Journal of Antimicrobial Chemotherapy (1977) 3, 57-63
Microbiological assay of ethambutol
P. R. J. Gangadharam and E. R. Candler
Microbiological assay for ethambutol has been standardized using Mycobacterium smegmatis ATCC 607 as the assay organism. The optimal conditions for the assay including the age and size of the inoculum, volume and pH of the medium and time of reading were standardized. Few essential criteria like the nature of the strain, and the medium were also indicated. Introduction Ethambutol is widely used for the treatment of mycobacterial diseases. In the routine management of patients with this drug, as is the case with other drugs, it is necessary to have information on the levels attained in serum and other biological fluids not only to be able to correlate with clinical response but also as a possible warning of the onset of toxicity. Furthermore, a series of recent findings from our laboratories (Gangadharam & Gonzales, 1970) have shown marked influence of certain commonly used agar media (e.g. 7H10) on the in vitro activity of this drug on Mycobacterium tuberculosis. Measurements of ethambutol levels were also done under several experimental conditions in order to understand the reason for this phenomenon (Gangadharam & Candler, 1974, 1975). Chemical methods of assay of ethambutol are tedious, time consuming and are not sufficiently sensitive. A microbiological method for the assay of this drug using Mycobacterium smegmatis ATCC 607 was mentioned in the reports by Place & Thomas (1963) and by Place, Peets, Buyske & Little (1966) though full description of the procedure was only contained in the brochure on this drug issued by the manufacturer (Medical Advisory Department, Lederle Laboratories, 1967). Satisfactory results were not obtained in our laboratories, in spite of carefully following their procedure. We have therefore reconsidered the microbiological assay of ethambutol, using the initial suggestion from the work of the Lederle group of investigators. In this process, consideration was also given to investigate the value of a close variant of the assay organism, M. smegmatis 607B (which is widely used for phage studies on mycobacteria) and the agar media (7H10 and 7H11) which are widely used for the cultivation of mycobacteria. This paper presents the results on the standardization of the microbiological assay for ethambutol, denning the optimal conditions. 57
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
Mycobacteriology Laboratories, Jefferson Davis Hospital and Department of Pathology, Baylor College of Medicine, Houston, Texas 77030, U.S.A.
58
P. R. J. Gangadharam and E. R. Candler Materials and methods
Test organisms Mycobacterium smegmatis ATCC 607 was obtained from the American Type Culture Collection. A variant of this organism, M. smegmatis 607B, was obtained from Dr W. B. Redmond, Atlanta, Georgia. Assay broth This consisted of: dextrose 100 mg; beef-extract 5-2 g; peptone 6-2g; yeast extract 2-0 g; sodium chloride 2-4 g; Tween 80 140 ml. Final volume to 1000 ml pH 70±0-2 before autoclaving. The medium was sterilized by autoclaving for 15 min at 121°C.
7H10 and 7H11 agar medium Middlebrook and Cohn's 7H10 agar medium and Cohn's 7H11 agar medium were prepared from dehydrated powders and sterile Oleic Acid-Albumin Dextrose Complex (OADC) enrichments. Seeding with the assay organism was made before pouring the plates, as was done in the case of Sauton medium. Paper discs
Standard discs of 5 mm diameter, obtained from Schleicher and Schuell, Inc., Keene, N.H. 03431 were used. They were sterilized by autoclaving for 15 min at 121°C. Bio-assay plates The stainless steel Bio-assay plates, along with the special forceps to be used in the cupplate method were obtained from Lab Line Instruments, Inc. The plates were sterilized by autoclaving at 121 °C for 15 min. Method of preparing the innoculum Growth of M. smegmatis ATCC 607 from Lowenstein-Jensen medium was transferred to a sterile tube containing 1 -0 ml of assay broth and 4 glass beads. A uniform suspension was made using a vortex mixer and an aliquot incubated at 37°C for 24 h. From this, another 24 h old subculture of this growth was made as above and used as the inoculum, and calculated amounts were transferred to flasks containing 100 ml of Sauton medium before pouring into Petri dishes. The same procedure was adopted for obtaining the inoculum of M. smegmatis 6O7B. Method of microbiological assay ofethambutol Paper disc method. Aliquots of 20 ul of the test sera or standards, at least in duplicate, were transferred to paper discs which were placed on the seeded Sauton's agar plates. With each batch of assays, a set of standards in duplicate was also set up in the same
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
Modified Sauton's medium used for assay plates This consisted of: iron ammonium citrate 0-05 g; dipotassium hydrogen phosphate 0-50g; magnesium sulphate anhydrous 0-50 g; citric acid 200g; L-asparagine 400g; Bacto agar (Difco) 2000g; glycerol 4000ml; Tween 80 20 ml; distilled water Q.S. to 1000 ml; pH7-0±0-2 before autoclaving. The medium was sterilized at 12PC for 15 min and when cooled to 45°C the inoculum was added, mixed by gentle shaking and 20 ml was poured into sterile disposable (15 mm) plastic Petri dishes. After solidification, the plates were stored at 4°C until used.
Microbiological assay of ethambutanol
59
Results Optimum age and size of the inoculum Using inocula ranging from 1 to 5 days age, it was observed that 1-day-old inoculum was sufficient to give a good assay. Similarly, a volume of 1-5 ml/100 ml of the Sauton medium was found to be optimal among several volumes tested. Optimal volume of test material to be applied Assays carried out using several volumes, with both the methods revealed that 20 ul and 0-3 ml were optimal for the paper and cup-plate methods, respectively. Optimal amount of media to be used Using several volumes of the medium ranging from 5 0 ml to 300 ml, it was found that 200 ml was optimal to be used in a standard (15 mm) Petri dish. Optimal pH of the medium Sauton medium was prepared with the final pH ranging from 6 0 to 80, with increases of 0-2 units in between: Microbiological assays were carried out in duplicate both by the cup-plate and the paper-disc methods. Medium with pH of 6-8 gave the largest zone sizes. Optimum time of reading the results The results obtained indicated that an optimum time of reading is at 40 h of incubation. Effect of prior diffusion on the zone size It was found that a prior period of diffusion for 3 h of the drug results in increased zone sizes. Usefulness of seeded plates after storage Microbiological assays carried out using seeded plates stored at 4°C for 1, 2, 3 or 4 weeks gave very similar results to those obtained using fresh plates. Consistency of the zone sizes with the paper-disc and cup-plate methods Zone sizes (in mm) along with the mean and standard deviation in sets of five assays, using known concentrations of ethambutol, both with the paper-disc and cup-plate methods, showed highly consistant values (Table I).
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
manner. The concentrations used for the standards using the paper disc method were 0, 5, 10, 20, 40, 80 ug/ml. The plates were then allowed to stand on the laboratory bench at room temperature (28°C±2°C) for 3 h, after which time they were transferred to an incubator (37°C). The zones of inhibition were read after 40 h of incubation at 37°C. Cup-plate method. The solutions to be assayed were transferred to the individual bioassay plates positioned on agar plates. The concentrations used for the standards were 0 0 , 0-4, 0-8, 1-6, 3-2 and 6-4 ug/ml of ethambol. Unless otherwise indicated, the volume to be transferred to each of the cups is 0-30 ml. Plates were left on the bench for 3 h and the zone of diameter read after 40 h incubation at 37°C. Calculation of the concentrations. A standard graph was constructed by plotting the known concentrations versus the mean zone diameters on semi-logarithmic paper. Using this graph, the concentration of the test sera was calculated from the mean diameter.
69
P. R. J. Gangadharam and E. R. Candler Table I. Inhibition zone sizes on M. smegmatis at different concentrations of ethambutol Zone diameter (mm)
Assay
Bio-assay plates
Paper discs
no.
0
0-4
0-8
1-6
3-2
6-4
5
10
20
40
80
1 2 3 4 5
0 0 0 0 0
10 12 11 12 10
14 13 12 14 14
16 16 15 16 17
22 22 23 24 23
28 27 28 29 28
8 7 8 8 8
13 12 12 11 12
20 20 21 21 22
27 27 26 27 28
37 37 36 38 39
Mean S.D.
110
±10
13-4 160 280 22-8 ±0-89 ±0-71 ±0-84 ±0-71
120 270 7-8 20-8 37-4 ±0-45 ±0-71 ±0-84 ±0-71 ±114
Recovery of ethambutol from serum Parallel estimations using the paper-disc method of known concentrations of ethambutol (ranging from 0 to 80 ug/ml) prepared in sterile distilled water or in sterile horse serum (Difco Laboratories, Detroit, Michegan) gave virtually identical zone sizes. The recovery of ethambutol from serum was 96% at 10 ug/ml, 100% at 20 ug/ml and 98% at 40 and 80 ug/ml concentrations. Agreement between duplicate assays on sera from patients Agreement of a very high degree (95 to 100%) between duplicate assays was noted with several hundred sera obtained at 2, 4 and 6 h after standard test doses. Differences of assay results using Mycobacterium smegmatis ATCC 607 and Mycobacterium smegmatis 607B Parallel assays with both the organisms revealed that the variant M. smegmatis 6O7B is less sensitive than the original 607 culture (Figure 1). For example, the reductions of 40 30
Bio-assay plates
20
Poper disks
607 B
I 10
I o o
0-2). Optimal conditions for the microbiological assay of ethambutol in serum The optimal conditions for the microbiological assay of this drug, as discussed in earlier sections, are summarized in Table II (a representative standard graph is shown in
Bio-ossoy plo es
Poper disks
rh
20
„ o 1 30 V
t
: J
.20-
§
7HII
J-p" I I 11
M
ft
i
40 -
i
30 -
-i-
rr-
i
20 -
ti
it] Sauton
4- -f
i
10 -
0-4
1-6 6-4 5 20 80 0-8 3 2 10 40 Concentration of ethombutol (/ig/ml)
Figure 2. Zones of inhibition of Mycobacterium smegmatis ATCC 607 by different concentrations of ethambutol in 7H10, 7H11 and Sauton media using the cup-plate and the paper disc methods.
Table II. Summary of optimal conditions for microbiological assay of ethambutol 1. Age of the inoculum 2. Size of the inoculum 3. Medium to be used 4. Volume of the medium per plate 5. pH of the medium 6. Volume of test sample 7. Pre-diffusion 8. Time of reading
24h 1-5 ml for 100 ml of Sauton medium Modified Sauton 20 ml for 15 mm Petri plate 6-8 20 nl in paper-disc method 0.30 ml for cup-plate method 3 h at room temperature 40h
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
30
62
P. R. J. Gangadbaram and E. R. Candler
Figure 3). Since levels of ethambutol after conventional therapeutic dosages of this drug (15 mg/kg or 25 mg/kg body weight) are usually in the range of 2 to 5 ug/ml, it is necessary to use the cup-plate method.
50
Figure 3. A representative standard graph for the microbiological assay of ethambutol using the cupplate method. Organism used is Mycobacterium smegmatis ATCC 607 under optimal conditions. Concentrations of ethambutol are plotted on a logarithmic scale on the y axis and the zone sizes in arithmetic scale on the x axis.
Discussion
Taking the necessary leads from earlier work (Place & Thomas, 1963; Place et ah, 1966) a microbiological assay procedure for ethambutol in serum and other biological fluids has been standardized. This method has been found to give highly satisfactory and reproducible results. These studies have also denned several essential criteria to be fulfilled for satisfactory assay. For example, use of a variant of the same organism, M. smegmatis 607B, gives a poorer result. Similarly, use of Sauton medium seems to be essential, and use of other commonly used media (e.g. 7H10 and 7H11), however powerful they might be, for promoting the growth of mycobacteria, invariably results in reduced sensitivity. Since estimations of the drug concentration are essential both with respect to monitoring clinical response as well as in predicting the onset of toxicity, availability of such a standardized procedure will be of great value. In fact, in our institution, these have been satisfactorily conducted in several hundreds of patients taking this drug. Using this procedure, serum levels of this drug were also assayed in both slow and rapid inactivators of isoniazid, as well as in tuberculosis patients with renal failure and who were on dialysis programs. Finally, this procedure has been found to be useful in estimations of the levels of this drug in cerebrospinal fluid in normal and meningeal tuberculosis patients. Acknowledgements
We are grateful to Dr M. Forbes for useful suggestions and to the Lederle Laboratories (Dr C. R. Hodgson) for a gift of pure ethambutol. Mr E. R. Gonzales and Mr P. V. Ramakrishna rendered excellent technical assistance in some parts of this work and Mr Erwin Bourhofer gave statistical advice.
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
20 30 40 Zone diometer (mm)
Microbiological assay of ethambutanol
63
References
{Manuscript accepted 16 July 1976)
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
Gangadharam, P. R. J. & Candler, E. R. Action of ethambutol on Mycobacterium tuberculosis and Mycobacterium smegmatis under several experimental conditions. Paper presented at the 14th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology. September 1974. Abstract no. 363. Gangadharam, P. R. J. & Candler, E. R. Influence of ions on the antimycobacterial activity of ethambutol. Paper presented at the 23rd International Tuberculosis Conference, Mexico City. September 1975. Gangadharam, P. R. J. & Gonzales, E. R. Influence of the medium on the in vitro susceptibility of Mycobacterium tuberculosis to ethambutol. American Review of Respiratory Disease 102: 653-5 (1970). Medical Advisory Department. Lederle Laboratories. Myambutol ethambutol hydrochloride. Oral Antituberculosis Therapy. A brochure published by the Lederle Laboratories. Pearl River, New York (1967). Place, V. A., Peets, E. A., Buyske, D. A. and Little, R. R. Metabolic and special studies of ethambutol in normal volunteers and tuberculous patients. Annals of New York Academy of Sciences 135: 775-95 (1966). Place, V. A. & Thomas, J. P. Clinical pharmacology of ethambutol. American Review of Respiratory Disease 87: 901-6 (1963).
Microbiological assay of ethambutol
P. R. J. Gangadharam and E. R. Candler
Microbiological assay for ethambutol has been standardized using Mycobacterium smegmatis ATCC 607 as the assay organism. The optimal conditions for the assay including the age and size of the inoculum, volume and pH of the medium and time of reading were standardized. Few essential criteria like the nature of the strain, and the medium were also indicated. Introduction Ethambutol is widely used for the treatment of mycobacterial diseases. In the routine management of patients with this drug, as is the case with other drugs, it is necessary to have information on the levels attained in serum and other biological fluids not only to be able to correlate with clinical response but also as a possible warning of the onset of toxicity. Furthermore, a series of recent findings from our laboratories (Gangadharam & Gonzales, 1970) have shown marked influence of certain commonly used agar media (e.g. 7H10) on the in vitro activity of this drug on Mycobacterium tuberculosis. Measurements of ethambutol levels were also done under several experimental conditions in order to understand the reason for this phenomenon (Gangadharam & Candler, 1974, 1975). Chemical methods of assay of ethambutol are tedious, time consuming and are not sufficiently sensitive. A microbiological method for the assay of this drug using Mycobacterium smegmatis ATCC 607 was mentioned in the reports by Place & Thomas (1963) and by Place, Peets, Buyske & Little (1966) though full description of the procedure was only contained in the brochure on this drug issued by the manufacturer (Medical Advisory Department, Lederle Laboratories, 1967). Satisfactory results were not obtained in our laboratories, in spite of carefully following their procedure. We have therefore reconsidered the microbiological assay of ethambutol, using the initial suggestion from the work of the Lederle group of investigators. In this process, consideration was also given to investigate the value of a close variant of the assay organism, M. smegmatis 607B (which is widely used for phage studies on mycobacteria) and the agar media (7H10 and 7H11) which are widely used for the cultivation of mycobacteria. This paper presents the results on the standardization of the microbiological assay for ethambutol, denning the optimal conditions. 57
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
Mycobacteriology Laboratories, Jefferson Davis Hospital and Department of Pathology, Baylor College of Medicine, Houston, Texas 77030, U.S.A.
58
P. R. J. Gangadharam and E. R. Candler Materials and methods
Test organisms Mycobacterium smegmatis ATCC 607 was obtained from the American Type Culture Collection. A variant of this organism, M. smegmatis 607B, was obtained from Dr W. B. Redmond, Atlanta, Georgia. Assay broth This consisted of: dextrose 100 mg; beef-extract 5-2 g; peptone 6-2g; yeast extract 2-0 g; sodium chloride 2-4 g; Tween 80 140 ml. Final volume to 1000 ml pH 70±0-2 before autoclaving. The medium was sterilized by autoclaving for 15 min at 121°C.
7H10 and 7H11 agar medium Middlebrook and Cohn's 7H10 agar medium and Cohn's 7H11 agar medium were prepared from dehydrated powders and sterile Oleic Acid-Albumin Dextrose Complex (OADC) enrichments. Seeding with the assay organism was made before pouring the plates, as was done in the case of Sauton medium. Paper discs
Standard discs of 5 mm diameter, obtained from Schleicher and Schuell, Inc., Keene, N.H. 03431 were used. They were sterilized by autoclaving for 15 min at 121°C. Bio-assay plates The stainless steel Bio-assay plates, along with the special forceps to be used in the cupplate method were obtained from Lab Line Instruments, Inc. The plates were sterilized by autoclaving at 121 °C for 15 min. Method of preparing the innoculum Growth of M. smegmatis ATCC 607 from Lowenstein-Jensen medium was transferred to a sterile tube containing 1 -0 ml of assay broth and 4 glass beads. A uniform suspension was made using a vortex mixer and an aliquot incubated at 37°C for 24 h. From this, another 24 h old subculture of this growth was made as above and used as the inoculum, and calculated amounts were transferred to flasks containing 100 ml of Sauton medium before pouring into Petri dishes. The same procedure was adopted for obtaining the inoculum of M. smegmatis 6O7B. Method of microbiological assay ofethambutol Paper disc method. Aliquots of 20 ul of the test sera or standards, at least in duplicate, were transferred to paper discs which were placed on the seeded Sauton's agar plates. With each batch of assays, a set of standards in duplicate was also set up in the same
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
Modified Sauton's medium used for assay plates This consisted of: iron ammonium citrate 0-05 g; dipotassium hydrogen phosphate 0-50g; magnesium sulphate anhydrous 0-50 g; citric acid 200g; L-asparagine 400g; Bacto agar (Difco) 2000g; glycerol 4000ml; Tween 80 20 ml; distilled water Q.S. to 1000 ml; pH7-0±0-2 before autoclaving. The medium was sterilized at 12PC for 15 min and when cooled to 45°C the inoculum was added, mixed by gentle shaking and 20 ml was poured into sterile disposable (15 mm) plastic Petri dishes. After solidification, the plates were stored at 4°C until used.
Microbiological assay of ethambutanol
59
Results Optimum age and size of the inoculum Using inocula ranging from 1 to 5 days age, it was observed that 1-day-old inoculum was sufficient to give a good assay. Similarly, a volume of 1-5 ml/100 ml of the Sauton medium was found to be optimal among several volumes tested. Optimal volume of test material to be applied Assays carried out using several volumes, with both the methods revealed that 20 ul and 0-3 ml were optimal for the paper and cup-plate methods, respectively. Optimal amount of media to be used Using several volumes of the medium ranging from 5 0 ml to 300 ml, it was found that 200 ml was optimal to be used in a standard (15 mm) Petri dish. Optimal pH of the medium Sauton medium was prepared with the final pH ranging from 6 0 to 80, with increases of 0-2 units in between: Microbiological assays were carried out in duplicate both by the cup-plate and the paper-disc methods. Medium with pH of 6-8 gave the largest zone sizes. Optimum time of reading the results The results obtained indicated that an optimum time of reading is at 40 h of incubation. Effect of prior diffusion on the zone size It was found that a prior period of diffusion for 3 h of the drug results in increased zone sizes. Usefulness of seeded plates after storage Microbiological assays carried out using seeded plates stored at 4°C for 1, 2, 3 or 4 weeks gave very similar results to those obtained using fresh plates. Consistency of the zone sizes with the paper-disc and cup-plate methods Zone sizes (in mm) along with the mean and standard deviation in sets of five assays, using known concentrations of ethambutol, both with the paper-disc and cup-plate methods, showed highly consistant values (Table I).
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
manner. The concentrations used for the standards using the paper disc method were 0, 5, 10, 20, 40, 80 ug/ml. The plates were then allowed to stand on the laboratory bench at room temperature (28°C±2°C) for 3 h, after which time they were transferred to an incubator (37°C). The zones of inhibition were read after 40 h of incubation at 37°C. Cup-plate method. The solutions to be assayed were transferred to the individual bioassay plates positioned on agar plates. The concentrations used for the standards were 0 0 , 0-4, 0-8, 1-6, 3-2 and 6-4 ug/ml of ethambol. Unless otherwise indicated, the volume to be transferred to each of the cups is 0-30 ml. Plates were left on the bench for 3 h and the zone of diameter read after 40 h incubation at 37°C. Calculation of the concentrations. A standard graph was constructed by plotting the known concentrations versus the mean zone diameters on semi-logarithmic paper. Using this graph, the concentration of the test sera was calculated from the mean diameter.
69
P. R. J. Gangadharam and E. R. Candler Table I. Inhibition zone sizes on M. smegmatis at different concentrations of ethambutol Zone diameter (mm)
Assay
Bio-assay plates
Paper discs
no.
0
0-4
0-8
1-6
3-2
6-4
5
10
20
40
80
1 2 3 4 5
0 0 0 0 0
10 12 11 12 10
14 13 12 14 14
16 16 15 16 17
22 22 23 24 23
28 27 28 29 28
8 7 8 8 8
13 12 12 11 12
20 20 21 21 22
27 27 26 27 28
37 37 36 38 39
Mean S.D.
110
±10
13-4 160 280 22-8 ±0-89 ±0-71 ±0-84 ±0-71
120 270 7-8 20-8 37-4 ±0-45 ±0-71 ±0-84 ±0-71 ±114
Recovery of ethambutol from serum Parallel estimations using the paper-disc method of known concentrations of ethambutol (ranging from 0 to 80 ug/ml) prepared in sterile distilled water or in sterile horse serum (Difco Laboratories, Detroit, Michegan) gave virtually identical zone sizes. The recovery of ethambutol from serum was 96% at 10 ug/ml, 100% at 20 ug/ml and 98% at 40 and 80 ug/ml concentrations. Agreement between duplicate assays on sera from patients Agreement of a very high degree (95 to 100%) between duplicate assays was noted with several hundred sera obtained at 2, 4 and 6 h after standard test doses. Differences of assay results using Mycobacterium smegmatis ATCC 607 and Mycobacterium smegmatis 607B Parallel assays with both the organisms revealed that the variant M. smegmatis 6O7B is less sensitive than the original 607 culture (Figure 1). For example, the reductions of 40 30
Bio-assay plates
20
Poper disks
607 B
I 10
I o o
0-2). Optimal conditions for the microbiological assay of ethambutol in serum The optimal conditions for the microbiological assay of this drug, as discussed in earlier sections, are summarized in Table II (a representative standard graph is shown in
Bio-ossoy plo es
Poper disks
rh
20
„ o 1 30 V
t
: J
.20-
§
7HII
J-p" I I 11
M
ft
i
40 -
i
30 -
-i-
rr-
i
20 -
ti
it] Sauton
4- -f
i
10 -
0-4
1-6 6-4 5 20 80 0-8 3 2 10 40 Concentration of ethombutol (/ig/ml)
Figure 2. Zones of inhibition of Mycobacterium smegmatis ATCC 607 by different concentrations of ethambutol in 7H10, 7H11 and Sauton media using the cup-plate and the paper disc methods.
Table II. Summary of optimal conditions for microbiological assay of ethambutol 1. Age of the inoculum 2. Size of the inoculum 3. Medium to be used 4. Volume of the medium per plate 5. pH of the medium 6. Volume of test sample 7. Pre-diffusion 8. Time of reading
24h 1-5 ml for 100 ml of Sauton medium Modified Sauton 20 ml for 15 mm Petri plate 6-8 20 nl in paper-disc method 0.30 ml for cup-plate method 3 h at room temperature 40h
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
30
62
P. R. J. Gangadbaram and E. R. Candler
Figure 3). Since levels of ethambutol after conventional therapeutic dosages of this drug (15 mg/kg or 25 mg/kg body weight) are usually in the range of 2 to 5 ug/ml, it is necessary to use the cup-plate method.
50
Figure 3. A representative standard graph for the microbiological assay of ethambutol using the cupplate method. Organism used is Mycobacterium smegmatis ATCC 607 under optimal conditions. Concentrations of ethambutol are plotted on a logarithmic scale on the y axis and the zone sizes in arithmetic scale on the x axis.
Discussion
Taking the necessary leads from earlier work (Place & Thomas, 1963; Place et ah, 1966) a microbiological assay procedure for ethambutol in serum and other biological fluids has been standardized. This method has been found to give highly satisfactory and reproducible results. These studies have also denned several essential criteria to be fulfilled for satisfactory assay. For example, use of a variant of the same organism, M. smegmatis 607B, gives a poorer result. Similarly, use of Sauton medium seems to be essential, and use of other commonly used media (e.g. 7H10 and 7H11), however powerful they might be, for promoting the growth of mycobacteria, invariably results in reduced sensitivity. Since estimations of the drug concentration are essential both with respect to monitoring clinical response as well as in predicting the onset of toxicity, availability of such a standardized procedure will be of great value. In fact, in our institution, these have been satisfactorily conducted in several hundreds of patients taking this drug. Using this procedure, serum levels of this drug were also assayed in both slow and rapid inactivators of isoniazid, as well as in tuberculosis patients with renal failure and who were on dialysis programs. Finally, this procedure has been found to be useful in estimations of the levels of this drug in cerebrospinal fluid in normal and meningeal tuberculosis patients. Acknowledgements
We are grateful to Dr M. Forbes for useful suggestions and to the Lederle Laboratories (Dr C. R. Hodgson) for a gift of pure ethambutol. Mr E. R. Gonzales and Mr P. V. Ramakrishna rendered excellent technical assistance in some parts of this work and Mr Erwin Bourhofer gave statistical advice.
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
20 30 40 Zone diometer (mm)
Microbiological assay of ethambutanol
63
References
{Manuscript accepted 16 July 1976)
Downloaded from http://jac.oxfordjournals.org/ at University of California, San Diego on June 4, 2015
Gangadharam, P. R. J. & Candler, E. R. Action of ethambutol on Mycobacterium tuberculosis and Mycobacterium smegmatis under several experimental conditions. Paper presented at the 14th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology. September 1974. Abstract no. 363. Gangadharam, P. R. J. & Candler, E. R. Influence of ions on the antimycobacterial activity of ethambutol. Paper presented at the 23rd International Tuberculosis Conference, Mexico City. September 1975. Gangadharam, P. R. J. & Gonzales, E. R. Influence of the medium on the in vitro susceptibility of Mycobacterium tuberculosis to ethambutol. American Review of Respiratory Disease 102: 653-5 (1970). Medical Advisory Department. Lederle Laboratories. Myambutol ethambutol hydrochloride. Oral Antituberculosis Therapy. A brochure published by the Lederle Laboratories. Pearl River, New York (1967). Place, V. A., Peets, E. A., Buyske, D. A. and Little, R. R. Metabolic and special studies of ethambutol in normal volunteers and tuberculous patients. Annals of New York Academy of Sciences 135: 775-95 (1966). Place, V. A. & Thomas, J. P. Clinical pharmacology of ethambutol. American Review of Respiratory Disease 87: 901-6 (1963).
