Vaccination against leprosy at Ben San Leprosy Centre, Ho Chi Minh City, Vietnam

Vaccine 19 (2001) 3451– 3458 www.elsevier.com/locate/vaccine

Vaccination against leprosy at Ben San Leprosy Centre, Ho Chi Minh City, Vietnam Le Van Truoc a, Ho Minh Ly b, Nguyen Kim Thuy b, Dang Duc Trach b, C.A. Stanford c, J.L. Stanford c,* a Ben San Leprosy Centre, Ho Chi Minh City, Vietnam National Institute for Hygiene and Epidemiology, 1 Pho Yersin, Hanoi, Vietnam c Department of Medical Microbiology, Windeyer Institute of Medical Sciences, Royal Free and Uni6ersity College Medical School, 46 Cle6eland Street, London W 1T 4JF, UK b

Received 9 August 2000; received in revised form 17 January 2001; accepted 30 January 2001

Abstract Three vaccines, BCG alone, BCG +107 killed Mycobacterium 6accae and 108 killed M. 6accae alone, were studied in children living in close contact with leprosy. In the year before vaccination, 14/446 (3.1%) children had developed leprosy. Among those who were not vaccinated, 9/74 (12.2%) developed the disease in the first 4 years of the study and 5/65 (7.7%) developed the disease in the second 4 years. In comparison with this, among those vaccinated, 20/343 (5.8%) developed leprosy in the first 4 years and 5/323 (1.5%) developed leprosy in the second 4 years. This represents 52.5% protection in the first 4 years and 80.5% in the second 4 years. There were no significant differences in protection afforded by each of the three vaccines but the success of the killed preparation of M. 6accae is an important finding. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Leprosy; Vaccination; Vietnam

1. Introduction There have been many attempts to develop a vaccine against leprosy, but until recently only BCG had been shown to reduce the numbers of new cases [1,2]. Last year the first results were released of the comparative study in south India of four vaccines [3], BCG alone, BCG + killed M. leprae, killed M. w. and killed Indian Cancer Research Centre (ICRC) bacilli, the last two being preparations of similar strains of M. intracellulare. ICRC bacilli were the most effective (67% protection), BCG+ killed M. leprae was next (65% protection), BCG alone was much less effective (33% protection) and M. w. was least effective (29% protection). The first two produced significantly better protection than either of the last two (P B0.01). Meanwhile this prospective study, based on different principles, was being carried out in Vietnam, in which * Corresponding author. Tel.: + 44-20-76799488; fax: +44-2076368175. E-mail address: [email protected] (J.L. Stanford).

three vaccines were compared. BCG alone, BCG+ killed M. 6accae (BCG + M.6.) and killed M. 6accae (M.6.) alone. We give an account of the results obtained. Several preliminary studies have been performed with these three vaccines in Iran [4], India [5] and Argentina [6], using surrogate markers of protective immunity in small numbers of individuals. In each study, the data obtained supported the value of their use, but practical demonstration was impossible because of the very small number of cases that might have been prevented within the confines of the populations studied. In the Ben San Leprosy Centre, the number of new cases arising amongst the healthy children living in close contact with their parents was lamentably high but offered us a practical measure of vaccine efficacy. Whilst our study was in progress, a case control study of the efficacy of BCG given in the Vietnamese vaccination programme was carried out in the Ho Chi Minh City amongst leprosy patients and controls aged between 5 and 25 years [7]. This study found a non-significant 29% of protection overall but with significant

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L.V. Truoc et al. / Vaccine 19 (2001) 3451–3458

protection of 52% against borderline forms of the disease. Evidence for vaccination was from scars and it was not certain whether the BCG had been given before or after the development of leprosy.

2. Subjects, materials and methods

2.1. Subjects Subjects were the children (aged 3– 20 years; 55% girls) of leprosy patients living in the Ben San Leprosy Centre a few miles outside Ho Chi Minh City in Vietnam. There are four villages of the leprosy centre, Ben San itself, Phuoc Tan, Binh Minh and Thanh Binh. There were 432 healthy children living with their parents in these villages in September1988 when our study was set up. Of these 358 children attended preliminary examination and those aged 5 years and above were skin-tested. The great majority (96%) of the children skin tested returned for reading the results after 72 h. Fifteen children were positive to both Tuberculin and Leprosin A, were considered to be already fully protected and were not vaccinated. The remaining 343 children received vaccines according to the schedule set out below. The 74 children who did not attend for the examination were left as an unvaccinated control group.

2.2. Ethical considerations The ethical committee of the Dermatology Centre in Ho Chi Minh City, gave permission for our study, on the understanding that all the children found to be suitable for vaccination should be vaccinated. Thus, no randomised non-vaccinated control group was acceptable.

2.3. Initial skin testing Following the procedures used in our preliminary studies, each child aged 5 years or more was tested with four new tuberculins [8]. Tuberculin from M. tuberculosis, Leprosin A from M. leprae extracted from the tissues of experimentally infected armadillos, Scrofulin from M. scrofulaceum and Vaccin from M. 6accae. These were administered as intradermal injections of 0.1 ml given approximately 10 cm apart on the volar aspects of the two forearms. Results were read in mm as 2 crossing diameters of the area of induration palpable 72 h after injection, at which time a search was made for a BCG scar. The 2-mm mean induration was taken as a positive reaction to any of the tuberculins, but only reactions to Tuberculin or Leprosin A of 5 mm or more were used to determine the suitability for vaccination.

The results for the four tuberculins allowed children to be allocated to responder categories [9]. These were those producing a positive response to all the four reagents and in general reacting to group i, common mycobacterial antigens [10], category 1. Those responding to none of the reagents and who may be genetically restricted [11] were category 2. Those responding to at least one but not all the reagents, reacting to group ii, slow-grower associated antigens, or group iv, speciesspecific antigens were category 3.

2.4. The 6accines used The BCG employed was Evans (Glaxo freeze-dried) BCG supplied by Medeva Ltd. This was made up for injection in the standard water provided according to the manufacturers recommendation when BCG was to be used alone, or with the same volume of a suspension of 108 killed M. 6accae per ml for BCG +M.6. For M.6. alone, the M. 6accae was at a concentration of 109 killed bacilli per ml. The M. 6accae used was strain R877R, NCTC 11659, grown on Sauton’s medium solidified with 1.3% agar, incubated aerobically for 1 month at 32°C. Harvested bacilli were suspended in sterile M/15 borate buffered saline at pH 8, at a concentration of 1010/ml, and autoclaved at 15 lbs. for 15 min. These sterile suspensions were further diluted 1 in 10, or 1 in 100 with further sterile borate buffer to constitute M.6. alone, or the diluent for BCG to make BCG +M.6., respectively. Each vaccine was mixed thoroughly just before use, and a dose of 0.1 ml was given by intradermal injection over the upper part of a deltoid muscle.

2.5. Vaccination schedule This was the same as that used to select persons for vaccination in the Argentine study [6]. Only children without BCG scars were considered for vaccination with BCG or BCG + M.6. Children with BCG scars (40), or without a BCG scar (31) but reacting to Tuberculin by 5 mm or more of induration, were given M.6. alone, if they needed further vaccination (71). For the above-mentioned children, the decision whether to vaccinate or not depended on whether they reacted to Leprosin A with less than 5 mm, or more than 5 mm of induration, respectively. The children without a BCG scar and with reactions to Tuberculin and Leprosin A of less than 5 mm induration, together with those aged less than 5 years who were not skin-tested, were randomised in groups of nine or ten to receive BCG alone (100), or BCG+M.6. (172). Since we wished to vaccinate more children with BCG+ M.6. than with BCG alone, each vaccinating session started with BCG+ M.6., explaining the excess receiving this vaccine.

L.V. Truoc et al. / Vaccine 19 (2001) 3451–3458

2.6. Clinical examination This was carried out at least twice a year when skin patches etc., thought to be due to leprosy, were routinely sought. Small biopsies were taken to confirm diagnosis and treatment with MDT was started. The number of cases of leprosy detected in the previously healthy children in the 12 months before the time of vaccination in 1988 was taken as the baseline and in subsequent years new cases were related to their vaccination groups.

2.7. Repeat skin testing One, two and three years after vaccination as many children as possible, including those too young to be tested at the first visit, were tested with the same tuberculins.

2.8. Analyses of results Student’s t-test and Fisher’s exact tests were used as being appropriate for the evaluation of the results obtained.

3. Results In the year immediately preceeding the beginning of our study 14/446 (3.1%) of the previously healthy children developed leprosy and Table 1 shows details of further cases of the disease developing over the 8 years of the study in relation to the vaccination received. Over the first 4 years, 1989–1992, 9 of the 74 (12.2%) unvaccinated controls and 20/343 (5.8%) vaccinated

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children developed leprosy (PB 0.02). In the next 4 years, 1993–1996, 5/65 (7.7%) unvaccinated controls and 5/323 (1.5%) vaccinated children developed the disease (PB 0.02). Over the entire 8 years 14/74 (18.9%) unvaccinated controls and 25/343 (7.3%) vaccinated children developed leprosy (PB 0.001). All the three vaccines were equally effective. Of the 15 children considered protected and not vaccinated, only one (6.7%) developed leprosy, in the 6th year of the study. Of the 40 cases of leprosy detected during the study, two were lepromatous, four borderline lepromatous, 11 borderline, five borderline tuberculoid, five tuberculoid, 12 were indeterminant and data was not available for one case. Tables 2 and 3 give the initial skin-test results (1988) for those about to be vaccinated and the results for the 2 succeeding years after BCG alone, or BCG+M.6. (excluding the results for those who subsequently developed leprosy). It can be seen that initially the proposed recipients of BCG alone were more responsive to the tuberculins than were those to receive BCG+ M.6. and this reached significance in both the numbers responding to Leprosin A (PB 0.03) and in the size of their responses (PB 0.005) and in numbers responding to Vaccin (PB 0.05). This was despite those receiving BCG alone being a little younger than BCG+M.6. recipients (PB 0.05). Both the vaccines induced similar increases in skin test positivity in the 1st year after vaccination which increased in each successive year and the same was true for the proportions of category 1 responders to common, group i, mycobacterial antigens. The proportion of children producing positive responses to each of the skin test reagents rose very significantly in the first year after vaccination (PB 0.0000 in every case), and even more so by the 3rd year

Table 1 Numbers of cases of leprosy detected amongst the study groups during the 8 years of the investigation and the percentages of protection afforded by each of the vaccinesa Year

Controls

‘Protected’

BCG alone

BCG+M.6.

M.6. alone

Numbers 1989 1990 1991 1992 Subtotal 1 1993 1994 1995 1996 Subtotal 2 Total Protection afforded BCG alone BCG+M.6. M.6. alone

(74) 2 3 3 1 9/74 (12%) 2 0 3 0 5/65 (8%) 14/74 (19%) 1st 4 years 51% 57% 42%

(15) 0 0 0 0 0/15 0 1 0 0 1/15 (7%) 1/15 (7%) 2nd 4 years 72% 84% 80%

(100) 3 1 2 0 6/100 (6%) 0 0 1 1 2/94 (2%) 8/100 (8%) Overall 58% 66% 55%

(172) 4 2 2 1 9/172 (5%) 0 1 1 0 2/163 (1%) 11/172 (6%)

(71) 1 3 0 1 5/71 (7%) 0 0 0 1 1/66 (2%) 6/71 (9%)

a

The ‘protected’ group were those considered not to need additional immunity and were not vaccinated.

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Table 2 Skin test results for recipients of BCG alone and BCG+M.6., by yeara

Tuberculin a BCG alone BCG+M.6. Leprosin A a BCG alone BCG+M.6. Vaccin a BCG alone BCG+M.6. Scrofulin a BCG alone BCG+M.6.

1988

1989

1990

1991

13/82 (16%) 2.54 90.78 mm 17/143 (12%) 2.7691.15 mm

73/89 (82%) 4.96 9 2.74 mm 99/140 (71%) 4.539 2.79 mm

64/75 (85%) 4.66 92.34 mm 93/120 (78%) 5.35 9 2.59 mm

62/69 (90%) 4.53 9 2.75 mm 103/122 (84%) 4.28 92.13 mm

19/82 (23%)b 4.729 2.93 mmc 20/143 (14%)b 2.559 0.69 mmc

63/89 (71%) 3.4091.81 mm 83/140 (59%) 3.0891.45 mm

66/75 (88%) 3.97 91.60 mm 100/120 (83%) 4.07 9 1.55 mm

57/69 (83%) 4.64 9 1.93 mm 104/122 (85%) 4.85 91.98 mm

18/82 (22%)d 3.11 91.41 mm 20/143 (14%)d 3.9091.94 mm

52/89 (58%) 3.88 9 1.71 mm 76/140 (54%) 3.829 1.80 mm

62/75 (83%) 4.11 91.98 mm 88/120 (73%) 4.59 9 2.41 mm

56/69 (81%) 3.86 9 1.37 mm 96/122 (79%) 4.53 91.92 mm

28/82 (34%) 3.049 1.14 mm 48/143 (34%) 3.6992.10 mm

74/89 (83%) 4.559 2.10 mm 107/140 (76%) 4.169 1.70 mm

67/75 (89%) 4.39 91.46 mm 97/120(81%) 4.87 9 1.85 mm

65/69 (94%) 3.43 9 1.05 mm 112/122 (92%) 3.90 9 1.56 mm

a Results are shown as numbers and percentages producing positive reactions (2 mm or more) and the mean sizes of positive reactions 9 S.D. ‘New tuberculin’ preparations of M. tuberculosis, M. leprae, M. 6accae and M. scrofulaceum. b PB0.03 (Fisher’s exact test). c PB0.005 (Student’s t-test). d PB0.05 (Fisher’s exact test)

after vaccination. Also shown in Table 2 are the categories of skin test responders. Initially and finally there were no differences between the recipients of the two vaccines. Both showed a large increase in category 1 responders (PB0.0001), a large reduction in category 2 non-responders (PB 0.0001) and a smaller reduction in category 3 responders to species-specific mycobacterial antigens. Tables 4 and 5 show the pre- and post-vaccination skin-test data for children receiving M.6. alone (excluding results for those who subsequently developed leprosy). The results are shown separately for those with and without scars of previous BCG vaccination. There were no significant differences in the pre-vaccination (1988) data except in responses to Tuberculin, reflecting the ways in which the children were selected. With the exception of those selected for positivity to Tuberculin who remained positive to this reagent throughout the study, vaccination resulted in significant increases in the proportion of positive responders to each skin test reagent (PB0.03). After vaccination, both the groups showed a fall in the size of positive responses to Tuberculin, significant by 1991 (P B0.05), and significant rises in response sizes to Leprosin A over the same time period (P B 0.05). In both the groups, proportions of category 1 responders rose from single figures to about 80% by 3 years after vaccination (P B0.0000). Over the

same time period proportions of category 2 non-responders amongst those with BCG scars fell markedly (PB 0.001). Category 3 responders in those with and without BCG scars fell sharply following vaccination (PB 0.005). There were no differences in the ages between those with or without a BCG scar. Table 3 Skin test responder categories for recipients of BCG alone and BCG+M.6., by yeara 1988 Category 1 BCG alone 3/79 (4%) BCG+M.6. 1/137 (1%)

1989

1990

1991

37/88 (42%) 47/75 (63%) 47/69 (68%) 46/143 (32%) 66/118 (56%) 80/123 (65%)

Category 2 BCG alone 39/79 (49%) 3/88 (3%) 2/75 (3%) BCG+M.6. 77/137 (56%) 19/143 (13%) 7/118 (6%)

0/69 (0%) 3/123 (2%)

Category 3 BCG alone 37/79 (47%) 48/88 (55%) 26/75 (35%) 22/69 (32%) BCG+M.6. 59/137 (43%) 78/143 (55%) 45/118 (38%) 40/123 (33%) a Results are shown as numbers and percentages falling into each responder category (category 1 are those responding to all the four skin tests; category 2 are those responding to none of the skin tests; category 3 are those responding to 1–3 of the skin tests). The age and sex distributions of the children studied are shown at the bottom of the table. Age and sex distribution (including those too young to be skin tested in 1988). BCG alone 9.2 9 4.2 years (41% boys). BCG 9 M.6. 10.3 94.7 years (42% boys).

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Table 4 Skin test results for recipients of M.6. alone by year, divided according to whether or not they had scars of past BCGa

Tuberculin BCG+ve No scar Leprosin A BCG +ve No scar Vaccin BCG +ve No scar Scrofulin BCG +ve No scar

1988

1989

1990

1991

15/34 (44%) 7.319 4.71 mmb 27/29 (93%) 9.1494.58 mmc

17/31 (55%) 6.4493.97 mm 19/19 (100%) 8.0093.96 mm

20/23 (87%) 4.86 9 4.30 mm 22/22 (100%) 7.48 9 3.40 mm

22/24 (92%) 4.59 92.63 mmb 15/16 (94%) 5.06 9 2.46 mmc

6/34 (18%) 2.679 0.82 mmd 6/29 (21%) 3.009 1.67 mme

19/31 (61%) 3.209 1.06 mm 14/19 (74%) 3.2791.39 mm

19/23 (83%) 3.30 9 1.30 mm 20/22 (91%) 4.00 9 1.38 mm

22/24 (92%) 4.59 9 1.65 mmd 14/16 (88%) 5.38 92.33 mme

6/34 (18%) 5.1493.72 mm 9/29 (31%) 4.809 3.82 mm

17/31 (55%) 3.71 9 1.61 mm 11/19 (58%) 4.8392.12 mm

14/23 (61%) 3.93 9 1.87 mm 21/22 (96%) 5.09 9 2.51 mm

19/24 (79%) 4.39 91.98 mm 14/16 (88%) 5.25 9 3.19 mm

15/34 (44%) 4.0091.41 mm 18/29 (62%) 4.5392.48 mm

22/31 (71%) 4.2791.52 mm 17/19 (90%) 5.289 2.40 mm

21/23 (91%) 4.68 9 1.62 mm 22/22 (100%) 5.09 9 1.73 mm

23/24 (96%) 3.74 9 1.29 mm 15/16 (94%) 4.47 9 1.66 mm

Results are shown as numbers and percentages producing positive reactions (2 mm or more) and the mean sizes of positive reactions 9 S.D. PB0.05. c PB0.005 for the reduction in response sizes to Tuberculin (Student’s t test). d PB0.02. e PB0.05 for the increase in response sizes to Leprosin A (Student’s t test). a

b

Table 6 shows skin test responses of children developing leprosy who had received vaccines in comparison with those who remained healthy. Significantly fewer children going on to develop disease produced positive responses to Tuberculin in the 1st, 2nd and 3rd years after vaccination (P B0.05, P B 0.01, P B 0.02) and to Scrofulin (P B0.003) in the 1st year after vaccination, than did those who stayed healthy. Among the 25 becoming diseased during the study, significantly more were category 2 non-responders 1 year after vaccination, than were those who did not develop the disease (P B 0.007). This reflected reduced proportions of category 3 responsive children. The initial skin test results according to the village of domicile of the children, and the distribution of BCG alone and BCG + M.6. by village are shown in Table 7 and Table 8. The proportions responding to Tuberculin and Leprosin A were greater (P B0.04) in those living in Ben San village itself than in the other villages. There were proportionately more responders to Scrofulin in Thanh Binh village than in the other villages (PB 0.005). Neither were the vaccines evenly distributed, far more recipients of BCG alone came from Phuoc Tan village (47/99 vs. 40/173; P B 0.00005) and far fewer from Ben San (2/99 vs. 40/173; P B0.00001). Amongst those vaccinated with BCG alone or BCG+ M.6., significantly more cases of leprosy arose in Phuoc Tan and Thanh Binh than in the other two villages (P B0.003).

4. Discussion All the three vaccines used in our study were effective in preventing new cases of leprosy. This was significant in both the 4-year periods, and over the entire 8 years only 25 cases of leprosy arose amongst the 343 (7.3%) vaccinees, compared with 14 amongst the 74 (18.9%) in the control group (P B 0.0001). Those selected as alTable 5 Responder categories for recipients of M.6. alone by year, divided according to whether or not they had scars of past BCGa 1988

1989

1990

Category 1 BCG +ve No scar

1/34 (3%) 2/29 (7%)

Category 2 BCG +ve No scar

15/34 (44%) 2/29 (7%)

Category 3 BCG +ve No scar

18/34 (53%) 12/31 (39%) 10/23 (44%) 25/29 (86%) 10/19 (53%) 3/22 (14%)

a

1991

12/31 (39%) 12/23 (52%) 19/24 (79%) 9/19 (47%) 19/22 (86%) 14/16 (88%) 7/31 (23%) 0/19 (0%)

1/23 (4%) 0/22 (0%)

1/24 (4%) 0/16 (0%) 4/24 (17%) 2/16 (13%)

Results are shown as numbers and percentages falling into each category. The age and sex distributions of the children studied are shown at the bottom of the table. Age and sex distribution. With BCG scar 12.5 94.7 years (51% boys). Without BCG scar 12.3 9 4.3 years (62% boys).

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Table 6 Skin test results and responder categories of children receiving BCG alone, BCG+M.6., or M.6. alone in 1988 remaining healthy, or becoming leprosy patients during the study

Tuberculin Healthy Patients Leprosin A Healthy Patients Vaccin Healthy Patients Scrofulin Healthy Patients Category 1 Healthy Patients

1988

1989

1990

1991

72/288 (25%) 5/23 (22%)

208/279 (75%)c 10/17 (59%)c

199/240 (83%)a 9/15 (60%)a

202/231 (87%)b 3/5 (60%)b

51/288 (18%) 2/23 (9%)

179/279 (64%) 11/18 (61%)

205/240 (85%) 15/16 (94%)

197/231 (85%) 4/5 (80%)

53/288 (18%) 4/23 (17%)

156/279 (56%) 6/18 (33%)

185/240 (77%) 12/16 (75%)

185/231 (80%) 4/5 (80%)

109/288 (38%) 6/23 (26%)

220/279 (79%)a 8/18 (44%)a

207/240 (86%) 12/16 (75%)

215/231 (93%) 5/5 (100%)

7/279 104/281 (3%) (37%) 0/22 (0%) 6/17 (35%)

144/238 (61%) 8/15 (53%)

160/232 (69%) 3/5 (60%)

Category 2 Healthy 133/279 (48%) Patients 14/22 (64%)

29/281 (10%)a 5/17 (29%)a

10/238 (4%) 0/15 (0%)

4/232 (2%) 0/5 (0%)

Category 3 Healthy 139/279 (50%) Patients 8/22 (36%)

148/281 (53%) 6/17 (35%)

84/238 (35%) 7/15 (47%)

68/232 (29%) 2/5 (40%)

a

PB0.01. PB0.02. c PB0.05 by Fisher%s exact test for differences between vaccinees remaining healthy or developing leprosy. b

ready showing parameters of protective immunity produced a single case of leprosy (1/15; 6.7%). Though the study was small, an estimated 40 cases of leprosy were prevented by our vaccination procedures. Most cases arose during the first 4 years after vaccination in both the vaccinees and controls (20/343 and 9/74, respectively; PB 0.02) whereas in the next 4 years only five cases arose amongst 323 vaccinees and five cases arose among the 65 controls (P B0.02). This may have been due to the introduction of multi-drug therapy in the first years of the study, reducing the infectious challenge to healthy children, or to the progression to clinical leprosy of those already developing disease at

the time of vaccination. The vaccines are thought to work by enhancing the cellular response to common mycobacterial antigens, thus lowering the threshold of immune recognition of casually encountered mycobacteria so that small challenge doses of potential pathogens are recognised before they can impose disease. Consequently, increasing levels of skin test positivity are seen each year after vaccination and infection with leprosy bacilli that has already occurred may not be reversed, allowing the disease to develop in the 1st year or so after vaccination [12]. Though there were no significant differences between the results for the different vaccines, the best protection (66%) was seen in recipients of BCG+ M.6. which was significantly better than the control group during both the first 4 years of the study (PB 0.02) and the second 4 years (P= 0.022). Our vaccines induced better protection than that observed in the case control study [7] but it is not possible to be sure whether this is a real difference, reflects different manufacturers vaccines, different study sites, or other differences between prospective and retrospective studies. All three vaccines significantly increased the numbers of recipients responding to each of the skin test reagents and the numbers becoming category 1 responders to common mycobacterial antigens. Surprisingly, the profiles of skin test response in the last year of testing (1991) were remarkably similar whichever vaccine was used and whatever their skin test status prior to vaccination. M.6. alone resulted in more recipients becoming category 1 responders (82.5%) in 1991, than did the other vaccines (PB0.02). In general, the skin test findings after BCG alone and BCG +M.6. agree with those obtained in similar studies in Argentina [6], India [5], Iran [4,13,14] and the Lebanon [15]. Values are boosted, notably to Tuberculin, and responses to the other reagents depend on proportions of category 1 responders induced and the frequency with which mycobacteria are met in the environment. Our Vietnamese children, children at Baba Baghi in Iran [4,13] and the adults in Argentina [6] were close contacts of leprosy, whereas the others were children living in areas of different endemicity for leprosy and tuberculosis, the highest being in India. Reactions to Leprosin A in the Argentinian leprosy contacts increased from 27/131 (20.6%) to 52/88 (59.1%) in 2–3 years after BCG alone and BCG+M.6. and in our Vietnamese children, reactivity to Leprosin A increased from 39/225 (17.3%) to 166/195 (85.1%) after 2 years, probably reflecting a much greater degree of contact with leprosy bacilli. The current study is the only one in which new cases of leprosy developed during the follow-up period. The vaccinated children developing leprosy showed surprisingly few differences in initial skin test response from those that remained healthy, though more of them

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Table 7 Distribution of initial skin test results for recipients of BCG alone and BCG+M.6. by village of domecilea Phuoc Tan

Binh Minh

Thanh Binh

Ben San

Tuberculin 10/7 (13%) 2.6090.97 mm

7/68 (10%) 2.57 9 0.79 mm

5/47 (11%) 3.20 91.30 mm

8/35 (23%) 2.50 9 1.07 mm

Leprosin A 10/76 (13%) 2.609 0.84 mm

8/68 (12%) 2.7591.16 mm

10/47 (21%) 4.60 93.27 mm

9/35 (26%) 3.89 9 +2.76 mm

Vaccin 14/76 (18%) 3.2991.38 mm

10/68 (15%) 3.009 1.25 mm

7/47 (15%) 3.43 91.51 mm

7/35 (20%) 4.86 9 2.67 mm

Scrofulin 25/76 (33%) 2.8091.73 mm

20/68 (29%) 4.45 9 2.33 mm

23/47 (49%) 3.39 9 1.27 mm

8/35 (23%) 3.50 9 1.51 mm

a Results are shown as numbers and percentages producing positive reactions (2 mm or more) and the mean sizes of positive reactions 9 S.D. There were more responders to Tuberculin (PB0.03) and Leprosin A (PB0.04) in Ben San than in the other villages and more responders to Scrofulin (PB0.005) in Thanh Binh than in other villages.

were category 2 non-responders, both initially and a year after vaccination and they showed reduced responses to Tuberculin and Scrofulin at some time points. This suggests either that they were children who did not respond adequately to vaccination or that they were already incubating disease. The identical skin test results obtained in 1991, 3 years after vaccination with any of the three vaccines, for children remaining healthy throughout the study supports the concept that category 1 responsiveness correlates with protection. Table 7 shows that children from different villages in the leprosarium complex showed different patterns of skin test reactivity reflecting their experience of slightly different environments and that recipients of BCG alone and BCG+ M.6. were differently distributed be-

tween the villages. There were significantly more new cases of leprosy developing after vaccination in Phuoc Tan and Thanh Binh than in Binh Minh and Ben San (PB 0.003). There was no obvious explanation for this and within villages, cases were similarly distributed for either vaccine and also for M.6. alone (not shown). Further studies are required of BCG+ M.6. in a situation where BCG alone is inadequate, such as Myanmar (Burma) [16], and M.6. alone needs to be tested in children selected to receive BCG alone or BCG + M.6. in this study. An entirely killed vaccine would have many advantages in populations with an increasing proportion of children infected with the human immunodeficiency virus.

Table 8 Distribution of recipients of BCG alone and BCG+M.6. by village of domecile and distribution of cases of leprosy arising amongst vaccinees in the four villagesa

References

Phuoc Tan

Binh Minh

Thanh Binh Ben San

Vaccine distribution BCG alone 47/99 (48%) PB0.00005 BCG+M.6. 40/173 (23%)

30/99 (30%) n.s. 59/173 (34%)

20/99 (20%) n.s. 34/173 (20%)

2/99 (2%) PB0.00001 40/173 (23%)

Disease distribution Vaccinees 8/87 (9%) PB0.05 BCG alone 4/47 (9%) BCG+M.6. 4/40 (10%)

2/89 (2%) PB0.01 0/30 (0%) 2/59 (3%)

8/54 (15%) PB0.04 4/20 (20%) 4/34 (12%)

1/42 (2%)

a

Differences shown are by Fisher’s exact test.

0/2 (0%) 1/40 (3%)

[1] Brown JAK, Stone MM, Sutherland I. BCG vaccination of children against leprosy in Uganda. Br Med J 1966;1:7 –14. [2] Fine PEM. Variation in protection by BCG: implications of and for heterologous immunity. Lancet 1995;346:1339 – 45. [3] Gupte MD, Vallishayee RS, Anantharaman DS, Nagaraju B, Sreevatsa BS, de Britto RL, Elang N, Uthayakumaran N, Mahalingam VN, Lourdusamy G, Ramalingam A, Kannan S, Arokiasamy J. Comparative leprosy vaccine trial in South India. Indian J Leprosy 1998;70:369 – 88. [4] Ghazi Saidi K, Stanford JL, Stanford CA, Dowlati Y. Vaccines containing Mycobacterium vaccae and their use in the children of leprosy patients in Iran. Med J Islamic Republic of Iran 1994;8:87 – 91. [5] Ganapati RP, Revankar CR, Lockwood DNP, Wilson R, Price J, Nye PM, Ashton P, Ashton L, Holmes R, Bennett C, Stanford JL, Rees RJW. A pilot study of three potential vaccines for leprosy in Bombay. Int J Leprosy 1989;57:33 – 7. [6] Bottasso O, Merlin V, Cannon L, Cannon H, Ingledew N, Keni M, Hartopp R, Stanford CA, Stanford JL. Studies of vaccination of persons in close contact with leprosy patients in Argentina. Vaccine 1998;16:1166 – 71.

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L.V. Truoc et al. / Vaccine 19 (2001) 3451–3458

[7] Nguyen VT, Abel L, Vu DL, Oberti J, Lagrange P. Protective effect of BCG against leprosy and its subtypes: a casecontrol study in Southern Vietnam. Int J Leprosy 1994;62: 532 – 8. [8] Shield MJ, Stanford JL, Paul RC, Carswell JW. Multiple skin testing of tuberculosis patients with a range of new tuberculins, and a comparison with leprosy and Mycobacterium ulcerans infection. J Hyg (Cambridge) 1977;78:331 –48. [9] Lockwood DNJ, McManus IC, Stanford JL, Thomas A, Abeyagunawardana DVP. Three types of human response to mycobacterial antigen: population studies in India and Sri Lanka. Eur J Resp Dis 1987;71:348 –55. [10] Stanford JL, Grange JM. The meaning and structure of species as applied to mycobacteria. Tubercle 1974;55:143 –52. [11] van Eden W, de Vries RR, Stanford JL, Rook GAW. HLA-DR3 associated genetic control of response to multiple skin tests with new tuberculins. Clin Exp Immunol 1983;52:287 –92. [12] Stanford JL, Rook GAW. Environmental mycobacteria and immunisation with BCG. In: Easmon CSF, Jeljaszewicz J,

[13]

[14]

[15]

[16]

editors. Medical microbiology. London: Academic Press, 1983:43 – 69. Stanford JL, Stanford CA, Ghazi-Saidi K, Dowlati Y, Weiss F, Farshchi Y, Madlener F, Rees RJW. Vaccination and skin test studies on the children of leprosy patients. Int J Leprosy 1989;57:38 – 44. Ghazi-Saidi K, Stanford JL, Stanford CA, Dowlati Y, Farshchi Y, Rook GAW, Rees RJW. Vaccination and skin test studies on children living in villages with differing endemicity for leprosy and tuberculosis. Int J Leprosy 1989;57:45 – 53. Bahr GM, Stanford JL, Rook GAW, Rees RJW, Abdelnoor AM, Frayha GJ. Two potential improvements to BCG and their effect on skin test reactivity in the Lebanon. Tubercle 1986;67:205 – 18. Bechelli LM, Lwin K, Garbajosa PG, Gyi MM, Uemura K, Sundaresan T, Tamondong C, Matejka M, Sansarricq H, Walter J. BCG vaccination of children against leprosy: nine-year findings of the controlled WHO trial in Burma. Bull World Health Org 1974;51:93 – 9.

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