Report of the 1966-67 cholera vaccine field trial in rural East Pakistan*

Bull. Org. mond. Sante 1969, 40, 177-185Bull. Wid Hith Org. Report of the 1966-67 Cholera Vaccine Field Trial in Rural East Pakistan* 1. Study Design and Results of the First Year of Observation WILEY H. MOSLEY,1 WILLIAM M. McCORMACK,2 M. FAHIMUDDIN, K. M. A. AZIZ, A. S. M. MIZANUR RAHMAN, A. K. M. ALAUDDIN CHOWDHURY, ALBERT R. MARTIN,2 JOHN C. FEELEY 3 & ROBERT A. PHILLIPS 4 A controlled cholera vaccine field trial was carried out in rural East Pakistan during the 1966-67 cholera season. A commercial cholera vaccine of average potency was tested in 40 000 children aged 3 months to 14 years in 1- and 2-dose schedules. In the cholera season extending for 8 months following immunization, a single dose produced an over-all protection of46 %; 2 doses at an interval of I month provided 64 % protection. The single dose was virtually ineffective in children under S years, but provided significant protection in older children. The enhanced effect of the 2-dose schedule was primarily due to the boosting of protection in children under the age of S years. The duration of significant protection, even with the 2-dose schedule, did not appear to extend beyond the first 3 months of the 8-month cholera season. The 2 cholera vaccine field trials conducted by the Pakistan-SEATO Cholera Research Laboratory (PSCRL) in rural East Pakistan in 1963-64 and 1964-65 demonstrated that the use of a whole-cell cholera vaccine of unusually high antigenic potency (Oseasohn et al., 1965; Benenson et al., 1968) * From the Pakistan-SEATO Cholera Research Labora- tory, Dacca, East Pakistan. The Pakistan-SEATO Cholera Research Laboratory is a part of the SEATO Cholera Re- search Program and is supported by the US Department of State, Agency for International Development; the National Institutes of Health, the National Communicable Disease Center, Public Health Service, US Department of Health, Education, and Welfare; and by the Governments of Pakistan and other SEATO nations. The NIH Cholera Advisory Com- mittee co-ordinates the research programme. The studies were supported in part by Research Agreement No. 196802 between the National Institutes of Health, Bethesda, Md., USA, and the Pakistan-SEATO Cholera Research Labora- tory, Dacca, East Pakistan. I Chief, Epidemiology Section, Pakistan-SEATO Cholera Research Laboratory, Dacca, East Pakistan. ' Formerly Epidemic Intelligence Service Officer, National Communicable Disease Center, Public Health Service, US Department of Health, Education, and Welfare, assigned to Epidemiology Section, Pakistan-SEATO Cholera Research Laboratory. ' Chief, Section on Bacterial Vaccines, Division of Biolo- gics Standards, National Institutes of Health, Bethesda, Md., USA. ' Director, Pakistan-SEATO Cholera Research Labora- tory, Dacca, East Pakistan. resulted in protection of 60 %-80% effectiveness during the short cholera seasons that ensued. The duration of protection provided fell markedly in the second year, particularly among children who, in this cholera endemic area, have the highest case rate. Serological studies in the field trial population, however, demonstrated a correlation of case rate with vibriocidal antibody titres suggesting that this serological test might be a useful tool for guiding future vaccine evaluations. It seems reasonable that by more intensive immunization, with either multiple injections or larger doses, the antibody level of children might be raised to that of adults, providing them with a correspondingly greater degree of protection (Mosley et al., 1968). This hypothesis was the basis for the third vaccine trial conducted during the cholera season of 1966-67. In the present study, begun in October 1966, it was decided to limit the population under observa- tion to children aged from 3 months to 14 years. A commercial cholera vaccine of average antigenic potency was employed and 1-dose and 2-dose schedules were followed in order to measure the level and duration of protection. In the under 5 years age-group, 2 doses appeared to be highly effective for a short period of about 2286 -177- 178 W. H. MOSLEY AND OTHERS 3 months, after which further protection was not observed. In children 5-14 years of age, 1 dose gave as much protection as 2. The duration of effective- ness was somewhat longer, but showed a decline after 3 months. Three random serological surveys were conducted at the beginning, during the middle, and at the end of the cholera season. These surveys permitted: (1) the evaluation of antibody response in the population at large; (2) the study of the relation of vibriocidal antibody to the incidence of cholera; and (3) the estimation of the inapparent infection rate in the study populations. This first paper in the series reports on the design and results of the field trial. The 2 subsequent papers present an analysis of the serological data and a clinical evaluation of the cases occurring in the various vaccine groups (Mosley et al., 1969; McCormack et al., 1969). METHODS Vaccines The cholera vaccine was prepared by a commercial manufacturer in the USA. The organisms included in the vaccine were classical Vibrio cholerae of the Ogawa (NIH 41) and Inaba (NIH 35A3) serotypes. The organisms were grown on nutrient agar, suspended in an isotonic sodium chloride solution, and killed and preserved with 0.5% phenol. The vaccine was standardized by opacity measurement to contain 4000 million organisms of each serotype per millilitre. Potency assays Protective activity against Inaba strain NIH 35A3 and Ogawa strain NIH 41 was determined relative to the homologous serotype reference vaccine by the mouse-protection test (Feeley & Pittman, 1962; Pittman & Feeley, 1963). This assay is prescribed as the official US potency test for cholera vaccine.' The control preparation was "Tetanus and Diphtheria Toxoids (for adult use)". The vaccines were supplied in identical 50-ml vials for use with jet injectors. The cholera vaccine was designated vac- cine X and the control vaccine, vaccine 0, with colour-coded labels. I Recommendations relating to the manufacture of cholera vaccine, 1964, Division of Biologics Standards, National Insti- tutes of Health, Public Health Service, US Department of Health, Education, and Welfare, Bethesda, Md., USA. Assignment and administration of vaccines The trial covered 132 villages in the Matlab area of Comilla District, East Pakistan. This included 58 villages which had participated in the previous trials, as well as an additional 74 villages. A house- to-house census was performed during March 1966. Every village was given a separate code number and each individual within the village was assigned a serial number which was used for identification purposes. Each family was issued with a census card giving the village number and the serial numbers of the family members. Upon completion of the census, census books were prepared from the family census sheets, in geographical sequence. Two copies were used for identification of the individuals in the field. The vaccine assignment was made in advance in the third copy. This was done by first stratifying the popula- tion into the following age-groups: 0-4 years, 5-9 years and 10-14 years. Individuals within these 3 age-strata were then assigned to each of the follow- ing vaccine groups in strict alternation: (1) Group 00-2 injections of vaccine 0; (2) Group XO- 1 injection of vaccine X followed by I injection of vaccine 0; (3) Group XX-2 injections of vaccine X. There were 2 assignments to group XX for each assignment to group 00 and group XO in order that group XX should be twice as large as the others. The vaccine was administered by 4 teams, each equipped with 2 foot-operated hypodermic jet in- jectors2-one for vaccine X and the other for vaccine 0. The dosage of vaccine given was 0.5 ml per injection to all participants in the trial. The teams went from house to house, located the children, identified them by census number, administered the designated vaccine and stamped the date of vacci- nation in the census book. The first round of vaccination extended from 20 September to 18 Octo- ber 1966. The second round of vaccination extended from 20 October to 17 November 1966. The progress of the teams through the villages was planned so that there were not less than 25 nor more than 35 days between the 2 injections. Surveillance for cases Field surveillance for acute diarrhoea was main- tained by daily house-to-house visits by a female field assistant who was usually a resident of the village 2Ped-O-Jet; Scientific Equipment Manufacturing Cor- poration, 99 Dell Glen Avenue, Lodi, New Jersey 07644, USA. THE 1966-67 CHOLERA VACCINE FIELD TRIAL IN RURAL EAST PAKISTAN. I under observation and she was supervised by male field assistants who visited each house at least twice a week. A rectal swab culture was obtained from all individuals with acute severe diarrhoea-defined as a diarrhoeal illness with acute onset resulting in the person being unable to perform his usual activities. All deaths were registered by the field workers. To identify suspect cholera deaths, rectal swab cultures were taken in all deaths associated with diarrhoea. If the body had been removed, cultures were taken from soiled clothes or bedding of the deceased, and family contacts were also tested. If any of these cultures were positive for V. cholerae, the death was considered to have been due to cholera. Patients with severe diarrhoea were treated in a centrally located field hospital. The methods of treatment are described in detail in the third report in this series (McCormack et al., 1969). Five speed- boat ambulances were stationed at various points along the major river and canals to provide rapid transportation to the hospital. Bacteriological methods All hospitalized cases had daily rectal swab cul- tures made until 3 consecutive cultures negative for V. cholerae were obtained. The swabs were first plated directly on tellurite-taurocholate-gelatin agar (TTGA) (Monsur, 1963) and plain gelatin agar and then placed in liquid alkaline-taurocholate-peptone medium and plated on the same media after over- night incubation. Cultures from field diarrhoeas were obtained with a tellurite-impregnated rectal swab which was placed in liquid alkaline-tauro- cholate-peptone medium and plated after overnight incubation on TTGA and gelatin agar. Vibrio cholerae was identified by agglutination of suspicious colonies in specific absorbed antisera. A case of diarrhoea was classified as cholera only if a positive culture for Vibrio cholerae was obtained. Serological studies Random sample serological surveys were carried out in the field trial population prior to, and 3 months and 6 months following, vaccination. The details of this part of the study are covered in the second paper in this series (Mosley et al., 1969). Statistical analysis Vaccine effectiveness was estimated by calculating the percentage reduction in the case rate in the vaccinated group compared with the control group. RESULTS The total population in the study villages was 111 673. Table 1 shows the population arranged by age and sex. The children under 15 years of age numbered 53 862, or 48.3% of the total. Vaccine was assigned only to this age-group. TABLE 1 TOTAL POPULATION IN MATLAB VACCINE FIELD TRIAL AREA BY AGE AND SEX Age- group Male Female Total (years) K 0-4 99913 9669 19582 5-9 10 581 10 007 20 588 10-14 7 456 6 236 13 692 15-29 10 387 12 733 23120 30+ 18 782 15 909 34 691 Total 57119 54554 111673 The study population for this field trial includes only those persons who received both assigned injections. The children who received only the first or only the second injection are excluded from this analysis. Table 2 shows the study population arranged by age, sex and vaccine group. Altogether, 39 862 children received both injections, which represents 74.0% of those assigned vaccine. Partici- pation was best in the 0-4-years age-group (77.9%) and least in the 10-14-years age-group (64.2%), mainly because the older boys were away at work. In the study population, 9923 (24.9%) were in vaccine group 00; 10 020 (25.1 %) were in vaccine group XO and 19 919 (50%) were in vaccine group XX. As indicated in Table 2, the various groups were essentially identical in their age and sex structure. The accompanying figure illustrates the time re- lationships of the vaccine programme to the cholera season. The first cholera cases appeared in Novem- ber 1966 towards the close of the vaccination campaign, and the season extended to May 1967. There were no cases in the subsequent 6 months. Altogether there were 79 cholera cases in the study population, with 1 death from cholera in vaccine group 00. All but 4 of the cholera cases were caused 179 W. H. MOSLEY AND OTHERS TABLE 2 STUDY POPULATION BY AGE, SEX AND VACCINE GROUP Age- Male Female Total group(years) No. % No. % No. % Vaccine 00 0-4 1 901 19.2 1 892 19.0 3793 38.2 5-9 1 868 18.8 2 052 20.7 3 920 39.5 10-14 1 010 10.2 1 200 12.1 2210 22.3 Total 4 779 48.2 5 144 51.8 9 923 100.0 Vaccine XO 0-4 1 946 19.4 1 872 18.7 3818 38.1 5-9 1 983 19.8 2 034 20.3 4 017 40.1 10-14 1 022 10.2 1163 11.6 2185 21.8 Total 4 951 49.4 5 069 50.6 10 020 100.0 Vaccine XX 0-4 3 877 19.5 3 759 18.9 7 636 38.3 5-9 3 840 19.3 4 050 20.3 7 890 39.6 10-14 2 019 10.1 2 374 11.9 4 393 22.1 Total 9736 48.9 10183 51.1 119919 100.0 -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ MONTHLY CHOLERA CASES IN THE 1966 VACCINE FIELD TRIAL POPULATION IN RURAL EAST PAKISTAN, SHOWING THE RELATIONSHIP OF SEROLOGICAL SURVEYS AND THE VACCINATION PROGRAMME 25- 20- u) cn IS- 0 cr m 10- D z 5- 0 ED 90132 2 3 A-SEROLOGICAL A- SURVEYS-A VACCII PROGRAI DOSE I DC II 4E MSE ISE 2 An I _ _ fI __A _ *A a SEPT. OCT NOV. 1966 by the Inaba serotype of V. cholerae. The 4 Ogawa cases occurred in April 1967. No organisms of the El Tor biotype were isolated. Table 3 shows the pattern of appearance of cholera cases in the hospital and in the field by month of onset in each vaccine group. There were 62 hospitalized cholera cases for an over-all rate of 15.6 per 10000 persons. There were 28 cases (28.2 per 10 000) in vaccine group 00, 15 cases (15.0 per 10 000) in vaccine group XO and 19 cases (9.5 per 10000) in vaccine group XX. These differences are highly significant (P= <0.001). Only 17 of the acute diarrhoea cases in the study population, which were cultured by field-surveillance personnel, were positive for V. cholerae; 16 were mild and did not require hospitalization or intravenous fluids; 1 was an acute diarrhoea death in group 00, which was confirmed as cholera by a positive culture from the clothing of the deceased. The over-all rate for field cases was 4.3 per 10 000. The distribution of field cases in the 3 vaccine groups gave a rate of 7.1 per 10 000 for group 00, 4.0 per 10 000 for group XO and 3.0 per 10 000 for group XX. While these differences are not significant statistically, the distri- bution of field cases by vaccine group follows the same pattern as the distribution of hospitalized cases. For comparative purposes, Table 4 gives the hospitalized and field acute non-cholera diarrhoea cases by month for each vaccine group. There were 63 hospitalized non-cholera diarrhoea cases for an over-all rate of 15.8 per 10 000 persons. The case rates by vaccine group range from 15.1 to 17.1 per 10 000 persons with no significant differences. Similarly, 628 acute diarrhoea cases were detected by field surveillance for an over-all rate of 157.5 per 10 000 persons. The rates for each vaccine group ranged from 149.6 to 169.3 per 410000 persons. Again, there was no significant difference in the acute diarrhoea experience for the various vaccine groups. DEC.I JAN. FEB. MAR. APR. MAY 1967 180 - THE 1966-67 CHOLERA VACCINE FIELD TRIAL IN RURAL EAST PAKISTAN. I TABLE 3 HOSPITALIZED AND FIELD CHOLERA CASES BY MONTHS OF ONSET IN EACH VACCINE GROUP ~~~~~~~~~~~~~~~~~~Rate Vaccine Nov. Dec. Jan. Feb. March April May June Total per group 10 000 Hospitalized cases a 00 1 10 9 3 4 1 ° 0 28 28.2 XO 2 3 6 2 0 1 1 0 15 15.0 0 4 2 4 4 4 1 0 19 9.5 Total 3 17 17 9 8 6 2 0 62 15.6 Field cases b 00 2 2 2c1 0 1 0 0 0° 7c 7.1 XO 0 0 1 0 2 1 0 0 4 4.0 XX 0 1 1 1 3 0 0 0 6 3.0 Total 2 3 4 1 6 1 0 0 17 4.3 a X2= 14.9 (2 degrees of freedom); P<0.001. b X2= 3.13 (2 degrees of freedom); P not significant. e Includes 1 cholera death detected by field surveillance. TABLE 4 HOSPITALIZED AND FIELD ACUTE NON-CHOLERA DIARRHOEA CASES BY MONTH IN EACH VACCINE GROUP i ~~~~~~~~~~Rate Vaccine Nov. Dec. Jan. Feb. March April May June Total per group 10 00o Hospitalized cases a 00 0 3 1 2 2 I5 3 17 17.1 XO 2 0 3 3 2 3 3 0 16 16.0 XX 1 8 5 1 6 4 2 3 30 15.1 Total 3 9 11 5 10 9 10 6 63 15.8 Field cases b 00 7 29 30 26 18 19 14 25 1681 169.3 XO 11 1 18 26 20 17 24 16 030 162 161.7 XX 23 43 47 35 29 40 341 47 1 298 149.6 Total 41 90 103 81 64 83 64 102 628 157.5 a X2 = 0.74 (2 degrees of freedom); P not significant. b x2 = 1.81 (2 degrees of freedom); P not significant. 181 W. H. MOSLEY AND OTHERS TABLE 5 CHOLERA CASES AND RATES BY AGE IN EACH VACCINE GROUP, AND VACCINE EFFECTIVENESS Vaccine Age- Cases Rate per 10 000 effectiveness group (%)(year) x 0 x x00arO XO |XX 0 XO |XX XO |XX Hospitalized cases 0-4 16 13 11 42.2 I 34.0 14.4 5-9 7 2 7 17.9 5.0 8.9 10-14 5 0 1 22.6 - 2.3 Total 28 15 19 28.2 15.0 9.5 Field cases 0-4 5 |3 3 13.2 7.9 3.9 5-9 1 1 3 2.6 2.5 3.8 10-14 1 0 0 4.5 - - Total 7 4 6 7.1 4.0 3.0 Total cases 0-4 21 16 14 55.4 41.9 18.3 24.4 67.0 5-9 8 3 10 20.41 7.5 12.7 ~78.9 60.5 10-14 6 0 1 27.1 2.3 Total 35 19 25 35.3 19.0 12.6 46.2 64.3 Table 5 gives an analysis of the cholera cases by age for each vaccine group, and the vaccine effective- ness. A single injection of cholera vaccine reduced the cholera case rate by 46% while the 2-injection schedule had an effectiveness of 64 %. Examining the case rates and vaccine effectiveness by age, it is evident that a single injection of cholera vaccine resulted in only minimal protection in children under 5 years of age; however, it produced substan- tial protection in the 5-14-years age-groups. The enhancement in over-all protection produced by the 2-injection schedule was entirely due to the lowering of the case rate in the children under 5 years of age. There were no significant differences in protection produced by a 1- or a 2-dose schedule in the 5-14- years age-groups. It should be noted that, in spite of the protection provided by a 2-dose injection sche- dule in children under 5 years of age, the case rate in these children remained higher than in children aged 5-9 and 10-14 years receiving a single dose of cholera vaccine. Table 6 summarizes the cholera case rates and the protection achieved in 3-month periods following the vaccination programme. While the numbers of cases are small for this analysis, the data suggest that a significant level of protection was maintained for only about 3 months, even in the children who received 2 doses of vaccine. The loss of protection seems definite in children under 5 years of age; in older children, the numbers of cases are too small to evaluate any per- sistence of protection throughout the cholera season. TABLE 6 CHOLERA CASE RATES, AND PROTECTION IN THE FIRST 3 MONTHS AND LAST 4 MONTHS OF THE CHOLERA SEASON, BY AGE-GROUP Age-group 0-4 years Age-group 5-14 years All ages Vaccinete Protec- Rate/ Protec- Rt/ Protec-group Cases 10e000 tion Cases| 10000 tion Cases Rate/ tion November 1966-January 1967 00 16 1 42.2 - 10 16.3 - 26 26.2 - XO 11 28.8 31.8 1 1.6 93.2 12 12.0 54.2 XX 3 3.9 90.8 5 4.1 74.8 8 4.0 84.7 February 1966-May 1966 00 5 13.2 - 4 6.5 - 9 9.1 - XO 5 13.1 0 2 3.2 50.7 7 7.0 23.1 XX 11 14.4 0 6 4.9 24.6 17 8.5 6.6 182 THE 1966-67 CHOLERA VACCINE FIELD TRIAL IN RURAL EAST PAKISTAN. 1 Table 7 gives a comparison of the potency in active mouse-protection tests of vaccine X with vaccine CRL used in earlier field trials (Oseasohn et al., 1965; Benenson et al., 1968). On a millilitre basis, vaccine X was considerably less potent than vaccine CRL, especially against Inaba challenge. Since different dose levels were used for the children, however, 0.5 ml of vaccine X should be compartd with 0.2 ml of vaccine CRL. At these levels, vaccine X was one-half as potent as vaccine CRL against Ogawa challenge and one-sixth as potent against Inaba challenge. These differences would be reduced by a factor of 2 for those children in the XX group who received 2 0.5-ml doses of vaccine X. Vaccine CRL was administered in a single dose. TABLE 7 RELATIVE POTENCY IN ACTIVE MOUSE-PROTECTION TESTS OF CHOLERA VACCINES EMPLOYED IN FIELD TRIALS Relative potency a per single Vaccine Serotype human dose b of:challenge 0.5 ml 0.4 ml 0.2 ml CRL c Ogawa 3.42 2.73 1.37 |Inaba 12.20 9.72 4.86 X Ogawa 0.62 _ - Inaba 0.72 _ - a Based on the Potency of US Reference Cholera Vaccines Ogawa and Inaba; 1.0 ml = 1.0 for both vaccines. b For vaccine CRL, single dose was 0.4 ml for adults, and 0.2 ml for children; for vaccine X, single dose for childrerl was 0.5 ml. c Designated as vaccine B and vaccine R in 1963 and 1964 field trials, respectively. DISCUSSION In this study, and in the 1963-64 and 1964-65 cholera vaccine field trials carried out by the PSCRL in the endemic cholera area in rural East Pakistan, several consistent patterns have emerged (Oseasohn et al., 1965; Benenson et al., 1968). In the control populations the cholera case rates were highest in children under 5 years of age and fell sharply with increasing age. Cholera vaccine has had the least effect in children under 5 years, and when protection was achieved, it was short-lived. By contrast, in older children and adults, the 2 whole-cell vaccines tested since 1963 have produced protection of 60%- 80% with a single injection and, as the present study indicates, this protection was not enhanced by the 2-dose schedule. It is evident from these observa- tions that a number of factors should be considered in interpreting the results of field trials in the endemic cholera area. Among these are vaccine potency, the injection schedule used, the ages and previous cholera experiences of the population. The effect of vaccine potency seems to be reflected only in the youngest age-group. The whole-cell vaccine CRL tested in the 1963-64 trial (Oseasohn et al., 1965) and again in the 1964-65 trial (Benenson et al., 1968) was unusually potent on a per-millilitre basis according to the mouse-protection test (Feeley & Pittman, 1965). However, based on the single human doses administered to the youngest age- group, vaccine CRL was approximately twice as potent against Ogawa challenge and 6 times as potent against Inaba challenge as vaccine X. The differences would be reduced by a factor of 2 for those children who received 2 doses (total 1.0 ml) of vaccine X. While the differences in potency against Ogawa challenge were less striking, the greater differences in potency against Inaba challenge were reflected in the results of the field trials in children under 5 years of age. A single dose (0.1 ml-0.2 ml) of vaccine CRL gave 80% and 75% protection in the first cholera seasons following the 1963-64 and 1964-65 trials, respectively. A single dose (0.5 ml) of vaccine X gave only 24% protection in the 1966-67 trial. However, with 2 injections (total 1.0 ml) of vaccine X at an interval of 1 month, the level of protection was raised to 67%, which approximates that obtained from 1 dose (0.2 ml) of vaccine CRL. Therefore, the prophylactic efficacy of these vaccines appears to be related to their potency assayed against Inaba challenge. Since there were only a few Ogawa infections in the study population, there is no basis for evaluating the relationship of laboratory potency and actual field efficacy for this serotype. The effect of age was important in two respects. In children under 5 years of age, any protection achieved seemed quite transitory. Thus, in spite of the relatively high level of protection produced by the 2-dose schedule (91 % in the first 3 months), this appears to have been lost toward the end of the cholera season 4-8 months after injection. There had been a similar loss of effectiveness in children under 5 years of age with the high potency vaccine; during the second cholera season of the 1963-64 trial, the protection in this age-group fell to 37% (Benenson et al., 1968). In older children in this endemic cholera area, there was evidence that the vaccine was 183 W. H. MOSLEY AND OTHERS acting as a booster. Thus, while the high potency vaccine gave 75% and 59% protection to children in the 5-14-year age-group in the 1963-64 and 1964-65 trials, respectively, similar results were obtained with vaccine X in 1966-67; 1 dose gave 79% protection and this was not improved by a 2-injection schedule. Data from the 1963-64 study indicate that protec- tion was also more sustained in the older children and adults (Benenson et al., 1968). The data from the field trials in East Pakistan have conclusively established that cholera vaccine can provide measurable protection against cholera. At the same time, it is clear that the duration of protec- tion provided either by a vaccine of high antigenic concentration or by a 2-dose injection schedule of commercial vaccine is inadequate for practical public health purposes. Further cholera vaccine develop- ment will require major modifications in the present vaccine, such as its use with adjuvants, or the exploration of new vaccines and new routes of immunization, for example, a living attenuated oral vaccine or possibly a toxoid preparation. ACKNOWLEDGEMENTS We wish to acknowledge the co-operation of Colonel S. A. Mallick, Director of Health Services, East Pakistan, Dr N. R. Majumder, District Health Officer, and Dr Majid, Civil Surgeon, Comilla, East Pakistan, in making the study possible. The help of Mr Imdadul Huq, Chief, Bacteriology Section, and the staff of the Bacteriology Section, Mr M. Shafiqul Islam, Deputy Field Surveillance Supervisor, and the Matlab Field Staff, the Matlab Hospital Staff, the Statistical Unit, the Maintenance Section and the Administrative Section of the Pakistan-SEATO Cholera Research Laboratory is gratefully acknowledged. The advice and interest of Dr Alexander Langmuir, Chief, Epidemiology Program, National Communicable Disease Center, Atlanta, Ga., USA, throughout this study are also gratefully acknowledged. Mr Wallis DeWitt, Microbiologist, Epidemiology Pro- gram, National Communicable Disease Center, Atlanta, Ga., USA, assisted in the establishment of the field labo- ratory. The suggestions of Dr Margaret Pittman, Chief, Laboratory of Bacterial Products, Division of Biologics Standards, National Institutes of Health, Bethesda, Md., USA, in the preparation of this paper are appreciated. RESUME RAPPORT SUR L'ESSAI PRATIQUE D'UN VACCIN ANTICHOLIRIQUE DANS UNE RtGION RURALE DU PAKISTAN ORIENTAL (1966-1967): 1. CONCEPTION DE L'5SSAI ET RtSULTATS ENREGISTRtS DURANT LA PREMItRE ANNtE On a proced6 en 1966-1967 a un essai pratique controle de vaccin anticholerique pendant la poussee saisonniere de l'affection dans une region rurale du Pakistan Oriental. Le vaccin utilise etait une preparation commerciale contenant par millilitre 4 milliards d'organismes de chacun des sero- types Ogawa (souche NIH 41) et Inaba (souche NIH 35A3) de Vibrio cholerae. De l'anatoxine tetanique et de l'anatoxine diphterique ont servi de preparation temoin. L'essai a porte sur un total de 39 862 enfants ages de 3 mois ai 14 ans. On a administre la preparation temoin a 9923 d'entre eux, cependant que 10 020 enfants rece- vaient une injection et 19 919 deux injections de vaccin anticholerique. Le depistage des choleriques a ete assure a la fois par les services hospitaliers fonctionnant au centre de la region de l'enquete et par des visites a domi- cile quotidiennes pour la recherche des cas d'affections diarrheiques graves. Tous les cas de cholera ont re;u une confirmation bacteriologique. Pendant la poussee saisonniere de cholera qui a sevi au cours des huit mois suivant la vaccination, on a releve chez les sujets ayant requ une seule dose de vaccin un taux de protection global de 46%. L'administration de deux doses a un mois d'intervalle a eu pour resultat de proteger 64% des sujets vaccines. Apres la vaccination i dose unique, le taux de protection n'etait que de 24% chez les enfants de moins de 5 ans, mais il atteignait 79% dans le groupe d'age 5-14 ans. Apres injection de deux doses de vaccin, la proportion des sujets immunises a depasse 60% aussi bien chez les jeunes enfants que chez les enfants plus ag6s. La protection conf6rde par l'admi- nistration d'une dose unique ou double de vaccin anti- cholerique s'est manifestee essentiellement pendant les trois premiers mois suivant la vaccination. Au cours des quatre demiers mois de la poussee saisonniere de cholera, elle s'est completement dissipee chez les enfants de moins de 5 ans, qu'ils aient recu une ou deux doses de vaccin. 184 THE 1966-67 CHOLERA VACCINE FIELD TRIAL IN RURAL EAST PAKISTAN. 1 185 REFERENCES Benenson, A. S., Mosley, W. H., Fahimuddin, M. & Oseasohn, R. 0. (1968) Bull. Wld Hlth Org., 38, 359-372 Feeley, J. C. & Pittman, M. (1962) In: SEATO confer- ence on cholera, Dacca, 1960, Bangkok, Post Publish- ing Co., p. 92 Feeley, J. C. & Pittman, M. (1965) Lancet, 1, 449-450 McCormack, W. M., Rahman, A. S. M. M., Chowdhury, A. K. M. A., Mosley, W. H. & Phillips, R. A. (1969) Bull. Wld Hith Org., 40, 199-204 Monsur, K. A. (1963) Bull. Wld Hlth Org., 28, 387-389 Mosley, W. H., Benenson, A. S. & Barui, R. (1968) Bull. Wld Hlth Org., 38, 327-334 Mosley, W. H., McCormack, W. M., Ahmed, A., Chowdhury, A. K. M. A. & Barui, R. K. (1969) Bull. Wld Hlth Org., 40, 187-197 Oseasohn, R. O., Benenson, A. S. & Fahimuddin, M. (1965) Lancet, 1, 450-453 Pittman, M. & Feeley, J. C. (1963) Bull. Wld Hlth Org., 28, 379-383