659 our study and the other three studies could be due in part to the relatively higher survival-rate in our control patients. The 3-year survival-rate of our control patients was 56% and ranged from 35 to 40% in the other three studies. Finally, if the results of our study and those of the other three are pooled, the difference in the survival-rates of patients with shunts and control patients is not significant.12 These pooled results strongly suggest that the benefit of therapeutic portacaval shunt in terms of longevity is either absent or very limited. Thus, therapeutic portacaval shunt in cirrhosis does not affect, or only slightly affects, the survival-rate and may induce chronic hepatic encephalopathy in a significant number of patients. For these reasons, portacaval shunt can no longer be accepted as a suitable treatment for cirrhotic patients with gastrointestinal bleeding. However, this pessimistic conclusion is valid only for patients selected according to current criteria and for the conventional end-to-side or side-to-side portacaval shunt. Our study and the other reported studies of therapeutic shunt were carried out before endoscopy had been extensively used to recognise the source of bleeding in cirrhotic patients; this means that some patients with non-variceal bleeding have had shunts. Hsemodynamic findings were not considered in selecting patients for portacaval shunt; the knowledge of portal venous pressure and liver blood-flow could aid the better selection of patients. Finally, our discouraging conclusion should not be extended to recently described shunting methods, such as combination of portacaval shunt and arterialisation of the portal vein" and selective distal splenorenal shunt.14 However, controlled studies should be carried out soon to assess these new surgical methods, to avoid the thirty-year delay which was needed for the correct evaluation of the conventional portacaval shunt. This work was presented in part at the 9th Meeting of the European Association for the Study of the Liver, Hemsedal, Norway, 1974. We thank all those members of the staff who participated in the care of the patients included in this study, especially Dr P. Clot, Dr P. Delagousie, Dr B. Estenne, Dr F. Fekete, Dr B. Goyer, Dr Giuli, and Dr M. Huguier who performed portacaval shunting, and Dr C. Atterbury for help in the preparation of this manuscript. Requests for reprints should be addressed to B.R., Hopital Beaujon, 92110 Clichy, France.
pancy between
REFERENCES
1. Grace,
N.
D., Muench, H., Chalmers, T. C. Gastroenterology, 1966, 50,
684. 2. Conn, H. O.,
Lindenmuth, W. W., May, C. J., Ramsby, G. R. Medicine, Baltimore, 1972, 51, 27. 3. Jackson, F. C., Perrin, E. B., Felix, W. R., Smith, T. Ann. Surg. 1971, 174, 672. 4.
Resnick, R. H., Iber, F. L., Ishihara, A. M., Chalmers, T. C., Zimmerman, H., Boston Inter-Hospital Liver Group. Gastroenterology, 1974, 67, 843. 5. Mikkelsen, W. P. Archs Surg. 1974, 108, 302. 6. Rueff, B., Benhamou, J. P. Clins Gastroenterology, 1975, 4, 425. 7. Prandi, D., Rueff, B., Roche-Sicot, J., Sicot, C., Maillard, J. N., Benhamou, J. P., Fauvert, R. Am. J. Surg. (in the press). 8. Schwartz, D., Flamant, R., Lellouch, J. L’Essai Thérapeutique chez l’Homme; p. 218. Paris, 1970. 9 Siegel, S. Non Parametric Statistics for the Behavioral Sciences; p. 96. New York, 1956. 10. Snedecor, G. W., Cochran, W. G. Statistical Methods; p. 329. Ames, Iowa, 1967. 11. Conn, H. O. Am. J. Gastroenterol. 1973, 59, 207. 12. Conn, H O. Gastroenterology, 1974, 67, 1065. 13 Maillard, J. N., Rueff, B., Prandi, D., Sicot, C. Archs Surg. 1974, 108, 315. 14 Warren, W. D., Salam, A. A., Huston, D., Zeppa, R. ibid. p. 306. 15 Garceau, A. J., Donaldson, R. M. Jr., O’Hara, E. T., Callow, A. D., Muench, H., Chalmers, T. C., Boston Inter-Hospital Liver Group. Gas-
troenterology, 1968, 54, 1057. Jackson, F. C., Pernn, E. B., Smith, A. G., Dagradi, A. E., Nadal, H. M. Am. J. Surg. 1968, 115, 22. 17 Resnick, R. H., Ishihara, A., Chalmers, T. C., Schimmel, E. M. Gastroenterology, 1968, 54, 1057. 18 Conn, H. O., Lindenmuth, W. W. New Engl. J. Med. 1965, 272, 1255. 16.
ENTEROTOXIGENIC ESCHERICHIA COLI AND REOVIRUS-LIKE AGENT IN RURAL BANGLADESH ROBERT W. RYDER Bacterial Diseases Division, Bureau of Epidemiology, Center for Disease Control, Public Health Service, Atlanta, Georgia 30333, U.S.A.
DAVID A. SACK
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. ALBERT Z. KAPIKIAN
Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. JYOTSNAMOY CHAKRABORTY A. S. M. MIZANUR RAHMAN
JAMES C. MCLAUGHLIN
Cholera Research Laboratory, Dacca, Bangladesh
MICHAEL H. MERSON
JOY G. WELLS Bacterial Diseases Division, Bureau of Epidemiology, Center for Disease Control, Atlanta, Georgia, U.S.A. 48 patients admitted to a rural Bangladesh hospital with dehydration secondary to diarrhœa were examined for infection caused by reovirus-like agent (R.L.A.) or enterotoxigenic Escherichia coli (E.T.E.C.). The diagnosis of R.L.A. infection was established by electron microscopy of stool filtrates and by a fourfold or greater rise in serum complement-fixing antibodies to the Nebraska calf diarrhœa virus. Evidence of infection by heat-labile-toxin (L.T.)-producing E.T.E.C. was sought by stool culture and serological testing using the adrenal-cell tissue-culture system. Infection by heat-stable-toxin (S.T.)-producing E.T.E.C. was sought by stool culture using the infant mouse test. 12 patients, all less than two years old, had evidence of R.L.A. infection, accounting for illness in 55% of the 22 patients who were less than two years old. None of these 22 children had evidence of E.T.E.C. infection. R.L.A. diarrhœa lasted five to six days, often led to serious dehydration, and was associated with vomiting and fever. 11 cases of E.T.E.C. diarrhœa were detected, accounting for 56% of the cases of diarrhœa in the 18 patients who were more than ten years old. Diarrhœa caused by E.T.E.C. was sudden in onset, shorter in duration, and caused pronounced dehydration. In a community survey E.T.E.C. was isolated with equal frequency in the stools of control and case family members. The data suggest that E.T.E.C. is a common cause of adult diarrhœa in Bangladesh, while R.L.A. is a common cause of diarrhœa in children.
Summary
Introduction
ENTEROTOXIGENIC Escherichia coli (E.T.E.C.) and reovirus-like agent (R.L.A.) are major causes of acute undifferentiated diarrhcea in many parts of the world.1-8 During December, 1974, and January, 1975, we investigated human diarrhoeal illness caused by these agents in rural Bangladesh where diarrhoea not caused by commonly recognised enteric pathogens (e.g., Vibrio
cholera, salmonella, shigella, &c.)
is
frequent.9
660 TABLE I-DIARRHOEAL ILLNESS IN FAMILY CONTACTS AND CONTROLS
Materials and Methods
Background Matlab Hospital, the field hospital of the Cholera Research Laboratory is located about 40 miles from Dacca in one of the most densely populated areas of Bangladesh. Patients are weighed on admission and daily thereafter. All patients are treated on cholera cots which permit liquid stool to be collected in calibrated containers. In hospital, patients receive oral and/or parenteral fluid rehydration. Stool cultures are plated on MacConkey’s, salmonella-shigella, and Monsur agars and incubated at 37° overnight. Suspect shigella or vibrio colonies are identified by agglutination with group and type specific
TABLE II—CHARACTERISTICS OF STUDY PATIENTS
antiserum.
Study Design Patients included in this study had the following characteristics : (1) they were in hospital with severe dehydration caused by diarrhoea; (2) they had become ill less than seventytwo hours before admission; (3) they had not received antecedent antibiotic therapy; (4) they had no leucocytes in their admission stool specimen;1O (5) they had stool cultures negative for V. cholera, shigella, and salmonella during the first two days of their hospital stay; and (6) they were the first patient admitted each day who satisfied the above criteria. All study patients remained in hospital and had daily rectal swabs taken for at least six days; none of them received antibiotics. The clinical course of 15 patients concurrently in hospital with cholera and receiving oral tetracycline was observed for comparison. Blood specimens were drawn from patients by venepuncture on admission and during convalescence ten to fourteen days later. We defined diarrhoea as loose stools occurring more frequently than normal. Clinical and bacteriological surveillance for diarrhoea in each patient’s family ("case family") and an adjacent "control family" was conducted by a trained field team within forty-eight hours of the patient’s admission to hospital. This team visited each case and control family on three alternate days inquiring about the presence of diarrhoea, recording demographic data, collecting water samples, and obtaining rectal swabs from each family member. Rectal swabs obtained in the field were collected with a sterile swab and placed in Cary-Blair medium for transport to the laboratory. Serum samples were also collected from individuals in control families of specified ages who had not had diarrhoea in the previous week.
Laboratory Studies Bacteriology.-From each MacConkey’s agar plated on admission, 10 typical E. coli colonies were selected and inoculated onto nutrient agar slants for further study; this is a reliable method of selecting E. coli.1’ A pool of these 10 colonies was also made. 10 E. coli colonies from subsequent stool cultures from patients, and all cultures from family contacts and controls were pooled and separate nutrient agar slants were inoculated from each pool. All slants were incubated at 370C overnight and then held at room temperature. TABLE III
-FREQUENCY OF
Enterotoxin assays.- The 10 E. coli isolates from each patient’s admission stool culture and the pooled cultures isolated from these patients, their family contacts, and controls (a total of 1142 cultures) were tested for heat-labile-enterotoxin (L.T.) production by the Y 1-adrenal-cell system using the miniculture technique.12 In addition, E. coli isolates from drinking bathing, and cooking water sources were also tested.5 E. coli isolates from each patient’s admission culture were also assayed for heat-stable-enterotoxin (s.T.) production using the infant mouse assay. 13 A ratio of intestinal weight to remaining body-weight of 0083 or greater was regarded as positive." All E.T.E.C. isolates were serotyped using 143 E. coli antisera in 19 pools. The antibiotic sensitivity of all E.T.E.C. isolates was determined using standard disc sensitivity methods." L.T.antitoxin titres from hospital patients and neighbourhood controls were assayed in the adrenal-cell system." Paired sera were not available from 6 study patients. Virology.—R.L.A. infection was established by: (1) examination of stools by immune electron microscopy using convales. cent-phase sera or human immune serum globulin as a source of antibody;’16 (2) a fourfold or greater rise in serum complement-fixing antibodies using the Nebraska calf diarrhea virus (N.C.D.V.) as a substitute antigen.’ Stools from patients at admission were prepared as a 2% suspension in veal infusion broth containing 0.5% bovine serum albumin. Each suspension was shaken with glass beads and centrifuged at 500 g for an hour and the sediment discarded. The faecal suspension was successively filtered through a 30 m and a 022 m Millipore filter before streptomycin and tetracycline were added to produce final concentrations of 1000 g/ml and 33 g/ml, respectively. Paired sera and/or stools were not obtained from 6 patients.
E.T.E.C. AND R.L.A. BY AGE IN
48 PATIENTS
IN HOSPITAL WITH DIARRHOEA
661 Results
TABLE IV-ANTIBODY TITRES AGAINST L.T. IN PATIENTS AND
SYMPTOM-FREE COMMUNITY CONTROLS
During the study period (December, 1974, to January, 1975) 423 patients were admitted to the hospital with diarrhoea; stool cultures from 242 (57%) of these patients were negative for shigella, salmonella, or V. choleræ. 48 (20%) of these 242 individuals were included in the investigation. The mean age of these 48 patients was twelve; the median age was two years. The frequency of diarrhoea in family contacts and controls was almost identical within each age-group (table i). Laboratory Studies There was evidence of diarrhoea caused by E.T.E.C. in 11 (23%) patients (L.T.[+]), 10 of whom were more than ten years old (table II). E.T.E.C. was found in stools of 8 patients, while 3 additional patients had a fourfold rise in antibody to L.T. (table III). None of the E.T.E.C. isolates belonged to a currently recognised enteropathogenic serotype. Using pooled specimens, enterotoxigenic organisms were no more common in family contacts of L.T.(+) patients than of L.T.(-) patients. E. coli from stools of 3 (36%) of 84 family contacts of L.T.(+) patients and none of 71 controls were enterotoxigenic, while stools from 4 (1.8%) of 219 family contacts of L.T.(-) patients and 3 (15%) of 201 controls contained L.T.-producing organisms. Only 1 of these 10 L.T.(+) individuals had diarrhoea at the time of culture. E. coli isolates from 1 of 39 contaminated water sources were enterotoxigenic. Pooled cultures from patients with diarrhoea were a satisfactory method for screening isolates, since no individual isolates were positive when pools were negative. Of the 8 cases with enterotoxigenic pools from admission cultures, all but one contained 9 or 10 of 10 enterotoxigenic single-colony isolates; in this one pool only 4 of 10 isolates produced L.T. All E.T.E.C. produced s.T. and L.T. Cases continued to shed E.T.E.C. in their stools for an average of 3 - 6 days after diarrhoea ceased. E.T.E.C. isolated from 3 of these patients demonstrated multiple antibioticresistance; the remainder were multiply sensitive. 6 (75%) of 8 patients with enterotoxigenic organisms and 3 (88%) of 35 other patients had a fourfold increase in antibody titre to L.T. enterotoxin. Initial antitoxin titres of individuals with diarrhoea caused by E.T.E.C. resembled those in patients with diarrhoea caused by other agents and symptom-free controls (table iv). Younger patients in all groups tended to have higher titres. In the 8 L.T.(+) patients there was no corTABLE
*Obtained 10-14 days after initial titre. tlnitial and second titres are significantly different
relation between antitoxin titre and length of stool carriage of the organism or severity of illness. There was evidence that diarrhoea was caused by R.L.A. in 12 (55%) of the 22 patients who were less than two years old and from whom sera and/or stool filtrates were collected and tested (tables n and III). None of the 20 patients who were more than two years old and from whom sera and/or stool filtrates were available had R.L.A. infection. R.L.A. was seen in the stools of 7 (44%) of 16. children who were less than two years old; 3 of these had a fourfold or greater increase in complementfixing antibody titre. 5 additional cases were confirmed serologically. Stool filtrates from only 2 of these cases were examined for R.L.A.; both were negative. Clinical Results
abdominal cramps, and raised temperature in patients with R.L.A. than in patients with E.T.E.C. (table ii). Although patients with E.T.E.C. waited a shorter time (mean nine hours) before coming to the hospital than did patients with R.L.A.
Vomiting,
were more common
(mean forty-six hours), E.T.E.C.-positive patients were dehydrated (mean admission serum-protein 11.1 on g/dl) arrival than patients with R.L.A. (mean admission serum-protein 9.8 gjdl) table v). Because of this pronounced dehydration E.T.E.c. patients required more fluid replacement (131.9 ml/kg) than did patients with R.L.A. (90.9 ml/kg). However, patients with R.L.A. had
more
V--CHARACTERISTICS REFLECTING THE SEVERITY OF ILLNESS OF STUDY PATIENTS, MATLAB HOSPITAL,
’All patients with cholera received5 days of tetracycline while in hospital. ffnterquarttte range.
by the Mann-Whitney U Test
(p
pancy between
REFERENCES
1. Grace,
N.
D., Muench, H., Chalmers, T. C. Gastroenterology, 1966, 50,
684. 2. Conn, H. O.,
Lindenmuth, W. W., May, C. J., Ramsby, G. R. Medicine, Baltimore, 1972, 51, 27. 3. Jackson, F. C., Perrin, E. B., Felix, W. R., Smith, T. Ann. Surg. 1971, 174, 672. 4.
Resnick, R. H., Iber, F. L., Ishihara, A. M., Chalmers, T. C., Zimmerman, H., Boston Inter-Hospital Liver Group. Gastroenterology, 1974, 67, 843. 5. Mikkelsen, W. P. Archs Surg. 1974, 108, 302. 6. Rueff, B., Benhamou, J. P. Clins Gastroenterology, 1975, 4, 425. 7. Prandi, D., Rueff, B., Roche-Sicot, J., Sicot, C., Maillard, J. N., Benhamou, J. P., Fauvert, R. Am. J. Surg. (in the press). 8. Schwartz, D., Flamant, R., Lellouch, J. L’Essai Thérapeutique chez l’Homme; p. 218. Paris, 1970. 9 Siegel, S. Non Parametric Statistics for the Behavioral Sciences; p. 96. New York, 1956. 10. Snedecor, G. W., Cochran, W. G. Statistical Methods; p. 329. Ames, Iowa, 1967. 11. Conn, H. O. Am. J. Gastroenterol. 1973, 59, 207. 12. Conn, H O. Gastroenterology, 1974, 67, 1065. 13 Maillard, J. N., Rueff, B., Prandi, D., Sicot, C. Archs Surg. 1974, 108, 315. 14 Warren, W. D., Salam, A. A., Huston, D., Zeppa, R. ibid. p. 306. 15 Garceau, A. J., Donaldson, R. M. Jr., O’Hara, E. T., Callow, A. D., Muench, H., Chalmers, T. C., Boston Inter-Hospital Liver Group. Gas-
troenterology, 1968, 54, 1057. Jackson, F. C., Pernn, E. B., Smith, A. G., Dagradi, A. E., Nadal, H. M. Am. J. Surg. 1968, 115, 22. 17 Resnick, R. H., Ishihara, A., Chalmers, T. C., Schimmel, E. M. Gastroenterology, 1968, 54, 1057. 18 Conn, H. O., Lindenmuth, W. W. New Engl. J. Med. 1965, 272, 1255. 16.
ENTEROTOXIGENIC ESCHERICHIA COLI AND REOVIRUS-LIKE AGENT IN RURAL BANGLADESH ROBERT W. RYDER Bacterial Diseases Division, Bureau of Epidemiology, Center for Disease Control, Public Health Service, Atlanta, Georgia 30333, U.S.A.
DAVID A. SACK
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. ALBERT Z. KAPIKIAN
Laboratory of Infectious Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. JYOTSNAMOY CHAKRABORTY A. S. M. MIZANUR RAHMAN
JAMES C. MCLAUGHLIN
Cholera Research Laboratory, Dacca, Bangladesh
MICHAEL H. MERSON
JOY G. WELLS Bacterial Diseases Division, Bureau of Epidemiology, Center for Disease Control, Atlanta, Georgia, U.S.A. 48 patients admitted to a rural Bangladesh hospital with dehydration secondary to diarrhœa were examined for infection caused by reovirus-like agent (R.L.A.) or enterotoxigenic Escherichia coli (E.T.E.C.). The diagnosis of R.L.A. infection was established by electron microscopy of stool filtrates and by a fourfold or greater rise in serum complement-fixing antibodies to the Nebraska calf diarrhœa virus. Evidence of infection by heat-labile-toxin (L.T.)-producing E.T.E.C. was sought by stool culture and serological testing using the adrenal-cell tissue-culture system. Infection by heat-stable-toxin (S.T.)-producing E.T.E.C. was sought by stool culture using the infant mouse test. 12 patients, all less than two years old, had evidence of R.L.A. infection, accounting for illness in 55% of the 22 patients who were less than two years old. None of these 22 children had evidence of E.T.E.C. infection. R.L.A. diarrhœa lasted five to six days, often led to serious dehydration, and was associated with vomiting and fever. 11 cases of E.T.E.C. diarrhœa were detected, accounting for 56% of the cases of diarrhœa in the 18 patients who were more than ten years old. Diarrhœa caused by E.T.E.C. was sudden in onset, shorter in duration, and caused pronounced dehydration. In a community survey E.T.E.C. was isolated with equal frequency in the stools of control and case family members. The data suggest that E.T.E.C. is a common cause of adult diarrhœa in Bangladesh, while R.L.A. is a common cause of diarrhœa in children.
Summary
Introduction
ENTEROTOXIGENIC Escherichia coli (E.T.E.C.) and reovirus-like agent (R.L.A.) are major causes of acute undifferentiated diarrhcea in many parts of the world.1-8 During December, 1974, and January, 1975, we investigated human diarrhoeal illness caused by these agents in rural Bangladesh where diarrhoea not caused by commonly recognised enteric pathogens (e.g., Vibrio
cholera, salmonella, shigella, &c.)
is
frequent.9
660 TABLE I-DIARRHOEAL ILLNESS IN FAMILY CONTACTS AND CONTROLS
Materials and Methods
Background Matlab Hospital, the field hospital of the Cholera Research Laboratory is located about 40 miles from Dacca in one of the most densely populated areas of Bangladesh. Patients are weighed on admission and daily thereafter. All patients are treated on cholera cots which permit liquid stool to be collected in calibrated containers. In hospital, patients receive oral and/or parenteral fluid rehydration. Stool cultures are plated on MacConkey’s, salmonella-shigella, and Monsur agars and incubated at 37° overnight. Suspect shigella or vibrio colonies are identified by agglutination with group and type specific
TABLE II—CHARACTERISTICS OF STUDY PATIENTS
antiserum.
Study Design Patients included in this study had the following characteristics : (1) they were in hospital with severe dehydration caused by diarrhoea; (2) they had become ill less than seventytwo hours before admission; (3) they had not received antecedent antibiotic therapy; (4) they had no leucocytes in their admission stool specimen;1O (5) they had stool cultures negative for V. cholera, shigella, and salmonella during the first two days of their hospital stay; and (6) they were the first patient admitted each day who satisfied the above criteria. All study patients remained in hospital and had daily rectal swabs taken for at least six days; none of them received antibiotics. The clinical course of 15 patients concurrently in hospital with cholera and receiving oral tetracycline was observed for comparison. Blood specimens were drawn from patients by venepuncture on admission and during convalescence ten to fourteen days later. We defined diarrhoea as loose stools occurring more frequently than normal. Clinical and bacteriological surveillance for diarrhoea in each patient’s family ("case family") and an adjacent "control family" was conducted by a trained field team within forty-eight hours of the patient’s admission to hospital. This team visited each case and control family on three alternate days inquiring about the presence of diarrhoea, recording demographic data, collecting water samples, and obtaining rectal swabs from each family member. Rectal swabs obtained in the field were collected with a sterile swab and placed in Cary-Blair medium for transport to the laboratory. Serum samples were also collected from individuals in control families of specified ages who had not had diarrhoea in the previous week.
Laboratory Studies Bacteriology.-From each MacConkey’s agar plated on admission, 10 typical E. coli colonies were selected and inoculated onto nutrient agar slants for further study; this is a reliable method of selecting E. coli.1’ A pool of these 10 colonies was also made. 10 E. coli colonies from subsequent stool cultures from patients, and all cultures from family contacts and controls were pooled and separate nutrient agar slants were inoculated from each pool. All slants were incubated at 370C overnight and then held at room temperature. TABLE III
-FREQUENCY OF
Enterotoxin assays.- The 10 E. coli isolates from each patient’s admission stool culture and the pooled cultures isolated from these patients, their family contacts, and controls (a total of 1142 cultures) were tested for heat-labile-enterotoxin (L.T.) production by the Y 1-adrenal-cell system using the miniculture technique.12 In addition, E. coli isolates from drinking bathing, and cooking water sources were also tested.5 E. coli isolates from each patient’s admission culture were also assayed for heat-stable-enterotoxin (s.T.) production using the infant mouse assay. 13 A ratio of intestinal weight to remaining body-weight of 0083 or greater was regarded as positive." All E.T.E.C. isolates were serotyped using 143 E. coli antisera in 19 pools. The antibiotic sensitivity of all E.T.E.C. isolates was determined using standard disc sensitivity methods." L.T.antitoxin titres from hospital patients and neighbourhood controls were assayed in the adrenal-cell system." Paired sera were not available from 6 study patients. Virology.—R.L.A. infection was established by: (1) examination of stools by immune electron microscopy using convales. cent-phase sera or human immune serum globulin as a source of antibody;’16 (2) a fourfold or greater rise in serum complement-fixing antibodies using the Nebraska calf diarrhea virus (N.C.D.V.) as a substitute antigen.’ Stools from patients at admission were prepared as a 2% suspension in veal infusion broth containing 0.5% bovine serum albumin. Each suspension was shaken with glass beads and centrifuged at 500 g for an hour and the sediment discarded. The faecal suspension was successively filtered through a 30 m and a 022 m Millipore filter before streptomycin and tetracycline were added to produce final concentrations of 1000 g/ml and 33 g/ml, respectively. Paired sera and/or stools were not obtained from 6 patients.
E.T.E.C. AND R.L.A. BY AGE IN
48 PATIENTS
IN HOSPITAL WITH DIARRHOEA
661 Results
TABLE IV-ANTIBODY TITRES AGAINST L.T. IN PATIENTS AND
SYMPTOM-FREE COMMUNITY CONTROLS
During the study period (December, 1974, to January, 1975) 423 patients were admitted to the hospital with diarrhoea; stool cultures from 242 (57%) of these patients were negative for shigella, salmonella, or V. choleræ. 48 (20%) of these 242 individuals were included in the investigation. The mean age of these 48 patients was twelve; the median age was two years. The frequency of diarrhoea in family contacts and controls was almost identical within each age-group (table i). Laboratory Studies There was evidence of diarrhoea caused by E.T.E.C. in 11 (23%) patients (L.T.[+]), 10 of whom were more than ten years old (table II). E.T.E.C. was found in stools of 8 patients, while 3 additional patients had a fourfold rise in antibody to L.T. (table III). None of the E.T.E.C. isolates belonged to a currently recognised enteropathogenic serotype. Using pooled specimens, enterotoxigenic organisms were no more common in family contacts of L.T.(+) patients than of L.T.(-) patients. E. coli from stools of 3 (36%) of 84 family contacts of L.T.(+) patients and none of 71 controls were enterotoxigenic, while stools from 4 (1.8%) of 219 family contacts of L.T.(-) patients and 3 (15%) of 201 controls contained L.T.-producing organisms. Only 1 of these 10 L.T.(+) individuals had diarrhoea at the time of culture. E. coli isolates from 1 of 39 contaminated water sources were enterotoxigenic. Pooled cultures from patients with diarrhoea were a satisfactory method for screening isolates, since no individual isolates were positive when pools were negative. Of the 8 cases with enterotoxigenic pools from admission cultures, all but one contained 9 or 10 of 10 enterotoxigenic single-colony isolates; in this one pool only 4 of 10 isolates produced L.T. All E.T.E.C. produced s.T. and L.T. Cases continued to shed E.T.E.C. in their stools for an average of 3 - 6 days after diarrhoea ceased. E.T.E.C. isolated from 3 of these patients demonstrated multiple antibioticresistance; the remainder were multiply sensitive. 6 (75%) of 8 patients with enterotoxigenic organisms and 3 (88%) of 35 other patients had a fourfold increase in antibody titre to L.T. enterotoxin. Initial antitoxin titres of individuals with diarrhoea caused by E.T.E.C. resembled those in patients with diarrhoea caused by other agents and symptom-free controls (table iv). Younger patients in all groups tended to have higher titres. In the 8 L.T.(+) patients there was no corTABLE
*Obtained 10-14 days after initial titre. tlnitial and second titres are significantly different
relation between antitoxin titre and length of stool carriage of the organism or severity of illness. There was evidence that diarrhoea was caused by R.L.A. in 12 (55%) of the 22 patients who were less than two years old and from whom sera and/or stool filtrates were collected and tested (tables n and III). None of the 20 patients who were more than two years old and from whom sera and/or stool filtrates were available had R.L.A. infection. R.L.A. was seen in the stools of 7 (44%) of 16. children who were less than two years old; 3 of these had a fourfold or greater increase in complementfixing antibody titre. 5 additional cases were confirmed serologically. Stool filtrates from only 2 of these cases were examined for R.L.A.; both were negative. Clinical Results
abdominal cramps, and raised temperature in patients with R.L.A. than in patients with E.T.E.C. (table ii). Although patients with E.T.E.C. waited a shorter time (mean nine hours) before coming to the hospital than did patients with R.L.A.
Vomiting,
were more common
(mean forty-six hours), E.T.E.C.-positive patients were dehydrated (mean admission serum-protein 11.1 on g/dl) arrival than patients with R.L.A. (mean admission serum-protein 9.8 gjdl) table v). Because of this pronounced dehydration E.T.E.c. patients required more fluid replacement (131.9 ml/kg) than did patients with R.L.A. (90.9 ml/kg). However, patients with R.L.A. had
more
V--CHARACTERISTICS REFLECTING THE SEVERITY OF ILLNESS OF STUDY PATIENTS, MATLAB HOSPITAL,
’All patients with cholera received5 days of tetracycline while in hospital. ffnterquarttte range.
by the Mann-Whitney U Test
(p
