- Email: [email protected]
CYTOTOXIN PRODUCTION BY MEMBERS OF GENUS VIBRIO
1217
IgG FRACTION OF TOTAL ANTIBODY (IgG+IgM) ON DAYS 28 AFTER
14 AND
POST-EXPOSURE VACCINATION’*
*effective antigemc vaiues’ PDEV 2- 5, 4’ 2, and 4 - 5 IU per dose; HDCV 6’ 3 IU per dose.
reduced vaccination schedule recommended by the World Health Organisation for HDCV. In 71 cases (PDEV 52, HDCV 19) enough of the serum taken on days 14 and 28 was available for IgG fractionation of total rabies antibody. This was done by enzymelinked immunosorbent assay, with enzyme-conjugated anti-y and anti- antibodies. The results were compared with a standard human serum of known anti-rabies IgG and IgM content. All vaccinees had titres of 1 - 5 IU/ml or more by day 14 (0 - 5 IU/ml or more is generally considered to be protective). The IgG fractions of the total antibodies (IgG+IgM) were similar in both vaccine groups. However, on day 14 the IgG fraction in the serum of 5 vaccinees accounted for less than one-quarter of the total antibodies and in the serum of 22 volunteers for less than half of the total antibodies. By day 28, the IgG fraction accounted for more than one-quarter of the total antibodies in the serum of all vaccinees and for more than half in 69 of the 71 sera investigated. The fact that on day 14 some of the vaccinees had a low IgG fraction of total antibody suggests that active immunisation alone might not guarantee protection. Therefore, the initial application of rabies immunoglobulin as recommended by the WH03 and by the Immunisation Practices Advisory Committee, US Centers for Disease Control,4is indispensable. Two recent cases of fatal rabies encephalitis in exposed persons treated with HDCV but without rabies immunoglobulin5,6 strongly support this point of view. R. GLÜCK A. WEGMANN R. GERMANIER
Swiss Serum and Vaccine Institute, CH 3000 Berne, Switzerland Medical
Propaedeutical Clinic, University of Berne
H. KELLER H. U. DETTWILER
Department of Veterinary Bacteriology, University of Berne
A. I. WANDELER
1 Turner GS.
Immunoglobuhn (IgG) and (IgM) antibody responses to rabies vaccine 1978; 40: 595-604. H, Gluck R, Wegmann A, Wandeler AI. Immunogenität einer neuen,
J Gen 2 Keller
Virol
hochkonzentrierten
Tollwut-Entenembryovakzine.
Schweiz Med Wschr 1984; 29:
648-53 3. World Health 4
5
6.
Organisation Sixth report of the Expert Committee on Rabies Techn Rep Ser WHO 1973; no 523. Immunization Practices Advisory Committee (Centers for Disease Control). Rabies prevention-United States, 1984. MMWR 1984; 33: 393-408. Devriendt J, Staroukine M, Costy F, Vanderhaeghen J-J. Fatal encephalitis apparently due to rabies: Occurrence after treatment with human diploid cell vaccine but not rabies immune globulin. JAMA 1982, 248: 2304-06. Wattanasri S, Boonthai P, Thongcharoen P Human rabies after late administration of human diploid cell vaccine without hyperimmune serum. Lancet 1982; ii. 870.
CAMPYLOBACTER-LIKE ORGANISMS IN GASTRITIS
SIR,-While it is certainly attractive
to
take the report of Dr
O’Connor and his colleagues (Nov 10, p 1091) as providing indirect evidence
that Campylobacter-like organisms (CLO) may be aetiologically important in chronic antral gastritis (rather than secondary colonisers), such a conclusion, tentative though it is, may be justified. The association of chronic gastritis with CLO (detected histologically or on culture) is beyond doubt and in eight published studies approaches 100% concordance if one allows for sampling error, satisfying the first of Koch’s four postulates for an aetiological not
relationship between a given microorganism and a given disease. Koch’s second postulate-microorganism must be grown in pure culture-is also fulfilled, but his third and fourth postulates, which require the microorganism to be inoculated into a susceptible animal, reproduce the disease, and be observed and be recoverable from the experimentally diseased animal, are impossible to apply in the present very limited state of our knowledge of CLO. What is known, however, is that CLO seem to be found only in association with inflamed epithelium of gastric typel-4-either true gastric epithelium or the gastric metaplastic epithelium found in the duodenal mucosa in some cases of duodenal ulcer-and they are absent from non-metaplastic duodenal mucosa and from gastric biopsies showing intestinal metaplasia. O’Connor et al found intestinal metaplasia in biopsy tissue from 11of 14 patients with pernicious anaemia (PA) (1 had numerous CLO and 2 scanty CLO) and in 7 of 14 patients with peptic ulcer (1 had scanty and 2 numerous CLO). It is not clear whether the presence or absence of metaplasia or CLO was an all-or-none phenomenon among replicate samples in individual patients but, allowing for sampling error, it is certainly possible that the rarity of CLO in PA patients is explained by the increased prevalence of intestinal metaplasia in this group. Their information is valuable in our present state of ignorance of the significance and aetiological importance of CLO but I am not sure that the rarity of gastric CLO in PA can really be accepted as indicative of a pathogenic role for these organisms in chronic
.
gastritis. Department of Surgery, Whittington Hospital,
JOHN MEYRICK THOMAS
London N19 5NF
1. Steer HW, Colin-Jones DG. Mucosal changes in gastric ulceration and their response to carbenoxolone sodium, Gut 1975; 16: 590-97. 2. Meyrick Thomas J, Poynter D, Gooding C, et al. Gastric spiral bacteria. Lancet 1984; ii. 100.
Phillips AD, Hine KR, Holmes GKT, Woodings DF. Gastric spiral bacteria. Lancet
3.
1984; ii: 100-01. 4. Steer HW. Surface
morphology of
the
gastroduodenal
mucosa
in duodenal ulceration
Gut 1984; 25: 1203-10.
CYTOTOXIN PRODUCTION BY MEMBERS OF GENUS VIBRIO
SIR,- The Lancet has carried several letters about the production of
a toxin distinct from cholera toxin (CT) by Vibrio cholerae serogroup 01 or the naming/misnaming of V cholerae Serogroup non-01 as V cholerae. 1-4 Members of serogroup non-01 produce an enterotoxin,which has been demonstrated immunologically to be identical to or similar to cholera enterotoxin.6,7 Members of both serogroups may cause diarrhoea without the production of CT .8-11 This form of diarrhoea may, in many cases, be distinguished from that caused by CT-producing vibrios by the presence of red blood cells and leucocytes in the stoo1.8,9,I2 We have isolated and characterised a toxin produced by a clinical isolate of V cholerae non-01 that appeared to be responsible for pathogenicity. The toxin was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary tissue culture cells. The activity of the cytotoxin in tissue culture was not neutralised by antibodies directed against the cytotoxins produced by Shigella dysenteriae 1 or V vulnificus. The cytotoxin, a heat-labile protein, insensitive to trypsin and destroyed by freezing, had a relative molecular weight of 52 000, an ionisation point of 8-65, and no subunit structure. The purified cytotoxin induced fluid accumulation in the 5 h rabbit ileal loop model, was lethal to adult mice (20 g) by intravenous injection (2’ 5 pg caused death within 2 min), and had a median lethal dose (LD5o) of 0’68 pg for suckling mice injected intragastrically. Sublethal doses injected intragastrically did not elicit fluid accumulation in suckling mice killed 3 h after infection. Eleven clinical isolates (ten V cholerae non-Ol and one V mimicus), tested immunologically, produced the cytotoxin but did not produce CT. This cytotoxin was also produced by one CTproducing clinical isolate each of V cholerae 01, classical biotype, V cholerae non-01, and V mimicus. We are continuing our investigations to determine the relatedness of this cytotoxin to that produced by V cholerae 01, as reported by Spira et al 13 in a clinical isolate of V cholerae 01, El Tor biotype, which lacks the potential to’
1218 and in a mutant of an El Tor biotype strain from which the CT gene had been selectively deleted. The ability of both serogroups to produce or not produce CT and/or the described cytotoxin further supports biochemical data and DNA hybridisation studies which dictate the maintenance of both serogroups in the same genus and species. Definition of a genus and species solely on the basis of the degree of pathogenicity is unreliable because of possible gain or loss of the ability to produce toxin and hence virulence.
PLANAR LATTICES IN FAECES OF BABIES
produce CT,
Division of Microbiology, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington DC 20204, USA
Division of Microbiology, Center for Food Safety and FDA, Cincinnati, Ohio 1. 2
3. 4. 5. 6
7.
8. 9
10 11. 12.
13
J. M. MADDEN B. A. MCCARDELL
Applied Nutrition,
Sanyal SC, Alam K, Neogi PBK, Kaper JB, Levine MM Toxins
D. B. SHAH et in
al. A
new cholera toxin. Lancet 1983, i: 1337. environmental Vibrio cholerae Lancet 1983,
ii
217-18 Kay BA, Sack RB, Spira WM, et al Vibrio cholerae non-O1 isolated from five people with diarrhoea in Lima Lancet 1984; i: 218 Preston NW Vibrio cholerae in South America. Lancet 1984, i 624-25 Zinnaka T, Carpenter CCJ, Jr. An enterotoxin produced by noncholera vibrios. Hopkins Med J 1972, 131: 403-11. Yamamoto K, Takeda Y, Miwatani T, et al. Purification and some properties of a nonO1 Vibrio cholerae enterotoxin that is identical to cholera enterotoxin Infect Immun 1983, 39: 1128-35 Yamamoto K, Takeda Y, Miwatani T, et al Evidence that a non-O1 Vibrio cholerae produces enterotoxin that is similar but not identical to cholera enterotoxin. Infect Immun 1983; 41: 896-901 Blake PA, Weaver RE, Hollis DG. Diseases of humans (other than cholera) caused by vibrios. Annu Rev Microbiol 1980; 34: 341-67 Bonner JR, Coker AS, Berryman CR, et al. Spectrum of vibrio infections in a Gulf coast community. Ann Intern Med 1983, 99: 464-69. Kenyon JE, Gillies DC, Piexoto DR, et al. Vibrio cholerae (non-O1) isolated from California coastal waters. Appl Environ Microbrol 1983, 46: 1232-33 Levine MM, Kaper JB, Black RE, et al. New insights into the pathogenesis of cholera and its prevention 19th Joint Conference on Cholera, 1983; abstr 56 Madden JM, Nematollahi WP, Hill WE, et al. Virulence of three clinical isolates of Vibrio cholerae non-O1serogroup in experimental enteric infections in rabbits. Infect Immun 1981; 33: 616-19 Spira MW, Sack D, Sanyal S, et al Description of a possible new extracellular virulence factor in non-toxigenic V cholerae O1. 19th Joint Conference on Cholera, 1983, abstr 80-81.
SIR,-An aetiological role for a new rotavirus-like agent in several outbreaks of non-bacterial diarrhoea in the People’s Republic of China has been proposed by Tao et al.1,2 The morphological features of both viruses as well as of putative viral components were described.’ A pattern of virus particle degradation was also proposed, involving the formation of a regular hexagonal lattice thought to be comprised of detached viral morphological subunits (see Tao et al,l fig IE). The centre-to-centre period of the lattice, as calculated from our measurements of the print, is about 11.5 nm. We report here the observation of similar structures in negatively stained preparations of sediments from faeces of infants up to 3 days old. We conclude that these structures do not necessarily correspond to rotavirus components. We studied the first faecal matter expelled by ten symptom-free babies (1-3 days old)-collected by gently scraping paper diapers with wooden tongue depressors. The faeces were stored at -80°C until processed for electron microscopy. For negative staining the samples were thawed, suspended in phosphate-buffered saline (PBS) pH 7 - 2, and centrifuged for 30 min to remove larger debris; the supernatant was centrifuged at 100 000 g for 1 h over 20% sucrose-PBS cushion. The sediment was studied by negative staining with 2% ammonium molybdate, pH 7 - 0. The dimensions of the lattice (see below) were determined by comparison with the molecular spacings in catalase crystals.33 In nine out of ten samples we found lattices with a characteristic hexagonal network. This network was not penetrated by the negative stain and seemed to consist of a planar, sheet-like structures (figure, A-C). It was never observed as a three-dimensional structure (eg, a basket of a coated vesicle) and was not associated with the membrane fragments commonly seen in these preparations. In each hexagon the vertices were slightly thicker than the arms (figure, D), a feature that was prominent in single structures. The centre-tocentre distance in the hexagonal lattice was 11 nm. To rule out the possibility that the hexagonal lattices represented extraneous contamination we looked at pellets (prepared by the same centrifugation schedule) of soaks of unused diaper material from the neonatal wards, in the same buffer and sucrose solutes.
DIARRHOEA AND SIMULTANEOUS EXCRETION OF CLOSTRIDIUM DIFFICILE CYTOTOXIN AND C PERFRINGENS ENTEROTOXIN
SIR,-A case of antibiotic-associated diarrhoea, consisting of five loose stools per day and beginning within a day of the completion of a course of piperacillin and lasting for 6 days, resolved without treatment. A stool sample collected on the last day of diarrhoea yielded cytotoxigenic Clostridium difficile on culture but no other routine enteric pathogens. A cytotoxin was also detected in the stool by tissue culture but the cytopathic effect was more characteristic of C perfringens enterotoxin than C dlfficile cytotoxin. This anomaly was resolved with the demonstration that the cytopathic effect was completely neutralised only if antitoxins to both Cdifficile cytotoxin and C enterotoxin were used. High counts of C perfringens (10 /g) were found in the stool. Use of the individual antitoxins allowed the amount of heterologous toxin to be estimated: titres were 4000 for C dlfficile cytotoxin and 8000 for C perfringens enterotoxin. This finding demonstrates the need to consider additional antitoxins when only partial neutralisation of a cytotoxin is achieved. The role of enterotoxigenic strains of C perfringens and its cytopathic enterotoxin in antibiotic-associated diarrhoeal and the finding of two different clostridial toxins in the same stool specimen highlight the multiple aetiology of antibioticassociated diarrhoea and the value of tissue culture in the investigation of diarrhoeal diseases. S. P. BORRIELLO A. R. WELCH Division of Communicable Diseases, H. E. LARSON Clinical Research Centre, Harrow HA1 3UJ FIONA BARCLAY
perfringens
SP, Larson HE, Welch AR, Barclay F, Stringer MF, Bartholomew BA. Enterotoxigenic Clostridium perfringens a possible cause of antibiotic-associated diarrhoea. Lancet 1984; i: 305-07.
Planar hexagonal lattices as of faeces of infants up to 3
seen
days
in negatively stained old.
preparations
1. Borriello
At
A, B,
higher magnification (D) thickening of the vertices becomes apparent. (In and C bar equals 20 nm; in D bar equals 10 nm.)
IgG FRACTION OF TOTAL ANTIBODY (IgG+IgM) ON DAYS 28 AFTER
14 AND
POST-EXPOSURE VACCINATION’*
*effective antigemc vaiues’ PDEV 2- 5, 4’ 2, and 4 - 5 IU per dose; HDCV 6’ 3 IU per dose.
reduced vaccination schedule recommended by the World Health Organisation for HDCV. In 71 cases (PDEV 52, HDCV 19) enough of the serum taken on days 14 and 28 was available for IgG fractionation of total rabies antibody. This was done by enzymelinked immunosorbent assay, with enzyme-conjugated anti-y and anti- antibodies. The results were compared with a standard human serum of known anti-rabies IgG and IgM content. All vaccinees had titres of 1 - 5 IU/ml or more by day 14 (0 - 5 IU/ml or more is generally considered to be protective). The IgG fractions of the total antibodies (IgG+IgM) were similar in both vaccine groups. However, on day 14 the IgG fraction in the serum of 5 vaccinees accounted for less than one-quarter of the total antibodies and in the serum of 22 volunteers for less than half of the total antibodies. By day 28, the IgG fraction accounted for more than one-quarter of the total antibodies in the serum of all vaccinees and for more than half in 69 of the 71 sera investigated. The fact that on day 14 some of the vaccinees had a low IgG fraction of total antibody suggests that active immunisation alone might not guarantee protection. Therefore, the initial application of rabies immunoglobulin as recommended by the WH03 and by the Immunisation Practices Advisory Committee, US Centers for Disease Control,4is indispensable. Two recent cases of fatal rabies encephalitis in exposed persons treated with HDCV but without rabies immunoglobulin5,6 strongly support this point of view. R. GLÜCK A. WEGMANN R. GERMANIER
Swiss Serum and Vaccine Institute, CH 3000 Berne, Switzerland Medical
Propaedeutical Clinic, University of Berne
H. KELLER H. U. DETTWILER
Department of Veterinary Bacteriology, University of Berne
A. I. WANDELER
1 Turner GS.
Immunoglobuhn (IgG) and (IgM) antibody responses to rabies vaccine 1978; 40: 595-604. H, Gluck R, Wegmann A, Wandeler AI. Immunogenität einer neuen,
J Gen 2 Keller
Virol
hochkonzentrierten
Tollwut-Entenembryovakzine.
Schweiz Med Wschr 1984; 29:
648-53 3. World Health 4
5
6.
Organisation Sixth report of the Expert Committee on Rabies Techn Rep Ser WHO 1973; no 523. Immunization Practices Advisory Committee (Centers for Disease Control). Rabies prevention-United States, 1984. MMWR 1984; 33: 393-408. Devriendt J, Staroukine M, Costy F, Vanderhaeghen J-J. Fatal encephalitis apparently due to rabies: Occurrence after treatment with human diploid cell vaccine but not rabies immune globulin. JAMA 1982, 248: 2304-06. Wattanasri S, Boonthai P, Thongcharoen P Human rabies after late administration of human diploid cell vaccine without hyperimmune serum. Lancet 1982; ii. 870.
CAMPYLOBACTER-LIKE ORGANISMS IN GASTRITIS
SIR,-While it is certainly attractive
to
take the report of Dr
O’Connor and his colleagues (Nov 10, p 1091) as providing indirect evidence
that Campylobacter-like organisms (CLO) may be aetiologically important in chronic antral gastritis (rather than secondary colonisers), such a conclusion, tentative though it is, may be justified. The association of chronic gastritis with CLO (detected histologically or on culture) is beyond doubt and in eight published studies approaches 100% concordance if one allows for sampling error, satisfying the first of Koch’s four postulates for an aetiological not
relationship between a given microorganism and a given disease. Koch’s second postulate-microorganism must be grown in pure culture-is also fulfilled, but his third and fourth postulates, which require the microorganism to be inoculated into a susceptible animal, reproduce the disease, and be observed and be recoverable from the experimentally diseased animal, are impossible to apply in the present very limited state of our knowledge of CLO. What is known, however, is that CLO seem to be found only in association with inflamed epithelium of gastric typel-4-either true gastric epithelium or the gastric metaplastic epithelium found in the duodenal mucosa in some cases of duodenal ulcer-and they are absent from non-metaplastic duodenal mucosa and from gastric biopsies showing intestinal metaplasia. O’Connor et al found intestinal metaplasia in biopsy tissue from 11of 14 patients with pernicious anaemia (PA) (1 had numerous CLO and 2 scanty CLO) and in 7 of 14 patients with peptic ulcer (1 had scanty and 2 numerous CLO). It is not clear whether the presence or absence of metaplasia or CLO was an all-or-none phenomenon among replicate samples in individual patients but, allowing for sampling error, it is certainly possible that the rarity of CLO in PA patients is explained by the increased prevalence of intestinal metaplasia in this group. Their information is valuable in our present state of ignorance of the significance and aetiological importance of CLO but I am not sure that the rarity of gastric CLO in PA can really be accepted as indicative of a pathogenic role for these organisms in chronic
.
gastritis. Department of Surgery, Whittington Hospital,
JOHN MEYRICK THOMAS
London N19 5NF
1. Steer HW, Colin-Jones DG. Mucosal changes in gastric ulceration and their response to carbenoxolone sodium, Gut 1975; 16: 590-97. 2. Meyrick Thomas J, Poynter D, Gooding C, et al. Gastric spiral bacteria. Lancet 1984; ii. 100.
Phillips AD, Hine KR, Holmes GKT, Woodings DF. Gastric spiral bacteria. Lancet
3.
1984; ii: 100-01. 4. Steer HW. Surface
morphology of
the
gastroduodenal
mucosa
in duodenal ulceration
Gut 1984; 25: 1203-10.
CYTOTOXIN PRODUCTION BY MEMBERS OF GENUS VIBRIO
SIR,- The Lancet has carried several letters about the production of
a toxin distinct from cholera toxin (CT) by Vibrio cholerae serogroup 01 or the naming/misnaming of V cholerae Serogroup non-01 as V cholerae. 1-4 Members of serogroup non-01 produce an enterotoxin,which has been demonstrated immunologically to be identical to or similar to cholera enterotoxin.6,7 Members of both serogroups may cause diarrhoea without the production of CT .8-11 This form of diarrhoea may, in many cases, be distinguished from that caused by CT-producing vibrios by the presence of red blood cells and leucocytes in the stoo1.8,9,I2 We have isolated and characterised a toxin produced by a clinical isolate of V cholerae non-01 that appeared to be responsible for pathogenicity. The toxin was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary tissue culture cells. The activity of the cytotoxin in tissue culture was not neutralised by antibodies directed against the cytotoxins produced by Shigella dysenteriae 1 or V vulnificus. The cytotoxin, a heat-labile protein, insensitive to trypsin and destroyed by freezing, had a relative molecular weight of 52 000, an ionisation point of 8-65, and no subunit structure. The purified cytotoxin induced fluid accumulation in the 5 h rabbit ileal loop model, was lethal to adult mice (20 g) by intravenous injection (2’ 5 pg caused death within 2 min), and had a median lethal dose (LD5o) of 0’68 pg for suckling mice injected intragastrically. Sublethal doses injected intragastrically did not elicit fluid accumulation in suckling mice killed 3 h after infection. Eleven clinical isolates (ten V cholerae non-Ol and one V mimicus), tested immunologically, produced the cytotoxin but did not produce CT. This cytotoxin was also produced by one CTproducing clinical isolate each of V cholerae 01, classical biotype, V cholerae non-01, and V mimicus. We are continuing our investigations to determine the relatedness of this cytotoxin to that produced by V cholerae 01, as reported by Spira et al 13 in a clinical isolate of V cholerae 01, El Tor biotype, which lacks the potential to’
1218 and in a mutant of an El Tor biotype strain from which the CT gene had been selectively deleted. The ability of both serogroups to produce or not produce CT and/or the described cytotoxin further supports biochemical data and DNA hybridisation studies which dictate the maintenance of both serogroups in the same genus and species. Definition of a genus and species solely on the basis of the degree of pathogenicity is unreliable because of possible gain or loss of the ability to produce toxin and hence virulence.
PLANAR LATTICES IN FAECES OF BABIES
produce CT,
Division of Microbiology, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington DC 20204, USA
Division of Microbiology, Center for Food Safety and FDA, Cincinnati, Ohio 1. 2
3. 4. 5. 6
7.
8. 9
10 11. 12.
13
J. M. MADDEN B. A. MCCARDELL
Applied Nutrition,
Sanyal SC, Alam K, Neogi PBK, Kaper JB, Levine MM Toxins
D. B. SHAH et in
al. A
new cholera toxin. Lancet 1983, i: 1337. environmental Vibrio cholerae Lancet 1983,
ii
217-18 Kay BA, Sack RB, Spira WM, et al Vibrio cholerae non-O1 isolated from five people with diarrhoea in Lima Lancet 1984; i: 218 Preston NW Vibrio cholerae in South America. Lancet 1984, i 624-25 Zinnaka T, Carpenter CCJ, Jr. An enterotoxin produced by noncholera vibrios. Hopkins Med J 1972, 131: 403-11. Yamamoto K, Takeda Y, Miwatani T, et al. Purification and some properties of a nonO1 Vibrio cholerae enterotoxin that is identical to cholera enterotoxin Infect Immun 1983, 39: 1128-35 Yamamoto K, Takeda Y, Miwatani T, et al Evidence that a non-O1 Vibrio cholerae produces enterotoxin that is similar but not identical to cholera enterotoxin. Infect Immun 1983; 41: 896-901 Blake PA, Weaver RE, Hollis DG. Diseases of humans (other than cholera) caused by vibrios. Annu Rev Microbiol 1980; 34: 341-67 Bonner JR, Coker AS, Berryman CR, et al. Spectrum of vibrio infections in a Gulf coast community. Ann Intern Med 1983, 99: 464-69. Kenyon JE, Gillies DC, Piexoto DR, et al. Vibrio cholerae (non-O1) isolated from California coastal waters. Appl Environ Microbrol 1983, 46: 1232-33 Levine MM, Kaper JB, Black RE, et al. New insights into the pathogenesis of cholera and its prevention 19th Joint Conference on Cholera, 1983; abstr 56 Madden JM, Nematollahi WP, Hill WE, et al. Virulence of three clinical isolates of Vibrio cholerae non-O1serogroup in experimental enteric infections in rabbits. Infect Immun 1981; 33: 616-19 Spira MW, Sack D, Sanyal S, et al Description of a possible new extracellular virulence factor in non-toxigenic V cholerae O1. 19th Joint Conference on Cholera, 1983, abstr 80-81.
SIR,-An aetiological role for a new rotavirus-like agent in several outbreaks of non-bacterial diarrhoea in the People’s Republic of China has been proposed by Tao et al.1,2 The morphological features of both viruses as well as of putative viral components were described.’ A pattern of virus particle degradation was also proposed, involving the formation of a regular hexagonal lattice thought to be comprised of detached viral morphological subunits (see Tao et al,l fig IE). The centre-to-centre period of the lattice, as calculated from our measurements of the print, is about 11.5 nm. We report here the observation of similar structures in negatively stained preparations of sediments from faeces of infants up to 3 days old. We conclude that these structures do not necessarily correspond to rotavirus components. We studied the first faecal matter expelled by ten symptom-free babies (1-3 days old)-collected by gently scraping paper diapers with wooden tongue depressors. The faeces were stored at -80°C until processed for electron microscopy. For negative staining the samples were thawed, suspended in phosphate-buffered saline (PBS) pH 7 - 2, and centrifuged for 30 min to remove larger debris; the supernatant was centrifuged at 100 000 g for 1 h over 20% sucrose-PBS cushion. The sediment was studied by negative staining with 2% ammonium molybdate, pH 7 - 0. The dimensions of the lattice (see below) were determined by comparison with the molecular spacings in catalase crystals.33 In nine out of ten samples we found lattices with a characteristic hexagonal network. This network was not penetrated by the negative stain and seemed to consist of a planar, sheet-like structures (figure, A-C). It was never observed as a three-dimensional structure (eg, a basket of a coated vesicle) and was not associated with the membrane fragments commonly seen in these preparations. In each hexagon the vertices were slightly thicker than the arms (figure, D), a feature that was prominent in single structures. The centre-tocentre distance in the hexagonal lattice was 11 nm. To rule out the possibility that the hexagonal lattices represented extraneous contamination we looked at pellets (prepared by the same centrifugation schedule) of soaks of unused diaper material from the neonatal wards, in the same buffer and sucrose solutes.
DIARRHOEA AND SIMULTANEOUS EXCRETION OF CLOSTRIDIUM DIFFICILE CYTOTOXIN AND C PERFRINGENS ENTEROTOXIN
SIR,-A case of antibiotic-associated diarrhoea, consisting of five loose stools per day and beginning within a day of the completion of a course of piperacillin and lasting for 6 days, resolved without treatment. A stool sample collected on the last day of diarrhoea yielded cytotoxigenic Clostridium difficile on culture but no other routine enteric pathogens. A cytotoxin was also detected in the stool by tissue culture but the cytopathic effect was more characteristic of C perfringens enterotoxin than C dlfficile cytotoxin. This anomaly was resolved with the demonstration that the cytopathic effect was completely neutralised only if antitoxins to both Cdifficile cytotoxin and C enterotoxin were used. High counts of C perfringens (10 /g) were found in the stool. Use of the individual antitoxins allowed the amount of heterologous toxin to be estimated: titres were 4000 for C dlfficile cytotoxin and 8000 for C perfringens enterotoxin. This finding demonstrates the need to consider additional antitoxins when only partial neutralisation of a cytotoxin is achieved. The role of enterotoxigenic strains of C perfringens and its cytopathic enterotoxin in antibiotic-associated diarrhoeal and the finding of two different clostridial toxins in the same stool specimen highlight the multiple aetiology of antibioticassociated diarrhoea and the value of tissue culture in the investigation of diarrhoeal diseases. S. P. BORRIELLO A. R. WELCH Division of Communicable Diseases, H. E. LARSON Clinical Research Centre, Harrow HA1 3UJ FIONA BARCLAY
perfringens
SP, Larson HE, Welch AR, Barclay F, Stringer MF, Bartholomew BA. Enterotoxigenic Clostridium perfringens a possible cause of antibiotic-associated diarrhoea. Lancet 1984; i: 305-07.
Planar hexagonal lattices as of faeces of infants up to 3
seen
days
in negatively stained old.
preparations
1. Borriello
At
A, B,
higher magnification (D) thickening of the vertices becomes apparent. (In and C bar equals 20 nm; in D bar equals 10 nm.)