Bull. Org. mond.

Sante 1969, 41, 21-43
Bull. Wld Hlth Org.

Advances in Techniques of Testing Mycobacterial
Drug Sensitivity, and the Use of Sensitivity Tests
in Tuberculosis Control Programmes
G. CANETTI,1 WALLACE FOX,2 A. KHOMENKO,3 H. T. MAHLER,'
N. K. MENON,5 D. A. MITCHISON,' N. RIST 1 & N. A. WMELEV 7

In a paper arising out of an informal international consultation of specialists in the
bacteriology of tuberculosis held in 1961, an attempt was made to formulate criteria, and
specify technical procedures, for reliable tests of sensitivity (the absolute-concentration
method, the resistance-ratio method and the proportion method) to the 3 main antitubercu-
losis drugs (isoniazid, streptomycin and p-aminosalicylic acid). Seven years later, a further
consultation was held to review the latest developments in the field and to suggest how
sensitivity tests might be put to practical use in tuberculosis control programmes. The
participants reached agreement on how to define drug sensitivity and resistance, and stressed
the importance of using a discrimination approach to the calibration of sensitivity tests.
Their views are contained in the present paper, which also includes descriptions of the
sensitivity tests used by the Medical Research Council of Great Britain for first- and second-
line drugs (minimal inhibitory concentration and resistance-ratio methods), the two main
variants of the proportion method developed by the Institut Pasteur, Paris, and a methodfor
calibrating sensitivity tests.

I. INTRODUCTION8
As the outcome of an international consultation of (1963) had said little about how sensitivity tests
specialists in the bacteriology of tuberculosis, ar- might be put to practical use in the control of
ranged by WHO in 1961, Canetti et al. (1963) de- tuberculosis. The present article reviews the pro-
scribed criteria and techniques for reliable tests of gress that has been made in methods of sensitivity
mycobacterial resistance to tuberculostatic drugs. testing and their calibration. Further, the ways in
Since then, considerable experience has been which the results of sensitivity tests may influence
gained in the use of the tests described and new the choice of regimens of chemotherapy for individ-
drugs have been introduced. The technical problems ual patients, and their use to estimate the prevalence
involved in standardizing such methods are now of drug-resistant strains in the community, are eva-
much better understood. The paper by Canetti et al. luated. An attempt is made also to formulate the
1 Chef de Service, Institut Pasteur, 25 rue du Docteur b Director, Tuberculosis Chemotherapy Centre, Madras,
Roux, Paris 15e, France. India.
' Director, Medical Research Council's Tuberculosis and ' Honorary Director, Medical Research Council's Unit
Chest Diseases Research Unit, Brompton Hospital, Fulham for Research on Drug Sensitivity in Tuberculosis, Royal
Road, London, S.W.3, England. Post-graduate Medical School, Ducane Road, London, W.12,
England.
3Formerly Medical Officer, Tuberculosis, Division of 7Director, Central Research Institute of Tuberculosis,
Communicable Diseases, World Health Organization, Moscow-128, USSR.
Geneva, Switzerland; present address: Professor and Head of
Tuberculosis Department, Postgraduate Medical Institute, 8 Prepared by the participants in an informal consultation
Kharkov, USSR. on the drug-resistance problem in tuberculosis, held in
Geneva, Switzerland, from 23 to 26 April 1968: Dr G.
' Chief Medical Officer, Tuberculosis, Division of Com- Canetti, Dr Wallace Fox, Dr A. Khomenko, Dr H. T. Mahler,
municable Diseases, World Health Organization, Geneva, Dr N. K. Menon, Professor D. A. Mitchison, Dr N. Rist
Switzerland. and Professor N. A. Smelev.

2353 -21-

22 G. CANETTI AND OTHERS

role of sensitivity tests in tuberculosis control pro- made in two directions. First, simplified direct tests,
grammes at widely varying stages of development. which are already available with certain methods
(see Part III), are being further developed, especially
DEFINITION OF DRUG RESISTANCE for isoniazid and streptomycin. Secondly, the slide-
culture method, which yields results in 7 days, is
Drug resistance may be defined in bacteriological being standardized in terms of established indirect
terms or in the light of the probable response of the tests.
patient to chemotherapy with the drug concerned.
As it is difficult to establish a precise clinical defini- THE CALIBRATION OF TESTS
tion of resistance, a definition in purely bacteriologi- The necessity to standardize any sensitivity test
cal terms has been adopted for the purposes of this method was emphasized by Canetti et al. (1963), but
paper. The following statement (Mitchison, 1962) is they gave no details of the ways in which this might
considered to be adequate: be done. Objective methods of calibration are im-
"Resistance is defined as a decrease in sensitivity of portant for several reasons. First, to clarify how the
sufficient degree to be reasonably certain that the strain present criteria of resistance in the methods de-
concerned is different from a sample of wild strains of scribed in Parts II and III have been obtained.
human type that have never come into contact with the Secondly, for technical reasons the most efficient
drug." criterion of resistance may be slightly different from
Such a definition assumes that the variation in one laboratory to another. In particular, drug activ-
sensitivity of sensitive strains to any one of the ity may vary because of heat instability of some
antibacterial drugs is small. (It does not apply to drugs during inspissation or storage (notably ethion-
thioacetazone, since natural resistance to this drug amide and cycloserine). The type of container and
occurs in a high proportion of strains obtained from the type of closure for slopes containing streptomy-
certain regions of the world (Thomas et al., 1961; cin also affect the result (Krebs et al., 1962; Interna-
Rist, 1968; Mitchison & Allen, to be published).) tional Union against Tuberculosis, 1964). Thirdly,
For most of the antituberculosis drugs, there is objective methods of calibration allow of compari-
evidence that a diminished clinical response may sons between methods of sensitivity testing in which
occur when resistance in the above-mentioned bac- the measures of sensitivity are expressed on different
teriological sense is demonstrated in the laboratory. scales.
In the past, a common approach was to test a
AVAILABLE METHODS OF TESTING sample of presumedly sensitive strains. Strains were
FOR DRUG SENSITIVITY considered to be resistant if they were slightly more
resistant than the great majority of the sensitive
Three widely used methods for testing sensitivity strains. Such a procedure has the defect that strains
to isoniazid, streptomycin and para-aminosalicylic considered to have natural or primary resistance
acid (PAS) on Lowenstein-Jensen medium-namely, must be removed from the population of sensitive
the absolute-concentration method, the resistance- strains, using necessarily arbitrary criteria.
ratio method and the proportion method-were de- An alternative approach is to consider the results
scribed by Canetti et al. (1963). Part II of the of the treatment of patients who have organisms of
present paper describes the minimal inhibitory varying degrees of sensitivity at the start of treat-
concentration method and the resistance-ratio ment, and to try to determine at what level of
method; Part III, a simplified variant and a standard sensitivity there is a change in the therapeutic re-
variant of the proportion method; and Part IV, a sponse. In practice, the number of patients with
method of calibrating sensitivity tests. initially resistant strains who are treated with the
There are, of course, other methods, including drug concerned, alone or in combination with only
tests with agar-based semi-synthetic media and one other drug, is too small to allow precise criteria
liquid media, as well as vertical diffusion methods. of resistance to be obtained.
There remains a need for more rapid and simple However, the best available approach is to com-
but accurate techniques of sensitivity testing. Direct pare the sensitivity of a sample of strains from
tests provide results more rapidly than indirect tests, untreated patients (a predominantly sensitive sam-
which are used in most laboratories. It is therefore ple) with a sample of strains from patients who have
encouraging that progress with direct tests is being been treated with the drug for at least a few months,

and to what extent each modifies the results of strains with borderline degrees of resistance. Unfortunately. dence is accumulating that there is a large difference nates between the two samples with the greatest from culture to culture in the number of colonies efficiency can then be chosen. Further evi- sample). Lefford & Mit- is unnecessary to have either a pure population of chison. which estimates the efficiency of each method in the detection of Second-line drugs resistance. there appears to be little to considerable work would be necessary. it would be choose between the minimal inhibitory concentra- certain that the efficiency of the tests performed in tion. the proportion method should compen- sensitive strains as resistant. a laboratory should make provision for the relative efficiency of the different methods in the calibration of these tests. Furthermore. it lowing provisional conclusions have been drawn. If the former interpretation in any method of testing. resistance-ratio and proportion methods. weight or opacity (Canetti et al. This will make it distinguishing a sample of predominantly sensitive possible to establish whether the criteria of resistance strains from a sample of predominantly resistant proposed by the initiators of the method are entirely strains. If the latter. it Union against Tuberculosis. The discrimination approach has been purpose. this advantage of the pro- of sensitive strains as resistant. would be desirable for large laboratories to repeat this calibration procedure at intervals. ied. tests to thioacetazone in strains from regions where ly sensitive and predominantly resistant groups of natural resistance to thioacetazone is prevalent. This discrimination that grow from a similar inoculum specified by approach is described fully in Part IV (" The calibra. and vice versa. buted either to differences in the actual number of in each sample. For such a comparison. For this investigated. using the valid in the laboratory in question or whether some discrimination approach or a variant of it. MYCOBACTERIAL DRUG SENSITIVITY TESTS: RECENT ADVANCES AND PRACTICAL USE 23 mostly without success (a predominantly resistant remains of the greatest importance. tests. Even though For streptomycin. some misclassification of is correct. the standardization of the size of the inoculum Evidence on comparisons of the minimal inhibitory . wild strains or a pure population of resistant strains: Variation in the number of culturable particles (for- it is essential only that there should be a substantial merly known as the " viable count ") can be attri- difference in the proportion of resistant strains with. The relative efficiency of the three methods for The discrimination approach should be used also testing PAS sensitivity does not appear to have been in comparing methods of sensitivity testing.. even with this dis. The criterion of resistance that discrimi. especially for First-line drugs tests that are difficult to perform. both these factors cause variation in the drug. 1966. using that laboratory remained high. The resistance-ratio method of measuring sensitiv- ity on Lowenstein-Jensen medium using serial 2-fold COMPARISON OF METHODS OF SENSITIVITY TESTING drug dilutions appears to be unsatisfactory. On the available the physician to deprive the patient of a valuable evidence. 1968). Since it is desirable to avoid misclassification inoculum. but further work is resistance that very rarely misclassifies a sensitive recommended to establish which is the more impor- strain. International tion of sensitivity tests "). a simi- samples of predominantly resistant cultures should lar conclusion can be drawn about the minimal be maintained in reference laboratories for issue to inhibitory concentration and proportion methods. mainly because the critical ratio that distinguishes sensitive Among the technical issues concerning sensitivity and resistant strains best lies between 2 and 4. any laboratory in which it is desired to calibrate but the resistance-ratio method has not been stud- sensitivity tests. owing to the errors inherent clumping of the bacilli. the fol- of the criteria require modification. For isoniazid. 1964. it is preferable to choose a final criterion of number of culturable particles. organisms in the inoculum or to the degree of crimination approach. It is desirable that the discrimination approach. irrespective of the method of testing retical considerations is the empirical comparison of employed. Bartmann & Galvez-Brandon. the best criterion for each method is chosen found to be unsatisfactory for analysing sensitivity and its ability to discriminate between predominant. 1963. tests. cultures is expressed as a percentage. although it may more often fail to detect tant. Largely from unpublished data. because this may lead portion method is not gained. As soon as possible after starting to carry out Of greater practical importance than these theo- sensitivity tests. tends to sate for variation in the total bacillary content of the occur.

CANETTI AND OTHERS concentration and proportion methods for ethion. provided that the from one group of workers to another. The minimal inhibitory concentra- tion and proportion methods might be equivalent. is most frequently obtained imme- vitro difference between sensitive and resistant diately before sputum conversion. with decreasing bacillary populations on serial smear a substantial proportion of patients with primary and culture rarely serves any useful purpose since. results of a recent investigation indicate that the For ethambutol. others. and some under these circumstances. and the patients whose organisms had previously acquired resistance-ratio method will probably prove to be isoniazid resistance did not improve the result. Furthermore. such strains are not only bacteriologically Only tentative suggestions can be made on resistant but also clinically resistant. The degree of methods to be adopted for testing sensitivity to two laboratory resistance is immaterial. Initial drug resistance tance to a single drug has little or no effect on the It is important to appreciate the difference be- outcome of treatment with the three drugs: isoniazid. For Acquired drug resistance pyrazinamide-sensitivity tests. but may also be strains is considerable. efficiency (Lefford & Mitchison. Nevertheless. Even so. and in all probability no further benefit is with the minimal inhibitory concentration and pro. ence of primary resistance to 2 first-line drugs. the only resistant strains patients achieve sputum conversion. The resistant culture. er. the in than 5 colonies. At the other extreme. isms. it may be concluded that the response is sensitivity tests during chemotherapy in patients often impaired by primary resistance. and it is likely that the resis. there regimen may emerge in the course of successful is evidence that in vitro resistance to ethambutol is chemotherapy. tween primary and initial drug resistance. the new drugs. course of chemotherapy. inadequate evidence is available to say wheth. to be obtained with long-term treatment with the portion methods. there is evidence that the presence of resis. This finding is tance-ratio method will be as efficient as the two due to the isolation of resistant. For other likely to be encountered are transitionally resistant drugs. i. Transitional resistance is in sharp contrast to acquired resistance in that significant Prinmary drug resistance multiplication of resistant organisms does not occur On the basis of the available evidence concerning in the former and there is no justification for a the influence of primary isoniazid resistance on the change of treatment. lesions. resistance show a bacteriological response. 1965). resistant organisms that are not multiply- ing in the lesions but have nevertheless survived PROGNOSTIC SIGNIFICANCE OF DRUG RESISTANCE (Canetti. Primary streptomycin and PAS (International Union Against resistance is resistance found in patients which has Tuberculosis. for rifampicin. persistent organ- other methods. 1964). Incidentally. a measure of response can be expected with treatment with the THE EPIDEMIOLOGY OF DRUG RESISTANCE drug concerned. Some workers bacteriological response is not infrequently obtained find that the methods are of similar discriminative with the 3 drugs. when given alone. the in vitro difference between addition of isoniazid to re-treatment regimens in sensitive and resistant strains is very small. even in the pres. usually com- often difficult to detect in cases of apparent clinical posed of only 1 or 2 colonies and seldom of more resistance.e. a amide and cycloserine is discrepant. kanamycin or viomycin. not resulted from treatment of those patients with . no further response is to be but. further tivity implies the growth of organisms within the studies are needed. obtained during the next few months. that the proportion method is the more efficient. This is true even for have been few comparisons between tests done isoniazid. unsatisfactory. again. there is agreement When drug resistance has emerged during the that the proportion method is the more efficient. strains.. Since there sputum is consistently positive. ethambutol and rifampicin. and no comparisons have been drug. 1966).24 G. Since the presence of persistent sputum posi- made for PAS. response of patients to treatment with this drug It is strongly emphasized that the performance of given alone. On the other hand. the extent of the advantage found differs expected from the drug concerned. in the presence of primary resistance. Transitional resistance but there should be only narrow intervals between Occasionally resistance to one or more drugs of a the drug concentrations to be tested.

clinics with good and with -poor treatment services tice in the area. as well as acquired resis. they have been repeated at intervals of ters. surveys of true primary drug resistance positive culture in such surveys is. since the dards of the tuberculosis service and of chemo. MYCOBACITERIAL DRUG SENSITIVITY TESTS: RECENT ADVANCES AND PRACTICAL USE 25 the drug concerned. have no reason for withholding the information. and acquired resis. Initial resistance occurs in patients attending a clinic for the first time for treatment. The available data provide no are fairly represented. and that resistance community. to only one drug is much more frequent than resis. Most drug resistance. who give no history of previous chemotherapy. all chronic excretors of drug-resis- community. In order to provide a measure of the problem that tance. total surveys of newly registered patients. isoniazid for a cough-with. where. the same clinic. they would not include patients already on tance in patients who have either concealed a history the clinic register who return for further treatment in of previous chemotherapy or have received chemo. pool during mass radiography surveys of the com- On the other hand. or even decrease. out being aware of the nature of the medicament. the results show that there has been no increase in the prevalence of primary one important index of the success of therapy in the resistance during the past decade. tious cases in the community. so that urban and rural communities and reflect the quality and extent of chemotherapy prac. the acceptable evidence of a progressive build-up of initial sample should include children under the age of drug resistance to alarming levels in any developing 15 years. The pool of chronic drug-resistant infectious cases Surveys of the prevalence of drug resistance A number of difficulties are involved in measuring Since it is very difficult to obtain a reliable history the size of the pool of chronic drug-resistant infec- of previous chemotherapy from patients in the deve. a drug-resistance survey a new way of assessing the amount of bacillary should be conducted within an entire country or transmission in the community. Such surveys will be referred to as therapy-for example. which may occur in a strain that has never all newly registered patients present for the treat- come into contact with the drug. It whether or not they have received treatment else- includes primary resistance. in the technically advanced munity. it is important to record the may become stable. a new epidemiological parameter. since there is evidence that the pattern of country where the initial interrogation of patients drug sensitivity of their organisms may give the best about the history of previous chemotherapy has been measure of the prevalence of primary resistance. Performance of surveys tance to more than one drug. . Surveys of primary resistance measure the tenden. surveys of such patients should be tance occurring as a result of exposure to the drug in made. There is increasing evidence that the level of primary resistance may be Whenever possible. If carefully conducted. as far as the trends can the community is multiracial or contains large be followed. Further attempts should be made to evolve know if they have had previous chemotherapy and accurate methods of characterizing the pool. in practice. usual- can more readily be performed because the patients ly small. In several of the technically advanced tant organisms are recorded in drug-resistance regis- countries. Thus it may provide region rather than in individual clinics or hospitals. Indeed. ment services. In general terms. surveys of primary resistance in vides an important indication of the total extent of these countries usually cannot be carried out. In developing countries. in practice. there is some evidence that the levels groups of immigrants. the total number of patients found to have a countries. groups may have different prevalences of drug resis- therapeutic practice improve. They act as a guide to the statistical sample of the clinics and hospitals in the formulation of chemotherapy programmes and regions. These surveys should include all patients another patient. race or the country of origin of the patient. as the stan. Surveys of initial drug resistance measure the Preliminary data on the notification of new cases of prevalence of resistance in patients who present tuberculosis should be obtained and used to draw a themselves for treatment. Drug-resistance registers cy for resistant strains to accumulate within the In some cities. so that the survey may be widely representative. a measurement that pro- loping countries. tance. Although it is possible to obtain surveys in patients disclaiming previous treatment information on the size and characteristics of the are. surveys of initial drug resistance. These registers are a valuable way of following years. It includes both natural resis.

including the number of culturable Furthermore. Also. The ratio between the In the developing countries. in ogy. a sample of pre. H37Rv) organisms in the community can be estimated. thus providing a check on the comparability of Too much stress has been laid on the last of these definitions of resistance.not a sensitivity test is performed. various antituberculosis drugs. and will facilitate comparisons with other be emphasized. Furthermore. or for comparisons between countries. measurements of Studies in some developing countries have demon- the prevalence of resistance to isoniazid have the strated the value of a careful interrogation of greatest epidemiological value. in time or at two different points in time in the same One or both of the following control methods are country. of a high standard of accuracy and. since isoniazid is patients about their previous history of chemothera- widely used in treatment throughout the world. especially in re- other survey using the same sensitivity test method. This information is likely to be closely comparable. whether at one point between different countries.those resistant to more than one drug is also of formed in a single laboratory capable of doing tests epidemiological importance. for the purposes of epidemiol- surveys using this approach. including. countries. initial drug resistance automatically limits the poten- Considering the prevalence of resistance to the tial value of wide-scale sensitivity testing. The harm that The application of the discrimination approach will could result to the individual patient from clinical then ensure that optimum definitions of resistance action taken on the basis of inaccurate results must are used. The wide scale on which unreliable sensitivity-test ically obtained in total surveys of newly registered results are reported. requirements for sen- number of strains resistant to a single drug and of sitivity testing necessarily vary according to the stage . dominantly resistant strains.concerning the regimen of chemotherapy to be given cin. the planning of wide-scale treatment or research. in particular. Secondly. which is often automat. full technical details of the inaccurate results are of very limited value. PAS. but streptomycin is more widely used in some to the patient. in order to decide compared with similar distributions obtained in any on regimens that might be effective. and the drugs used. good particles obtained on drug-free medium. the test is more difficult to is of importance in planning treatment. For this reason. advisable. a series of tests on the standard strain. indeed. its duration and the tests are comparatively easy to perform. CANETII AND OTHERS The sensitivity tests for a survey should be per.The uses of sensitivity tests ing a choice between the several accurate sensitivity test methods available.uses. bovis or some other mycobacterial species. method used. the distribution can then be For use in the individual patient. development line drugs are of less value. the use of well organized. gives cause for concern.ple. should be regimens of chemotherapy in communities with little given. tuberculosis. compared with the other three. and For scientific purposes: they have contributed comparability with surveys in other countries is also greatly to our understanding of the mechanisms of of value. and it is possible that the occur. the proportion of patients with fully sensitive ly. whether or perform reliably. treatment. It is also of value to available at the time when decisions have to be taken estimate the prevalence of resistance to streptomy. IN TUBERCULOSIS CONTROL PROGRAMMES ed should be identified as Myco. a detailed distribution of the sensi. even in the technically advanced patients (as described on page 25). may be tested. In mak. rence of strains with natural resistance to the drug may obscure the epidemiological value of the data. Myco. employed.26 G. should be obtained. thioacetazone or the second. less satisfactori. in order to allow accurate comparisons For epidemiological studies. All mycobacteria isolat. since. reporting the results. For planning wide-scale treatment. It is clearly desirable to reduce to a chemotherapy and of the reasons for success and minimum the number of different sensitivity tests failure. careful interrogation countries than in others. Sensitivity testing in countries at different stages of Sensitivity tests to. comparability with previous Sensitivity tests have four main uses: surveys in the same region is the first priority. for exam- tivity of a sample of wild strains (or. First. the py. if a sufficient standard cannot be obtained the survey THE ROLE OF SENSITIVITY TESTS should not be undertaken. to indicate whether pre-treatment drug results obtained by different methods of testing are resistance is likely to be present.

Such countries might need culosis has been newly diagnosed. only on the role of sensitivity testing. In developing countries existing knowledge does scale treatment programmes or. three poli- scale to maintain an adequate control over their cies of chemotherapy. In this treatment drug resistance is likely to occur only in connexion. the situation in many techni. less acceptable. treme choice of policy for an area might be to treat crease the number of laboratories should be carefully all patients with a standard regimen. countries that either already have. In some countries. sensitivity testing might even result single laboratory able to undertake a sufficient num. then the gains may be At the next stage of development. One ex- efficient laboratory is functioning. This has overshadowed what is often a more impor- try. even when sensi- to obtain limited reference work for epidemiological tivity testing is of the highest standard. MYCOBACTERIAL DRUG SENSITIVITY TESTS: RECENT ADVANCES AND PRACTICAL USE 27 of technical progress and the availability of laborato. Once one resistance encountered in the community. tant factor: the failure of the patient to co-operate ing until adequate staff. many using unsatis. especially the first-line drugs are low and where the available when the cultures yield abundant growth and there resources permit the use of therapy with the three has been a "fall and rise" phenomenon in the . where laboratories use uniform methods. such therapy policy to be adopted in the light of the a laboratory might be able to meet likely require. This is purposes and do so in a laboratory abroad. equipment. there are many negligible. ment patterns of sensitivity actually encountered in Only too often. whatever the stage resistance in pre-treatment tests for drug sensitivity of development of the laboratory services of a coun. for the planning of wide. either based on or ignoring the standards. or else require. ries undertaking sensitivity tests. most appropriate policies of chemotherapy to apply It is clearly desirable to make one laboratory a In the past. a position may be reached where. The sensitivity tests might be used for effective second-line regimens. in some instances. In such population and with limited resources there is no circumstances. in actual harm by leading to unnecessary changes ber of sensitivity tests to meet all needs for the of chemotherapy from effective and acceptable current stage of development of the tuberculosis regimens to more toxic. are being being made in some countries to centralize sensitivity compared. Pre-treatment sensitivity tests Sensitivity testing during treatment In technically advanced countries where it has been Patients who are consistently culture-positive at established that the levels of primary resistance to the end of 6 months or more of treatment. It is encouraging to note that efforts are results of pre-treatment sensitivity tests. sensitivity testing are lower. not provide adequate guidance on the best chemo- even for individual patients. laboratory services that are required. Indeed. The other extreme might be to apply several At the other extreme. the role ry services. cally. only too often because the supervisory intellectual skills are available to sustain a high staff do not adapt the organization and supervision standard of work. efforts are currently being made by the very small proportion of patients with strains WHO to establish inter-country regional reference resistant to two or three drugs. first-line drugs in all newly diagnosed cases. but also on the offers substantial advantages. and less programme. the failure of good regimens of chemo- national reference centre. not only according to be changed only when it was apparent that the the requirements of the tuberculosis programme but patient remained consistently positive bacteriologi- also in relation to competing health priorities. a paradoxically. widely different levels of initial and acquired drug ments for a number of years to come. interest and in treatment. no laboratory should embark on sensitivity test. In a study that is now in progress in an area with a factory methods or else doing tests on too small a substantial level of initial drug resistance. For some countries with a very small of pre-treatment sensitivity tests is limited. the organisms of individual patients. possibly because failure of the standard regimen due to pre- with the help of an international agency. the regimen to evaluated and fully justified. epidemiological purposes. different basic regimens according to the pre-treat- cally advanced countries today is unsatisfactory. Such studies can yield information not testing in a few regional laboratories: such central. If the standards of laboratories. of chemotherapy to the needs of the patient. and hence the ization. the need to in. there is little to be gained from pre- justification for establishing sensitivity testing in treatment sensitivity tests in patients in whom tuber- even a single laboratory. there are large numbers of laborato. therapy has often been explained by the finding of It should be emphasized that.

Further. Fluorescence microscopy offers the substantial ad- duced. because just defined. Sensitivity testing and second-line regimens The aim in a developing country is to have a smear For patients whose treatment with the first-line service on a large enough scale to grade the bacterial drugs has failed. important conclusions. or the and the possibility of instituting adequate supervi- finding that the patient's organisms were still sensi.. It may well be possible. the other not only the importance of concentrating the is in the presence of very low degrees of primary resources of the therapeutic services on patients isoniazid resistance (Canetti et al. In both these (d) sensitivity tests for individual patients. sion of the reading of the smears. . a The minimum aim of a bacteriological service in a direct test. their most tance have been encountered in two situations in important use is for diagnostic purposes. which is available with certain methods. ensure as far as possible that all patients brought One is unsuccessful treatment with thioacetazone under treatment are. in taking their medicament. can be regarded as (a) smears. smears. to formulate policies for further treat. for example. If drug sensitivity were common in the com. for diagnostic purposes in patients cal terms. If resistance were common. in order to patients who have never been treated with the drug. often entails some degree of centralization of the or evidence that the second-line regimen had itself examination of sputum specimens. at intervals. so From the point of view of their value in a tubercu. application of sensitivity testing could be of greater Therefore. If a further course of second-line drugs is research is needed to see to what extent the gains required and it is uncertain whether resistance might might be outweighed by the disadvantages of delay have been acquired to one or more of them. sensitiv. these patients may have drug. the likelihood smear examinations to permit an accurate diagnosis of finding sensitive or resistant organisms varies on smear and also to assess the progress of therapy according to the standards of treatment in the com. would be given. Smears. 1967). a single positive result on smear examination. and operational failed. has failed to respond adequately to a regimen of munity. high degrees of in vitro ethionamide resis. (b) cultures. There are also problems of cross-resistance. in the outlying services. frequently yield sensitive organisms. should not be reached on the basis of normally be resumed. Furthermore. the priority in the individual patient tivity tests may be ranked in the following order of is to test cultures from those who have "failed" priority: during or after treatment with first-line drugs. and at 6 and 12 months. clear-cut " failures " of the regimen in bacteriologi. cultures and sensi. referred to above. cultures and sensitivity tests in control pro. The treatment with second-line drug combinations. Sensitivity tests for epidemiological purposes (dis- grammes cussed earlier) have a somewhat lower priority. by the systematic of 6 and 12 months of therapy. when the results of smear examinations general value than testing in the individual patient. or with isoniazid plus thioacetazone which disease. treatment with the first-line drugs would chemotherapy. its use tive to more effective and acceptable first-line drugs. discussed above. However. is very informative. the grade of positivity should be recorded. developing country should be to perform enough would be of particular value. becomes possible to undertake cultures. for example. that a patient ment. patients who have had a relapse after stopping chemotherapy (c) sensitivity tests for epidemiological purposes. 2 smears collection of data in cases of treatment failure. who are bacteriologically negative on sputum smear sensitive organisms because they have been irregular examination. have sensitivity tests for the individual patient. as should be performed at each assessment. Such an epidemiological of the sputum. in fact. If possible. This recommendation takes into account has resulted in resistance to thioacetazone. For When. treatment with a second-line content of the sputum smears monthly in the first regimen can be safely initiated without undertaking few months of treatment. However. The indications for changing a second-line vantages of allowing more specimens to be examined regimen might be the occurrence of toxicity. circumstances a rapid sensitivity test. CANETrI AND OTHERS bacterial content of the sputum. actually suffering from the disease but also the prior- ity of bringing all infectious cases under treatment. are read. in reporting results and the possible loss of interest ity tests are likely to be of value. As losis control programme. if " fall and rise " phenomenon in the bacterial content available. 28 G. the minimum frequency being at the end munity. sensitivity tests to the second-line drugs to be intro. suffering from the alone. with the development of the service. it example.

MYCOBACTERIAL DRUG SENSITIVITY TESTS: RECENT ADVANCES AND PRACTICAL USE 29 II. after preparation. stored at 4°C.4 mm) Stock solutions of isoniazid. and then. The range required for the test strain is Nichrome loop.25-fl oz (7-mI) screw-capped bottles inhibited by either 8 pg/ml or 4 . STREPTOMYCIN AND PAS concentration inhibiting growth (as defined above) of the standard sensitive strain. but the definitive concentrations noted below. candle or sintered-glass . with a volume. After the test has been in use for a period. Tests are set up little loss of information. sterilized with a membrane.ug/ml of together with 6 glass beads 3 mm in diameter. Procedure with different drugs Isoniazid. with a 3-mm external Storage diameter 27 SWG (wire diameter 0. a loopful of the suspension is spread PAS are usually prepared at monthly intervals and on the surface of each slope of the sensitivity test. SENSITIVITY TESTS USED BY THE MEDICAL RESEARCH COUNCIL OF GREAT BRITAIN FOR FIRST. Mitchison. and older growths Medical School. The medium is reading is made at 4 weeks. A. Stock solutions are prepared in distilled water and 'British Standard Wire Gauge. prepared by shaking the 7-ml bottle for 1 minute on a mechanical shaker.4 ml of sterile distilled water more for resistant strains. of 2 mm3 (approximately 2 mg moist weight the need to determine a resistance ratio of 2 or less of bacilli). Short test referred to below as the standard inoculum. A reading may medium without potato starch (Jensen. is tested in each set of tests and again within each set if the batch of medium is changed. colonies. a drug-free slope is set up for each stored at 4°C and can be used for at least 2 months strain tested. The resistance ratio (RR) is the minimal concentration inhibiting growth (as defined above) of the test strain divided by the minimal TESTS FOR ISONIAZID. RR = 8 or more). A suspension is MIC = 8 jug/ml. in the same Inoculum set of tests. in the of the presence of resistant strains. However. The be made at 2 weeks to give a preliminary indication drugs are added before inspissation. streptomycin in a series of batches of tests. H37Rv. screw-capped bottles and is inspissated once for " growth " is d2fined as the presence of 20 or more 50 minutes at 85°C. a representative sweep from the determined by the variation in the minimal growth is taken on the loop. worker should initially weigh a number of such RR = 2 or less) and 32 jug/ml as the highest loopfuls of growth to ensure that there are actually concentration (test strain grows on 32 jug/ml.g/ml.tg/ml. H37Rv MIC = 4. it is possible that slightly inoculum is composed of young viable organisms.AND SECOND-LINE DRUGS 1 MEDIUM Incubation and reading of tests Sensitivity tests are done on Lowenstein-Jensen The slopes are incubated at 37°C.ug/ml as the lowest important and depends on estimating the amount of streptomycin concentration in the test strain range growth on the loop as 2 mm3: it is advisable that the (test strain MIC = 8 . London. H37Rv. are indicated with an are subcultured if necessary to ensure that the asterisk below. if H37Rv is contained in 0. 1955). the prepared from the growth on primary diagnostic range of drug concentrations may be shortened with Lowenstein-Jensen medium slopes. Drug-containing media as well are As a control. The sensitivity tests are set up with an inoculum. H37Rv 2 mg of growth on his loop.7 mm) laboratories. Thus. The standard sensitive strain. For all tests. and a report that a strain dispensed in about 5-ml amounts in 1-fl oz (28-ml) is sensitive should not be given earlier. different concentrations might be needed in other With a 22 SWG 2 (wire diameter 0. then it Standardization of the size of the inoculum is would be necessary to have 8 . The concentrations that not later than 2 weeks after the slopes become could be omitted in tests at the Royal Postgraduate positive (usually within 3 days). The growth taken on the loop is then for sensitive strains and a resistance ratio of 8 or discharged into 0. and by by eye. streptomycin and Nichrome loop. judged inhibitory concentration (MIC) of H37Rv. I Prepared by Professor D.

Ethionamide-containing medium should be used within 2 weeks of Streptomycin. 40 . set up on slopes * Not necessary in short test. Drug-containing medium Resistant: growth on 1 cg/ml isoniazid or growth should be transferred to a refrigerator as soon as on 0. Test strains are set up on a control.ug/ml sodium PAS concerned. 1 and 2 . preparation. 2. since tubercle bacilli growing on this concentration are catalase- negative. niazid * Not necessary in the short test. For these two drugs. 0. for * Not necessary in short test.2 . minimum time possible. of the usual 20-colony minimal inhibitory concentra- Sensitive: a resistance ratio of 2 or less tions obtained in these titrations is calculated. from sterile ampoules of sodium PAS dihydrate.tg/ml.07. 10.tg/ml. 32. but cycloserine-containing medium is Stock aqueous solutions are prepared with aseptic more stable. sulfate. 10 lbf/in2 (0.tg/ml sodium PAS slope and on one slope containing the lower. the concentrations should be 0. at all stages in the 0.1 N HCI.Ag/ml followed by growth on possible after inspissation and. if the geometric test done from the control slope or on mean minimal inhibitory concentration for ethiona- another culture from the same patient. Procedure Concentration in test medium. Thus. the inclusion of Stock solutions and storage of medium 50 gug/ml is helpful in the identification of atypical mycobacteria. However. Preliminary titration Concentrations (before inspissation) in test medium.25*.2 and 1 sg/ml iso. Kanamycin and viomycin are both precautions from sterile ampoules of streptomycin heat-stable. respectively. 5* and 50* Htg/ml isoniazid the same way as in tests for isoniazid. the concentrations would be 28 . and PAS. it should be kept at room temperature for the same patient. 16.tg/ml streptomycin usual end-point for sensitive strains. patients is done in several batches of medium mycin containing drug concentrations above and below the H37Rv: 2.ug/ml ethionamide. 4. trations are referred to below as the lower. 2 pre-treatment dihydrate control strains are also included. A preliminary titration of culture from 20-25 Test strain: 8. The geometric mean Definition of resistance. Sterile water and containers are used but the final Sensitive: no growth (less than 20 colonies) on solution is not sterilized. CANETTI AND OTHERS filter. drug-free Test strain: 1*. The results with these control Definition of resistance.2 fig/ml isoniazid. The standard inoculum is prepared and used in Test strain: 0. the dihydrate middle and the upper concentrations of the drug H37Rv: 0. should be made up fresh for each batch of medium. whereas atypical mycobacteria are often catalase-positive. strains are collected at intervals of 6 months to As for streptomycin.2 ug/ml in a further test done from the control slope or on a culture from the test. Cycloserine and especially ethionamide are both heat-labile. and the geometric means calculated as a . Seitz filtration should not KANAMYCIN AND VIOMYCIN be practised as it removes some of the isoniazid. The mean is then multiplied by 1. ethionamide.5. 0.05. containing the range of concentrations used in the preliminary titration. 40 and 80 .7 kgf/cm2)). 0. 1 year.025. 64 * and 1024 * tg/ ml strepto. In each batch of tests.tg/ml and 56 jug/ml. or by very brief autoclaving (5 minutes at TESTS FOR ETHIONAMIDE. streptomycin H37Rv: 0. Ethionamide is made up 0. 20. the Stock solutions are prepared as for isoniazid or middle and the upper concentrations.1. These concen- PAS. mide is 27 . CYCLOSERINE. 0. 1. to give the three drug concentrations to resistance ratio of 4 followed by a resistance ratio of 4 or more in a further be used in the routine test. 4. Thus. 8 and 16* . Inoculum Concentrations in test medium.1 (a 1.30 G.2. 8 and 16 * .4- Resistant: a resistance ratio of 8 or more or a fold series). stock solutions Definition of resistance. and diluted in 0.5 and 2. 1.

In these areas. However.sg/ml or even 50 ug/ml pyrazinamide). Medium TESTS FOR THIOACETAZONE Acid medium is prepared by adding N HCl very Tests for thioacetazone sensitivity are of little gradually and with continuous shaking to the prognostic value in areas of the world where a medium before inspissation (approximately 50 ml proportion of wild strains are naturally resistant to N HCl is required for 1. middle and upper concentra. Hence. the pyrazinamide. (Growth is usually inhibited by 25 jug/ml. 1968).0 litre medium) until the the drug since it is difficult to distinguish between medium is at pH 4. . Once acidified. MYCOBACTERIAL DRUG SENSITIVITY TESTS: RECENT ADVANCES AND PRACTICAL USE 31 check that the lower. Inoculum. Readings should be made with a lens.g. The test may be simplified by using following method can be used. and read after 4 weeks' incubation. 50 and 100 . As for the first-line drugs. When inoculating the slopes. Sensitive: no growth on the lower concentration Resistant: growth of 10 colonies or more on (e. 25. 100 jig/ml pyrazinamide. Medium at pH 4. The medium may be stored with a regimen of isoniazid and thioacetazone for I month. Resistant: growth on the middle concentration or It is probable that strains with acquired resistance the upper concentrations or growth on to pyrazinamide always have very high degrees of the lower concentration followed by a resistance.tg/ml pyrazin- amide) but a duplicate pair of drug-free and drug. or in the water of (particularly important for ethionamide tests).85 0. strains and the response of patients to treatment for 50 minutes at 85°C.. similar growth in a further test done on Medium at a pH more alkaline than 4.ug/ml pyrazinamide. regional variation in the characteristics of The medium must be acid and colonies take longer Myco. natural thioacetazone sensitivity of pre-treatment ml screw-capped bottles and inspissated. resistant strains. readings should be made at 6 weeks the optimum pH for strains from different areas of after inoculation. as usual. slopes containing 25 . Fewer studies using the Medical Research Council Test strains and strain H37Rv are inoculated on to sensitivity test methods have been done in areas slopes containing 0. Incubation and reading of tests Definition of resistance. the world. tuberculosis renders it necessary to determine to grow.85 is a little the control slope or on another culture less efficient in distinguishing between sensitive and from the same patient. in the example. the medium should because there is no strong association between be distributed without delay in 7-ml amounts in 28. amide). occasional sensitive strains may grow on Definition of resistance. the not to dig the loop into the soft surface of the results on these controls also serve as a check that medium and not to inoculate the margins of the the batch of medium used has not deteriorated slope near the bottle walls. care must be taken tions are still correct.ug/ml where natural resistance is rare.95) was necessary to get satisfactory Pyrazinamide sensitivity tests are set up with a growth of many strains from patients from Uganda. All tests are incubated at 37°C and read at 4 weeks. as measured with a strains with natural and acquired resistance. only one drug concentration (100 . it is therefore Procedure unprofitable to do sensitivity tests to the drug. (Mitchison. incubation and reading of tests containing slopes should then be set up from each Slopes are inoculated with the standard inoculum strain. In any batch of tests. preliminary Sensitive: growth of less than 10 colonies on readings at 2 weeks may indicate the presence of a 100 .05. incubation and reading of tests pH medium (4. 1:10 dilution of the standard inoculum suspension. 28 ug/ml ethion. taking into account the most minute colonies. but resistant strain. This is glass electrode. Thus. a slightly more alkaline Inoculum.85 has been found to support the growth of the great majority of strains TEST FOR PYRAZINAMIDE from British. However. condensation. South Indian and Chinese (Hong Kong) patients.

CANETTI AND OTHERS Stock solutions and storage of medium Procedure A stock solution of 6400 . Below a certain proportion. that are resistant to antibacillary drugs. Rist.32 G. except for research All strains of tuberculosis contain some bacilli purposes. The triethylene glycol should be warmed at strain is not fully sensitive to the drug. III. a high and a low one. in resistant strains. and fresh pipettes control strain is not recommended. above. thioacetazone and rifampicin. more. Strain triethylene glycol (Trigol. should be used only in laboratories that are highly specialized ' Canetti et al. Canetti and Dr N. After the the strain is classified as sensitive.2 The simplified variant is suitable for cotton-wool plugs and are then covered with rubber current practice: with the criteria adopted. I solubility in water is very low. Grosset. the There exist two variants of the proportion medium is coagulated at 85°C for 50 minutes. but since this glycol. stock solutions and medium should be stored in the Resistant: growth on 2 /ugfml thioacetazone or dark. as drug has been added to the medium and after resistant. are inoculated on drug-containing and L6wenstein-Jensen medium with or without drug-free medium. dissolved in distilled water. The ratio of the number of slopes used are 17 mm x 170 mm and contain 7 ml colonies obtained on the drug-containing medium to of medium.uglml and 0.25 . which requires a wider range The concentrations are: of drug-containing media and more work. 2. medium indicates the proportion of resistant bacilli The control medium without drugs is prepared at the present in the strain. medium. for media with almost negligible proportion of sensitive strains to be ethionamide.tg/ml. same time as the drug-containing media. 4 and 8 . THE PROPORTION METHOD (THE SIMPLIFIED AND THE STANDARD VARIANTS) 1 PRINCIPLE OF THE METHOD in sensitivity testing. The drugs. the proportion of such bacilli is THE SIMPLIFIED VARIANT 3 considerably higher than in sensitive strains. it has relatively little advan- tage over the simplified variant. All 2 yg/ml thioacetazone. However. the medium has been distributed in the slopes. . 1. but are heat-stable. whose Prepared by Dr G.4 the number of colonies obtained on the drug-free are incorporated in the medium before coagulation. 1 month.g/ml thioacetazone. The method: the simplified variant and the standard slopes are left at room temperature for 24 hours with variant. its use caps and stored at 4°C. British Drug Houses). in distilled water. concentrations of drugs employed Two appropriate bacillary dilutions. 1 % solutions in pure ethylene 2 Both variants of the proportion method were worked glycol are first prepared.5. Only one concentration is employed per drug.tg/ml thioacetazone is Test strains are inoculated on to slopes containing prepared by dissolving 64 mg thioacetazone in 10 ml 0.12 . The subsequent dilutions are made out in collaboration with Dr J. The proportion method consists in calculating the Culture medium. The stock solutions are self-sterilizing. L6wenstein-Jensen medium to give a final concentra. The colonies on both media. misclassified as resistant and a very small proportion Concentrations of drugs employed of resistant strains to be misclassified as sensitive. The standard variant. and H37Rv may also be inoculated on to slopes dilutions of this solution are also made in triethylene containing 0. The period of validity of the under adequate technical conditions causes an media stored at 4°C is 2 months. (1964). and are added to Definition of resistance. its use as a 37°C to reduce its viscosity. should be used for each dilution step. incorporation of drugs into the proportion of resistant bacilli present in a strain. Sensitive: no growth (of 20 colonies or more) on tion of 0.5% triethylene glycol in the medium. in order to provide numerable potato starch is used for all the resistance tests. 0. 'For ethionamide.

36).01 ml. The drug are inoculated with a loopful of each dilution. slopes. The slopes are put in a stand at a very slight angle from Inoculum the horizontal and placed in the incubator at 37°C. Criteria of resistance 'If sodium PAS is used to prepare the solution. flat-bottomed flask containing wool plug. The loop should be of platinum (wire diam.1 ml of the 2 chosen dilutions. the 2 inocula are drug is required: the strain is classified as resistant.. the inoculum for If. produces PAS 2 0. as resistant. MYCOBACTERIAL DRUG SENSITIVITY TESTS: RECENT ADVANCES AND PRACTICAL USE 33 Isoniazid 0. 5 ml of distilled water are added ered with rubber caps and left in the incubator at slowly under continuous shaking. The results are read for the first time on the For further processing and for inoculation. Below a of distilled water and shaking. down Cycloserine 30 ug/ml to 10-5 mg/ml). The opacity of the 370C. no further reading of the test for that 10-3 mg/ml and 10-5 mg/ml.7 mm) and have an internal diameter of 3 mm. The 2 bacterial bacilli) for the control slopes and the high inoculum dilutions required for inoculation with the loop are 10-2 (10-4 mg of bacilli) for the drug-containing slopes. With a spatula. no rubber caps are placed on them. The colonies are counted only on the a calibrated loop or pipettes may be used. The dilutions are prepared by 10-fold dilution reading is made on the 42nd day: this provides the definitive result. 0.5 ml of distilled Ethionamide 20 ug/ml Kanamycin 20 . the 2 inocula are respectively The average number of colonies obtained for the 10-4 mg and 10-6 mg of bacilli for each slope. without touching the cotton- placed in a spherical. 1 Dihydrostreptomycin sulfate.9 is used (see under bacilli equal or superior to that indicated below is " Test of sensitivity to pyrazinamide ". Streptomycin1 4 .38 g of Any strain showing a proportion of resistant the salt is employed for 1 g of p-aminosalicylic acid. 1. either 28th day. Pipettes.2 . at a concentration corre- sponding to 4 zg/ml base. ' Lowenstein-Jensen medium at pH 4.ug/ml water. respectively. This inoculum either may be the Loop. The ratio between the dilution 10-2 mg/ml into a small tube containing 2 ml second figure and the first indicates the proportion of resistant bacilli existing in the strain. according to the criteria indicated below. the dilution 10-4 slopes indicates the number of resistant bacilli mg/ml is produced by discharging 2 loopfuls of the contained in the inoculum.g/ml discharged into 4. the each slope is 0. produces the dilution 10-2 mg/ml. bacterial suspension is then adjusted by the addition of distilled water to that of a standard suspension Reading of tests containing 1 mg/ml of tubercle bacilli (or BCG). for each If the result at the 28th day is " sensitive ". etc. The pipette is changed for each dilu- Viomycin 30 jug/ml tion. p. Similarly.5 ml of the bacterial sus- Thioacetazone 2 pg/ml pension 10-1 mg/ml. classified as resistant to the corresponding drug: . proportions are reported in terms of percentages. discharged into 4. a representative sample of The liquid should cover as much surface of the 5 mg-10 mg is taken from the primary culture and medium as possible. When the liquid part of the inoculum 30 glass beads 3 mm in diameter. The 2 control slopes indicates the number of culturable dilution 10-2 mg/ml is produced by discharging particles contained in the inoculum.5 ug/ml the dilution 10-1 mg/ml.g/ml steps (0. a second slope.5 ml of the bacterial suspension 1 mg/ml. again. The flask is shaken has evaporated (24-48 hours) the slopes are cov- for 20-30 seconds. Ethambutol 2 pg/ml The inoculated slopes are plugged with cotton- Rifampicin 40 pg/ml wool. same for the control slopes and the drug-containing eter 0. Two slopes of certain proportion (the critical proportion) the strain medium without drug and 2 slopes of medium with is classified as sensitive. 10-4 mg and 10-6 mg of bacilli.1 ml. the 2 bacterial result of the reading made on the 28th day is dilutions required for inoculation with the pipette are " resistant ". or it may be the low inoculum (10-6 mg of such a loop delivers 0. Two slopes of medium without drug and Capreomycin 20 tg/ml 2 slopes of medium with drug are inoculated with Pyrazinamide 3 1oo jLg/ml 0. above. Accordingly. mg/ml and 10-4 mg/ml. If pipettes are used. The average 2 loopfuls of the bacterial suspension standardized at number of colonies obtained for the drug-containing 1 mg/ml into a small tube containing 2 ml of distilled water and shaking. slopes seeded with the lowest inoculum that has produced growth.5 ml of distilled water.

30 10 40 1 Concentrations of drugs employed Viomycin 20 50 The 3 (or sometimes 2) concentrations of drugs 30 10 employed are shown in the middle column of 40 1 Table 1. . CANETTI AND OTHERS Drug concentration Critical proportion TABLE 1 (jig/ml) for resistance (%) CRITERIA OF RESISTANCE APPLICABLE Isoniazid 0. concentration) if the average number of colonies on the control- slopes seeded with the low inoculum is less than 20.25 10 bacilli. 2The rationale for these criteria is discussed by Canetti c The criterion indicated is tentative.2 1 IN THE STANDARD VARIANT OF THE PROPORTION Streptomycin 4 10 METHOD a PAS 0.1 b taken. b This criterion cannot be employed for the diagnosis of I The criterion indicated is tentative. Ethambutol c 1 50 Criteria of resistance 2 10 The critical proportions indicating resistance are 3 1 shown in Table 1. The concentrations within boxes.1b For the calculation of the proportion of resistant PAS 0. owing to limited ex- et al. (1963). The only difference consists in the greater number of drug concentrations tested. 33) 1 slope of the main drug 1100 10 concentration and 1 of each subsidiary drug 400 1 concentration are inoculated. 10 The preparation of the culture medium.1 1 Viomycin 30 10 0. owing to limited sensitivity (<0. on the 42nd day. regardless of whether this count is obtained Thioacetazone 1 50 on the 28th day. perience. or on the 28th day 2 10 with one medium and on the 42nd with the other. With each 40 1 of the 2 bacillary dilutions used as inoculum (see Pyrazinamide 50 50 under 'Inoculum ". the highest count obtained on the drug-free 0.2 Rifampicin 20 10 40 1 Reading of the results The results are read for the first time on the a The main drug concentrations and the criteria for them are 28th day.1 % bacilli resistant to the corresponding drug experience.5 1 Thioacetazone 2 10 Concentration Critical proportion Ethionamide 20 10 Drug (ig/ml) I_ for resistance Kanamycin 20 10 Cycloserine 30 10 Isoniazid 0. are the main concentrations. which are Capreomycin 20 10 those used in the simplified variant.21 b Pyrazinamide 100 10 Ethambutol 1 2 10 Streptomycin 4 10 Rifampicin 40 1 8 0. 4 1 Ethionamide 10 50 THE STANDARD VARIANT 20.2 1 Capreomycin 20 10 1 0. p. the 40 1 preparation of the inoculum. the bacillary dilutions Kanamycin 10 50 employed for inoculation and the numbers of 20 10 control slopes inoculated with each dilution are 30 1 exactly the same as for the simplified variant (see Cycloserine 20 50 pp. 32-33). the others are subsidiary.51 1 and on the drug-containing medium should be 1 0. The procedure of counting the number of boxed.34 G.

a direct test may be is classified as such: no further reading of the test is made. in simplified variant. provide satisfactory numbers of culturable particles However. If the result is " resistant ". examination demonstrates a sufficient number of 2The criteria for ethambutol are not valid in direct tests. If they indicate Inoculum " resistance " for no subsidiary drug concentration (nor for the main drug concentration) the strain is The inocula indicated above (p. ing criteria for resistance are applied for definitive classification of the strain. criteria variant (inoculation of one concentration per drug) of resistance for the subsidiary drug concentrations or as the standard variant (inoculation of more than may be slightly less reliable than those for the main one concentration per drug) in exactly the same way drug concentrations. second test. The mixture the slopes without drug and on the slopes containing is triturated with the pestle for 1-2 minutes. the ing of the colonies on the other drug concentra. smaller. is obtained. wrapped the main drug concentration. and the correspond. the reading of the results and the Application of criteria of resistance for subsidiary application of the criteria for resistance 2 are exactly drug concentrations the same as in the previously described tests. strains for the test: more than 10 and less than 100 colonies showing resistance at one subsidiary drug concentra. the 2 dilutions chosen for inoculation are 10-1 subsidiary drug concentrations. owing to different growth conditions. In THE DIRECT TEST I If a method involving centrifugation of the treated sputum specimen is used. in sterile paper. required for that drug. the criteria again indicate " resistance " for one subsidiary drug concentration (or for both. If the result for the main drug concentration is If the smear has shown more than 10 bacilli per " sensitive ". as indicated below. owing to a dearth of culturable particles in the tions. the colonies are also counted on the field. mortar. incubated at 37°C forN 50 minutes for the calculation of the proportion of resistant and neutralized with a few drops of 15% sulfuric bacilli the highest count obtained on the drug-free acid. However. tion are classified as resistant without further However. is 3-4 weeks. 33) usually definitively classified as sensitive. applied under the conditions specified above indicate since the frequency of failures of the direct test "resistance" for both subsidiary drug concentra. the strain oil-immersion-objective field. The product thus obtained constitutes and on the drug-containing medium should be dilution 1. the yield may prove retesting. If. dilution 1 will be provided by the centrifugation-sediment resuspended in 4 ml of distilled Any sputum specimen in which microscopic water. and 10-3. the strain is ADDITIONAL RECOMMENDATIONS definitively classified as resistant. The saving of time. for the slopes with comparison to the indirect test." using pipettes. for the sake of drug concentration. in some laboratories. If the result is " sensitive ". the strain is retested. An equal quantity of 4 % NaOH and On the 42nd day. may also be inoculated on 2 tubes without drugs). MYCOBACTERIAL DRUG SENSITIVITY TESTS: RECENT ADVANCES AND PRACTICAL USE 35 colonies and calculating the proportion of bacilli bacilli may be submitted to a direct test by the resistant to a given drug is the same as with the proportion method. per control slope seeded with the smaller inoculum. the simplified variant If they indicate " resistance " for one subsidiary should be generally preferred. if the criteria as the indirect tests described previously. at the economy. If the result is " resistant ". The direct test may be used as the simplified For mathematical and biological reasons. regardless of the date at which this count steps.1 Further dilutions are prepared by 10-fold taken. As stated above. If the smear shows at least 1 bacillus per concentration. the counting on the 28th day may be A smear is prepared from the purulent part of the restricted to those containing the main drug sputum. If the smear has shown 1-10 bacilli per field. drugs. The number of tubes inoculated with each dilution. . or for the main drug concentration). Consequently. inoculum is not negligible. 2 dilutions chosen for inoculation are 1 and 10-2 (10-3 tions is needed: the strain is classified as resistant. no count. However. the colonies are counted first on 3 drops of tincture of litmus are added. for isoniazid and streptomycin. the strain is definitively classified as resistant. Two millilitres of sputum are placed in a sterile the test is read again on the 42nd day.

negative influence of dysgony on the number of colonies that develop is usually more pronounced in the slopes seeded with the smaller inoculum than in CONTROL TESTS those seeded with the larger inoculum (and may vary considerably between slopes seeded with the same Routine check of drug-containing media inoculum) the proportions calculated from one Each new batch of drug-containing medium inoculum are sometimes highly inconsistent with should be checked for drug activity. Appropriate dilutions are inoculated on ment may be highly misleading. the test should be The amount of growth produced by a known repeated (from the control slope). If the number of bacilli growing on the acid controls amounts to less than 10 % of the Any indirect or direct test showing an average of number of bacilli growing on the standard controls. stronger than the usual ones: 10-3 mg of bacilli and Dysgonic growth 10. some are less reliable than those of tests of sensitivity to strains grow very dysgonically in the drug.8). an amount of the tests). The use of direct tests often leads to the containing and in the drug-free slopes. acid 5x10-4 mg and 5x10-6 mg L6wenstein-Jensen medium at pH 4.mg of bacilli. in order to those calculated from the other. If the situation has not changed. the smaller inoculum should be repeated (from the The 2 inocula used for the test are 10 times controls). and principally tightness of the cap). The number of culturable particles on which the calculation of the proportion A certain amount of dysgony on the drug. the more growth. the set of inocula abundant than on standard L6wenstein-Jensen employed should always be medium (pH 6. For tests of sensitivity to 10-3 mg and 10-5 mg pyrazinamide. and its results are illustrated in available in different medium containers (slopes. surface area of the medium. H37Rv-should be used throughout.9 is used.36 G. Table 2. For the control test. inoculum on the new drug-containing medium should fall within a range of values established Test of sensitivity to streptomycin previously. controls. controls. a different set of inocula may be adopted Test ofsensitivity to pyrazinamide for current use. CANETrI AND OTHERS such cases. the oxygen supply available in the container: the less The type of control test used at the Institut oxygen. dilution 1 contains 5 to 100 x 10-6 The criteria for streptomycin resistance indicated culturable particles per ml (approximately 90% of above apply to a type of container. other drugs. and no reliable disclose errors in the amount of drug dispensed or in figures can be given. the same wild strain reading of the test should be postponed for 2 weeks. 2 slopes (per dilution) of the new batch of drug- 1 With streptomycin. etc. the oxygen effect is even more containing medium and on 2 control slopes: the pronounced. but the results may be slightly different culture conditions without previous assess. of resistant bacilli is based is the number counted on containing media. control slopes with acid medium owing to the smaller yield of culturable particles should be inoculated. for instance: For tests of sensitivity to pyrazinamide. Growth from a Lowenstein-Jensen medium and a type of slope closure that have been culture standardized to 1 mg/ml may be used instead specified (p. The control test is done only once for Growth on dihydrostreptomycin-containing each batch (a repetition each time the same medium Lowenstein-Jensen medium is highly dependent on is employed is useless). is a frequent occurrence in resistance tests The results of tests of resistance to pyrazinamide and does not impair the reading. However. -for instance.) depends on several factors (amount of liquid medium is standardized opacimetrically to medium. 33).' The amount of oxygen Pasteur. the definitive heating. Paris. size of the 1 mg/ml (= dilution 1): under the growth conditions containers. growth on such a medium is less For tests on single colonies. prevailing. With or 10-3 mg and 10-5 mg many strains. Since the misclassification of resistant strains as sensitive. In such cases. less than S colonies in the control slopes seeded with the test should be repeated. The use of the same criteria under of a Dubos culture. different. A 6-8-day-old culture of H37Rv in Dubos bottles. dilutions most suitable for each drug and for the . in addition to the normal from single colonies. with normal growth on the the acid controls.

sometimes minute. the strains for due mainly to fluctuations of the test strain. an error is than one concentration per drug (standard variant of sure to have occurred. streptomycin.2 ml of greater size. a preliminary reading of drug concentration is close to 0 with the chosen the test is made. concentration. concentration per drug (simplified variant of the containing media are often considerably smaller proportion method). which the batch has already been employed should Insufficient drug concentrations (through errors in the be retested on a new batch.tLgl/mI) I (4mAg/mI) cn (0.2 . and the batch is discarded. MYCOBACTERIAL DRUG SENSITIVITY TESTS: RECENT ADVANCES AND PRACTICAL USE 37 TABLE 2 NORMAL RANGE OF RESULTS a OBTAINED AT THE INSTITUT PASTEUR WITH CONTROL TEST FOR NEW BATCHES OF DRUG-CONTAINING LWWENSTEIN-JENSEN-MEDIUM. since for most drugs the lower limit of growth on a control slope inoculated with a 10-6 dilution. slightly less sensitive than H37Rvto most drugs.5 Ag/mI) (20 pg/mI) serine (30 Ag/mI) i(30 ug/ml) (20 Ag/ml) I I~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 2-50 1-20 ++ ++ oc <100 <50 10-1 0-5 0-2 0-10 <100 ++ <100 >100 ++ 0 0-10 0-5 10-2 0 0-10 <100 0-10 <100 >100 10-3 0 0-10 <100 10-5 5->100 106 < 1-20 a Results expressed as growth (number of colonies) on the 28th day. In addition. b Dilution I = 6-8-day-old Dubos culture standardized at I mg/mi. (instead of the routine 0. the colonies on the drug. ++ = innumer- able distinct colonies. or excessive heating) are The findings indicated in Table 2 apply to the disclosed by considerably more growth on the drug. viomycin and the control test described above.1 ml). In laboratories using more slopes is equal to growth on the controls. + ++ + = confluent eugonic growth.2 ml. c Reading at 42 days: 0-50 colonies. If the concentration in the medium. If growth on the drug-containing inoculum (see Table 2). of growth of strain H37Rv compatible with the streptomycin and PAS. of the strain H37Rv compatible with the expected At the 18th to 21st day. The range of findings for increasing several times the inoculum of dilution 1 in isoniazid. 3 subcolumns in Table 2 strain. Table 2 indicates for each drug the normal revealed by insufficient growth on the medium with range of results obtained at the Institut Pasteur: they the lowest concentration used. these differences are test shows that an error has occurred. or by running a kanamycin is narrow. the colonies are usually given in Table 2. quantity of drug added. none of The new batch of medium is obviously used before which is incompatible with the expected drug the results of the control test are available. controls are those against which data have been containing slopes. USING STRAIN H37Rv AND THE MAIN DRUG CONCENTRATIONS SHOWN IN TABLE 1 Dilution Controls soniazid Strepto-i PAS Ethionamide 1 Cyclo. culture conditions (duration of inspissation of the . summarize the different types of findings. cycloserine. parallel series of control tests with a second wild the range is wider: for each. Inoculum per slope: 0. For PAS and ethionamide. and 40-60 batches of medium expected drug concentration is higher on that for the other drugs (main drug concentrations). In laboratories using only one drug On the 28th day. in order to obtain Excessive drug concentrations are more difficult to more culturable particles: it produces < 1-20 colonies detect. the proportion method) an excessive amount of drug The definitive reading of the test is made on the 28th added to all the corresponding media may be day. The inoculum per slope is 0. Viomycin Kanamycin inoculatedbb no (0. since the lower limit are based on 100-120 batches of medium for isoniazid. an excessive drug concentration than those on the controls: they may be of in the new batch may be disclosed either by borderline visibility.

36) should be results for wild strains. conditions tend to change. Some of the (2) No correlation with the clinical progress of the statistical procedures were suggested by Dr S. Even if the culture conditions are the very beginning of testing practice. under the existing culture conditions may be made in ly. each laboratory producing drug-containing media two ways. The risk is minimal with isoniazid. . 32-33): the eventually in all laboratories. H37Rv (Pasteur variant) is more sensitive to prepared. Under different requires a modification in the laboratory concerned. slope However. has to build up its own experience with regard to results of control tests for drug-containing media. They apply to specific culture conditions indicated by the reference laboratory. instead of 10%. it cannot be used at conditions. THE CALIBRATION OF SENSITIVITY TESTS1 The method for obtaining definitions of drug that best distinguish between two populations. 1 Prepared by Professor D. ethionamide Jensen medium. resistance needs to be changed does not interfere The best way of assessing the validity of resistance with the principles of the proportion method. IV. A preliminary apparently the same. carefully drugs. size of slopes. the findings may vary to some estimation of the validity of the proposed criteria extent from one laboratory to another. Growth of the H37Rv strain Table 2 is given merely for preliminary orientation. Consequent. i. A. the their validity under these conditions has been corresponding criterion of resistance most likely confirmed in many laboratories. but criteria to be used in a given laboratory is to apply merely with the validity of certain figures given in the calibration method described in Part IV below. amount of medium. The new criterion should be elaborated with the Attention has already been drawn to the importance calibration method described in Part IV. the criteria of resistance. the criterion of degree.) indicated above (pp. at least with regard to certain performed with H37Rv on 3 different. confirmation of the definition obtained.ia because of its relative stability. 32-33): technical shortcomings can be excluded. and of this factor in the case of streptomycin (see under expressed in terms of proportions. indicated in Table 2. (For instance.. Mitchison. CANETTI AND OTHERS medium. culture conditions they may require modification.) The fact that. batches of drug-containing Lowenstein- streptomycin and less sensitive to PAS. so that no preconceptions resistant.e. though such information may provide Miss Ruth Tall of the Medical Research Council's Tuber- culosis and Chest Diseases Research Unit. are probably valid for the containing med. requires familiarity findings may be different under different culture with the method of testing. culture 5% on 20 ug/ml. p. The results obtained with H37Rv are not average A control test (as described on p. and if which have been described previously (pp. and necessary. London. 36). etc. although to a lesser under certain culture conditions. the " Test of sensitivity to streptomycin ". and obtains the definitions of resistance about definitions of resistance are necessary. In the new criterion of resistance to ethionamide might be case of ethionamide and thioacetazone also. Table 1.38 G. The resistance to be described compares a population of method has the following advantages: strains that are predominantly sensitive with a (1) Pure populations of sensitive and resistant population of strains that are predominantly strains are not required. If the results are within the limits and thioacetazone than most other wild strains. given in Table 2. laboratory concerned. as H37Rv is nevertheless used for checking the drug. which should be applied closure. this approach. Modification of the criteria of resistance Testing of sensitive and resistant strains received from a highly qualified reference laboratory using the The resistance criteria indicated above have been resistance criteria indicated in Table I arrived at by comparing large numbers of wild strains and of strains from patients treated with the If the proportions of strains classified as sensitive corresponding drug during increasing lengths of and as resistant are significantly different from those time. Madras. Radhakrishna patient under treatment with the drug concerned is of the Tuberculosis Chemotherapy Centre.

if tests of the procedure is improved by the inclusion of are available on at least 2 strains from a patient. is set out in Table 3 (R. not essential. personal communication). between a central reference laboratory and an outlying laboratory) it is unnecessary to take (2) A large sample of strains that may have special precautions to guard against instability of acquired resistance must be available. subtracted from the corresponding cumulative percentage for the probably resistant strains. including a range of inoculum dilutions data should be recognized. concentration of 16 .5 % or more and of 1 % or more. POPULATIONS OF STRAINS EVALUATION OF RESULTS The following groups of strains are required. and a value so as to diminish the chance of misclassifying few that may well still be sensitive. should be obtained from of sensitivity in distinguishing between sensitive and patients who have remained sputum-positive during resistant strains. and preferably as many as the percentage is a measure of the efficiency of each level probably sensitive strains. a single " population sensitivity-test methods. Correspondingly.4%) was obtained by using should contain a similar ratio of probably sensitive a definition of resistance of a minimal inhibitory to probably resistant strains. The latter At least 50 strains. Alternatively. For each level of different patient. the effect of grouping of the study " test. At least 100 (and preferably tests. the cumulative primary or acquired resistance to the drug concerned percentage for the probably sensitive strains is is immaterial. The results of the sensitivity tests are set out as Probably sensitive strains cumulative distributions of the results with the These strains are conveniently obtained from any probably sensitive and the probably resistant patient who has not been treated with the drug populations. though proportion methods. The aim should be to include often advisable to choose a definition at a level of not only strains that are very likely to be resistant. the tests should be peak difference between percentages (55. for each strain to be obtained from a Riddell. when feasible. to reach a peak. the difference between these strains it is possible to use more than 1 strain percentages usually increases. The efficiency sensitive strains as resistant. absolute-concentration and proportion methods. In some methods of . It has the following disadvantages: allowing a comparison of these two methods. the peak difference each on a separate batch of medium. instance. sensitivity very slightly higher than this optimum but also some in which resistance is emerging. can be read by the sensitivity testing may be compared. As the level of sensitivity in the treatment with the drug concerned. the numerous strains of doubtful resistance in the definition of resistance giving the peak difference probably resistant population. to give Probably resistant strains the " difference between percentages ". and from the same patient provided that they are then decreases again. between percentages may be used as an indication of resistance provided that it shows all the strains from SENSITIVITY TESTS the same patient to be resistant. Each batch between percentages (52. resistance in the probably resistant strains. The peak value indicates the obtained at different intervals of time following the most efficient definition of resistance. In (1) Large numbers of sensitivity tests must be making comparisons between laboratories (for done. It is advisable. MYCOBACTERIAL DRUG SENSITIVITY TESTS: RECENT ADVANCES AND PRACTICAL USE 39 (3) The efficiency of radically different methods of and drug concentrations. The presence of a few strains with sensitivity in these distributions. Considering first the minimal inhibitory concen- If the method is to be used for the calibration of tration obtained in the streptomycin sensitivity tests one sensitivity test. tomycin of 0. In selecting distribution increases. several sets of tests are set up. of the example (Table 3). W. or.tg/ml dihydrostreptomycin or When several methods of testing sensitivity (to the more.ug/ml dihydrostrep- probably sensitive and probably resistant strains. read by the absolute-concentration and the more) strains are required. the different methods can be made In the application of this procedure to different part of one larger test. with proportion tests. it is start of chemotherapy. the same drug) are being compared.4 %) was set up simultaneously on the same populations of obtained with proportions on 4 . Thus. An example for streptomycin sensitivity concerned previously. However.

the measure of sensitivity can be taken Cleaily. % No.6 84 47.5 44. If the two dihydrostreptomycin were not used in the tests. in the absolute-concentration method. Bearing this in mind. CANETI1 AND OTHERS TABLE 3 CUMULATIVE DISTRIBUTIONS OF SENSITIVITY TO DIHYDROSTREPTOMYCIN Number and cumulative percentage of strains Probably Probably Difference Measure of sensitivity sensitive Wild resistant between (PS) (PR) percentages (PR-PS) No.3 9 5.0 0.ug/ml instance. definition of resistance. of strains tested 192 170 177 reading tests.6 0.8 118 66.4 16 or more 9 4. On the other hand. 9 drostreptomycin or more might have been more probably sensitive strains would be considered as efficient in detecting resistance than a minimal " resistant " if they had a minimal inhibitory inhibitory concentration of 16 jug/ml or more.1 51. One way of allowing for this difficulty is as readings of the minimal inhibitory concentration follows.3 32 18. concentration of 16 .1 0 0.ug/ml and 8 tg/ml and. between 8 . The distributions of the probably sensitive cannot be obtained between the drug concentrations strains for the two types of test are examined in the used in the test.4 98 55. many between methods with continuous distributions of methods of reading sensitivity are discontinuous.2 7 4. For 4 .ug/ml dihydrostreptomycin or However. though it is desirable in A further feature of the grouping effect is that it practice to choose a round number that can be tends to make accurate comparisons difficult remembered easily.7 50 % or more 4 2.ug/ml with concentrations between 4 .7 4 2.0 Total no.1 23 13.9 149 84.7 4 2.1 % or more 29 15.1 25. sensitivity and those with discontinuous distribu- For instance.ug/ml and 8 jug/ml dihydrostreptomycin of 5% or more.0 0. tions.3 111 62. methods are of fairly similar efficiency. it is possible for and 16 .7 55.ug/ml dihy.ug/ml should have been included if the routine use to choose any value of the proportion absolute-concentration method for streptomycin (such as. % No.2 42. no such readings are available since slopes more or if they had a proportion on 4 . say. in the proportion method.4 5 % or more 9 4.4 101 57.2 73 42. tant" by both methods.4 50.0 48 27.23 %) to examine as a potential were to have been calibrated with greater efficiency.4 32 or more 5 2.01 % or more 81 42. % Minimal inhibitory concen- tration (Mg/ml dihydro- streptomycin) 8 or more 39 20.6 55.4 1 % or more 12 6. 1. the difference . possibly.1 109 61.5 % or more 14 7.1 52. Thus. it is evident range of optimum discrimination.5 117 66.7 46. to find some from Table 3 that a definition of resistance of a measure of sensitivity which yields the same number minimal inhibitory concentration between 8 ttg/ml of probably sensitive strains consid'ered as " resis- dihydrostreptomycin or more and 16 . intermediate concentrations between anywhere on a continuous scale of sensitivity. in Table 3.40 G.9 Proportion on 4 AgIml dihydrostreptomycin 0.

This procedure " difference between percentages ") obtainable when removes strains which are clearly resistant from comparing these strains with a probably sensitive the probably sensitive population. If not. COMPARISONS BETWEEN LABORATORIES Finally. An (for instance. The second laboratory of strains assumed to be wild. However. Such a procedure has the strains from the 4 patients concerned may have advantage that it requires less work to be done by been resistant by one measure but not by the other. the results of the comparison may not whether the provisional definition of resistance be representative of those obtained in routine tests. should be done under identical the probably sensitive population. or during the a drug. For an accurate comparison. many bacteriologists will wish to know what proportion of wild strains will be classified as The most efficient method of comparing the work resistant by any of the calculated definitions of of different laboratories. it is envisaged that it will first carry out tests period before the sensitivity test is set up in the for a trial period by whichever method it favours. leaving a group population in its own tests. keeping records of whether the strains have been the sensitivity tests. it has the In any case. mycin or more. In of sensitivity which it has tested. (1) Great care must be taken to see that no reversion towards sensitivity occurs in the strains APPLICATIONS during storage in the central laboratory. would each to send out a number of strains with varying degrees classify 4 (2. 4 . been found difficult so far to get a sufficiently good fit to all of the curves. for testing at the interpreting this result. For this purpose laboratories. a peripheral laboratory resistance.e.4y%) of the wild strains as resistant. or have been conditions in both laboratories-conditions which obtained after treatment for several months with are difficult to fulfil. MYCOBACTERIAL DRUG SENSITIVITY TESTS: RECENT ADVANCES AND PRACTICAL USE 41 between percentages at these cuts (52. would alternative statistical procedure is to attempt to fit be changed. Having accumulated laboratory.. an analysis would than in the performance of routine tests.7 % for the proportion definition) may be possibly the detailed design of the sensitivity tests considered to be approximately equivalent. peripheral laboratory. the value of the method of measuring sensitivity. and therefore belong to the (2) The tests. since they will wish to know how the and a central laboratory. e.g. but it has should be done at intervals of. and the efficiency of its tests would be definitions of resistance of either a minimal measured by the value of the " difference between inhibitory concentration of 16 . or a proportion on 4 jug/ml An alternative method is for the central laboratory dihydrostreptomycin of 5% or more. percentages" obtained. the number of wild strains found following disadvantages: resistant is satisfactorily low. one should recall that the peripheral laboratory. sive checks on the efficiency of the definitions in use ages for each method of sensitivity testing. In be done by the procedure indicated above to see consequence. In practice. In the example would then test the same probably resistant with streptomycin (Table 3) it is evident that population. especially those in the peripheral probably resistant population. and all previous subculturing of obtained before treatment. Once the tests are established. the peripheral laboratory. is to carry out the definition obtained by this discrimination procedure calibration procedure described above in both compares with previous experience. the definition of resistance. say.. a few years. succes- curves to the cumulative difference between percent.g/ml dihydrostrepto. are likely to be done with greater care a sufficient number of results.4% for the adopted for these preliminary tests was of maximum minimal inhibitory concentration definition and efficiency. the drug concentrations used). and 50. and therefore belong to the exchange strains. the central laboratory it is useful to " purify " the probably sensitive would maintain batches of probably resistant strains population by removal of any strain found resistant for each drug and would have established what was by all of the definitions of resistance chosen for each the degree of discrimination (i. during When a laboratory is initiating sensitivity tests to transport to the peripheral laboratory. the drug concerned.

On peut en revanche recourir aux deux autres programmes de chimiotherapie efficace dans ce territoire. les mycine.42 G. L'institution d'un traitement par des drogues dans la proportion des bacilles isoles et celle des bacilles de seconde ligne peut etre faite sans test de resistance a groupes en amas. la r6sistance primaire. telle methode de mesure de la sensibilite. teneur en unit6s viables varie dans des limites relative. Lorsqu'un laboratoire adopte telle ou ces drogues. etudiee. dans la pratique des quelques rares villes. En cas de en matiere de mesure de la pharmacoresistance des resistance acquise . Dans les pays en voie de d6veloppement. ments de premiere ligne. La mise en ceuvre de tests de resistance chez des malades ment etroites et soit au moins approximativement connue. bien que les avis different au sujet de leur La connaissance du nombre total de malades infec- valeur comparative et que de nouvelles recherches soient tieux 'a bacilles resistants qui existent dans une commu- ndcessaires 'a cet egard. traitement . ce qui positive apres 6 mois de traitement. d'autre part. complexe. redevient d'unites viables pr6sent. de nuire au malade. placant un regime efficace par un regime plus toxique Le pronostic qui s'attache a la resistance est different ou moins facilement accepte. sur la foi de resultats lonnage des tests permettant d'y parvenir est decrite errones ou mal interpr6tes. naute serait fort utile: mais on ne la possede que pour II est particulierement important. qu'"a un certain point la diffusion et la qualitd de la La m6thode des rapports de r6sistance ne semble pas chimiotherapie pratiqu6e dans le territoire correspondant. L'epidemiologie de la resistance se pr6sente differem- culeuse. On peut egalement appliquer les deux premi&res enquetes correspondantes apportent une donnee plus de ces methodes a la mesure de la resistance a l'isoniazide. Celle-ci tuberculeux la resistance au sens bacteriologique a n'atteint nulle part des taux alarmants. utilis6e a cet effet. L'etude comparative des pro. en cours de traitement est surtout utile dans deux circons- Des semences de poids et d'opacite identiques peuvent tances: chez les malades qui gardent une expectoration donner des nombres de colonies tres differents. mais egalement a des differences positive.il est vain d'attendre du medicament lites pratiques de recours aux epreuves de sensibilit6 homologue la moindre action th6rapeutique. apres s'etre negativee. et chez les malades tient non seulement aL des differences dans le nombre dont l'expectoration. methodes. aux differents stades des operations de lutte antituber. Dans les pays d6veloppes. assures d'une activite primaire sur les resultats therapeutiques est pratiquement suffisante pour pouvoir maintenir un niveau technique . en rem- dans le rapport. Le present document expose leurs conclusions. n'ont guere d'utilite therapeutique. Quant a la valeur comparative des la resistance acquise lors de traitements non avoues trois procedds pour l'evaluation de la sensibilite au PAS. laire. drogue et que tous les malades peuvent etre traites par cedes de recherche de la sensibilite fait apparaitre que les trois drogues de premiere ligne a la fois. ment dans les pays d6veloppes et dans les pays en voie II est preferable de definir la resistance en se fondant de developpement. d'opdrer avec des semences dont la registre des cas r6sistants. Elles fournissent la methode des proportions et la methode des rapports cependant une indication sur l'intensite des transmis- de r6sistance (resistance ratio) permettent avec un egal sions bacillaires qui se produisent dans la collectivite succes d'evaluer la resistance d'une souche a la strepto. renseignent reellement sur la r6sistance primaire. la resistance initiale. il importe qu'il I1 est tout a fait inutile de faire des tests de resistance verifie au bout d'un certain temps que les criteres de en cours de traitement lorsque les examens directs et les resistance proposes par les auteurs de la methode sont cultures montrent une diminution de la population bacil- valables dans les conditions locales: une methode d'eta. EMPLOI DES EPREUVES DE SENSIBILITE AU COURS DES PROGRAMMES DE LUlTE ANTITUBERCULEUSE Un groupe de consultants de l'OMS s'est reuni a nulle s'il s'agit de resistance a un seul des trois medica- Geneve du 23 au 26 avril 1968. selon qu'il s'agit de resistance primaire ou de resistance Les tests de resistance ne devraient etre pratiques que acquise. ou ignor6s du malade. les enquetes sur des criteres purement bacteriologiques. et comme il s'agit souvent comme corollaire clinique une diminution de dans la grande majorite des cas de resistance a une seule l'efficacite therapeutique. La resistance initiale reflate jus- elle n'a apparemment fait l'objet d'aucune recherche. avantageuse pour l'etude des medicaments de (' deuxieme Sa connaissance est tres utile pour la formulation de ligne *.c'est-a-dire apparue en cours de mycobacteries tuberculeuses et de suggerer des moda. si le malade ne les a encore jamais reques. ces enquetes la methode de la concentration inhibitrice minimale. I1 est en effet sur la resistance chez les malades non encore traites demontr6 que pour la plupart des medicaments anti. Ces specialistes se pro. CANETI AND OTHERS RI-SUMI PROGRES DES TECHNIQUES DESTINE-ES A EVALUER LA SENSIBILITt DES MYCOBACtRIES AUX MEDICAMENTS. On risque meme alors. II a ete demontre que l'incidence de la resistance dans des laboratoires bien equipes. oiu ces malades figurent sur un tests de resistance. ofu se melent deux 616- la methode des rapports de resistance n'a jamais et ments: d'une part. ai condition que le malade soit posaient de faire le point des progres recents intervenus trait6 par ces trois medicaments ai la fois.

687-703 Jensen. & Selkon. Hauduroy. 83 Canetti.. les divers examens de laboratoire en voie de developpement. 47. International Union against Tuberculosis (1964) Bull. Grosset. (1962) Beitr. K. Un. (1968) Tubercle (Edinb. D... H. L. 38 (1967) Rev. 4) les epreuves de sensi- de leur utilit6 globale dans un programme de lutte bilite pratiqu6es pour des cas individuels. Tuberc. 747-758 . 25.. 2) les cultures pratiquees dans un but diagnos. & Galvez-Brandon. REFERENCES Bartmann. 126. 217-272 J.. Rev. J. V.. Joseph. Wld Hlth Org. MYCOBACTERIAL DRUG SENSITIVITY TESTS: RECENT ADVANCES AND PRACTICAL USE 43 eleve. Dis. J. N..... Dans un pays contre la tuberculose. S... (1968) Scand. P.. 84-97 son.. N. R. 34. & Grosset.. Tuberc. Lefford. Wld Hith Org. Subbaiah. Krebs. T. K. (1963) Bull. A. 25. (1963) Rev. Dis. Tuberc.). (1966) Tubercle 565-578 (Edinb. & Ferreira. (1961) Bull. S. J. (1965) Amer. K. J.. A. 0. Dans la perspective dans un but 6pid6riiologique.. P. il est particulierement impor- peuvent etre classes dans l'ordre suivant: 1) les examens tant de respecter cet ordre de priorit6s lorsqu'on procede directs. 31. Thomas. A. & gula. 49. & Le Lirzin. Kappler. 433-474 Rist.. Tuberc. D. et soucieux de controler constamment ce niveau. Meissner. (1968) Tubercle (Edinb. resp. 49. G. Froman. Mitchison. 92. Thibier. 14 Mitchison. G. Langerovi. B. 29.). Tuberk. p. & Cetrangolo... G. pp.. int. a l'implantation des organes d'ex6cution n6cessaires. resp. (Paris). A.. G. G. in!. J. D. (Paris). 27.. T. T. D.). int. Gay. J. Mahler. 89 Canetti. 3) les dpreuves de sensibilite pratiqu6es devrait etre fortement encouragee. L. A. 36-46 Canetti.. 49. Suppl.. M. Grosset. 81-99 Inst. Mitchi. A. Un. (1962) Bull. tique chez les malades dont 1'expectoration est negative La centralisation de la pratique des tests de resistance a 1'examen direct. Canetti. W. Klin. B. (1964) Arch. 42. Suppl. & Mitchison. 250-262 Canetti. Rist. M.. Pasteur Alger. J. 32. M. Tuberc. 141-152 Un. G. (1955) Bull. Kreis.