856

antigen. Specimens from the remaining 17 type-2 patients have not yet been analysed because of insufficient anti-HC. Anti-HC was found in only 4 cases of type-2 hepatitis, and nowhere else in this material. Discussion

During the period 1970-77 apparent non-B hepatitis found in 116 out of the 1082 patients (10.7%) who received blood-transfusions at the surgical clinic of Sendai National Hospital. Successively obtained sera from 7 of these cases were examined for anti-HA rise, but none showed a significant increase. Furthermore, seroepidemiological evidence from Japan clearly shows that hepatitis A does not occur exclusively after blood-transfusion,12 so these 116 patients can be called non-A, was

non-B hepatitis.4 On clinical grounds these hepatitis patients can be divided into two groups2 with a third type in which the

characterised by a plateau pattern, but with an incubation period (7-4 weeks) and duration (13-3 3 weeks) resembling type 2 rather than type 1. Type 2 can be called non-A, non-B hepatitis of longer incubation and duration. The HC antigen was detected by immunodiffusion in 178 out of 268 serum specimens sequentially obtained throughout the incubation period and during the clinical course of 13 patients with type-2 post-transfusion hepatitis. HC antigen, once detected, persisted for 8-24 weeks. During this positive period, the qualitative immunodiffusion test revealed no striking differences in antigen concentrations; in other words, the peak period of antigen detection was difficult to find. However, the constant detection in sera obtained sequentially during the critical period of the disease suggests a close correlation between this HC antigen and type-2 post-transfusion hepatitis. In 11 cases out of 13 HC antigen was first detected 1 or 2 weeks before or coincided with the rise S.G.P.T. was

in

S.G.P.T.

Surprisingly antibodies against HC antigen were detected in only 4 of the 13 patients, despite the availability of sequential serum specimens during the recovery phase. HC antigen was also found in 4 out of 10 type-1 hepatitis patients. However, the antigen, first found 3 or 4 weeks after transfusion, disappeared in a week. This transience may be an additional discriminating factor between type-1 hepatitis and type-2 hepatitis. Perhaps both types have the same xtiological basis, type 1 being acute and type 2 chronic. However, this does not fully explain the aetiology of type-1 hepatitis. Another unexpected finding was the detection of HC antigen in some patients before blood-transfusion and in patients not transfused at all (fig. 5). Although the numbers are small, these cases may indicate the presence of symptom-free carriers and lessen the astiological significance of HC antigen as an indicator of post-transfusion hepatitis. HC antigen migrated in the (3-globulin region on immunoelectrophoresis but could easily be distinguished from HA antigen 13 and HB antigen 14 15 on the basis of its small molecular weight. HC antigen did not react with human anti-HB, or anti-e’6 by immunodiffusion. Sera with high titres against HA and HBc antigens did not react with three different concentrations of HC

antigen.

The lower S value and later elution by sepharose 6B gel filtration indicate that HC is smaller than HB,Ag. Its buoyant density (1 .30) suggests that it is not a lipoprotein. Thus, it differs from HB, Ag and HA antigen. Perhaps HC antigen is not a whole virus but a virusassociated substance. However, the correlation between the infectivity and antigenicity of this antigen is not yet clear. Alter et a1.9 and Tabor et a1.lO have reported that chimpanzees inoculated with plasma or sera from acute or chronic ,non-A, non-B post-transfusion hepatitis patients developed the disease. However, they did not suggest the presence of a serological marker for non-A, non-B hepatitis. A serological survey of post-transfusion hepatitis with HC antigen as the marker may throw light on the aetiology of this disease. There may be yet more agents in the aetiology of posttransfusion hepatitis; HC antigen was not detected in every patient with type-1 non-A, non-B post-transfusion

hepatitis. This work was supported by grants from the Japanese Ministry of Health and Welfare, Hepatitis Research Committee and the Japanese Ministry of Education, Science and Culture. Requests for reprints should be addressed to R. S. 4-7-2 Saiwai-cho, Sendai, Japan 983. REFERENCES 1. 2.

Ishida, N., Tateda, A. J. Clin. Sci. 1977, 13, 219 (in Japanese). Tateda, A., Kikuchi, K., Numazaki, Y., Shirachi, R., Ishida, N. J. infect.

Dis. (in the press). 3. David, J., Gocke, D. J. J. Am. med. Ass. 1974, 219, 1165. 4. Prince, A. M., Grotman, B., Grady, G. F., Kuhns, W. J., Hazzi, C., Levine, R. W., Millian, S. J. Lancet, 1974, ii, 241. 5. Purcell, R. H., Alter, H. J., Dienstag, J. L. Yale J. Biol. Med. 1976, 49, 243. 6. Alter, H. J., Purcell, R. H., Holland, P. V., Feinstone, S. M., Morrow, A. G., Moritsugu, Y. Lancet, 1975, ii, 838. 7. Feinstone, S. M., Kapikian, A. Z., Purcell, R. H., Alter, H. J., Holland, P. V. New Engl. J. Med. 1975, 292, 767. Purcell, R. H., Walsh, J. H., Holland, P V., Morrow, A. G., Wood, S., Chanock, R. M. J. infect. Dis. 1971, 123, 406. 9. Alter, H. J., Purcell, R. H., Holland, P. V., Popper, H. Lancet, 1978, i, 459. 10. Tabor, E., Gerety, R. J., Drucker, J. A., Seefe, L. B., Hoofnagle, J. H., Jackson, D. R., April, M., Barker, L. F., Tamondong, G. T. ibid. 1978, i, 463. 11. Graber, P. U. Ann. Inst. Pasteur, 1960, 99, 13. 12. Ohori, H., Yamada, E., Tateda, A., Ishida, N. J. infect. Dis. (in the press). 13. Feinstone, S. M., Kapikian, A. Z., Purcell, R. H. Science, 1973, 182, 1026. 14. Blumberg, B. S., Alter, H. J., Visnich, S. J. Am. med. Ass. 1965, 191, 541. 15. Barker, L. F., Almeida, J. D., Hoofnagle, J. H., Gerety, R. J., Jackson, D. J., McGrath, P. P. J. Virol. 1974, 14, 1552. 16. Magnius, L. O., Espmark, J. A.J. Immun. 1972, 109, 1017. 8.

CHOLERA, NON-VIBRIO CHOLERA, AND STOMACH ACID DAVID R. NALIN RICHARD J. LEVINE MYRON M. LEVINE DAVID HOOVER ERIC BERGQUIST JAMES MCLAUGHLIN JOE LIBONATI JAINUL ALAM RICHARD B. HORNICK Center for Vaccine Development,

University of Maryland,

U.S.A.; and Johns Hopkins Center for Medical Research and Training, and Cholera Research Hospital, Dacca, Bangladesh

Fasting and postprandial stomach acid production were low in 16 of 37 Bangalees convalescing from cholera or non-vibrio cholera. Gastric juice of hypochlorhydric patients did not kill

Summary

cholera vibrios in vitro, whereas that from normochlorhydric patients rapidly killed vibrios in concentrations

857

1010/ml.

To determine whether

hypoacidity predisposing factor, basal and betazole-hydrochloride-stimulated acid production were measured before and after cholera in a second group of patients consisting of American volunteers participating in a vaccine development proup to

resulted from cholera

or was a common

gramme. Cholera did not alter the stomach acid secretion of American volunteers, but low precholera basal acid production predisposed to severe cholera. The results indicate that hypochlorhydria observed in convalescent Bangalee cholera patients is not caused by cholera, and must therefore have preceded it. Idiopathic tropical hypochlorhydria may be a major factor accounting for the high incidence of diarrhœa due to acid-sensitive pathogens in developing countries. Introduction NORMAL stomach acid is vibriocidal, and protects against Vibrio cholerce and other acid-sensitive pathogens; 12 is normochlorhydric volunteers induction of cholera is facilitated by neutralisation of stomach acid with oral bicarbonate.3 Hypochlorhydria predisposes to cholera and other enteric infections,2 and exists in patients with classical or El Tor cholera,4-6 but data on its incidence and importance are in conflict. Only 1 of 16 convalescent East Bengal cholera patients was hypochlorhydric,7 compared with 16 of 39 such patients in West Bengal, where hypoacidity was regarded as both an effect of cholera and a possible antecedent disorder.6 89 In the study reported here we examined Bangladesh patients for hypochlorhydria, tested the vibriocidal activity of their gastric secretions, and, in American volunteers, determined whether cholera causes hypochlorhydria.

Patients and Methods Stomach acid secretion was studied in 37 Bangalee patients convalescing from cholera or non-vibrio cholera (N. V .c.), 10 and in 45 American volunteers before and after cholera.

Studies in

Bangalee Patients

Patients were treated at the Cholera Research Hospital (C.R.L.), Dacca, Bangladesh, for severe dehydration due to uncomplicated cholera (16 cases) or N.v.c. (21 cases). Patients with conditions linked with hypochlorhydria’ were excluded. Mean stated age of subjects was 30±4 years, cholera, and 40±4 years, N.V.C. (all figures given as mean±s.E.M.). Sex ratio (M/F) was 10/6 (cholera) and 15/6 (N.v.c.). Mean diarrhoea volume (litres) was 12±2 and 1-5+0.6for cholera and N.v.c. patients, respectively, and body-weight (pre-/postrehydration, kg) was 3 8 ±3/40 ±3 (cholera) and 3 9 + 2/42 ±1

(N.V.C.).

Cholera was confirmed by culture of Vibrio cholerce on Monsur’s agar" and agglutination with type-specific antisera. These cultures revealed V. ’Cholera biotype El Tor, serotype Inaba or Ogawa (8 cases each). Cholera-negative patients’ rectal swabs plated on salmonella-shigella and MacConkey’s agars revealed Escherichia coli but no salmonella or shigella. During diarrhoea, dehydration was corrected with intravenous electrolyte solutions,’2 and patients received a rice and curry diet. After diarrhoea ceased, study procedures were explained in Bengali, and patients who consented to participate were fasted overnight and received a soft, 2 mm bore polyvinyl nasogastric tube which was allowed to pass the estimated nose-to-antrum distance. After removal of gastric residuum, fasting gastric juice was aspirated for pH measurements, acid titration, bacterial cultures, and in-vitro inoculation with V. cholerae. If gastric juice pH was more than

was adjusted up and down and several aspirexamined. In all studies tube position was further verified by gastric auscultation during injection of air through the tube. Then 300 ml rice gruel (9 g of boiled rice/dl) was given by tube. At 0, 20, and 60 min afterwards, gastric aspirates from 20 patients (16±1 ml/sample) were obtained for measurement of total acid, by titration to neutrality with NaOH, and pH by Corning pH meter (and by pH paper at the bedside). Single aspirates were obtained from another 17 N.v.c. patients 50 min postprandially for acid titration. After completion of the studies, tetracycline was given for 5 days. For in-vitro inoculation of gastric aspirates from 18 patients (14 cholera and 4 N.V.C.), V. cholera Inaba 569B cells were harvested by centrifugation of an overnight T1N1 broth culture’3 and were washed and resuspended in buffered saline (pH 7-3). Paired 5 ml samples of gastric aspirates were agitated and incubated (37°C) after inoculation with 0.1 ml of vibrio suspension containing 2.6x 107 or 2-6xl0’" cells. Subsamples of these mixtures taken before and 1, 5, 20, 60, 120, and 240 min after vibrio inoculation were plated on Monsur’s agar before and 24 h after incubation in 1% NaCI, 1% peptone water (pH 8.5). Cultures were incubated overnight to detect surviving vibrios.

2.4, tube position ates were

Studies in American Volunteers After passing medical and psychological examinations and after satisfactorily completing a written test about cholera, volunteers participating in a vaccine development programme consented to the procedures described. After fasting 2 h, volunteers (32 males, 13 females; 23±0.6y; 71±lkg) ingested 150 ml water with 105 or 106 V. cholerae and 2 g NaHC03. 11 volunteers received Inaba 569B, 20 received Ogawa 395 (both classical biotype), and 14 received El Tor biotype (105 or 106 Inaba N16191 or P27459, or 105 Ogawa N15870 or N16117). Diarrhoea developed in 34 volunteers 34±2 h later and was treated with oral and intravenous rehydration solutions and tetracycline. Diarrhoea volume after 105 V. cholerce was similar to that after 106 (4.6±2.011 vs 3.1 :to.1). Stomach acid of volunteers was measured 24 h before ingestion of vibrios and 9 days later, 24-72 h after cholera (and therapy) had ceased. To measure stomach acid, a plastic orogastric tube was passed 23-5±0-4 inches (incisor-to-port distance). This changed by only 0-7±0-8 inches in individuals’ serial studies. By continuous syringe suction, basal gastric secretions were collected for 1 h, after aspiration of gastric residuum. Then betazole (ametazole) hydrochloride (0.5 mg/kg) was given subcutaneouslyl4 and samples of gastric juice were collected every 15 min for 2 more hours. Peak acid production (mmol HCl/h) was calculated from the 15 min of maximal acid production. Free acid was titrated with Toepfer’s indicator. Additional serial basal acid measurements were made on 10 immune, non-ill volunteers.

Results Convalescent Bangalee Patients

38% of convalescent cholera patients (3/8 Inaba, 3/8

Ogawa)

had no free basal stomach acid (pH>30); their acid secretion changed little after the rice meal (fig. 1). Postprandial secretion was also low (pH>3-0 20 min after the meal) in 3 of 4 low-basal-acid N.v.c. patients tested the same way (2 with fsecal E. coli producing both thermostable and thermolabile enterotoxins15) and in 3 other cholera patients whose pre-meal gastric-fluid pH was less than 3-0. In contrast, 13 cholera patients whose basal gastricjuice pH was 2.4,

Fig. 3-Relation of pre-cholera basal acid to volume of diarrhrea.

8 of the 17 aspirates obtained 50 min postprandially from convalescent N.V.C. patients had low acid (7-7+2-0 mmol HCl/1, pH 4·5±0·5). The remaining 9 patients’ gastric fluid had pHs averaging 1-0±0’1, with 30.2±3.8mmol HC1/1.

American Volunteers

Fig. 1--Gastric secretion in convalescent patients. Postprandial change was significant only in patients initially present (P=0-05).

with free acid

Cholera did not reduce stomach acid secretion. Mean basal acid values before and after cholera were in the normal range and did not change significantly (fig. 2): However, basal acid production measured 24 h before vibrio ingestion correlated with the log of total diarrhoea volume, low acid being linked with severe illness (r for all individuals’ values=-0.33, n=33, p0.05). The change in peak acid before and after cholera correlated with diarrhoea severity (r=0.39, n=45, p

Cholera, non-vibrio cholera, and stomach acid.

856 antigen. Specimens from the remaining 17 type-2 patients have not yet been analysed because of insufficient anti-HC. Anti-HC was found in only 4...
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