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Polymerase chain reaction detection of Cryptosporidium parvum in intestinal samples
GASTROENTEROLOGY Vol. 114, No. 4
AI00S AGA ABSTRACTS • G4126
MANNOSE BINDING LECTIN GENOTYPE AS A RISK FACTOR FOR INTESTINAL PROTOZOAL INFECTION IN AIDS. Kelly P, Jack D, Naeem A, Tumer M, Farthing MJG. Digestive Diseases Research Centre, St Bartholomew's and Royal London School of Medicine, London; Institute of Child Health, London. Background Mannose binding lectin (MBL) is a multi-chain serum lectin with up to six 96 kDa subunits which plays an important role in non-specific defence against invasive organisms expressing polysaccharides terminating in mannose or N-acetyl glucosamine residues. Deficiencies of MBL predominantly arise from mutations in exon 1 of the MBL gene, at codons 52, 54 or 57. MBL gene mutations are over-represented in H1V positive populations, and are associated with shortened survival in AIDS. We examined MBL gene mutations in Zambian patients with AIDS related diarrhoea in order to determine whether they are associated with enhanced risk of enteric protozoal infections which might confer shortened survival. Methods 73 HIV seropositive patients with diarrhoea of over three weeks duration underwent stool microscopy, duodenal biopsy for evaluation of infection, and serum sampling. DNA was extracted from one frozen biopsy and used as the template for PCR amplification of exon 1; mutations were detected by controlled annealing of the products with a universal heteroduplex generator followed by polacrylamide electrophoresis. MBL concentrations in serum were assayed by ELISA. Results C. parvum was detected in 27%, L belli in 29%, and microsporidia in 32%. 46 of these patients had wild type MBL gene sequences, 20 were heterozygotes for the 57 codon mutation and 6 were homozygotes. MBL concentrations in serum were 1137 ~tg/l in wild types, 326 in heterozygotes, and 108 in homozygotes (p=0.0001). Individuals with MBL homozygous 57 codon mutations or 57/52 double mutations were at higher risk of cryptosporidiosis (Risk Ratio 3.1; 05%CI 1.6,6.0; p=0.006) but not of the other infections. Genotype predicted cryptosporidiosis, but serum MBL concentration did not. Conclusion In these AIDS patients, MBL mutations conferred higher risk for cryptosporidiosis than the wild type. No extra risk of other protozoal infection was observed. This extra risk for cryptosporidiosis could partially explain the shortened survival in AIDS experienced by individuals with MBL gene mutations. G4127 POLYMERASE CHAIN REACTION DETECTION OF CRYPTOSPORIDIUM PARVUM IN INTESTINAL SAMPLES. P Kelly, FA Makumbi, N. Luo, YK Seedat, MJG Farthing. Digestive Diseases Research Centre, St Bartholomew's and Royal London School of Medicine, London, UK; Gastrointestinal unit, University of Natal, Congella, South Africa. Background C. parvum is associated with considerable morbidity and mortality in AIDS patients and children, but no effective therapy is available and the pathophysiology of the condition is poorly understood. Previous studies have revealed that diarrhoea may be a feature of the infection without any evidence of a secretory lesion in the proximal small intestine. In gnotobiotic piglets, C. parvum infection moves caudally along the intestine during the course of infection. We set out to use PCR to characterise the distribution of infection in the gut as this may explain variability in the clinical course and in pathophysiological studies. Methods In the first study, 75 AIDS patients were studied with stool examination and duodenal biopsy for light microscopy, electron microscopy and PCR. In the second study, biopsies were taken for PCR only from duodenum, terminal ileum and colon in eight patients with AIDS and persistent cryptosporidiosis. PCR was used to amplify a portion of specific C. parvum DNA of unkown function, and PCR products were detected by electrophoresis and hybridisation of Southern blots. Results The PCR was sensitive and detected the equivalent of 5 genome copies. No positive reactions were obtained with human, bacterial or protozoal DNA. 18 of 75 (24%) of the AIDS patients in the first study had cryptosporidiosis diagnosed by conventional techniques, and 7 had duodenal infection detected microscopically. All of these microscopy positive cases were found to be positive by PCR, and 4 additional microscopy-negative cases were positive by PCR. In the second study, 1 patient had pan-enteric infection, 1 had localised duodenal infection and 2 had predominantly colonic infection. In the remainder, none of the biopsies were positive by PCR. Conclusion PCR confirms that C. parvum infection can persist in any part of the gut, that proximal small intestinal infection occurs in about half of all persistent cases, and it seems likely that some infections are confined to mid small intestine. This variability must be borne in mind when designing pathophysiological studies. G4128 IODOACETAMIDE-INDUCED COLITIS IN RATS IS INHIBITED BY THALIDOMIDE. G. Kenet, Y. Avni, H. Aeed, H. Shirin, Z. Matas, L. Zaidel, S. Galor, R. Hershkovitz and R, Brnck. Dept. of Gastroenterolgy, The E. Wolfson Medical Center, Holon, Israel. Thalidomide, a non-barbiturate hypnotic drug, has well known anti inflammatory effects. It suppresses TNFct production by accelerating the degradation of TNFtx messenger RNA. There have been isolated reports of beneficial effects of thalidomide in ulcerative colitis patients. The aim of the present study, was to evaluate the effect of thalidomide on experimentally-
induced colitis. Colitis was induced in Wistar rats by intracolonic administration of 0.1 ml iodoacetamide (IA) 3%. In one group, thalidomide (50 mg/kg) was administered 2 days before the induction of colitis, and continued daily until 7 days after. Blood was drawn on day 2 and 7. Rats were sacrificed 7 days after the induction of colitis. Colons were isolated, rinsed with saline, I0 cm segment weighed and tissue were processed for light microscopy, myeloperoxidase (MPO) activity and serum TNFa levels were determined by bioassay. Results:
Treatment n weight (g/cm) Iodoacetamide 4 0.37 _+0.15 IA + Thalidomide 6 0.14_+0.06"
lesion area (cm2) 4.01 _+2.40 0.78_+1.0"
MPO (U/gin) 1.7 _+1.49 0.14_+0.11"*
Treatment n TNFt~ $50 Iodoacetamide 4 5.3 _+1.07 IA + Thalidomide 6 2.2 _+0.74* Mean _+SD, * p < 0.01, ** p < 0.02 Conclusions: Iodoacetamide induced colitis in rats is ameliorated by thalidomide, probably by enhancing degradation of TNF messenger RNA. (34129
PATIENT PREFERENCES REGARDING PROPHYLAXIS FOR THE PREVENTION OF POST-OPERATIVE CROHN'S DISEASE (CD). ED Kennedy, BI O'Connor, M Varkul, A Detsky, RS McLeod. Departments of Surgery, Medicine. Mount Sinai Hospital, University of Toronto. Introduction: Although evidence suggests that 5-ASA is effective in delaying recurrence of CD post-operatively, it has been our anecdotal experience that many patients refuse prophylaxis with 5-ASA. This study was performed to elicit patient preferences for the risk of recurrence and 5-ASA. Methods: Patients with CD attending outpatient medical and surgical clinics were invited to participate in the study. Demographic and clinical information was obtained. The standard gamble (SG) was used to determine the utilities of two health states: (1) 40% recurrence of CD at 3 years not taking 5-ASA and (2) 25% recurrence of CD at 3 years taking 5-ASA. Perfect health was assigned a utility of "1" and death was assigned a utility of "0". The health states were derived from the results of a previous randomized controlled trial. Results: One hundred patients with CD (48 males, 52 females, mean age 35.7 _+ 12.5 years) were studied. The mean utilities for the desirability of the two health states were similar 0.97 _+0.09 vs 0.96 _+0.09. Subgroup analysi s showed no significant differences in the mean utilities for each health state when patients were stratified for duration of disease, level of education, whether they had previous surgery and whether they had past experience with 5-ASA products. Discussion: This study suggests that patients may view the reduced risk of recurrence attributable to 5-ASA as being of very little value. Larger sample sizes and more sensitive devices are needed to confirm this conclusion. G4130 ANTIOXIDANTS VITAMIN E & VITAMIN CARE EFFECTIVE THERAPY FOR RADIATION PROCTITIS. M. Kennedy, A. Keshavarzian. Department of Medicine (GI) Loyola University Medical Center, Maywood, IL; Medical Service, Hines VA Hospital, Hines, Illinois. Pelvic radiation is still commonly used for the treatment of prostatic and gynecologic malignancies. Radiation proctitis is a common sequelae of pelvic radiation with significant morbidity. Recent evidence suggest that radiation induced injury is due to oxidative damage. The aim of this study was to evaluate the effect of antioxidants Vitamin E & Vitamin C supplementation in symptoms of radiation proctitis. Methods: 12 patients with radiation proctitis due to pelvic radiation (received 1 to 36 years prior) for treatment of prostatic (n=6) or gynecologic (n=6) malignancies were given Vitamin E (400 nag TID) & Vitamin C (500 mg TID). Severity (grade 0-4) & frequency (grade 0-4) of 4 GI symptoms (rectal bleeding, rectal pain, diarrhea, urgency/incontinence) were assessed by a questionnaire before and after supplementation. A symptom severity index was calculated by the addition of severity + frequency (Max=8). The impact of symptoms on lifestyle (grade 0-4) was also questioned before and after therapy. Results: All patients received multiple medication prior to supplementation. 10 patients had multiple symptoms (6 had rectal bleeding, 11 diarrhea, 4 rectal pain, 10 urgency, 5 incontinence). Symptoms were severe enough to affect the lifestyle in 11, including 8 with daily inconvenience (grade 3). 11 patients noted improvement of or resolution of symptoms 0.5 to 2 months after therapy. There was a significant improvement in symptom index for bleeding, (5.2_+0.5 vs 2.8_+0.9), diarrhea (5.6_+0.5 vs 2.1_+0.8) and urgency (6.1 _+0.5 vs 0.7). Rectal pain was improved (5.8 _+0.8 vs 3.2 _+ 1.7) but not significantly. Bleeding resolved in 2 patients; diarrhea in 6 (X2=6.75, p=0.01), pain in 1 and urgency in 3 patients. Incontinence was resolved in all patients. Lifestyle improved in 8 including 4 who reported a return in normal lifestyle (grade 0). The 2 patients with no change in their daily inconvenience, had radiation enteritis as well. Conclusion: Vitamin E & Vitamin C appear to be effective therapy for radiation proctitis. A double blind controlled study is now needed to confirm this observation.
AI00S AGA ABSTRACTS • G4126
MANNOSE BINDING LECTIN GENOTYPE AS A RISK FACTOR FOR INTESTINAL PROTOZOAL INFECTION IN AIDS. Kelly P, Jack D, Naeem A, Tumer M, Farthing MJG. Digestive Diseases Research Centre, St Bartholomew's and Royal London School of Medicine, London; Institute of Child Health, London. Background Mannose binding lectin (MBL) is a multi-chain serum lectin with up to six 96 kDa subunits which plays an important role in non-specific defence against invasive organisms expressing polysaccharides terminating in mannose or N-acetyl glucosamine residues. Deficiencies of MBL predominantly arise from mutations in exon 1 of the MBL gene, at codons 52, 54 or 57. MBL gene mutations are over-represented in H1V positive populations, and are associated with shortened survival in AIDS. We examined MBL gene mutations in Zambian patients with AIDS related diarrhoea in order to determine whether they are associated with enhanced risk of enteric protozoal infections which might confer shortened survival. Methods 73 HIV seropositive patients with diarrhoea of over three weeks duration underwent stool microscopy, duodenal biopsy for evaluation of infection, and serum sampling. DNA was extracted from one frozen biopsy and used as the template for PCR amplification of exon 1; mutations were detected by controlled annealing of the products with a universal heteroduplex generator followed by polacrylamide electrophoresis. MBL concentrations in serum were assayed by ELISA. Results C. parvum was detected in 27%, L belli in 29%, and microsporidia in 32%. 46 of these patients had wild type MBL gene sequences, 20 were heterozygotes for the 57 codon mutation and 6 were homozygotes. MBL concentrations in serum were 1137 ~tg/l in wild types, 326 in heterozygotes, and 108 in homozygotes (p=0.0001). Individuals with MBL homozygous 57 codon mutations or 57/52 double mutations were at higher risk of cryptosporidiosis (Risk Ratio 3.1; 05%CI 1.6,6.0; p=0.006) but not of the other infections. Genotype predicted cryptosporidiosis, but serum MBL concentration did not. Conclusion In these AIDS patients, MBL mutations conferred higher risk for cryptosporidiosis than the wild type. No extra risk of other protozoal infection was observed. This extra risk for cryptosporidiosis could partially explain the shortened survival in AIDS experienced by individuals with MBL gene mutations. G4127 POLYMERASE CHAIN REACTION DETECTION OF CRYPTOSPORIDIUM PARVUM IN INTESTINAL SAMPLES. P Kelly, FA Makumbi, N. Luo, YK Seedat, MJG Farthing. Digestive Diseases Research Centre, St Bartholomew's and Royal London School of Medicine, London, UK; Gastrointestinal unit, University of Natal, Congella, South Africa. Background C. parvum is associated with considerable morbidity and mortality in AIDS patients and children, but no effective therapy is available and the pathophysiology of the condition is poorly understood. Previous studies have revealed that diarrhoea may be a feature of the infection without any evidence of a secretory lesion in the proximal small intestine. In gnotobiotic piglets, C. parvum infection moves caudally along the intestine during the course of infection. We set out to use PCR to characterise the distribution of infection in the gut as this may explain variability in the clinical course and in pathophysiological studies. Methods In the first study, 75 AIDS patients were studied with stool examination and duodenal biopsy for light microscopy, electron microscopy and PCR. In the second study, biopsies were taken for PCR only from duodenum, terminal ileum and colon in eight patients with AIDS and persistent cryptosporidiosis. PCR was used to amplify a portion of specific C. parvum DNA of unkown function, and PCR products were detected by electrophoresis and hybridisation of Southern blots. Results The PCR was sensitive and detected the equivalent of 5 genome copies. No positive reactions were obtained with human, bacterial or protozoal DNA. 18 of 75 (24%) of the AIDS patients in the first study had cryptosporidiosis diagnosed by conventional techniques, and 7 had duodenal infection detected microscopically. All of these microscopy positive cases were found to be positive by PCR, and 4 additional microscopy-negative cases were positive by PCR. In the second study, 1 patient had pan-enteric infection, 1 had localised duodenal infection and 2 had predominantly colonic infection. In the remainder, none of the biopsies were positive by PCR. Conclusion PCR confirms that C. parvum infection can persist in any part of the gut, that proximal small intestinal infection occurs in about half of all persistent cases, and it seems likely that some infections are confined to mid small intestine. This variability must be borne in mind when designing pathophysiological studies. G4128 IODOACETAMIDE-INDUCED COLITIS IN RATS IS INHIBITED BY THALIDOMIDE. G. Kenet, Y. Avni, H. Aeed, H. Shirin, Z. Matas, L. Zaidel, S. Galor, R. Hershkovitz and R, Brnck. Dept. of Gastroenterolgy, The E. Wolfson Medical Center, Holon, Israel. Thalidomide, a non-barbiturate hypnotic drug, has well known anti inflammatory effects. It suppresses TNFct production by accelerating the degradation of TNFtx messenger RNA. There have been isolated reports of beneficial effects of thalidomide in ulcerative colitis patients. The aim of the present study, was to evaluate the effect of thalidomide on experimentally-
induced colitis. Colitis was induced in Wistar rats by intracolonic administration of 0.1 ml iodoacetamide (IA) 3%. In one group, thalidomide (50 mg/kg) was administered 2 days before the induction of colitis, and continued daily until 7 days after. Blood was drawn on day 2 and 7. Rats were sacrificed 7 days after the induction of colitis. Colons were isolated, rinsed with saline, I0 cm segment weighed and tissue were processed for light microscopy, myeloperoxidase (MPO) activity and serum TNFa levels were determined by bioassay. Results:
Treatment n weight (g/cm) Iodoacetamide 4 0.37 _+0.15 IA + Thalidomide 6 0.14_+0.06"
lesion area (cm2) 4.01 _+2.40 0.78_+1.0"
MPO (U/gin) 1.7 _+1.49 0.14_+0.11"*
Treatment n TNFt~ $50 Iodoacetamide 4 5.3 _+1.07 IA + Thalidomide 6 2.2 _+0.74* Mean _+SD, * p < 0.01, ** p < 0.02 Conclusions: Iodoacetamide induced colitis in rats is ameliorated by thalidomide, probably by enhancing degradation of TNF messenger RNA. (34129
PATIENT PREFERENCES REGARDING PROPHYLAXIS FOR THE PREVENTION OF POST-OPERATIVE CROHN'S DISEASE (CD). ED Kennedy, BI O'Connor, M Varkul, A Detsky, RS McLeod. Departments of Surgery, Medicine. Mount Sinai Hospital, University of Toronto. Introduction: Although evidence suggests that 5-ASA is effective in delaying recurrence of CD post-operatively, it has been our anecdotal experience that many patients refuse prophylaxis with 5-ASA. This study was performed to elicit patient preferences for the risk of recurrence and 5-ASA. Methods: Patients with CD attending outpatient medical and surgical clinics were invited to participate in the study. Demographic and clinical information was obtained. The standard gamble (SG) was used to determine the utilities of two health states: (1) 40% recurrence of CD at 3 years not taking 5-ASA and (2) 25% recurrence of CD at 3 years taking 5-ASA. Perfect health was assigned a utility of "1" and death was assigned a utility of "0". The health states were derived from the results of a previous randomized controlled trial. Results: One hundred patients with CD (48 males, 52 females, mean age 35.7 _+ 12.5 years) were studied. The mean utilities for the desirability of the two health states were similar 0.97 _+0.09 vs 0.96 _+0.09. Subgroup analysi s showed no significant differences in the mean utilities for each health state when patients were stratified for duration of disease, level of education, whether they had previous surgery and whether they had past experience with 5-ASA products. Discussion: This study suggests that patients may view the reduced risk of recurrence attributable to 5-ASA as being of very little value. Larger sample sizes and more sensitive devices are needed to confirm this conclusion. G4130 ANTIOXIDANTS VITAMIN E & VITAMIN CARE EFFECTIVE THERAPY FOR RADIATION PROCTITIS. M. Kennedy, A. Keshavarzian. Department of Medicine (GI) Loyola University Medical Center, Maywood, IL; Medical Service, Hines VA Hospital, Hines, Illinois. Pelvic radiation is still commonly used for the treatment of prostatic and gynecologic malignancies. Radiation proctitis is a common sequelae of pelvic radiation with significant morbidity. Recent evidence suggest that radiation induced injury is due to oxidative damage. The aim of this study was to evaluate the effect of antioxidants Vitamin E & Vitamin C supplementation in symptoms of radiation proctitis. Methods: 12 patients with radiation proctitis due to pelvic radiation (received 1 to 36 years prior) for treatment of prostatic (n=6) or gynecologic (n=6) malignancies were given Vitamin E (400 nag TID) & Vitamin C (500 mg TID). Severity (grade 0-4) & frequency (grade 0-4) of 4 GI symptoms (rectal bleeding, rectal pain, diarrhea, urgency/incontinence) were assessed by a questionnaire before and after supplementation. A symptom severity index was calculated by the addition of severity + frequency (Max=8). The impact of symptoms on lifestyle (grade 0-4) was also questioned before and after therapy. Results: All patients received multiple medication prior to supplementation. 10 patients had multiple symptoms (6 had rectal bleeding, 11 diarrhea, 4 rectal pain, 10 urgency, 5 incontinence). Symptoms were severe enough to affect the lifestyle in 11, including 8 with daily inconvenience (grade 3). 11 patients noted improvement of or resolution of symptoms 0.5 to 2 months after therapy. There was a significant improvement in symptom index for bleeding, (5.2_+0.5 vs 2.8_+0.9), diarrhea (5.6_+0.5 vs 2.1_+0.8) and urgency (6.1 _+0.5 vs 0.7). Rectal pain was improved (5.8 _+0.8 vs 3.2 _+ 1.7) but not significantly. Bleeding resolved in 2 patients; diarrhea in 6 (X2=6.75, p=0.01), pain in 1 and urgency in 3 patients. Incontinence was resolved in all patients. Lifestyle improved in 8 including 4 who reported a return in normal lifestyle (grade 0). The 2 patients with no change in their daily inconvenience, had radiation enteritis as well. Conclusion: Vitamin E & Vitamin C appear to be effective therapy for radiation proctitis. A double blind controlled study is now needed to confirm this observation.