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Tubercle 60 (1979) 239-243 PRIMARY AND SECONDARY RESISTANCE OF MYCOBACTERIUM TUBERCULOSIS IN EASTERN BOTSWANA Niels Jacob Nielsen * Mahalapye Government Hospital, Botswana Summary Of 51 patients in Eastern Botswana who denied previous anti-tuberculosis treatment, 6 (11.8 %) were excreting tubercle bacilli resistantto one of in regular use : 4 patients (7.8 %) showed resistance to isoniazid, 2 (3.9 %) to thiacetazone and none to streptomycin. Of 44 patients known to have been on previous anti-tuberculosis treatment, 31 (70.5 %) were found to show resistance to one or more of the first line drugs: 31 (70.5 %) to isoniazid, 12 (27.2 %) to streptomycin and 11 (25.9 %) to thiacetazone. No appreciable resistance was found to second line drugs. These resistance patterns, which correspond quite well with other published results from Africa, are related to the overall problem in Botswana, namely the failure of a high proportion of patients, to complete a full course of first line treatment. R&urn6 Sur 51 malades du Botswana oriental qui affirmaient n’avoir jamais recu de traitement antituberculeux anterieurement, 6 (soit 11.8 %) excretaient des bacilles resistants a I’un des medicaments de premiere ligne utilise couramment: 4 malades (7.8 %) se montraient resistants a I’isoniazide, 2 (3.9 %) au thiacetazone; aucun n’a 6te trouve resistant B la streptomycine. Sur 44 malades dont on savait qu’ils avaient recu anterieurement un traitement antituberculeux, 31 (soit 70.5 %) ont bte trouves resistants a un ou plus d’un medica- ment de premiere ligne: 31 (70.5 %) Btaient resistants a I’isoniazide, 12 (27.2 %) a la streptomycine et 11 (25.9 %) a la thiacetazone. Aucune resistance appreciable n’a ete trouvee vis-a-vis des medicaments de deuxieme ligne. Ces profils de resistance, qui correspondent tres bien a d’autres resultats publies concernant I’Afrique, sont en rapport avec la probleme d’ensemble qui existe au Botswana, a savoir I’bchec d’une proportion dlevee des malades a terminer completement la prise d’un traitment de premiere ligne. Resumen De 51 enfermos de Botswana del Este que habian declarado no haber tenido trata- mientos antituberculosos previos. 6 (11.8 %) fueron eliminadores de bacilos resistentes a una de las drogas de primera linea de uso corriente : 4 pacientes (7.8 %) mostraron resistencia a la isoniacida, 2 (3.9 %) a la tiacetazona y ninguno a la estrepto- micina. De 44 pacientes que se sabia habian sido tratados previamente por tuberculosis, en 31 (70.5 %) se demostrd una resistancia a una o m&s de las drogas de pnmera linea : *Present address: Helseverj 4. Ith, 3700 Rsnne, Denmark.

Primary and secondary resistance of mycobacterium tuberculosis in Eastern Botswana

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Tubercle 60 (1979) 239-243

PRIMARY AND SECONDARY RESISTANCE OF MYCOBACTERIUM TUBERCULOSIS IN EASTERN BOTSWANA

Niels Jacob Nielsen * Mahalapye Government Hospital, Botswana

Summary Of 51 patients in Eastern Botswana who denied previous anti-tuberculosis treatment, 6 (11.8 %) were excreting tubercle bacilli resistantto one of in regular use : 4 patients (7.8 %) showed resistance to isoniazid, 2 (3.9 %) to thiacetazone and none to streptomycin.

Of 44 patients known to have been on previous anti-tuberculosis treatment, 31 (70.5 %) were found to show resistance to one or more of the first line drugs: 31 (70.5 %) to isoniazid, 12 (27.2 %) to streptomycin and 11 (25.9 %) to thiacetazone.

No appreciable resistance was found to second line drugs. These resistance patterns, which correspond quite well with other published results from Africa, are related to the overall problem in Botswana, namely the failure of a high proportion of patients, to complete a full course of first line treatment.

R&urn6 Sur 51 malades du Botswana oriental qui affirmaient n’avoir jamais recu de traitement antituberculeux anterieurement, 6 (soit 11.8 %) excretaient des bacilles resistants a I’un des medicaments de premiere ligne utilise couramment: 4 malades (7.8 %) se montraient resistants a I’isoniazide, 2 (3.9 %) au thiacetazone; aucun n’a 6te trouve resistant B la streptomycine.

Sur 44 malades dont on savait qu’ils avaient recu anterieurement un traitement antituberculeux, 31 (soit 70.5 %) ont bte trouves resistants a un ou plus d’un medica- ment de premiere ligne: 31 (70.5 %) Btaient resistants a I’isoniazide, 12 (27.2 %) a la streptomycine et 11 (25.9 %) a la thiacetazone.

Aucune resistance appreciable n’a ete trouvee vis-a-vis des medicaments de deuxieme ligne. Ces profils de resistance, qui correspondent tres bien a d’autres resultats publies concernant I’Afrique, sont en rapport avec la probleme d’ensemble qui existe au Botswana, a savoir I’bchec d’une proportion dlevee des malades a terminer completement la prise d’un traitment de premiere ligne.

Resumen De 51 enfermos de Botswana del Este que habian declarado no haber tenido trata- mientos antituberculosos previos. 6 (11.8 %) fueron eliminadores de bacilos resistentes a una de las drogas de primera linea de uso corriente : 4 pacientes (7.8 %) mostraron resistencia a la isoniacida, 2 (3.9 %) a la tiacetazona y ninguno a la estrepto- micina.

De 44 pacientes que se sabia habian sido tratados previamente por tuberculosis, en 31 (70.5 %) se demostrd una resistancia a una o m&s de las drogas de pnmera linea :

*Present address: Helseverj 4. Ith, 3700 Rsnne, Denmark.

240 Nielsen

31 (70.5 %) a la isoniacida, 12 (27.2 %) a la estreptomicina y 11 (25.9 %) a la tiacetazona.

La resistencia a las drogas de segunda linea fue despreciable. Estos niveles de resis- tencia que corresponden perfectamente bien a otras resultados publicados, proven- ientes del Africa, tienen relacidn con el problema global de Botswana, principalmente con la alta proportion de pacientes que no logran complir un period0 complete de tratamiento de primera linea.

Resistencia primaria y secundaria del micobacterio de la tuberculosis en Botswana del Este.

Introduction So far no reports have been published on initial and acquired resistance patterns of lMyco- bacterium tuberculosis in Botswana, where tuberculosis is one of the main health problems. There were 459 newly registered cases per 100 000 population in 1976 (Tuberculosis Office, 1977). In a retrospective study by the author of the tuberculosis files at Mahalapye hospital in Eastern Botswana from 1975 to 1976, it was found that less than 25 % of the patients had completed a full course of treatment with follow up. Consequently a high incidence of drug resistance was to be expected.

The present survey was carried out at Serowe and Mahalapye hospitals in Serowe Medical Region, Eastern Botswana from October 1977 to April 1978. The purpose was to investigate the incidence of primary and secondary resistance in the area.

Material Serowe Medical Region in Central District (Eastern Botswana) has a population of approxi- mately 100 000. The incidence of tuberculosis in the area is representative of Botswana as a whole with 453 new cases registered in 1976 (Tuberculosis Office, 1977). Serowe and Mahalapye are the centres for registration, diagnosis and treatment of new cases in the area. During the time of the survey all patients appearing at one of these 2 hospitals with smear positive sputum (Ziehl-Nielsen microscopy in the local laboratories) had sputum specimens collected for culture and sensitivity testing. Both old and new tuberculosis patients were included in the survey, and the usual procedure of questioning about prior treatment was done. In most cases the sputum was collected before the start of treatment. None of the patients registered as new cases had sputum for sensitivity testing collected later than 2 weeks after the start of treatment.

The standard treatment regimen in Botswana is streptomycin, isoniazid and thiacetazone daily for the first 2 months, followed by isoniazid and thiacetazone (given as Thiazina) daily for between 1 and 2 years, depending on the severity of the disease.

Methods The sputum samples were stored in local refrigerators for up to 3 weeks before being sent to the Statens Seruminstitute (Tuberculosis Laboratory). A total of 208 specimens of sputum collected from 103 patients were examined in the laboratory in Copenhagen by microscopy and culture, with sensitivity testing against streptomycin, isoniazid, thiacetazone, ethionamide, rifampicin, ethambutol and pyrazinamide.

The sputum samples were homogenized by the sodium lauryl-sulphate method (Engbaek, Vergmann and Bentzon, 1967) and plated on Liiwenstein-Jensen medium with 3/4 % glycerine. They were incubated at 37” C and from all growths subcultures were made for secondary resistance examination.

Resistance of M. tuberculosis 241

Subcultures were made to ensure that the strains were NI. tuberculosis and not atypical mycobacteria. Cultures at 22” C were all negative, niacin tests were all strongly positive and no strains were photochromogenic.

The sensitivity tests were performed by the absolute concentration method (World Health Organisation, 1950), comparing minimal inhibitory concentrations between the test strain and a control strain on Lowenstein-Jensen medium. The control strain was a standard strain used at Statens Seruminstitute, which was isolated from a patient with newly acquired tuberculosis and was selected as suitable after extensive laboratory tests. The test strain and control strain were cultured on series of tubes with a fourfold increase in drug concentration from one tube to the next. If the resistance ratio was 4 or more, the strain was said to be resistant to that drug.

Results

Of the sputum samples from 103 patients, 98 were positive on microscopy in Copenhagen, while 5 were microscopy negative. None of these 5 proved to be positive on culture and 2 others were also culture negative, so positive cultures were obtained from a total of 96 patients. These figures show a good correlation between sputum smear positivity as found in the laboratory in Botswana and microscopy/culture positivity as found in the laboratory in Denmark. Also bacterial death during storage and transport must have been minimal.

In 51 patients who denied previous anti-tuberculosis treatment, 6 (11.8 %) were found to show resistance to one of the first-line drugs. No patients showed initial resistance to more than one drug, 4 patients (7.8 %) being resistant to isoniazid 2, (3.9 %) to thiacetazone and no patient showing resistance to streptomycin (Table I). Of 5 patients under 18 years of age none carried resistant bacilli.

In 44 patients known to have been on previous anti-tuberculosis treatment, 31 (70.5 X) were found to show resistance to one or more of the first line drugs : 31.8 % to one drug, 25 % to 2 drugs and 13.5 % to all 3 drugs (Table II). All of these 31 patients were found to show resistance to isoniazid, 12 (27.2 %) to streptomycin and 11 (25 %) to thiacetazone. No patient showed any significant resistance to ethionamide. In one patient there was resistance (second- ary) to rifampicin and ethambutol as well as to the first line drugs, while all other patients were sensitive to these two drugs. Sensitivity testing to pyrazinamide presented technical problems and the results are not considered to be reliable enough to quote.

Of the 35 patients showing resistance to isoniazid, in 26 the resistance ratio was 8 or more, 24 of them in previously treated patients (Table Ill).

Table I. Resistance to individual first line drugs (resistance ratio of 4 or more)

Streptomycin lsoniazid Thiacetazone

Number % Number % Number %

New patients Sensitive Resistant

Old patients Sensitive Resistant

Total Sensitive Resistant

51 100.0 47 92.2 49 96. I 0 0.0 4 7.8 2 3.9

32 72.8 13 29.5 33 75.0 12 27.2 31 70.5 11 25.0

83 87.4 60 63.2 82 86.3 12 12.6 35 36.8 13 13.7

242 Nielsen

Table II. Frequency of primary and secondary resistance to 1, 2 and 3 first-line drugs

Resistance New patients (57) Old patients (44) Al/patients (95) _______-

No % No % No % ----

1 Drug 6 11.8 14 31.8 20 21.1 2 Drugs 0 0.0 11 25.0 11 11.6 3 Drugs 0 0.0 6 13.7 6 6.3

Total 6 11.8 31 70.5 37 39.0

Table III. Resistance to the first line drugs in 95 “old” and “new” patients with differentiation between 2 degrees of resistance (resistance ratio)

Resistance ratio Patients Streptomycin lsoniazid Thiacetazone - -_

1 or less New 51 47 49 (sensitive) Old 32 13 33

Total 83 60 82

4 New 0 2 2 Old 4 7 7 Total 4 9 9

8 or more New 0 2 0 Old 8 24 4 Total 8 26 4

Discussion The figures quoted for primary resistance may be artificially inflated by a hidden acquired resistance: the patients might not have been interrogated closely enough about previous treatment, they might have forgotten about previous treatment, or simply they might not have been aware of being on treatment before. No initial resistance to streptomycin was found, which is surprising since streptomycin is widely used in Botswana in the treatment of other illnesses besides tuberculosis.

As in most other surveys, it is mainly resistance to isoniazid that is high. This is partly caused by the speed with which tubercle bacilli develop resistance to isoniazid (within weeks during monotherapy) and partly by the very extensive use of this drug in the treatment of tuberculosis over the world.

Frequencies of primary resistance in different surveys vary considerably depending on methods of susceptibility testing and criteria used for calling a strain resistant. The present figures for Eastern Botswana, although based on a very few patients, seem to correspond quite well with what is found in other African countries (Horne, 1969 ; Fawcett et al. 1975), with primary resistance around 10 % and with isoniazid resistance in the majority of cases.

The incidence of acquired resistance is a good mirror of the therapeutic practice and rate of success in a given country. In this Botswana survey44 of the 95 patients with positive sputum proved to have been on treatment before (46.5 %), and of these 31 (70.5 %) showed resis- tance to one or more of the first line drugs. The frequency of acquired resistance in the Botswana survey corresponds quite well with the figures published for the Far East, but is slightly higher than figures published for Africa (Horne, 1969).

Resistance of M. tuberculosis 243

The real problem in Botswana today, therefore, seems to be inconsistent treatment (default- ing) with the development of secondary resistance to first line drugs in a high proportion of the patients on treatment. One of the ways to overcome this problem is to consider modern short course regimens, in which the treatment time is considerably reduced and thereby the chance of defaulting is reduced as well.

Acknowledgements I would like to thank H. C. Engbaek, M.D. and B. Vergmann, Pharmacist of the Tuberculosis

Department, Statens Seruminstitute in Copenhagen, for receiving the sputum samples for testing. Money for the survey was granted by the Danish Volunteer Service and the staff of the tuberculosis departments of Serowe and Mahalapye hospitals took an active part in conducting the survey.

References

Engbaek, H. C., Vergmann, B., 8 Bentzon, M. W. (1967). The sodium lauryl sulphate method in culturing sputum for Mycobacteria. Scandinavian Journal of Respiratory Disease, 48, 268.

Fawcett, I. W., Watkins, 6. J., Davies, D. L. J., Et Phillips, I. (1975). Initial resistance of Mycobacterium tuberculosis in Northern Nigeria. Tubercle, 57, 71.

Horne, N. W. (1969). Drug-resistant tuberculosis: a review of the world situation. Tubercle, 50,2. Supplement. Tuberculosis Manual (7977). Tuberculosis Office, Gaborone, Botswana. 2nd. edition. World Health Organisation (1950). Tuberculosis. Report on the fourth session of the Expert Committee, 1950. Technical

Report Series. No. 7.