 in 10 to 1 in 640 were prepared, and agglutination tests were repeated with them. The titre of the toxin was recorded as the highest dilution of the faecal supernatant which gave a positive agglutination reaction using C. sordelli antitoxincoated latex beads. The advantages of using C. sordellicoated beads for CDT assay have been described in detail earlier [5]. 2.3. FLLA assay
The FLLA assay was also carried out as described earlier [4] on the same faecal samples. Based on our previous experience, latex beads were coated with anti-lactoferrin serum [6]. Briefly, anti-human lactoferrin (Sigma Chemical Company, USA) was coated onto 0.81 µm latex beads (Difco Labs, USA) after pre-titration of the optimum strength of anti-lactoferrin in phosphate-buffered saline, pH 7.2. The FLLA was carried out similarly to the C. difficile toxin assay, using anti-lactoferrin-coated beads thus prepared. The positive control consisted of a known positive faecal sample from a breast-fed infant. Uncoated latex beads plus the diluted faecal supernatant constituted the negative control. All positive faecal samples for lactoferrin were further titrated, and results were recorded as for the CDT assay. The cut-off value for intestinal inflammation was an FL titre of 1 in 40 or more. 2.4. Statistical analysis
The data were expressed by descriptive statistics. The statistical analysis for comparison of the CDT and FL assays between the group receiving antibiotics and the group not receiving antibiotics was carried out by the chi-square test for qualitative data. The quantitative data were analysed by an unpaired Student’s t-test. The relationship between CDT status and FL status was analysed by the McNemar’s test. P < 0.05 was considered a statistically significant difference. 1828
< 0.01
< 0.01
3. Results Of the 231 patients whose faecal samples we analysed, there were 129 males (55.8%) and 102 females (44.2%); 211 (91.3%) of the 231 patients had diarrhoea, while 191 (82.7%) had received antibiotics. Forty-one (17.7%) of the 231 samples were C. difficile toxin positive, and 100 (43.3%) were FLLA positive. No statistically significant difference (P > 0.05) in any of the above parameters was seen according to sex. Of the 191 patients receiving antibiotics, 179 (93.7%) had diarrhoea, while 12 (6.3%) did not. There was a statistically significant difference (P < 0.05) between antibiotic usage and occurrence of diarrhoea. However, CDT positivity was not influenced by prior antibiotic usage, as 37 (19.4%) patients receiving antibiotics were CDT positive compared with four (10.0%) of those who had not received antibiotics (P > 0.05), although diarrhoea was present in 40/41 CDT-positive patients (97.6%). This relationship was statistically significant (P < 0.01) (table I). When we analysed FLLA positivity, 97% of these patients had diarrhoea, while only 3% did not. This difference was highly significant statistically (P < 0.01). However, there was no statistical difference (P > 0.05) in FLLA positivity between patients who had received antibiotics (44%) and those who had not (40%) (table I). 3.1. Relationship between CDT status and FL status
In 123 patients (53.2%) both FL and CDT were negative, and in 33 patients (14.3%) both the assays were positive; but in six patients the FL titres were below cut-off value (< 1 in 40) and therefore not significant. However in 67 patients (29%) FL was positive but CDT was negative, whereas in eight patients (3.5%) FL was negative and CDT was positive (table II). This relationship in both negative and positive status is highly significant statistically (P < 0.001). Other pathogens like salmonella, shigella and campylobacter were not found in any of these patients.
4. Discussion Like virtually all bacterial pathogens, C. difficile causes a spectrum of conditions ranging from asymptomatic carMicrobes and Infection 2000, 1827-1830
Clostridium difficile toxin and faecal lactoferrin
Original article
Table II. The relationship between CDT status and FL status. CDT status CDT positive CDT negative Total
FL positive
FL negative
Total
≥ 1 in 40 < 1 in 40 significant insignificant 27 6 21 46 48 52
8 123 131
41 190
riage to full-blown clinical disease. Many people may have mild to profuse diarrhoea that may last for several weeks or months after administration of the implicated drugs is discontinued. In a recent report it was found that FLLA is a good predictor of symptomatic C. difficile diarrhoea [7]. Therefore, FLLA can be used as an adjunct for diagnosing C. difficile-induced enteric disease in adults in the absence of idiopathic inflammatory colitis. There are numerous reports of cases of C. difficileinduced enteric disease in nursing homes and hospitals [8, 9]. The rate of asymptomatic carriage is 20–30% in patients receiving antibiotics and in hospitalised patients [8, 10]. All of our patients were hospitalised and a majority of them (82.7%) received one or several antibiotics, and of these 93.7% developed diarrhoea. In our study, CDT was present in only 37 patients (19.4%) who received antibiotics. Diarrhoea was present in 97.6% of CDT-positive patients. At times, however, CDT may be present in an individual without the presence of clinical manifestations [11], especially after treatment, as seen in some of our patients. The quantity of lethal toxin produced by different C. difficile strains may vary and might reflect the degree of colitis involved [5], as seen in patients whose CDT titre ranged from 1 in 5 to 1 in 320. There was a highly significant correlation between CDT positivity and FL positivity. Twenty-seven samples were both CDT positive and had significant levels of FL. These probably represented true symptomatic infection, as diarrhoea was present in 97.6% of these patients. Only three patients without prior antibiotic treatment had significant FL titres (i.e. ≥ 1 in 40) with low titres of CD toxin (1 in 10 (n = 2); 1 in 20 (n = 1)). These patients had clinically diagnosed diabetes mellitus with hypothyroiditis, relapsed ulcerative colitis and cirrhosis, respectively, and were over 30 years of age. Diarrhoea was present in all three cases. Here it may be noted that occasionally we may come across C. difficile colitis that cannot be ascribed to antibiotic exposure [12] even though antibiotic therapy accounts for 98% of all cases of C. difficile-associated diarrhoea [13]. The six CDT-positive patients with insignificant levels of FL had rheumatic heart disease, pancreatitis, AIDS, pneumonitis, cirrhosis or encephalitis. It should be remembered that C. difficile may also be present without actually causing harm to the individual. In contrast, nine CDT-negative patients with significant FL levels were ulcerative colitis patients, where FL levels are expected to rise due to intestinal inflammation. In the Microbes and Infection 2000, 1827-1830
same group, nine other patients had septicaemia, where cross-gut border infection can give rise to increased FL titres. One tuberculous meningitis patient also had a high FL titre, probably because of an intestinal focus. Another had portal hypertension, where oedema of the gut wall is expected. It is difficult to say whether this was responsible for the significant FL titre in this case. We are also unable to explain one more case of a patient with herpes simplex viral encephalitis who had significant FL titre. The incidence of C. difficile infection increases with age and is particularly high in the elderly. This could be due to increased use of antibiotics in older patients and also due to the presence of predisposing factors such as gastrointestinal malignancy and major surgical procedures [14, 15]. In our study 51% of the CDT-positive patients were over 45 years of age. C. difficile pseudomembraneous colitis may result from nosocomial infection, since clustering of cases is observed, and contacts of infected patients become carriers of the organism [16, 17]. Studies that examine only colonisation with C. difficile may not be identifying the critical virulence factors associated with diarrhoea [18]. Therefore assays of in vitro CDT production would be more beneficial, along with FL assay. However, interpretation cannot be made using FL titres in neutropenic patients. We believe that FLLA, which is a very simple in vitro test for intestinal inflammation, should be used as an adjunct for diagnosing C. difficile-associated enteritis. It can help rule out asymptomatic carriage. It would be more appropriate to evaluate paired samples for CDT and FL simultaneously, once at the time of admission of the patient and the other one after the development of diarrhoea, for final interpretation of the diagnosis.
Acknowledgments The authors express their gratitude to the World Health Organisation, Geneva and Statens Seruminstitut, Denmark for the gift of C. sordelli antitoxin. We are also thankful to Dr K. Thennarasu for his help in statistical evaluation of this study.
References [1] Bartlett J.G., Chang T.W., Gurwith M., Gorbach S.L., Onderdonk A.B., Antibiotic associated pseudomembraneous colitis due to toxin producing clostridia, N. Engl. J. Med. 298 (1978) 531–534. [2] Choi S.W., Park C.H., Silva T.M.J., Zaenker E.I., Guerrant R.L., To culture or not to culture: Fecal lactoferrin screening for inflammatory bacterial diarrhea, J. Clin. Microbiol. 34 (1996) 928–932. [3] Guerrant R.L., Araujo V., Soares E., Kotloff K., Lima A.A.M., Cooper W.H., Lee A.G., Measurement of fecal lactoferrin as a marker of fecal leucocytes, J. Clin. Microbiol. 30 (1992) 1238–1242. 1829
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