Sources of variation in studies of the early bactericidal activity of antituberculosis drugs

Frik Sirgela, Amour Ventera and Denis Mitchisonb,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The early bactericidal activity (EBA) of antituberculosis drugs can be measured as the daily fall in cfu counts of Mycobacterium tuberculosis in sputum, usually during the first 2 days of treatment. In studies of low potency drugs, it is necessary to compare the treated group of patients with a group who receives no chemotherapy (Nil group). Over the past 10 years, five Nil groups of between five and 13 patients have been studied in Cape Town and two Nil groups in Hong Kong. There was a suggestion of an increase in variation within the Cape Town groups, as shown by a regression of variance size against study date (P = 0.06), which could not be attributed to technical causes. It might indicate increasing host resistance in the Western Cape epidemic of tuberculosis. Since the weighted mean of all Nil groups at Cape Town was 0.00036, very close to zero, it would seem safe to test means of treated groups against zero thus increasing precision and avoiding ethical problems in delaying treatment. In contrast to Nil groups, the variation found in five groups who received 300 mg isoniazid daily (INH 300) was uniform and homoscedastic, possibly because the additional variation was caused mainly by individual differences in plasma isoniazid concentrations and patient body weights. The mean EBA in the INH 300 series was 0.575 with 95% confidence limits of 0.515 and 0.636.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The early bactericidal activity (EBA) of an antituberculosis drug measures its ability to kill rapidly multiplying bacilli present in the cavitating lesions of patients with pulmonary tuberculosis. It is determined by measuring the rate at which colony forming units (cfu) of Mycobacterium tuberculosis in sputum decrease during the first 2 days of treatment with the drug in question.1,2 In a series of studies at the University of Stellenbosch, Cape Town, two types of control group have been included in some investigations. These are: a low control group consisting of patients admitted to hospital but not treated with any drug known or likely to have antituberculosis activity during the 2 days of the study (Nil group); and a high control group consisting of patients given 300 mg isoniazid daily (INH 300). The importance of the Nil groups arises when a drug of low potency is explored. Its EBA must be compared with the EBA of a Nil group. The inclusion of Nil groups poses ethical problems and also increases the number of patients in each study. Furthermore, the precision of the comparison is limited by the number of patients in the Nil group as well as in the treated group. The design and analysis of studies incorporating one or only a few treatment groups would be improved if the mean of the treated groups could be tested against a single known value for Nil groups. The bacillary content of patients who are taken into hospital but receive no chemotherapy for 2 days might be expected to be unchanged so that the EBA is then zero. This theoretical value has been tested against actual estimates in successive studies of Nil groups at Cape Town.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Studies

The seven studies at Cape Town were carried out over the period 1992–1999. 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 IGo) and INH 300 groups in another five studies (Table IIGo), several of which overlapped with those in Table IGo. 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 1990–1991 and 1995–1998, respectively. Studies 5 and 9 (Table IGo) 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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Table I. Studies of EBA in untreated patients EBA
 

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Table II. Studies of EBA in patients treated with 300 mg isoniazid daily
 
Estimation of the EBA

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 = {Sigma}wiYi/{Sigma}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 {chi}2 = {Sigma}wiYi2 –({Sigma}wiYi)2/{Sigma}wi where k = number of groups. If homogeneous, the 95% confidence limits (95% CL) Ym ±= 1.96 (1/{Sigma}Vi), Shapiro–Wilk 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 {sigma}4/ (n – 1) where {sigma} 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The EBA results obtained in the five Nil groups at Cape Town are set out in Table IGo. A Bartlett's test carried out over all five studies gave {chi}2 = 38.3, 4 DF, indicating a highly significant (P < 0.00001) degree of heterogeneity of variance (heteroscedasticity) between the groups. The mean of the five studies was therefore calculated using weightings for the differences in intra-group standard s.e.m.s, as 0.00036 with 95% confidence limits from 0.0222 to –0.0215. The standard deviation (s.d.) within each group increased from 0.013 in study 1, performed in 1992, to 0.099 for the combined results of studies in studies 6 and 7, performed in 1996–1999. A regression, weighted by the reciprocals of the variances of the variances, was then calculated, relating the variances in the five groups to the mean day between the start and end of the study. This regression was just not significant (F = 8.70, 1,3 DF; P = 0.060). However, the estimate of the variance of a variance is affected by departures from normality11 and the Shapiro–Wilk test for departures from normality in study 4, but in no other study, was significant (P = 0.033). The regression without weighting for the size of the variance was clearly not significant (P = 0.3). The s.d.s (0.31 and 0.39) for the Nil groups in the two Hong Kong studies are considerably greater than any found at Cape Town.

In the five INH groups at Cape Town (Table IIGo) 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 {cong} 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 IIIGo. 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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Table III. Values of the EBAs obtained with isoniazid 300 or 18.5 mg, rifampicin 600 mg or ofloxacin 800 mg administered daily in study 5 at Cape Town
 
In seeking a reason for the different s.d.s in Nil groups in Cape Town and Hong Kong, a retrospective analysis of the results of clinical trials using the same treatment regimens in East Africa and Hong Kong is set out in Table IVGo. In Hong Kong, the patients had less cavitation and less extensive disease than in East Africa. Sputum conversion to culture negativity also occurred more rapidly in Hong Kong. However, despite the smaller amount of disease and the more rapid conversion, the relapse rate was higher in the Hong Kong patients in all three studies. Thus sterilization of the lesions by the chemotherapy occurred more slowly in Hong Kong.


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Table IV. Response to the same treatment regimens in East Africa and Hong Kong
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The first issue concerns the testing of EBAs in a group of treated patients against zero. The value of zero in untreated patients is to be expected as the mean cfu count in sputum should not alter during the 2 days of the EBA test. This is a reasonable expectation for a chronic disease, which usually progresses slowly. The finding that the weighted mean of the Nil group was estimated as the low value of 0.00036 supports the conclusion that the test of the mean of the treated group should be against zero, though it cannot prove the point. When there is more than one dosage group, a regression analysis comparing the means in the groups with a Nil group may seem appropriate. However, the finding of heteroscedasticity in the Nil groups at Cape Town and the larger variance found within the INH 300 and other treated groups makes it inadvisable to use least squares methods for comparing treated and control groups unless these are weighted for the different s.e.m.s. Furthermore, the need to choose particular Nil groups for the comparison may lead to bias and render the calculation suspect. A less controversial procedure would be to avoid carrying out Nil groups altogether and to test the mean of each dosage group against zero.

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 IVGo), 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 IIIGo). 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.


    Notes
 
* Corresponding author. Tel: +44-20-8725-5704; Fax: +44-20-8672-0234; E-mail: dmitchis{at}sghms.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 21 March 2000; returned 4 August 2000; revised 18 October 2000; accepted 3 November 2000