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Multiple comparison of dried BCG vaccines: stability at 37°C and persistence of strains in the mouse spleen
Multiple comparison of dried BCG vaccines: stability at 37 °C and persistence of strains in the mouse spleen L Lugosi The Gabriel test was used for mul@le comparison of the stability of vaccines stored at 37°C over a 28 day period and o f the long-term relative persistence capacity (RPC) in mice spleen (residual virulence) over a 547 day period following intravenous vaccination with 12 BCG dried products. Multiple comparisons were performed after rejected null hypotheses (Anova, Regranal), with ranked mean viable units 106 ml -~ estimates (VU) of vaccines and with ranked mean R P C values in spleen determined with five sampling and on nine autopsy days, respectively. Results showed that VU x 106 ml -~ values o f the compared vaccines at day O varied between 1.1 and27.0 x 106 ml -~ and formed two homogeneous subsets. The heat stability test showed that the V U x lO6 mi-~ values of the vaccines decreased parallely but at day 28 only six vaccines showed a survival rate over 20°/5. The RPC model showed that the trends of early mul~plication and of late persistence capacity depended on the Lv. BCG VU doses. The Regranal of late R P C indicated that the decrease o f BCG VU in mice spleen from 84 to 547 days was parallel and that six strains persisted over 20% at day 360. The overall R P C on the nine autopsy days of the BCG strains was evaluated with Kendall concordance test In contrast with pairwise comparison, the Gabriel test reduces the risk o f type I error in statistical inference at the 5% experimental level and selects the homogeneous subsets o f ranked parameter estimates o f the compared products. Multiple comparison makes the statistical quality control o f BCG vaccines more exact and provides a more objective interpretation of the experimental results. It also helps the progress of the standardization and the explanation of the present controversies in BCG vaccination.
Keywords:Statisticalquality control; BCG vaccine:heat stability:residual virulence;persistencein mouse spleen; multiple comparison.
Introduction The aim of the statistical quality control of BCG vaccine is to select the most efficacious product(s) from the tested samples for immunization programmes with minimal frequency of adverse reactions. To compare the in vitro and in vivo potency of BCG preparations reliable statistical methods, unbiased point and confidence interval estimation of parameters and appropriate transformations of data should be used to fulfil validity criteria of hypothesis tests. When comparing the parameters of laboratory controls of more than two BCG products after rejected null hypothesis, multiple comparison is needed to diminish the risk of type I error. This study uses Gabriel's simultaneous test procedure (STP) for the statistical quality control of BCG products using the ranked point estimates of in vitro and in vivo animal experiments for the multiple comparison: (1) of the stability of the viable units (VU) of the tested BCG vaccines stored at 37°C over a 28 day period. expressed in terms of VU × 106 ml-~ decrease/day, and (2) of the residual virulence of the tested BCG strains by determining over 547 days the regression of the relative persistence capacity (RPC) in mice spleen, i.e. the ratio of BCG Laboratory, National Institute of Hygiene, Gyali ut 2, Budapest 1097, Hungary. (Received 22 June 1 983) 0264-410)(/84/020149-08 $03.00 © 1984 Butterworths 8- Co. (Publishers) Ltd.
the recoverable VU/i.v. inoculated VU (Rec-VU/Inoc-VU). Association among ranked mean RPC of the tested BCG products was finally evaluated with Kendall's coefficient of concordance.
Materials and methods Laboratory methods Tested vaccines. The dried BCG vaccines investigated were the same as those used in the collaborative assay of the International Association of Biological Standardization (IABS) 1-4. The origins and abbreviations of the tested BCG vaccines are as follows: Rijks Instituut voor de Volksgezondheid, Bilthoven (Bil), Connaught Medical Research Laboratories, Toronto (Con): Statens Seruminstitut, Copenhagen (Cop): Institut Pasteur, Dakar (Dak); Glaxo Research Laboratories, Greenford (Gla): Japan BCG Laboratory, Tokyo (Jap); Institut M~rieux, Lyon (M~r); Tarassevich State Institute, Moscow (Mos); Institut Pasteur, Paris (Par): Institute of Hygiene and Epidemiology, Prague (Pra): Reference Japan Strain (Ref): International Reference Preparation Japan Strain (IRef)L Determination o f viability and stability of the vaccines. The culturable particles (colony count) of the
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Multiple comparison of dried BCG vaccines: L Lugosi tested BCG vaccines were determined parallely on L6wenstein-Jensen (L-J) and blood-oleic acid-albumin (BOAA) media s. The VU × l06 ml -t values were estimated from the culturable particles. The thermostability, i.e. the VU decrease in the ampoules stored at 37°C was determined at 0.7, 14, 21 and 28 days from the daily VII decrease in slope and expressed as VU × 106 ml -~ decrease/day as previously described 7.
Determination of RPC: vaccination of mice, recoverable BCG colony counts from the spleen of mice. Determination of the viability on BOAA medium of vaccines stored at 4°C and sterility tests were performed 24 h before vaccination. A volume (0.2 ml) of the 10 × and 100 × dilutions of the 12 BCG vaccines was injected intravenously with an Omega syringe: 24 groups of 124 male and/or 124 female CFLP (Carworth Europe), 18-22 g, 4-6 week old mice were i.v. inoculated in random order. A total of 2976 mice (1488 male and 1488 female) were housed in 96 polypropylene boxes (38 × 27 × 11 cm) each containing 31 mice at the beginning of the experiment (124 mice in four boxes = one 'treatment' group, vaccinated from one dilution of one of the compared 12 vaccines). The animals were fed on a pellet diet and water adlibitum After vaccination at days 1, 8, 21, 42, 84, 184, 349, 450 and 547, eight mice from each vaccinated group were killed with chloroform. Spleens were removed aseptically then homogenized individually with a Potter-Elvehjem Teflon-glass homogenizer in 3.0 ml of 1 + 3 diluted Sauton medium containing Bovine albumin fraction V of 0.1%. From the three-fold diluted whole spleen homogenates and their two-fold dilutions (6 × diL) 10 drops each of 0.02 ml were seeded on BOAA medium in Petri dishes using injection needles (internal diameter, 0.6 ram; external diameter, 1.1 mm) mounted on glass pipettes ( ~ e t e r , 8 m-m~l¢-ngth,lO0 mm). After seeding, drying and sterility tests were carded out overnight in an incubator, the ten inverted Petri dishes were put in cylindrical aluminium boxes (diameter, 12 cm; heighL 26 cm) and covered with plastic bags for incubation at 37°C. After an incubation period of 15-17 days, BCG colony counts were made with a ! 0 × magni~ing binocular stereomicroscope. For reliability, an optimum of 20 and an upper limit of 50 colony counts was prescribed 6. Statistical methods Estimation of the parameters of stability and RPC. In order to have unbiased point and 95% confidence interval estimates of the VU × 10' ml -t values of vaccines and the individual Rec-VU values using recovered BCG colony counts from mice spleen, censored Poisson (CP) distribution was assumed. The CP formula includes the proportion of readable but uncountable colony counts of the dilution levels inoculated* and is an efficient BCG viability estimation method. For hypothesis tests, the VU × 106 ml -~ estimates and mean RPC values calculated by averaging the Rec-VU point estimates of eight mice and divided by i.v. Inoc-VU were used. Hypothesis testa For statistical inferences, analysis of variance (Anova) and linear regression analysis (Regranal) with VU × 106 ml -t values of vaccines stored at 37°C for 28 days and with mean RPC values were performed. Where necessary, to fulfil validity assumptions in the hypothesis tests, log or square root of the mean RPC values were used as transformed point estimates in the statistical analyses 9.
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Multiple comparison. After using the Anova and Regranal tests, in cases of rejected null hypotheses, Gabriel's STP was performed with the ranked VU x 106 m1-1 estimates, with estimated regression coefficients and with mean RPC values in order to diminish the risk of type I error ~°. Kendall test of concordance. To test the association between sets of ranked mean RPC values of the BCG strains obtained on the days mice were killed, Kendall's coefficient of concordance was computed". Computation. A CDC-3300 computer of the Computer and Automation Institute of the Hungarian Academy of Sciences was used for data processing and statistical analysis. The main results only of our studies are presented here, all computer printed tables are available on request from the BCG Laboratory of Budapest. Results
Stability a t 3 7 ° C Viability of the heat exposed vaccines. Table 1 summarizes the mean VU × l0 ~ ml -~ point estimates of the BCG vaccines stored at 37°C over 28 days. Three-WayAnova ofVU × l06 ml -~ values at day 0 and Gabriel's STP of the compared vaccines resulted in two homogeneous subsets included in Table 2.
Regression of the viability on exposure to heat. Table2 presents the separate Regranal of the 37°C heat stability of each vaccine (sampling days: 0, 7, 14, 21, 28). In contrast to the two point model (37°C/0, 28 day) used in routine in vitro assays of BCG products 12, with our five point regression model which included four repeated viability estimates, the linearity of the regression lines could be tested. Having fulfiled statistical validity assumptions, estimation of the regression coefficients with their 95% confidence limits and prediction of VU survival at day 28 become more exact. The 37°C resistance of VU at day 28 and its 95% confidence limits estimated from the regression equation, are the most important parameters when the potency of vaccines has to be judged before administration, after transportation and storage in tropical conditions. The 28/0 day estimates expressed in % show the relative heat-resistance capacity. The thermal degradation of the number of culturable particles in the vaccine after heating should not be less than 20% of the refrigerated Table I Viability expressed in mean a VU x 106 m1-1 values of the heat exposed BCG vaccines Time exposure (days) at 37°C
BCG vaccine Jap Gla Mos ~n ~k Bil ~p M6r Par Pra
0
7
27.0 20.1 7.1 6.9 6.5 4.2 2.9 2.8 2.7 1.1
12.6 14.8 6.7 4.0 3.9 2.5 3.6 2.2 2.7 0.5
14
21
28
23.3 13.9 5.5 0.4 2.9 1.3 3.3 1.7 1.7 0.3
18.0 11.5 4.8 0.2 2.0 1.5 2.1 0.7 1.9 0.2
16.6 10.9 3.6 0.2 1.8 1.3 1.9 0.3 1.3 0.2
*Each mean =s calculated trom four separate viability estimates parallely determined from two dilutions on two media as described in Methods. The non-listed 2 0 0 individual estimates and 9 5 % confidence limits can be obtained on request
Multiple comparison o f dried BCG vaccines: L Lugosi Table 2 Regranal of VU x 106 m1-1 values of BCG vaccines on days O. 7, 14, 21, 28 for testing 37°C heat stability followed by Anova and Gabriel's STP. Analysis of data from Table I Regranal
Anova: MS values
VU in vials at day 28
VU x 106 m1-1 BCG at day O a
+ s.e.
-b a
Linear (95% confidence limit) 10 s m1-1 (95% confidence limit) (%)b d.f. = 3
Residual d.f. = 1 5
Jap Gla Mos COn Dak Bil COp Met Par Pra
5.464 1.706 0.577 0.556 0.395 O.135 0.206 0.133 0.259 0.247
0.219 0.312" 0.129" 0.246* O.163" 0.099* 0.048* 0.093* 0.052* 0.030
0 -0.O10 -0.057 -0.177 -0.114 -0.083 -0.023 -0.077 -0.020 0
199.03 19.41 2.22 2.06 1.04 O.12 0.28 0.12 0.45 O.41
27.O19 20.131 7.148 6.926 6.513 4.227 2.885 2.805 2.737 1.110
22.019 18.584 7.342 5.796 5.692 3.555 3.465 2.843 2.782 0.904
-0.898) -0.524) -0.201) -0.315) -0.212) -0.116) -0.074) -0.110) -0.083) -0.060)
Anova for differences between the ten regression lines Source of variation
d.f.
Between (37°C expos, days)
MS
16.434 9.820 3.730 0 1.137 0.777 2.109 0.230 1.331 0.066
31.075) 13.437) 4.259) 2.611) 3.076) 2.569) 3.549) 0.634) 2.048) 0.547)
74.6 52.8 50.8 NC 20.0 21.9 60.9 9.1 47.8 7.3
141.10 8.75 O.18 9.00* 2.47 2.11 1.37 0.11 0.34 0.15
Gabriel's STP for ranked regression coefficients VU x 106 m1-1 values at day O and 28 F
Rank BCG - b
49
166.87
7.41 *
1
1
379.38
16.85*
17.37 793.82 16.56 22.51
0.77 35.27* 0.74
Residual
9 9 30 150
Total
199
2 3 4 5 6 7 8 9 10
Regression (common slope)c Parallelism (equality of slopes) Identity (equality of means) Linearity (curvature)
(1.793 (6.203 (3.201 (0 (0 (0 (0.669 (O (0.614 (O
HS BCG day O
Pra
0.030
Cop Par Mer Bil Mos Dak Jap Con Gla
0.048 0.052 0.093 0.099 O.129 0.163 0.219 0.246 0.312
Jap Gla Mos Con Dak Bil Cop M~r Par Pra
27.019 20.131 7.148 6.926 6.513 4.227 2.885 2.805 2.737 1.110
(b)
and
HS BCG d a y 2 8 HS Jap Gla Mos Cop Par Dak Bil Mer Pra Con
16.434 9.820 3.730 2.109 1.331 1.173 0.777 0.230 0.066 0
*Significant at 5% level; N C, Not computable; a(slope) daily VU decrease expressed in 108 m[-1; bRelative heat resistance capacity; the decrease of VU shall be not less than 20% of that in the refrigerated vaccine 13; c_b =_0.139 + 0.034; HS, Vertical bars indicate inhomogeneous subsets of Gabriel's STP
'Table 3
Estimates of mean RPCa of the compared 100 x diluted BCG vacines in mice spleen
BCG
i.v. inocc VU
Bil Con Cop Dak Gla IRef Jap Met Mos Par Pra Ref
6000 9343 5600 8743 3745 23771 297 6040 9657 4677 3533 2377
Time of death after vaccination (days) 1
8
21
29.9 5.6 8.0 3.9 36.6 4.0 3.1 19.4 3.7 3.9 2.9 0.2
41.9 17.4 1.5 0.8 22.5 1 .O 2.9 9.9 8.2 2.3 1.2 0.O
125.4 15.9 6.3 10.4 7.1 0.7 0.O 5.8 12.0 9.7 1.O O. 1
42
84
64.3 12.4 3.8 22.7 24.3 0.1 0.0 6.1 56.6 31.4 1.1 0.O
92.6 6.3 1.9 3.9 4.3 1.4 0.0 1.0 41.9 6.4 4.5 O.0
184 17.4 0.9 0.3 22.3 1.8 O.01 90.4 0.7 15.8 4.2 0.0 39.4
349 39.6 0.4 2.0 56.7 0.5 0.8 3.8 1.1 12.4 22.3 0.9 5.2
450
547
72.8 18.8 O.0 33.7 0.03 4.4 350.1 1.7 78.0 29.3 0.0 35.1
57.9 0.9 2.9 29.1 0.08 2.4 0.0 0.4 31.9 34.5 O.0 32.7
aMean RPC is expressed in %, i.e. mean recoverable VUb/inoculated VU estimated by censored Poisson distribution. bValues of mean recoverable VU from the whole spleens of mice determined on BOAA medium can be calculated from formula: mean Rec VU = (mean RPC x inoc VU)/100. Each mean is based on eight mice except those where there were missing values, recorded in experimental protocol, because the seeds on BOAA medium were contaminated; or colony counts were over limit (> 50) in all seeds, i.e., readable but not countable, therefore uncomputable; or colony counts were zero, i.e., the possibly persisting VU in spleens were not demonstrable. CThe mean-RPC values of the larger inoculum sizes (10 x diluted vaccines) are not listed but have been used for statistical analyses and can be obtained on request
vaccine ~3. Thus, vaccine parameters have immunobiological meaning in the statistical quality control within the biomathematical models. Using this model subjective interpretation of vaccine thermostability can be eliminated.
Anova and mul@le comparison of the regression of viability. In addition, Table2 presents the results of Anova
and Gabriel's STP. Anova tests c o m m o n slope, parallelism, identity and linearity of the ten regression lines. Linearizing transformation of the VU values was not needed. It can be seen that at the 5% level the F value of parallelism is not significant. Thus the slopes of VU-decrease of the vaccines during heat treatment can be considered similar. Gabriel's STP shows that the ranked b values form a homogeneous subset, Le., there is no
Vaccine, Vol. 2, J u n e 1 9 8 4
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Multiple comparison of dried BCG vaccines: L Lugosi Table 4 Regranal of mean RPC (%) of BCG strains on 8 4 - 5 4 7 days after i.v. vaccination of mice followed by Anova and Gabriel's STP. Analysis of data from Table 3 Regranal BCG
Inoc. VU
a
+ s.e.a
ba
Jap Par Dak Mos Ref Con IRef Cop Mer Pra Gla Bil
297 4676 8743 9657 2377 9343 23771 5600 6040 3533 3745 6000
-54.8 - 3.0 9.9 20.3 8.1 1.6 0.1 1.0 0.9 3.5 4.2 64.0
155.3 15.8 17.4 14.0 19.6 9.2 1.2 1.1 0.7 2.0 0.9 25.8
0.591 0.069 0.061 0.051 0.043 0.O16 0.OO5 0.002 0.0004 -0.007 -0.009* -0.027
BCG in spleen at day 360
(95% confidence limit) -0.423 -0.021 -0.039 -0.031 -0.067 -0.040 -0.001 -0.005 -0.003 -0.O18 -0.014 -0.188
1.605) O.159) 0.161) 0.134 0.154 0.073 0.012 0.008 0.004 0.004 -0.004 0.135
VU 469 1022 2794 3750 564 694 481 87 62 29 38 3268
Anova: MS values
inoc. VU Linear (95% confidence limit)(%) d.f. = 3 ( 0 ( 241 (1245 (2717 ( 95 ( O ( 213 ( 27 ( 21 ( 0 ( 4 (1545
991 1803 4342 4783 1033 1626 750 146 103 98 72 4992
157.8 21.9 32.0 38.8 23.7 7.4 2.0 1.5 1.0 0.8 1 .O 54.5
Residual
1 3 7 2 7 7 182351 123 2307 2565 2571 5757* 760 2797 3355 529 675 19 11 15 10 2 4 20 34 5 7 7867 4809
d.f. 28 32 32 26 33 29 34 34 31 34 34 25
Anova for differences between the 12 regression lines
Gabriel's STP for ranked regression coefficients (b) and VU % persisting in spleen at day 360
Source
MS
F
Rank
BCG
b
1 11 11 36
20322 21066 35490 13081
1.37 1.42 2.39* 0.88
Residual
372
14878
Total
431
1 2 3 4 5 6 7 8 9 10 11 12
Jap Par Dak Mos Ref Con IRef Cop Mer Pra Gla Bil
0.591 0.069 0.O61 0.O51 0.043 O.016 0.005 O.002 0.0004 -0.007 -0.009* -0.027
d.f.
Comm. slope Parallelism Identity Linearity
*Significant at 5% level; a(slope) daily RPC increase (-decrease) in %; HS, Vertical bars indicate homogeneous subsets of Gabriel's STP
significant difference in the daily decrease between 30 000 a n d 312 000 VU × 106 ml-' values in this e x p e r i m e n t a l design. As to the VU × 106 ml -~ values, at d a y 28 the vaccines form one h o m o g e n e o u s subset.
P e r s i s t e n c e in m o u s e s p l e e n Estimation o f the mean R P C o f B C G in mice spleen, Table 3 presents the c o m p a r e d B C G strains, the i.v. i n o c u l a t e d VU from 100 x diluted vaccines a n d the 108 m e a n R P C estimates. F o r all further statistical analyses these 108 m e a n R P C values a n d their log or square root values have been used. (Altogether 216 m e a n R P C values as an average o f eight mice expressed in percent at a given a u t o p s y d a y were c o m p u t e d from 34 560 B C G c o l o n y c o u n t d e t e r m i n a t i o n s o b t a i n e d from 12 vaccines, two i.v. doses, n i n e a u t o p s y days, eight mice from each group, two mice spleen dilutions, ten seeds per d i l u t i o n on B O A A medium.)*
Trends o f the R P C in mice splee~ F o r statistical inferences two m o d e l s were used: (l) R e g r a n a l t with m e a n R P C values between day 1-21 a n d 1-42 (early RPC, i.e., m u l t i p l i c a t i o n ) as well as between days 42-547 a n d 84-547 (late RPC, i.e. persistence) after v a c c i n a t i o n was *Results of large inoculums (10 × diluted vaccines) are not presented but are available on request "~Four periods of RPC trends were analysed; early (1-21 and 1-42 days) and late (42-547 and 84-547 days) with %, log and square root RPC values of the 10 x and 100 × diluted BCG vaccines. Thus, 24 trend tables were computer printed. From these, because of limited space, only the main results of ranked b values and the related Anova of six models are summarized in Table 5, viz., the 1--42 and 84-547 days trends of 100 × diluted vaccines.
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Vaccine, Vol. 2, June 1 9 8 4
HS
BCG
VU %
Jap Bil Mos Dak Ref Par Con IRef Cop Mer Gla Pra
157.8 54.5 38.8 32.0 23.7 21.9 7.4 2.0 1.5 1.0 1.0 0.8
HS
c o m p u t e d for each B C G strain in o r d e r to d e m o n s t r a t e the t r e n d o f m u l t i p l i c a t i o n a n d persistence capacities, then A n o v a a p p l i e d for R e g r a n a l was c o m p u t e d to c o m p a r e the regression lines o f the strain; (2) O n e - W a y - A n o v a $ with m e a n R P C was c o m p u t e d at each d a y mice were killed to m a k e cross-section c o m p a r i s o n between B C G strains d u r i n g the 547 day follow-up study. Table4 presents the results o f the Regranal, the A n o v a a n d G a b r i e l ' s STP. In this table o n l y the trends between 84 a n d 547 days o f the 100 × diluted vaccines are presented c o m p u t e d with m e a n R P C in % values. T h e u p p e r parts o f the tables show the estimated p a r a m e t e r s of the R e g r a n a l for each tested strain. T h e a values show the intercepts. T h e regression coefficients (b) express the daily increase or decrease o f the B C G VU in the spleen o f mice as a percentage of the i n o c u l a t e d VU. The e x t r a p o l a t e d B C G VU estimates at day 360 a n d their 95% c o n f i d e n c e limits show the absolute n u m b e r o f recoverable VU still persisting in the spleen. Values given in % o f i n o c u l a t e d VU express the R P C o f the B C G strains at day 360. T h e r e are strains showing R P C values u n d e r 20% h i n t i n g at a decreased RPC, i.e. residual virulence. The A n o v a shows that only B C G - M o s has significant deviation from linearity. T h e lower part o f the table shows two models. T h e A n o v a a p p l i e d for R e g r a n a l p e r f o r m e d with R P C (%) shows c o m m o n slope, p a r a l l e l i s m , identity a n d linearity of the 12 regression lines. T h e 0.88 F value o f linearity shows that the m o d e l is valid at 5% p r o b a b i l i t y level, thus this m o d e l is a c c e p t a b l e for valid inferences to c o m p a r e p a r a l l e l i s m SAltogetherwith log and square root metameters of RPC in % of 10 × and 100 × diluted vaccines six One-Way-Anova tables were computer printed. Table8 summarizes the full mean ranks of RPC (%), log and square root RPC values of the tested BCG vaccines from these six tables.
Multiple comparison of dried BCG vaccines: L Lugosi Table 5
Gabriel's STP of the ranked regression coefficients between 1-42 and 8 5 - 5 4 7 days of lOOx diluted BCG vaccines. b Values of 1-42 days; early RPC
Rank
ba BCG (%)
b HS BCG (log)
1 2 3 4 5 6 7 8 9 10 11 12
Mos Bil Par Dak Con Ref Pra Cop IRef Jap Mer Gla
Anova
d.f.
MS
Comm. b Linearity Parallel. Identity Residual
1 24 11 11 319
3630 10.71" ~903 2.66* 1764 5.20* 5974 17.62" 339
1.265" 0.936 0.701" 0.514* 0.082 -0.002 -0.034 -0.042 -0.076 =~ -0.084 -0.249~ -0.249 F
Mos Dak Con Par Bil Ref COp Jap Gla Mar Pra IRef d.f.
HS
0.022 0.019* 0.004 0.001 -0.001 -0.004 -0.011 -0.015 -0.017* -0.026-0.028 == -0.042 ~* MS
F
b Values of 8 4 - 5 4 7 days; late RPC
b BCG (square root)
HS
bb 8CG (%)
b HS BCG (log)
Mos Dak Par Bil COn Ref Cop Pra Jap IRef Gla Mar
0.0105* 0.0067* 0.0055 0.0038 0.0011 -0.0003 -0.0017 -0.0023* -0.0030* -0.0035* -0.0046* -0.0053*
Jap 0.591 Par 0.069 Dak 0.061 Mos 0.051 Ref 0.043 Con 0.016 IRef 0.005 COp 0.002 Mar 0.000 Pra -0.007 Gla -0.009* Bil -0.027
IRef Jap Ref Par Mos Dak Pra Cop Mer Bil Gla Con
d.f.
MS
d.f.
1 6.009 9.42* 1 24 1.135 1.78" 24 11 2.716 4.26* 11 11 1 7 . 7 8 2 2 7 . 8 9 " 11 319 0.638 319
F
0.017 0.60 1 0.064 2.24* 36 0.183 6.40* 11 0.915 31.99" 11 0.029 372
b HS BCG square root
0.0030* 0.0020 0.0016 0.0004 -0.0001 -0.0001 -0.0006 -0.0007 -0.0007 -0.0025 -0.0035* -0.0036*
MS
F
d.f. MS
20322 13081 21066 35490 14878
1.37 0.88 1.42 2.39*
1 36 11 11 372
F
HS
Jap Dak Par Ref Mos IRef Cop Mar Pra Con Gla Bil
0.00149 0.00044 0.00041 0.00039 0.00023 0.00022* 0.00000 -0.00002 -0.00012 -0.00017 -0.00035* -0.00044
d.f.
MS
3.807 3.32 1 2.250 1.96" 36 4.125 3.60* 11 2 4 . 4 4 3 2 1 . 3 3 * 11 1.146 372
F
0.112 0.87 0.241 1,87" 0.211 1.64 1.317 10.20" 0.129
*Significance at 5% level; a(Slope) daily increase or decrease (+ b) of BCG VU in mice spleen-expressed in % or in log or square root transformed form; bDetails see Table 4; HS, vertical bars indicate homogeneous subsets of Gabriel's STP. For Regranal, Anova and Gabriel's STP mean (%), respectively, log RPC and square root RPC values of Table 3 were used
and identity of the lines. Gabriers STP shows that the ranked regression coefficients (b) form one homogeneous subset, i.e, from 84 to 547 days after vaccination the trends of late RPC of the 12 BCG strains are parallel, although the VU % values still persisting in spleens at day 360 form two subsets. Table 5 summarizes the ranked regression coefficients of only six of the 24 models of the compared BCG strains from the computer printed trend tables +. The b values are listed according to the tested period (early. late) and to the transformations (log or square root). The following question arises in the comparison of RPC: which of the six models should be accepted for null hypothesis. The model in which the F value of Anova indicates non-significant deviation from linearity at the 5% probability level could be accepted as a valid decision in ranking the trend of RPC of the tested BCG strains. The non-significant variance ratio of linearity (F ----0.88) of ranked b% of late RPC demonstrates the case of the valid decision model. It can be seen that the tested BCG strains form one homogeneous subset. The log b of the late RPC model is invalid (Lin. F ----- !.96) and it can be seen that the log transformation of the RPC selects two additional subsets. The valid model of b% is the best example for interpreting the biological meaning of the rank position of the RPC of a particular BCG strain. One can understand that there is no fixed rank position in a test system for a biological character because the inherent variability, the environmental conditions and experimental factors may influence the RPC in the actual experiment. The vertical bars of Gabriel's STP indicate that several BCG strains could be members of the same subset when residual virulence is determined under particular experimental conditions of variables and in statistical assumptions predetermined for the test system. Altogether, there were eight valid models (four early and four late) out of 24 with immunobiologically interpretable effective VU % values still persisting on day 360. Gabriel's STP of Table4 shows one
of these valid models. Other transformations of RPC values ofthe invalid models need to be researched further. Further analysis is needed also to evaluate the associations between dose and effect, i.e., the relationships between inoculated VU and early, late or overall RPC trends depending on the residual virulence of the BCG substrains.
Multiple comparison o f the m e a n RPC at each autopsy day. Table6 presents the results of the Gabriel's STP with mean RPC after One-Way-Anova computed at each autopsy day expressi~ag in % the BCG VU persisting on the given day the mice were killed, in the spleen of i.v. vaccinated mice with 100 x diluted vaccines*. The mean RPC values are ranked for each day the mice were killed. The F values of Anova indicate significant and nonsignificant differences of the mean RPC values. H o m o geneous subsets of mean RPC of the tested strains are indicated by vertical bars. The full mean rank values express the overall mean RPC of the BCG strains. Association between ranks of m e a n R P C of the nine days mice were killed Table 7 shows the results of Kendall's test of the ranks of mean RPC (%) values taken from Table6. In this model nine independent rank orders are arranged and the extent of association of the ranked mean RPC of BCG of the nine days mice were killed can be calculated by the Kendall coefficient of concordance (W). As the significant F value of the W shows, there is agreement between the ranks of the nine dfiys mice were killed. Thus the null hypothesis of the Kendall's test, i.e., no agreement between the nine ranking sets of days mice were killed can be rejected. As in Table 6 full means indicate the overall rank order in the model. Table 8 lists these full mean ranks according to the inoculated vaccine dilutions and to the RPC transformatSee footnote to page 152
Vaccine, Vol. 2, June 1 9 8 4
153
Multiple comparison of dried BCG vaccines: L Lugosi Table 6 Gabriel's STP with mean RPC (%) taken from Table 4 after One-Way-Anova at each day a mouse was killed after i.v. vaccination for ranking and selecting the compared 100 x diluted BCG vaccines Days mice killed
1
Rank
BCG
%
1 2 3 4 5 6 7 8 9 10 11 12
Gla Bil M~r Cop Con IRef Dak Par Mos Jap Pra Ref
36.6 29.9 19.4 8.0 5.6 4.0 3.9 3.9 3.7 3.1 2.9 0.2
MS Betw./d.f. MS WithJd.f. F
8 HS BCG
I
I Con M~r ios Jap Par Cop Pra IRef Dak Ref
BCG
%
1 2 3 4 5 6 7 8 9 10 11 12
Jap Ref Dak Bil Mos Par Gla Con M~r Cop IRef Pra
19.4 39.4 22.3 17.4 15.8 4.2 1.8 0.9 0.7 0.3 0.0 0.0
MS Betw./d.f. MS WithJd.f. F
HS BCG
I
Bil Con Mos Dak Par Gla COp M~r Pra IRef Ref Jap
%
Dak Bil Par Mos Ref Jap COp M~r IRef Gla Con
5592/11 2956/82 1.89
3387/11 974/77 3.48*
HS BCG
350.1 78.0 72.8 35.1 33.7 29.3 18.8 4.4 1.7 0.0 0.0 0.0
%
HS
92.6 41.9 6.4 6.3 4.5 4.3 3.9 1.9 1,4 1.0 0.0 0.0 5832/11 474/82 12.31" Full mean rank
%
Bil Par Ref Mos Dak Cop IRef Con M~r Gla Jap Pra
75616/11 69242/73 1.09
2517/11 892/77 2.82*
Bil Mos Par Con Pra Gla Dak Cop IRef Mer Ref Jap
547
%
Jap Mos Bil Ref Dak Par Con IRef M6; Gla Cop Pra
HS BCG
64.3 56.6 31.4 24.3 22.7 12.4 6.1 3.8 1.1 0.9 0.0 0.0
450 HS BCG
84
%
Bil Mos Par ~'Gla Dak Con Mer Cop Pra IRef Ref Jap
3953.2/11 308.7/79 12.81"
56.7 39.6 22.3 12.4 5.2 3.8 2.0 1.1 0.9 0.8 0.5 0.4
Pra
HS BCG
125.8 1 5.9 12.0 10.4 9.7 7.1 6.3 5.8 1.0 0.7 O. 1 0.0
349 HS BCG
42
%
1266.1/11 67.6/84 18.74"
184
Rank
41.9 22.5 17.0 9.9 8.2 2.9 2.3 1.5 1.2 1.0 0.8 0.0
Gla
1040.5/11 39.0/79 26.71"
Days mice killed
%
Bil
I
21
HS BCG
57.9 34.5 32.7 31.9 29.1 2.9 2.4 0.9 0.4 0.1 0.0 0.0
Bil Mos Par Dak Con Gla Mer Cop Jap Ref IRef Pra
1.8 a 4.0 4.8 5.3 6.1 6.3 7.4 7.7 7.9 7.9 9.0 9.8
2062/11 2237/58 0.92
*Significant at 5% level; "See Table 7; HS, Vertical bars indicate homogeneous subsets of Gabriel's STP
Table 7 Ranks of Mean RPC (%) values taken from Table 6. Kendall's test for determining the association between nine sets of ranks (days mice killed) of the compared 1O0 x diluted BCG vaccines Days mice killed after vaccination Rank
BCG
1
8
21
42
84
184
349
450
547
1 2 3 4 5 6 7 8 9 10 11 12
Bil Mos Par Dak Con Gla M~r COp Jap Ref IRef Pra
2 9 8 7 5 1 3 4 10 12 6 11
1 5 7 11 3 2 4 8 6 12 10 9
1 3 5 4 2 6 8 7 12 11 10 9
1 2 3 5 6 4 7 8 12 11 10 9
1 2 3 7 4 6 10 8 12 11 9 5
4 5 6 3 8 7 9 10 1 2 11 12
2 4 3 1 12 11 8 7 6 5 10 9
3 2 6 5 7 10 9 11 1 4 8 12
1 4 2 5 8 10 9 6 11 3 7 12
Full means 1.8 a 4.0 4.8 5.3 6.1 6.3 7.4 7.7 7.9 7.9 9.0 9.8
Coefficient of concordance: W = 0.393 F = 5.17" d.f.1 = 11 d.f.2 = 86 *Significant at 5% level; ~See Table 6; Kendall's W values express the degree of agreement between the ranks of Mean RPC of the nine sacrifice days in ranking the 12 vaccines. Full means indicate the best estimate of the 'true' ranking of the averaged ranks of nine days mice were killed of mean RPC of BCG strains when the F value for W is significant
154
Vaccine, Vol. 2, J u n e 1 9 8 4
Multiple comparison of dried BCG vaccines: L Lugosi Table 8 Comparison of full mean ranks of RPC, log RPC and square root RPC of BCG vaccines tested in mice spleen from 1 to 547 days mice killed Rank
A o "o _= x o
RPC (%)
1 2 3 4 5 6 7 8 9 10 11 12
"o := -o x o 0
1 2 3 4 5 6 7 8 9 10 11 12
Square root RPC
BCG
Full mean ranks BCG
Full mean ranks BCG
Full mean ranks
Bil Mos Jap Con Gla Par Dak Ref Mer IRef Cop Pra
3.8 3.8 4.6 5.4 5.9 6.1 6.8 7.3 7.8 7.8 8.5 10.5
4.0 4.3 4.6 5.3 5.8 6.3 6.5 7.0 7.9 8.0 8.4 10.1
3.8 3.9 4.9 5.4 5.6 6.3 6.8 7.1 7.8 7.9 8.4 10.4
W F dfl df2 ~" ® ~" '~
Log RPC
Bil Mos Par Dak Con Gla M6r Cop Jap Ref IRef Pra W F dfl df2
Bil Mos Jap Gla Con Dak Par IRef Mer Ref Cop Pra
0.309 3.13* 11 75 1.8 t 4.0 4.8 5.3 6.1 6.3 7.4 7.7 7.9 7.9 9.0 9.8 0.393 5.17* 11 86
Bil Mos Jap Con Gla Par Dak Ref IRef Mrr Cop Pra
0.266 2.54* 11 75 Bil Mos Dak Gla Par Jap Con Mrr Cop Ref Pra I Ref
1.9 3.3 4.0 5.7 5.7 6.3 7.1 7.2 8.1 9.1 9.8 9.8 0.495 7.83* 11 86
0.291 2.87* 11 75 Bil Mos Dak Par Con Gla Mrr Cop Ref Jap Iref Pra
1.9 3.6 4.2 5.7 6.1 6.2 7.2 7.4 8.2 8.7 8.8 10.0 0.437 6.22* 11 86
*Significant at 5% level. Kendall's W express the degree of agreement between the ranks of Mean RPC of the nine days mice killed in ranking the 12 vaccines. Full means, taken from Table 6a and computer printed tables, indicate the best estimate of the "true' ranking of the 1 - 5 4 7 days averaged ranks of mean RPC of BCG strains when the F values for W are significant
tions. Significant F ratios of W values show that there is agreement between ranks in all six models in spite of the small fluctuation in rank position of the full means of the BCG strains. This could be due to the random variability occurring in this biological test system. Thus, the full mean ranks could represent the global BCG-RPC for the total observation period-
Discussion immunogenicity and reactogenicity Of the BCG vaccines are determined by two characters: the viability of the vaccine and the residual virulence of the strain used in preparation. The exact measure of these two characters allows analysis of the association between the laboratory control data and the post-vaccination host response (protective efficacy, immune and hypersensitivity reactions, frequency of side effects). Therefore, it is a prerequisite to test the dose-response relationship and to use sensitive laboratory techniques with exact staE~tical methods in order to demonstrate significant differences in the in vitro potency and in the in vivo residual virulence of the BCG strains expressed in VU × 10~ ml -: and in mean RPC in host organ(s). Biological products are controlled by standardized methods in valid bioassay models, but the BCG produc-
tion still lacks uniform statistical quality controlrequirements '~-17. We have studied: (1) the comparison of the stability of the viability at 37°C and the RPC in mice spleen of BCG vaccines and (2) the application of exact methods for statistical inference (unbiased parameter estimation, valid hypothesis tests, Gabriel's STP for multiple comparison of the ranked parameters) in order to achieve objective interpretation in laboratory control. Data show that the mean VU × 106 ml -~ values of the intradermal BCG vaccines of various substrains vary between 1.0 and 27.0 × 106 ml -~ at day 0 (Table 2) but 50-fold difference is also indicated ~3. The pragmatic question is: why should the immune system be overloaded with high VU when satisfactory dose-response can be obtained by giving 100 000-300 000 VU with BCG strain(s) of normal residual virulence ~s,~9. O f course, taking into account the residual virulence of the strains, sensitive and exact control methods are required to demonstrate small (1.0-3.0 × 10~ ml "~) VU differences. Evaluation of the stability at 37°C by Regranal, Anova and Gabriel's STP show that the VU x 106 ml -~ values of the compared vaccines (Table 2) decrease paraUely but at day 28 only six vaccines have, in valid model, the expected 20% survival rate t3. In contrast to previously published data concerning the RPC in mice spleen using short-term investigations 2s-2~, the experimental design presented is a long-term model which can be used to control the early and later RPC and to evaluate exactly the in vivo persistence of the living, metabolizing and multiplying BCG as conditions of acquired resistance and delayed hypersensitivityz3. As has been shown z*, the mean RPC estimates indicated that depending on the VU doses and on killing mice after vaccination: (1) with large i.v. VU dose (magnitude of 10 000) all strains show RPC under 20% during the whole follow-up (1-547 day) period; (2) with small i.v. VU dose (magnitude of 1000): (i) some strains have a progressive mutiplication then a persistence during the whole period; (ii) some strains have only an early multiplication capacity, (iii) some strains have only a late persistence capacity after the vaccination. The Regranal of the late RPC indicates (Table 4) that the trends are parallel from 84 to 547 days but there are only six BCG strains whose inoculated VU persist over 20% in the spleen at day 360 in a valid model. Ranking the mean RPC % according to the days mice were killed (Table 6): (i) Gabriel's STP shows the homogeneous subsets of the mean RPC values, (ii) the ranks of the BCG products from which the full mean ranks of the overall multiplication and persistence capacity can be computed- If Kendall's test is significant and full mean ranks are under 6.0 overall mean RPC of BCG can be considered appropriate (Tables 7 and 8). The second objective of the study was to introduce the Gabriel's STP for multiple comparison of ranked parameters of the stability of viability and persistence capacity of the BCG vaccines in mice spleen. In contrast to the Student test, which while performing pairwise comparison between experimental groups increases the risk of type I error, Gabriel's STP reduces the risk of type I error in inferences and prevents rejection of the null hypothesis when it is true, i.e., to state that there is a difference between the estimated parameters of groups (vaccines) whereas in fact such a difference does not exist. As a transitive t multiple comparison method, Gabriel's STP t If a subset (S) of ranked parametersof the comparedvaccinesis judged significant, everysubset that contains S will also be judged significant; conversely, if a S is not significant, no further subsets of S will be significant.
Vaccine, Vol. 2, J u n e
1984
155
M u l t i p l e c o m p a r i s o n o f d r i e d BCG vaccines: L L u g o s i
maintains the risk of type I error at a 5% experimental (study) level, it is easy to compute and can be used for parameters estimated from unequal sample sizes "s,'6. The models in Table 5 show Gabriel's test to be very selective. The selection of homogeneous subsets of BCG strains having the same trends (b values) of mean RPC values depends on the validity (linearity) of the Anova model. Control of the viability and the residual virulence of the BCG strains can be more exact and the interpretation more objective when multiple comparison and additional concordance tests are used. These methods can help with the choice of BCG strain when preparing an effective vaccine with the lowest possible frequency of adverse reactions "7-29, and can help with standardization of the vaccineaa,n, they can also explain the differing efficacies of certain BCG vaccines 3~.
Acknowledgements I would like to thank Mr P. Csaki and Mr G. Tusnady for their advice and help in the data processing and statistical analyses. The technical assistance of Mrs M. Drinoczy and Miss A. N~meth is gratefully acknowledged.
8
9 10 11 12 13
14 15 16 17 18
19
Note Details of statistical analysis, formulae of the BCG VU × 106 ml-' and mean RPC point and 95% confidence interval estimation, computer programs for Anova, Regranal and Gabriel's STP are available On request.
20
21
References 1
2
3
4 5 6 7
156
Cohen, H. Information of the Chairman of the BCG Steering committee of lABS reveal the code of the BCG vaccines used in phase I and II of the collaborative assay. Letter no.: U 2 0 7 / 7 9 Dir Si/ms/19.VU.1979. RIV. Bilthoven Sekhuis, V.M., Freudenstein, H. and Sirks, J.L. Report on results of a collaborative assay of BCG vaccines organized by the International Association of Biological Standardization. J. Biol. Stand 1977, 5, 85 Freudenstein, H., Pranter, W. and Schwinsberg, H. Assessment of several BCG vaccines in different animal test systems (additional studies to an lABS collaborative assay). J. Biol. Stand 1979, 7, 203-212 Smith, D. et el. Potency of 10 BCG vaccines as evaluated by their influence on the bacillemic phase of experimental airborne tubercolosis in guineapigs. J. BioL Stand 1979, 7, 179 WHO, List of international biological standards and international biological reference preparations, Biological substances. WHO, Geneva, 1979 WHO. Designs for in vitro assays of 8CG products. WHO/TB/ Techn. Guide/67.7. 1967 Lugosi, L, Stabilit6 de la viabilit6 du vaccin 8CG sec (souche Pasteur 1173-P2) atock6 a 4=C pendant 540 jours: ~tude statistique, AnrL Microbiol. (Paris) 1982, 133(B), 4 7 5
V a c c i n e , Vol. 2, J u n e 1 9 8 4
22 23 24 25 26
27 28 29 30 31 32
Lugosi, L. Biomathematical methods for the statistical quality control and standardization of the BCG vaccine. Report on the WHO BCG control program to WHO/TB Unit. BCG Laboratory. NIH, Budapest, 1978 Armitage, P. Statistical methods in medical research. Blackwell, Oxford, 1971 Gabriel, K.R. A procedure for testing the homogeneity of all sets of means in analysis of variance. Biometrics 1964, 20, 459 Siegel, S. Nonparametric statistics for the behavioral sciences, McGraw-Hill, New York, 1956 WHO, In vitro assays of BCG products, WHO/l'B/Technical Guide/77.9. 1977 WHO. Revised requirements for dried BCG vaccine (Requirements for Biological Substances No. 11 .) (Revised 1976) WHO. Expert committee on Biological Standardization, Thirtieth Report. WHO Techn. Rep. Ser. No. 638, 1979 Finney, D.J. Statistical method in biological assay. Griffin, London, 1964 Roberts, M. and Boyce, C.B.C. Principles of biological assay. In: Methods in microbiology lEd. Norris, J.R. and Ribbons, D.W.) Academic Press, London, 1972, p 1 5 3 - 1 9 0 Sarkadi, K. and Vincze, I. Mathematical methods of statistical quality control. Akademia~ Budapest 1974 Hewitt, W. Microbiological assay. An introduction to quantitative principles and evaluation, Academic Press, New York, 1977 Lugosi, L. Factors influencing the BCG policy in different countries. Children tuberculosis trends comparison between the Federal Republic of Germs,ny and Hungary. Arb. Paul Ehrlich Inst- Georg Speyer Haus Ferdinand Blum Inst Frankfurt am Main 1977, 71,1 Lehmann, H.G. et el. Investigations carried out to ascertain the dose-effect relationship of a BCG vaccine, strain 1331 Copenhagen, in neonates and young infants. ZentralbL BakterioL Paresitenkde Infektionskr. Hyg. Abt. 1: Orig. Reihe B. 1978, 188, 250 Benoit, J.-C. and Panisset, M. Survie et multiplication du BCG et du bacille tuberculeux chez la souris. IV. Survie et multiplication de souches-filles de BCG en fonction du temps et de la dose. Acta Tuberc, PneumoL Scand. 1963, 43, 125 Roger, F. and Roger, A. Inactivation du BCG in vivo. Ann. InstPasteur. Paris, 1965, 109, 754 Sher, N.A., Chaparas, S.D., Pearson, J. and Chirigos, M. Virulence of six strains of Mycobacterium bovis (BCG) in mice. Infect Immun. 1973, 8, 736 Lagrange, P. Present status of knowledge on immunity in tuberculosis. WHO/'I'B/80.110, 1980 Lefford, M.J. The effect of inoculum size on the immune response to BCG infection in mice. Immunology 1 9 7 1 , 2 1 , 3 6 9 - 3 8 1 Miller, R.G. Jr. Simultaneous statistical inference. McGraw-Hill, New York, 1966 Lugosi, L. Control of viability, thermostability and residuaIvirulence of BCG vaccines. Multiple comparison of laboratory data to select products for immunostimulation in the treatment of cancer. Dev. Biol. Stand 1978, 38, 45 WHO. The choice of a BCG strain. Bull. WHO 1976, 54, 118 WHO. Expanded programme on immunization. Progress Report by the Director-General. WHO A 3 1 / 2 1 , 1978 Gheorghiu, M., Carnus, H., Lagrange, P. and Chambon, L. Potency and suppurative adenitis in BCG vaccination. Dev. BioL Stand 1978, 41, 79 L0vy, F.M., Mande, R., Conge, G., Fillastre, C. and Orssaud, E. Perspectives for BCG standardization. Ad~. Tuberc. Res. 1968, 16, 63 WHO. Progress in standardization. Bull. WHO 1973, 49, 167 WHO. BCG vaccination policies. Report of a WHO Study Group. WHO Techn. Rep. Ser. 652, 1980
Keywords:Statisticalquality control; BCG vaccine:heat stability:residual virulence;persistencein mouse spleen; multiple comparison.
Introduction The aim of the statistical quality control of BCG vaccine is to select the most efficacious product(s) from the tested samples for immunization programmes with minimal frequency of adverse reactions. To compare the in vitro and in vivo potency of BCG preparations reliable statistical methods, unbiased point and confidence interval estimation of parameters and appropriate transformations of data should be used to fulfil validity criteria of hypothesis tests. When comparing the parameters of laboratory controls of more than two BCG products after rejected null hypothesis, multiple comparison is needed to diminish the risk of type I error. This study uses Gabriel's simultaneous test procedure (STP) for the statistical quality control of BCG products using the ranked point estimates of in vitro and in vivo animal experiments for the multiple comparison: (1) of the stability of the viable units (VU) of the tested BCG vaccines stored at 37°C over a 28 day period. expressed in terms of VU × 106 ml-~ decrease/day, and (2) of the residual virulence of the tested BCG strains by determining over 547 days the regression of the relative persistence capacity (RPC) in mice spleen, i.e. the ratio of BCG Laboratory, National Institute of Hygiene, Gyali ut 2, Budapest 1097, Hungary. (Received 22 June 1 983) 0264-410)(/84/020149-08 $03.00 © 1984 Butterworths 8- Co. (Publishers) Ltd.
the recoverable VU/i.v. inoculated VU (Rec-VU/Inoc-VU). Association among ranked mean RPC of the tested BCG products was finally evaluated with Kendall's coefficient of concordance.
Materials and methods Laboratory methods Tested vaccines. The dried BCG vaccines investigated were the same as those used in the collaborative assay of the International Association of Biological Standardization (IABS) 1-4. The origins and abbreviations of the tested BCG vaccines are as follows: Rijks Instituut voor de Volksgezondheid, Bilthoven (Bil), Connaught Medical Research Laboratories, Toronto (Con): Statens Seruminstitut, Copenhagen (Cop): Institut Pasteur, Dakar (Dak); Glaxo Research Laboratories, Greenford (Gla): Japan BCG Laboratory, Tokyo (Jap); Institut M~rieux, Lyon (M~r); Tarassevich State Institute, Moscow (Mos); Institut Pasteur, Paris (Par): Institute of Hygiene and Epidemiology, Prague (Pra): Reference Japan Strain (Ref): International Reference Preparation Japan Strain (IRef)L Determination o f viability and stability of the vaccines. The culturable particles (colony count) of the
Vaccine, Vol. 2, June 1 984
149
Multiple comparison of dried BCG vaccines: L Lugosi tested BCG vaccines were determined parallely on L6wenstein-Jensen (L-J) and blood-oleic acid-albumin (BOAA) media s. The VU × l06 ml -t values were estimated from the culturable particles. The thermostability, i.e. the VU decrease in the ampoules stored at 37°C was determined at 0.7, 14, 21 and 28 days from the daily VII decrease in slope and expressed as VU × 106 ml -~ decrease/day as previously described 7.
Determination of RPC: vaccination of mice, recoverable BCG colony counts from the spleen of mice. Determination of the viability on BOAA medium of vaccines stored at 4°C and sterility tests were performed 24 h before vaccination. A volume (0.2 ml) of the 10 × and 100 × dilutions of the 12 BCG vaccines was injected intravenously with an Omega syringe: 24 groups of 124 male and/or 124 female CFLP (Carworth Europe), 18-22 g, 4-6 week old mice were i.v. inoculated in random order. A total of 2976 mice (1488 male and 1488 female) were housed in 96 polypropylene boxes (38 × 27 × 11 cm) each containing 31 mice at the beginning of the experiment (124 mice in four boxes = one 'treatment' group, vaccinated from one dilution of one of the compared 12 vaccines). The animals were fed on a pellet diet and water adlibitum After vaccination at days 1, 8, 21, 42, 84, 184, 349, 450 and 547, eight mice from each vaccinated group were killed with chloroform. Spleens were removed aseptically then homogenized individually with a Potter-Elvehjem Teflon-glass homogenizer in 3.0 ml of 1 + 3 diluted Sauton medium containing Bovine albumin fraction V of 0.1%. From the three-fold diluted whole spleen homogenates and their two-fold dilutions (6 × diL) 10 drops each of 0.02 ml were seeded on BOAA medium in Petri dishes using injection needles (internal diameter, 0.6 ram; external diameter, 1.1 mm) mounted on glass pipettes ( ~ e t e r , 8 m-m~l¢-ngth,lO0 mm). After seeding, drying and sterility tests were carded out overnight in an incubator, the ten inverted Petri dishes were put in cylindrical aluminium boxes (diameter, 12 cm; heighL 26 cm) and covered with plastic bags for incubation at 37°C. After an incubation period of 15-17 days, BCG colony counts were made with a ! 0 × magni~ing binocular stereomicroscope. For reliability, an optimum of 20 and an upper limit of 50 colony counts was prescribed 6. Statistical methods Estimation of the parameters of stability and RPC. In order to have unbiased point and 95% confidence interval estimates of the VU × 10' ml -t values of vaccines and the individual Rec-VU values using recovered BCG colony counts from mice spleen, censored Poisson (CP) distribution was assumed. The CP formula includes the proportion of readable but uncountable colony counts of the dilution levels inoculated* and is an efficient BCG viability estimation method. For hypothesis tests, the VU × 106 ml -~ estimates and mean RPC values calculated by averaging the Rec-VU point estimates of eight mice and divided by i.v. Inoc-VU were used. Hypothesis testa For statistical inferences, analysis of variance (Anova) and linear regression analysis (Regranal) with VU × 106 ml -t values of vaccines stored at 37°C for 28 days and with mean RPC values were performed. Where necessary, to fulfil validity assumptions in the hypothesis tests, log or square root of the mean RPC values were used as transformed point estimates in the statistical analyses 9.
150
Vaccine, Vol. 2, J u n e 1 9 8 4
Multiple comparison. After using the Anova and Regranal tests, in cases of rejected null hypotheses, Gabriel's STP was performed with the ranked VU x 106 m1-1 estimates, with estimated regression coefficients and with mean RPC values in order to diminish the risk of type I error ~°. Kendall test of concordance. To test the association between sets of ranked mean RPC values of the BCG strains obtained on the days mice were killed, Kendall's coefficient of concordance was computed". Computation. A CDC-3300 computer of the Computer and Automation Institute of the Hungarian Academy of Sciences was used for data processing and statistical analysis. The main results only of our studies are presented here, all computer printed tables are available on request from the BCG Laboratory of Budapest. Results
Stability a t 3 7 ° C Viability of the heat exposed vaccines. Table 1 summarizes the mean VU × l0 ~ ml -~ point estimates of the BCG vaccines stored at 37°C over 28 days. Three-WayAnova ofVU × l06 ml -~ values at day 0 and Gabriel's STP of the compared vaccines resulted in two homogeneous subsets included in Table 2.
Regression of the viability on exposure to heat. Table2 presents the separate Regranal of the 37°C heat stability of each vaccine (sampling days: 0, 7, 14, 21, 28). In contrast to the two point model (37°C/0, 28 day) used in routine in vitro assays of BCG products 12, with our five point regression model which included four repeated viability estimates, the linearity of the regression lines could be tested. Having fulfiled statistical validity assumptions, estimation of the regression coefficients with their 95% confidence limits and prediction of VU survival at day 28 become more exact. The 37°C resistance of VU at day 28 and its 95% confidence limits estimated from the regression equation, are the most important parameters when the potency of vaccines has to be judged before administration, after transportation and storage in tropical conditions. The 28/0 day estimates expressed in % show the relative heat-resistance capacity. The thermal degradation of the number of culturable particles in the vaccine after heating should not be less than 20% of the refrigerated Table I Viability expressed in mean a VU x 106 m1-1 values of the heat exposed BCG vaccines Time exposure (days) at 37°C
BCG vaccine Jap Gla Mos ~n ~k Bil ~p M6r Par Pra
0
7
27.0 20.1 7.1 6.9 6.5 4.2 2.9 2.8 2.7 1.1
12.6 14.8 6.7 4.0 3.9 2.5 3.6 2.2 2.7 0.5
14
21
28
23.3 13.9 5.5 0.4 2.9 1.3 3.3 1.7 1.7 0.3
18.0 11.5 4.8 0.2 2.0 1.5 2.1 0.7 1.9 0.2
16.6 10.9 3.6 0.2 1.8 1.3 1.9 0.3 1.3 0.2
*Each mean =s calculated trom four separate viability estimates parallely determined from two dilutions on two media as described in Methods. The non-listed 2 0 0 individual estimates and 9 5 % confidence limits can be obtained on request
Multiple comparison o f dried BCG vaccines: L Lugosi Table 2 Regranal of VU x 106 m1-1 values of BCG vaccines on days O. 7, 14, 21, 28 for testing 37°C heat stability followed by Anova and Gabriel's STP. Analysis of data from Table I Regranal
Anova: MS values
VU in vials at day 28
VU x 106 m1-1 BCG at day O a
+ s.e.
-b a
Linear (95% confidence limit) 10 s m1-1 (95% confidence limit) (%)b d.f. = 3
Residual d.f. = 1 5
Jap Gla Mos COn Dak Bil COp Met Par Pra
5.464 1.706 0.577 0.556 0.395 O.135 0.206 0.133 0.259 0.247
0.219 0.312" 0.129" 0.246* O.163" 0.099* 0.048* 0.093* 0.052* 0.030
0 -0.O10 -0.057 -0.177 -0.114 -0.083 -0.023 -0.077 -0.020 0
199.03 19.41 2.22 2.06 1.04 O.12 0.28 0.12 0.45 O.41
27.O19 20.131 7.148 6.926 6.513 4.227 2.885 2.805 2.737 1.110
22.019 18.584 7.342 5.796 5.692 3.555 3.465 2.843 2.782 0.904
-0.898) -0.524) -0.201) -0.315) -0.212) -0.116) -0.074) -0.110) -0.083) -0.060)
Anova for differences between the ten regression lines Source of variation
d.f.
Between (37°C expos, days)
MS
16.434 9.820 3.730 0 1.137 0.777 2.109 0.230 1.331 0.066
31.075) 13.437) 4.259) 2.611) 3.076) 2.569) 3.549) 0.634) 2.048) 0.547)
74.6 52.8 50.8 NC 20.0 21.9 60.9 9.1 47.8 7.3
141.10 8.75 O.18 9.00* 2.47 2.11 1.37 0.11 0.34 0.15
Gabriel's STP for ranked regression coefficients VU x 106 m1-1 values at day O and 28 F
Rank BCG - b
49
166.87
7.41 *
1
1
379.38
16.85*
17.37 793.82 16.56 22.51
0.77 35.27* 0.74
Residual
9 9 30 150
Total
199
2 3 4 5 6 7 8 9 10
Regression (common slope)c Parallelism (equality of slopes) Identity (equality of means) Linearity (curvature)
(1.793 (6.203 (3.201 (0 (0 (0 (0.669 (O (0.614 (O
HS BCG day O
Pra
0.030
Cop Par Mer Bil Mos Dak Jap Con Gla
0.048 0.052 0.093 0.099 O.129 0.163 0.219 0.246 0.312
Jap Gla Mos Con Dak Bil Cop M~r Par Pra
27.019 20.131 7.148 6.926 6.513 4.227 2.885 2.805 2.737 1.110
(b)
and
HS BCG d a y 2 8 HS Jap Gla Mos Cop Par Dak Bil Mer Pra Con
16.434 9.820 3.730 2.109 1.331 1.173 0.777 0.230 0.066 0
*Significant at 5% level; N C, Not computable; a(slope) daily VU decrease expressed in 108 m[-1; bRelative heat resistance capacity; the decrease of VU shall be not less than 20% of that in the refrigerated vaccine 13; c_b =_0.139 + 0.034; HS, Vertical bars indicate inhomogeneous subsets of Gabriel's STP
'Table 3
Estimates of mean RPCa of the compared 100 x diluted BCG vacines in mice spleen
BCG
i.v. inocc VU
Bil Con Cop Dak Gla IRef Jap Met Mos Par Pra Ref
6000 9343 5600 8743 3745 23771 297 6040 9657 4677 3533 2377
Time of death after vaccination (days) 1
8
21
29.9 5.6 8.0 3.9 36.6 4.0 3.1 19.4 3.7 3.9 2.9 0.2
41.9 17.4 1.5 0.8 22.5 1 .O 2.9 9.9 8.2 2.3 1.2 0.O
125.4 15.9 6.3 10.4 7.1 0.7 0.O 5.8 12.0 9.7 1.O O. 1
42
84
64.3 12.4 3.8 22.7 24.3 0.1 0.0 6.1 56.6 31.4 1.1 0.O
92.6 6.3 1.9 3.9 4.3 1.4 0.0 1.0 41.9 6.4 4.5 O.0
184 17.4 0.9 0.3 22.3 1.8 O.01 90.4 0.7 15.8 4.2 0.0 39.4
349 39.6 0.4 2.0 56.7 0.5 0.8 3.8 1.1 12.4 22.3 0.9 5.2
450
547
72.8 18.8 O.0 33.7 0.03 4.4 350.1 1.7 78.0 29.3 0.0 35.1
57.9 0.9 2.9 29.1 0.08 2.4 0.0 0.4 31.9 34.5 O.0 32.7
aMean RPC is expressed in %, i.e. mean recoverable VUb/inoculated VU estimated by censored Poisson distribution. bValues of mean recoverable VU from the whole spleens of mice determined on BOAA medium can be calculated from formula: mean Rec VU = (mean RPC x inoc VU)/100. Each mean is based on eight mice except those where there were missing values, recorded in experimental protocol, because the seeds on BOAA medium were contaminated; or colony counts were over limit (> 50) in all seeds, i.e., readable but not countable, therefore uncomputable; or colony counts were zero, i.e., the possibly persisting VU in spleens were not demonstrable. CThe mean-RPC values of the larger inoculum sizes (10 x diluted vaccines) are not listed but have been used for statistical analyses and can be obtained on request
vaccine ~3. Thus, vaccine parameters have immunobiological meaning in the statistical quality control within the biomathematical models. Using this model subjective interpretation of vaccine thermostability can be eliminated.
Anova and mul@le comparison of the regression of viability. In addition, Table2 presents the results of Anova
and Gabriel's STP. Anova tests c o m m o n slope, parallelism, identity and linearity of the ten regression lines. Linearizing transformation of the VU values was not needed. It can be seen that at the 5% level the F value of parallelism is not significant. Thus the slopes of VU-decrease of the vaccines during heat treatment can be considered similar. Gabriel's STP shows that the ranked b values form a homogeneous subset, Le., there is no
Vaccine, Vol. 2, J u n e 1 9 8 4
1 51
Multiple comparison of dried BCG vaccines: L Lugosi Table 4 Regranal of mean RPC (%) of BCG strains on 8 4 - 5 4 7 days after i.v. vaccination of mice followed by Anova and Gabriel's STP. Analysis of data from Table 3 Regranal BCG
Inoc. VU
a
+ s.e.a
ba
Jap Par Dak Mos Ref Con IRef Cop Mer Pra Gla Bil
297 4676 8743 9657 2377 9343 23771 5600 6040 3533 3745 6000
-54.8 - 3.0 9.9 20.3 8.1 1.6 0.1 1.0 0.9 3.5 4.2 64.0
155.3 15.8 17.4 14.0 19.6 9.2 1.2 1.1 0.7 2.0 0.9 25.8
0.591 0.069 0.061 0.051 0.043 0.O16 0.OO5 0.002 0.0004 -0.007 -0.009* -0.027
BCG in spleen at day 360
(95% confidence limit) -0.423 -0.021 -0.039 -0.031 -0.067 -0.040 -0.001 -0.005 -0.003 -0.O18 -0.014 -0.188
1.605) O.159) 0.161) 0.134 0.154 0.073 0.012 0.008 0.004 0.004 -0.004 0.135
VU 469 1022 2794 3750 564 694 481 87 62 29 38 3268
Anova: MS values
inoc. VU Linear (95% confidence limit)(%) d.f. = 3 ( 0 ( 241 (1245 (2717 ( 95 ( O ( 213 ( 27 ( 21 ( 0 ( 4 (1545
991 1803 4342 4783 1033 1626 750 146 103 98 72 4992
157.8 21.9 32.0 38.8 23.7 7.4 2.0 1.5 1.0 0.8 1 .O 54.5
Residual
1 3 7 2 7 7 182351 123 2307 2565 2571 5757* 760 2797 3355 529 675 19 11 15 10 2 4 20 34 5 7 7867 4809
d.f. 28 32 32 26 33 29 34 34 31 34 34 25
Anova for differences between the 12 regression lines
Gabriel's STP for ranked regression coefficients (b) and VU % persisting in spleen at day 360
Source
MS
F
Rank
BCG
b
1 11 11 36
20322 21066 35490 13081
1.37 1.42 2.39* 0.88
Residual
372
14878
Total
431
1 2 3 4 5 6 7 8 9 10 11 12
Jap Par Dak Mos Ref Con IRef Cop Mer Pra Gla Bil
0.591 0.069 0.O61 0.O51 0.043 O.016 0.005 O.002 0.0004 -0.007 -0.009* -0.027
d.f.
Comm. slope Parallelism Identity Linearity
*Significant at 5% level; a(slope) daily RPC increase (-decrease) in %; HS, Vertical bars indicate homogeneous subsets of Gabriel's STP
significant difference in the daily decrease between 30 000 a n d 312 000 VU × 106 ml-' values in this e x p e r i m e n t a l design. As to the VU × 106 ml -~ values, at d a y 28 the vaccines form one h o m o g e n e o u s subset.
P e r s i s t e n c e in m o u s e s p l e e n Estimation o f the mean R P C o f B C G in mice spleen, Table 3 presents the c o m p a r e d B C G strains, the i.v. i n o c u l a t e d VU from 100 x diluted vaccines a n d the 108 m e a n R P C estimates. F o r all further statistical analyses these 108 m e a n R P C values a n d their log or square root values have been used. (Altogether 216 m e a n R P C values as an average o f eight mice expressed in percent at a given a u t o p s y d a y were c o m p u t e d from 34 560 B C G c o l o n y c o u n t d e t e r m i n a t i o n s o b t a i n e d from 12 vaccines, two i.v. doses, n i n e a u t o p s y days, eight mice from each group, two mice spleen dilutions, ten seeds per d i l u t i o n on B O A A medium.)*
Trends o f the R P C in mice splee~ F o r statistical inferences two m o d e l s were used: (l) R e g r a n a l t with m e a n R P C values between day 1-21 a n d 1-42 (early RPC, i.e., m u l t i p l i c a t i o n ) as well as between days 42-547 a n d 84-547 (late RPC, i.e. persistence) after v a c c i n a t i o n was *Results of large inoculums (10 × diluted vaccines) are not presented but are available on request "~Four periods of RPC trends were analysed; early (1-21 and 1-42 days) and late (42-547 and 84-547 days) with %, log and square root RPC values of the 10 x and 100 × diluted BCG vaccines. Thus, 24 trend tables were computer printed. From these, because of limited space, only the main results of ranked b values and the related Anova of six models are summarized in Table 5, viz., the 1--42 and 84-547 days trends of 100 × diluted vaccines.
152
Vaccine, Vol. 2, June 1 9 8 4
HS
BCG
VU %
Jap Bil Mos Dak Ref Par Con IRef Cop Mer Gla Pra
157.8 54.5 38.8 32.0 23.7 21.9 7.4 2.0 1.5 1.0 1.0 0.8
HS
c o m p u t e d for each B C G strain in o r d e r to d e m o n s t r a t e the t r e n d o f m u l t i p l i c a t i o n a n d persistence capacities, then A n o v a a p p l i e d for R e g r a n a l was c o m p u t e d to c o m p a r e the regression lines o f the strain; (2) O n e - W a y - A n o v a $ with m e a n R P C was c o m p u t e d at each d a y mice were killed to m a k e cross-section c o m p a r i s o n between B C G strains d u r i n g the 547 day follow-up study. Table4 presents the results o f the Regranal, the A n o v a a n d G a b r i e l ' s STP. In this table o n l y the trends between 84 a n d 547 days o f the 100 × diluted vaccines are presented c o m p u t e d with m e a n R P C in % values. T h e u p p e r parts o f the tables show the estimated p a r a m e t e r s of the R e g r a n a l for each tested strain. T h e a values show the intercepts. T h e regression coefficients (b) express the daily increase or decrease o f the B C G VU in the spleen o f mice as a percentage of the i n o c u l a t e d VU. The e x t r a p o l a t e d B C G VU estimates at day 360 a n d their 95% c o n f i d e n c e limits show the absolute n u m b e r o f recoverable VU still persisting in the spleen. Values given in % o f i n o c u l a t e d VU express the R P C o f the B C G strains at day 360. T h e r e are strains showing R P C values u n d e r 20% h i n t i n g at a decreased RPC, i.e. residual virulence. The A n o v a shows that only B C G - M o s has significant deviation from linearity. T h e lower part o f the table shows two models. T h e A n o v a a p p l i e d for R e g r a n a l p e r f o r m e d with R P C (%) shows c o m m o n slope, p a r a l l e l i s m , identity a n d linearity of the 12 regression lines. T h e 0.88 F value o f linearity shows that the m o d e l is valid at 5% p r o b a b i l i t y level, thus this m o d e l is a c c e p t a b l e for valid inferences to c o m p a r e p a r a l l e l i s m SAltogetherwith log and square root metameters of RPC in % of 10 × and 100 × diluted vaccines six One-Way-Anova tables were computer printed. Table8 summarizes the full mean ranks of RPC (%), log and square root RPC values of the tested BCG vaccines from these six tables.
Multiple comparison of dried BCG vaccines: L Lugosi Table 5
Gabriel's STP of the ranked regression coefficients between 1-42 and 8 5 - 5 4 7 days of lOOx diluted BCG vaccines. b Values of 1-42 days; early RPC
Rank
ba BCG (%)
b HS BCG (log)
1 2 3 4 5 6 7 8 9 10 11 12
Mos Bil Par Dak Con Ref Pra Cop IRef Jap Mer Gla
Anova
d.f.
MS
Comm. b Linearity Parallel. Identity Residual
1 24 11 11 319
3630 10.71" ~903 2.66* 1764 5.20* 5974 17.62" 339
1.265" 0.936 0.701" 0.514* 0.082 -0.002 -0.034 -0.042 -0.076 =~ -0.084 -0.249~ -0.249 F
Mos Dak Con Par Bil Ref COp Jap Gla Mar Pra IRef d.f.
HS
0.022 0.019* 0.004 0.001 -0.001 -0.004 -0.011 -0.015 -0.017* -0.026-0.028 == -0.042 ~* MS
F
b Values of 8 4 - 5 4 7 days; late RPC
b BCG (square root)
HS
bb 8CG (%)
b HS BCG (log)
Mos Dak Par Bil COn Ref Cop Pra Jap IRef Gla Mar
0.0105* 0.0067* 0.0055 0.0038 0.0011 -0.0003 -0.0017 -0.0023* -0.0030* -0.0035* -0.0046* -0.0053*
Jap 0.591 Par 0.069 Dak 0.061 Mos 0.051 Ref 0.043 Con 0.016 IRef 0.005 COp 0.002 Mar 0.000 Pra -0.007 Gla -0.009* Bil -0.027
IRef Jap Ref Par Mos Dak Pra Cop Mer Bil Gla Con
d.f.
MS
d.f.
1 6.009 9.42* 1 24 1.135 1.78" 24 11 2.716 4.26* 11 11 1 7 . 7 8 2 2 7 . 8 9 " 11 319 0.638 319
F
0.017 0.60 1 0.064 2.24* 36 0.183 6.40* 11 0.915 31.99" 11 0.029 372
b HS BCG square root
0.0030* 0.0020 0.0016 0.0004 -0.0001 -0.0001 -0.0006 -0.0007 -0.0007 -0.0025 -0.0035* -0.0036*
MS
F
d.f. MS
20322 13081 21066 35490 14878
1.37 0.88 1.42 2.39*
1 36 11 11 372
F
HS
Jap Dak Par Ref Mos IRef Cop Mar Pra Con Gla Bil
0.00149 0.00044 0.00041 0.00039 0.00023 0.00022* 0.00000 -0.00002 -0.00012 -0.00017 -0.00035* -0.00044
d.f.
MS
3.807 3.32 1 2.250 1.96" 36 4.125 3.60* 11 2 4 . 4 4 3 2 1 . 3 3 * 11 1.146 372
F
0.112 0.87 0.241 1,87" 0.211 1.64 1.317 10.20" 0.129
*Significance at 5% level; a(Slope) daily increase or decrease (+ b) of BCG VU in mice spleen-expressed in % or in log or square root transformed form; bDetails see Table 4; HS, vertical bars indicate homogeneous subsets of Gabriel's STP. For Regranal, Anova and Gabriel's STP mean (%), respectively, log RPC and square root RPC values of Table 3 were used
and identity of the lines. Gabriers STP shows that the ranked regression coefficients (b) form one homogeneous subset, i.e, from 84 to 547 days after vaccination the trends of late RPC of the 12 BCG strains are parallel, although the VU % values still persisting in spleens at day 360 form two subsets. Table 5 summarizes the ranked regression coefficients of only six of the 24 models of the compared BCG strains from the computer printed trend tables +. The b values are listed according to the tested period (early. late) and to the transformations (log or square root). The following question arises in the comparison of RPC: which of the six models should be accepted for null hypothesis. The model in which the F value of Anova indicates non-significant deviation from linearity at the 5% probability level could be accepted as a valid decision in ranking the trend of RPC of the tested BCG strains. The non-significant variance ratio of linearity (F ----0.88) of ranked b% of late RPC demonstrates the case of the valid decision model. It can be seen that the tested BCG strains form one homogeneous subset. The log b of the late RPC model is invalid (Lin. F ----- !.96) and it can be seen that the log transformation of the RPC selects two additional subsets. The valid model of b% is the best example for interpreting the biological meaning of the rank position of the RPC of a particular BCG strain. One can understand that there is no fixed rank position in a test system for a biological character because the inherent variability, the environmental conditions and experimental factors may influence the RPC in the actual experiment. The vertical bars of Gabriel's STP indicate that several BCG strains could be members of the same subset when residual virulence is determined under particular experimental conditions of variables and in statistical assumptions predetermined for the test system. Altogether, there were eight valid models (four early and four late) out of 24 with immunobiologically interpretable effective VU % values still persisting on day 360. Gabriel's STP of Table4 shows one
of these valid models. Other transformations of RPC values ofthe invalid models need to be researched further. Further analysis is needed also to evaluate the associations between dose and effect, i.e., the relationships between inoculated VU and early, late or overall RPC trends depending on the residual virulence of the BCG substrains.
Multiple comparison o f the m e a n RPC at each autopsy day. Table6 presents the results of the Gabriel's STP with mean RPC after One-Way-Anova computed at each autopsy day expressi~ag in % the BCG VU persisting on the given day the mice were killed, in the spleen of i.v. vaccinated mice with 100 x diluted vaccines*. The mean RPC values are ranked for each day the mice were killed. The F values of Anova indicate significant and nonsignificant differences of the mean RPC values. H o m o geneous subsets of mean RPC of the tested strains are indicated by vertical bars. The full mean rank values express the overall mean RPC of the BCG strains. Association between ranks of m e a n R P C of the nine days mice were killed Table 7 shows the results of Kendall's test of the ranks of mean RPC (%) values taken from Table6. In this model nine independent rank orders are arranged and the extent of association of the ranked mean RPC of BCG of the nine days mice were killed can be calculated by the Kendall coefficient of concordance (W). As the significant F value of the W shows, there is agreement between the ranks of the nine dfiys mice were killed. Thus the null hypothesis of the Kendall's test, i.e., no agreement between the nine ranking sets of days mice were killed can be rejected. As in Table 6 full means indicate the overall rank order in the model. Table 8 lists these full mean ranks according to the inoculated vaccine dilutions and to the RPC transformatSee footnote to page 152
Vaccine, Vol. 2, June 1 9 8 4
153
Multiple comparison of dried BCG vaccines: L Lugosi Table 6 Gabriel's STP with mean RPC (%) taken from Table 4 after One-Way-Anova at each day a mouse was killed after i.v. vaccination for ranking and selecting the compared 100 x diluted BCG vaccines Days mice killed
1
Rank
BCG
%
1 2 3 4 5 6 7 8 9 10 11 12
Gla Bil M~r Cop Con IRef Dak Par Mos Jap Pra Ref
36.6 29.9 19.4 8.0 5.6 4.0 3.9 3.9 3.7 3.1 2.9 0.2
MS Betw./d.f. MS WithJd.f. F
8 HS BCG
I
I Con M~r ios Jap Par Cop Pra IRef Dak Ref
BCG
%
1 2 3 4 5 6 7 8 9 10 11 12
Jap Ref Dak Bil Mos Par Gla Con M~r Cop IRef Pra
19.4 39.4 22.3 17.4 15.8 4.2 1.8 0.9 0.7 0.3 0.0 0.0
MS Betw./d.f. MS WithJd.f. F
HS BCG
I
Bil Con Mos Dak Par Gla COp M~r Pra IRef Ref Jap
%
Dak Bil Par Mos Ref Jap COp M~r IRef Gla Con
5592/11 2956/82 1.89
3387/11 974/77 3.48*
HS BCG
350.1 78.0 72.8 35.1 33.7 29.3 18.8 4.4 1.7 0.0 0.0 0.0
%
HS
92.6 41.9 6.4 6.3 4.5 4.3 3.9 1.9 1,4 1.0 0.0 0.0 5832/11 474/82 12.31" Full mean rank
%
Bil Par Ref Mos Dak Cop IRef Con M~r Gla Jap Pra
75616/11 69242/73 1.09
2517/11 892/77 2.82*
Bil Mos Par Con Pra Gla Dak Cop IRef Mer Ref Jap
547
%
Jap Mos Bil Ref Dak Par Con IRef M6; Gla Cop Pra
HS BCG
64.3 56.6 31.4 24.3 22.7 12.4 6.1 3.8 1.1 0.9 0.0 0.0
450 HS BCG
84
%
Bil Mos Par ~'Gla Dak Con Mer Cop Pra IRef Ref Jap
3953.2/11 308.7/79 12.81"
56.7 39.6 22.3 12.4 5.2 3.8 2.0 1.1 0.9 0.8 0.5 0.4
Pra
HS BCG
125.8 1 5.9 12.0 10.4 9.7 7.1 6.3 5.8 1.0 0.7 O. 1 0.0
349 HS BCG
42
%
1266.1/11 67.6/84 18.74"
184
Rank
41.9 22.5 17.0 9.9 8.2 2.9 2.3 1.5 1.2 1.0 0.8 0.0
Gla
1040.5/11 39.0/79 26.71"
Days mice killed
%
Bil
I
21
HS BCG
57.9 34.5 32.7 31.9 29.1 2.9 2.4 0.9 0.4 0.1 0.0 0.0
Bil Mos Par Dak Con Gla Mer Cop Jap Ref IRef Pra
1.8 a 4.0 4.8 5.3 6.1 6.3 7.4 7.7 7.9 7.9 9.0 9.8
2062/11 2237/58 0.92
*Significant at 5% level; "See Table 7; HS, Vertical bars indicate homogeneous subsets of Gabriel's STP
Table 7 Ranks of Mean RPC (%) values taken from Table 6. Kendall's test for determining the association between nine sets of ranks (days mice killed) of the compared 1O0 x diluted BCG vaccines Days mice killed after vaccination Rank
BCG
1
8
21
42
84
184
349
450
547
1 2 3 4 5 6 7 8 9 10 11 12
Bil Mos Par Dak Con Gla M~r COp Jap Ref IRef Pra
2 9 8 7 5 1 3 4 10 12 6 11
1 5 7 11 3 2 4 8 6 12 10 9
1 3 5 4 2 6 8 7 12 11 10 9
1 2 3 5 6 4 7 8 12 11 10 9
1 2 3 7 4 6 10 8 12 11 9 5
4 5 6 3 8 7 9 10 1 2 11 12
2 4 3 1 12 11 8 7 6 5 10 9
3 2 6 5 7 10 9 11 1 4 8 12
1 4 2 5 8 10 9 6 11 3 7 12
Full means 1.8 a 4.0 4.8 5.3 6.1 6.3 7.4 7.7 7.9 7.9 9.0 9.8
Coefficient of concordance: W = 0.393 F = 5.17" d.f.1 = 11 d.f.2 = 86 *Significant at 5% level; ~See Table 6; Kendall's W values express the degree of agreement between the ranks of Mean RPC of the nine sacrifice days in ranking the 12 vaccines. Full means indicate the best estimate of the 'true' ranking of the averaged ranks of nine days mice were killed of mean RPC of BCG strains when the F value for W is significant
154
Vaccine, Vol. 2, J u n e 1 9 8 4
Multiple comparison of dried BCG vaccines: L Lugosi Table 8 Comparison of full mean ranks of RPC, log RPC and square root RPC of BCG vaccines tested in mice spleen from 1 to 547 days mice killed Rank
A o "o _= x o
RPC (%)
1 2 3 4 5 6 7 8 9 10 11 12
"o := -o x o 0
1 2 3 4 5 6 7 8 9 10 11 12
Square root RPC
BCG
Full mean ranks BCG
Full mean ranks BCG
Full mean ranks
Bil Mos Jap Con Gla Par Dak Ref Mer IRef Cop Pra
3.8 3.8 4.6 5.4 5.9 6.1 6.8 7.3 7.8 7.8 8.5 10.5
4.0 4.3 4.6 5.3 5.8 6.3 6.5 7.0 7.9 8.0 8.4 10.1
3.8 3.9 4.9 5.4 5.6 6.3 6.8 7.1 7.8 7.9 8.4 10.4
W F dfl df2 ~" ® ~" '~
Log RPC
Bil Mos Par Dak Con Gla M6r Cop Jap Ref IRef Pra W F dfl df2
Bil Mos Jap Gla Con Dak Par IRef Mer Ref Cop Pra
0.309 3.13* 11 75 1.8 t 4.0 4.8 5.3 6.1 6.3 7.4 7.7 7.9 7.9 9.0 9.8 0.393 5.17* 11 86
Bil Mos Jap Con Gla Par Dak Ref IRef Mrr Cop Pra
0.266 2.54* 11 75 Bil Mos Dak Gla Par Jap Con Mrr Cop Ref Pra I Ref
1.9 3.3 4.0 5.7 5.7 6.3 7.1 7.2 8.1 9.1 9.8 9.8 0.495 7.83* 11 86
0.291 2.87* 11 75 Bil Mos Dak Par Con Gla Mrr Cop Ref Jap Iref Pra
1.9 3.6 4.2 5.7 6.1 6.2 7.2 7.4 8.2 8.7 8.8 10.0 0.437 6.22* 11 86
*Significant at 5% level. Kendall's W express the degree of agreement between the ranks of Mean RPC of the nine days mice killed in ranking the 12 vaccines. Full means, taken from Table 6a and computer printed tables, indicate the best estimate of the "true' ranking of the 1 - 5 4 7 days averaged ranks of mean RPC of BCG strains when the F values for W are significant
tions. Significant F ratios of W values show that there is agreement between ranks in all six models in spite of the small fluctuation in rank position of the full means of the BCG strains. This could be due to the random variability occurring in this biological test system. Thus, the full mean ranks could represent the global BCG-RPC for the total observation period-
Discussion immunogenicity and reactogenicity Of the BCG vaccines are determined by two characters: the viability of the vaccine and the residual virulence of the strain used in preparation. The exact measure of these two characters allows analysis of the association between the laboratory control data and the post-vaccination host response (protective efficacy, immune and hypersensitivity reactions, frequency of side effects). Therefore, it is a prerequisite to test the dose-response relationship and to use sensitive laboratory techniques with exact staE~tical methods in order to demonstrate significant differences in the in vitro potency and in the in vivo residual virulence of the BCG strains expressed in VU × 10~ ml -: and in mean RPC in host organ(s). Biological products are controlled by standardized methods in valid bioassay models, but the BCG produc-
tion still lacks uniform statistical quality controlrequirements '~-17. We have studied: (1) the comparison of the stability of the viability at 37°C and the RPC in mice spleen of BCG vaccines and (2) the application of exact methods for statistical inference (unbiased parameter estimation, valid hypothesis tests, Gabriel's STP for multiple comparison of the ranked parameters) in order to achieve objective interpretation in laboratory control. Data show that the mean VU × 106 ml -~ values of the intradermal BCG vaccines of various substrains vary between 1.0 and 27.0 × 106 ml -~ at day 0 (Table 2) but 50-fold difference is also indicated ~3. The pragmatic question is: why should the immune system be overloaded with high VU when satisfactory dose-response can be obtained by giving 100 000-300 000 VU with BCG strain(s) of normal residual virulence ~s,~9. O f course, taking into account the residual virulence of the strains, sensitive and exact control methods are required to demonstrate small (1.0-3.0 × 10~ ml "~) VU differences. Evaluation of the stability at 37°C by Regranal, Anova and Gabriel's STP show that the VU x 106 ml -~ values of the compared vaccines (Table 2) decrease paraUely but at day 28 only six vaccines have, in valid model, the expected 20% survival rate t3. In contrast to previously published data concerning the RPC in mice spleen using short-term investigations 2s-2~, the experimental design presented is a long-term model which can be used to control the early and later RPC and to evaluate exactly the in vivo persistence of the living, metabolizing and multiplying BCG as conditions of acquired resistance and delayed hypersensitivityz3. As has been shown z*, the mean RPC estimates indicated that depending on the VU doses and on killing mice after vaccination: (1) with large i.v. VU dose (magnitude of 10 000) all strains show RPC under 20% during the whole follow-up (1-547 day) period; (2) with small i.v. VU dose (magnitude of 1000): (i) some strains have a progressive mutiplication then a persistence during the whole period; (ii) some strains have only an early multiplication capacity, (iii) some strains have only a late persistence capacity after the vaccination. The Regranal of the late RPC indicates (Table 4) that the trends are parallel from 84 to 547 days but there are only six BCG strains whose inoculated VU persist over 20% in the spleen at day 360 in a valid model. Ranking the mean RPC % according to the days mice were killed (Table 6): (i) Gabriel's STP shows the homogeneous subsets of the mean RPC values, (ii) the ranks of the BCG products from which the full mean ranks of the overall multiplication and persistence capacity can be computed- If Kendall's test is significant and full mean ranks are under 6.0 overall mean RPC of BCG can be considered appropriate (Tables 7 and 8). The second objective of the study was to introduce the Gabriel's STP for multiple comparison of ranked parameters of the stability of viability and persistence capacity of the BCG vaccines in mice spleen. In contrast to the Student test, which while performing pairwise comparison between experimental groups increases the risk of type I error, Gabriel's STP reduces the risk of type I error in inferences and prevents rejection of the null hypothesis when it is true, i.e., to state that there is a difference between the estimated parameters of groups (vaccines) whereas in fact such a difference does not exist. As a transitive t multiple comparison method, Gabriel's STP t If a subset (S) of ranked parametersof the comparedvaccinesis judged significant, everysubset that contains S will also be judged significant; conversely, if a S is not significant, no further subsets of S will be significant.
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maintains the risk of type I error at a 5% experimental (study) level, it is easy to compute and can be used for parameters estimated from unequal sample sizes "s,'6. The models in Table 5 show Gabriel's test to be very selective. The selection of homogeneous subsets of BCG strains having the same trends (b values) of mean RPC values depends on the validity (linearity) of the Anova model. Control of the viability and the residual virulence of the BCG strains can be more exact and the interpretation more objective when multiple comparison and additional concordance tests are used. These methods can help with the choice of BCG strain when preparing an effective vaccine with the lowest possible frequency of adverse reactions "7-29, and can help with standardization of the vaccineaa,n, they can also explain the differing efficacies of certain BCG vaccines 3~.
Acknowledgements I would like to thank Mr P. Csaki and Mr G. Tusnady for their advice and help in the data processing and statistical analyses. The technical assistance of Mrs M. Drinoczy and Miss A. N~meth is gratefully acknowledged.
8
9 10 11 12 13
14 15 16 17 18
19
Note Details of statistical analysis, formulae of the BCG VU × 106 ml-' and mean RPC point and 95% confidence interval estimation, computer programs for Anova, Regranal and Gabriel's STP are available On request.
20
21
References 1
2
3
4 5 6 7
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