a National Tuberculosis Research Programme, Medical Research Council, PO Box 19070, Tygerberg, 7505 South Africa; b Department of Medical Microbiology, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK
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Abstract |
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Introduction |
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The second aim was to explore the variation encountered within and between the Nil groups and treated groups such as those given 300 mg isoniazid daily. This should throw light on the relative sizes of various sources of variation in EBA studies and give leads on acceptable statistical analysis. Furthermore, the reason for the increasing variation found in the Nil groups studied at Cape Town since 1992 was sought. Results from Nil groups in two studies performed in Hong Kong were also included since their variances differed substantially from those in Cape Town and helped determine the reasons for variation in Nil groups.
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Materials and methods |
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The seven studies at Cape Town were carried out over the period 19921999. The results of study 1,3 study 2,4 study 3,5 study 46 and study 5,7 have been published but studies 6 and 7 are unpublished. Nil groups were included in five of these studies (Table I) and INH 300 groups in another five studies (Table II
), several of which overlapped with those in Table I
. In the INH 300 group in study 2,4 the isoniazid was given in a combined preparation with rifampicin and pyrazinamide (Rifater; Aventis). It is included since evidence is available that other drugs given with isoniazid do not increase the exceptionally high EBA found with isoniazid alone.1,4 All patients were of mixed race living in the Western Cape district where there is a serious epidemic of tuberculosis. They were admitted for the study into Tygerberg Hospital, Cape Town or Brooklyn Hospital for Chest Diseases, South Africa. Studies 88 and 97 were carried out on Chinese patients resident in Hong Kong over the periods 19901991 and 19951998, respectively. Studies 5 and 9 (Table I
) were two parts of a multi-centre study carried out under the same protocol in Cape Town and Hong Kong. Ethical permission for all studies was obtained from local ethics committees and the patients gave informed consent.
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Details of the procedures have been given in reports of the studies. In all, a 16 h collection of sputum was made on the night of the first day (S1 collection). Soon after the completion of this collection the first dose of 300 mg isoniazid (or of other drugs studied) was given in the INH 300 groups, and daily dosage was continued for another day. On the night of the second day, a further 16 h sputum collection was made (S3 collection). Immediately after the end of the study period, the patient was given standard multi-drug chemotherapy. No drug resistance arose as a result of the monotherapy. The same sputum collections were made in the Nil group but the patients received no therapy with antituberculosis drugs. Sputum collections in the Nil groups were often examined for the presence of isoniazid. If it was found, their results were excluded, as were those from patients whose sputum contained insufficient M. tuberculosis or whose cultures were resistant to isoniazid, or were contaminated, or (very occasionally) when colonies were too small to count.
Sputum was sent by air courier from Cape Town to Pretoria in the first four studies, but in later studies, the bacteriological examination was done at Cape Town with the same equipment and by the same two workers (F.S. and A.V.). The sputum collections were homogenized by shaking with glass beads or, in later studies, by stirring with a magnetic bar. Samples were then digested with dithiothreitol and plated from serial 10-fold dilutions on to 7H11 medium made selective with the addition of selectatabs (Mast Laboratories, Bootle, UK) so that final concentrations in the medium were 200 U/mL polymyxin B, 100 mg/L carbenicillin, 20 mg/L trimethoprim lactate and 10 mg/L or 100 mg/L amphotericin B. Colony counts were read after incubation at 37°C for 3 weeks. Dilutions were inoculated on to 7H10 selective slopes in the first study, but on to 7H11 plates thereafter. In the later studies, the dilutions were made in five-fold steps over the most used range in order to increase the precision of the counts. Very similar techniques were used in Hong Kong.
Statistical analysis
The numbers of organisms in the S1, S3 and S6 collections were expressed as log10 cfu/mL of sputum. The standard EBA was defined as the fall in log counts/mL sputum/day (S1 log cfu/mL S3 log cfu/mL)/2. The values of the EBA were examined by ANOVA, and Bartlett's test in EPI INFO version 6.0d (Centers for Disease Control, Atlanta, GA, USA). The mean EBA in the Nil studies, weighted for the considerable differences in the standard errors of the mean (S.E.M.) in the treatment groups, was calculated as Ym = wiYi/
wi where wi = ni/Vi and ni, Yi and Vi are numbers of patients in a treatment group, its mean and its variance, respectively.9 Homogeneity of the means was tested with k 2 degrees of freedom (df) as
2 =
wiYi2 (
wiYi)2/
wi where k = number of groups. If homogeneous, the 95% confidence limits (95% CL) Ym ±= 1.96 (1/
Vi), ShapiroWilk tests10 for normality of the distributions of the EBAs and weighted regression of the group variances on mean day of the study were carried out with STATA version 6.0 (College Station, TX, USA). The analytical weights were the reciprocals of the variance of the variance = 2
4/ (n 1) where
is the estimated standard deviation (s.d.) and n is the number of patients in the group.11 A similar regression analysis weighted only for the numbers of patients in each group was also carried out.
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Results |
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In the five INH groups at Cape Town (Table II) the EBAs of all groups were normally distributed. However, they differed from the Nil group EBAs in that their variances appeared homogeneous (homoscedastic); Bartlett's test between the five groups was non-significant. The analysis of variance demonstrated no significant differences between their means; the mean squares were 0.044 between groups (4 DF) and 0.046 within groups (45 DF). The overall mean was 0.575 with 95% confidence limits from 0.636 to 0.515. The s.d. of the EBA values considered as a single series was 0.214. The within-groups variance (0.0458) was 4.7 greater than the variance (0.009801) for the combined results of the Nil groups in studies 6 and 7 (F = 4.9, 49, 14 DF, P
0.001). The s.d.s of the INH 300 groups in Hong Kong were higher than those in Cape Town.
The results of standard EBAs in study 5 are summarized in Table III. The s.d.s of the EBAs are similar for the four daily regimens given, which were 18.5 mg isoniazid (INH 18.5), 600 mg rifampicin (R 600), 800 mg ofloxacin (O 800), as well as INH 300. The pooled s.d. between patients within groups (the residual of the analysis of variance) was 0.17. Thus, high s.d.s were found not only in the INH 300 series but in groups receiving other antituberculosis treatment.
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Discussion |
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We then have to consider why the variation in the EBAs of the Nil groups was so much greater in Hong Kong than Cape Town. We also need to account for the suggestion of an increase in the s.d. of the Nil EBAs at Cape Town from 0.013 to 0.099 between 1992 and 1999. The main cause of variation in the Nil EBAs would seem to be the sampling of sputum during the 16 h collections. If lesions in the lungs liquefy readily and cavities interconnect, as in acute disease, there would be little variation in cfu count between the S1 and the S3 collections. However, if fibrosis occurred readily round cavities, as in chronic disease, so that they did not easily interconnect, it would be more difficult to expectorate large volumes of sputum and a greater degree of variation between the two collections would be expected. Thus, a possible explanation for the difference between the Cape Town and the Hong Kong Nil group results is that the disease in Western Cape province is more acute than the disease in Hong Kong. More chronic disease in Hong Kong than in East Africa could also account for less rapid sterilizing activity in the lesions of Hong Kong patients in clinical trials (Table IV), since the bacilli would be growing more slowly and therefore be killed less rapidly than in acute pneumonic lesions. The Cape Town studies might have been carried out during a period in the evolution of a serious epidemic of tuberculosis in the Western Cape,16 starting in a very susceptible population, with little variation in their EBAs. The epidemic may have continued with the gradual evolution of higher degrees of immunity and more variable EBAs, as the most susceptible members of the population were eliminated by successful treatment or death.17,18 Should further studies support this contention, we may have a method of estimating herd immunity which is easy to carry out and can provide absolute estimates, capable of being transported between countries.
The validity of this proposition certainly requires further study. Could the findings be due to variations in laboratory techniques? Considering the Cape Town data, the laboratory procedures were done by the same workers and were steadily improved over the 7 year interval between studies 1 and 7. Furthermore, the transit time for the sputa was decreased when the laboratory moved nearer to the patients. Improvements in the clinical handling of the patients also improved. For instance, detection of isoniazid in sputum, which accounted for exclusion of some 10% of patients in later studies, was not carried out in the earliest studies and was gradually introduced later. Improvements in the clinical and laboratory techniques would result in a decrease in variance as time progressed rather than an increase. Furthermore, no corresponding increase occurred in the INH 300 groups during the period. Thus the changes in the Nil group variance are unlikely to be due to techniques carried out after collection of sputum. Laboratory error was also unlikely to have accounted for the high s.d.s encountered in the Hong Kong Nil groups, since blind duplicate readings showed that laboratory variation was too small to account for differences between patients in study 8.8 In the multi-centre study 9, the highest correlation between total acid-fast bacilli and cfu counts was obtained in Hong Kong, indicating the accuracy of the laboratory measurements.7
Early reviews, written at a time when it was possible to watch the development of untreated pulmonary tuberculosis, have suggested that the disease was more acute in African populations than in other populations.19,20 Since observing the progress of untreated disease is now not permissible, these observations cannot be confirmed. Measurements of the acuteness or chronicity of disease at one point in its evolution are difficult to obtain. Patients tend to come to clinics when their disease reaches a certain severity, as suggested in an analogous study in which the pretreatment clinical characteristics of Madras patients failed to relate to the guinea pig virulence of their strains of M. tuberculosis.21 Most patients are vague in their assessments of the duration of symptoms and radiographic estimates of chronicity of disease in single radiographs of cavitary disease are unreliable. No attempt has been made in the present report to see whether any characteristic of the patients is related to the EBA or might influence its variability since, in previous reports, no association was found between the EBA and age, sex, weight, severity of disease or extent of cavitation.2 As the production of purulent sputum might be expected to be more difficult in chronic disease, dilution of sputum with saliva would also be expected more often. The number of patients, the average sputum volume and its s.d. in the Nil groups in study 5 were 7 mL, 30.6 mL and 48 mL for Cape Town and 12 mL, 12 mL and 7 mL for Hong Kong, respectively. The dilution of sputum by saliva was estimated in study 5 since total counts of acid-fast bacilli were done as well as cfu counts. With dilution, these would be correlated. The correlation coefficient, r, relating cfu EBAs to total EBAs was 0.01 (37 DF) for Cape Town and 0.76 (52 DF) for Hong Kong, indicating that sputum dilution was a common phenomenon in Hong Kong but rare in Cape Town. Thus estimates of sputum volumes and of sputum dilution both support the concept of more acute disease at Cape Town.
When treatment was given, an additional large source of variation between patients, above that found in the Nil groups, was present in the Cape Town data. This variation was similar irrespective of drug or dosage (Table III). The additional variation might be due to differences in the bactericidal action of drugs on the bacterial populations of patients. A source of possible importance is the large variation in the individual plasma drug concentrations following a single dose: this occurs with most drugs, whether given by mouth or by im injection. As only two doses were given during the standard EBA treatment period, this variation would not have been entirely evened out.
It is of interest that, unlike the findings in Cape Town, the s.d.s of Nil groups and INH 300 groups in Hong Kong patients were similar at 0.31 and 0.37, respectively, in study 8 and were identical at 0.39 in study 9. These similarities suggest that the variation in Hong Kong patients is dominated by variation in the sampling of lesions in the lungs so that the effect of drugs is more difficult to estimate than at Cape Town. It is probably for this reason that estimates of drug action by the EBA technique have not been satisfactory in Hong Kong. Indeed if Nil group s.d.s are appreciably greater than 0.15 in any patient population, it seems unlikely that the EBA method will be effective in measuring differences between drugs or dosages.
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Notes |
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References |
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2 . Mitchison, D. A. & Sturm, W. A. (1997). The measurement of early bactericidal activity. In Bailliere's Clinical Infectious Diseases: Mycobacterial Diseases Part II, (Malin, A. & McAdam, K. P. W. J., Eds), pp. 185206. Bailliere Tindall, London.
3 . Sirgel, F. A., Botha, F. J., Parkin, D. P., Van de Wal, B. W., Donald, P. R., Clark, P. K. et al. (1993). The early bactericidal activity of rifabutin in patients with pulmonary tuberculosis measured by sputum viable counts. A new method of drug assessment. Journal of Antimicrobial Chemotherapy 32, 86775.[Abstract]
4 . Botha, F. J. H., Sirgel, F. A., Parkin, D. P., Van de Wal, B. W., Donald, P. R. & Mitchison, D. A. (1996). Early bactericidal activity of ethambutol, pyrazinamide and the fixed combination of isoniazid, rifampicin and pyrazinamide (Rifater) in patients with pulmonary tuberculosis. South African Medical Journal 86, 1558.[ISI][Medline]
5
.
Donald, P. R., Sirgel, F. A., Botha, F. J., Seifart, H. I., Parkin, D. P., Vandenplas, M. L. et al. (1997). The early bactericidal activity of isoniazid related to its dose size in pulmonary tuberculosis. American Journal of Respiratory and Critical Care Medicine 156, 895900.
6
.
Sirgel, F. A., Botha, F. J., Parkin, D. P., Van de Wal, B. W., Schall, R., Donald, P. R. et al. (1997). The early bactericidal activity of ciprofloxacin in patients with pulmonary tuberculosis. American Journal of Respiratory and Critical Care Medicine 156, 9015.
7
.
Sirgel, F. A., Donald, P. R., Odhiambo, J., Githui, W., Umapathy, K. C., Paramasivan, C. N. et al. (2000). A multi-centre study of the early bactericidal activity of antituberculosis drugs. Journal of Antimicrobial Chemotherapy 45, 85970.
8 . Chan, S. L., Yew, W. W., Ma, W. K., Girling, D. J., Aber, V. R., Felmingham, D. et al. (1992). The early bactericidal activity of rifabutin measured by sputum viable counts in Hong Kong patients with pulmonary tuberculosis. Tuberculosis and Lung Disease 73, 338.
9 . Armitage, P. & Berry, G. (1987). Statistical Methods in Medical Research, 2nd edn, pp. 1946. Blackwell Scientific, Oxford.
10 . Royston, P. (1991). Estimating departure from normality. Statistics in Medicine 10, 128393.[ISI][Medline]
11 . Armitage, P. (1971). Statistical Methods in Medical Research, p. 93. Blackwell Scientific Publications, Oxford.
12 . East African/British Medical Research Councils. (1974). Controlled clinical trial of four short-course (6-month) regimens of chemotherapy for treatment of pulmonary tuberculosis. Third report. Lancet 2, 23740.[Medline]
13 . East African/British Medical Research Councils. (1976). Controlled clinical trial of four 6-month regimens of chemotherapy for pulmonary tuberculosis. Second report. American Review of Respiratory Disease 114, 4715.[ISI][Medline]
14 . Hong Kong Chest Service/British Medical Research Council. (1979). Controlled trial of 6-month and 8-month regimens in the treatment of pulmonary tuberculosis. The results up to 24 months. Tubercle 60, 20110.[ISI][Medline]
15 . Hong Kong Chest Service/British Medical Research Council. (1977). Controlled trial of 6-month and 9-month regimens of daily and intermittent streptomycin plus isoniazid plus pyrazinamide for pulmonary tuberculosis in Hong Kong. The results up to 30 months. American Review of Respiratory Disease 115, 72735.[ISI][Medline]
16 . Donald, P. R. (1998). The epidemiology of tuberculosis in South Africa. In Genetics and Tuberculosis. Novartis Foundation Symposium 217, (Cardew, G., Ed.), pp. 2441. Wiley, Chichester.
17 . Grigg, E. R. N. (1958). The arcana of tuberculosis with a brief epidemiologic history of the disease in the USA. American Review of Tuberculosis and Pulmonary Disease 78, 15172, 42653, 583603.
18 . Bates, J. H. & Stead, W. W. (1993). The history of tuberculosis as a global epidemic. Medical Clinics of North America 77, 120517.[ISI][Medline]
19 . Rich, A. R. (1946). The Pathogenesis of Tuberculosis, p. 131. Charles C. Thomas, Springfield, IL.
20 . Hutton, P. W., Lutalo, Y. K., Williams, A. W., Tonkin, I. M. & Fox, W. (1956). Acute pulmonary tuberculosis in East Africans: a controlled trial of isoniazid in combination with streptomycin or PAS. Tubercle 37, 15165.[Medline]
21 . Ramakrishnan, C. V., Bhatia, A. L., Fox, W., Mitchison, D. A., Radhakrishna, S., Selkon, J. B. et al. (1961). The virulence in the guinea-pig of tubercle bacilli isolated before treatment from South Indian patients with pulmonary tuberculosis. 3. Virulence related to pretreatment status of disease and to response to chemotherapy. Bulletin of the World Health Organization 25, 32338.[ISI][Medline]
Received 21 March 2000; returned 4 August 2000; revised 18 October 2000; accepted 3 November 2000