The ethionamide sensitivity of british pre-treatment strains of mycobacterium tuberculosis

Tubercle, Lond., (1966), 47, ! 98

198

THE ETHIONAMIDE SENSITIVITY OF BRITISH PRE-TREATMENT OF MYCOBACTERIUM T U B E R C U L O S I S t

STRAINS

By M. J. LI-FFORD* ~'om the Ah'dicul Research Council's Unit ~ r Research on Drug Sensitirit.v in Tuherculosis. Posc-grad, aw A,fedical School. Ducane Road., London. W.12 SUMMARY

Ethionamide sensitivity tests on L~wenstein-Jensen medium were performed on 144 strains of ,~lvco. tuberculosis obtained from patients who had not received anti-tuberculosis chemotherapy. Sensitivity was measured in terms of the minimal inhibitory concentrations and resistance ratios at two sizes of inoculum, and the proportion of each culture growing on medium containing 10. 20 and 40~g./ml. ethionamide. True variation in sensitivity to ethionamide was demonstrated by repeat testing of possibly resistant strains, and by testing two cultures from each of a sample of patients. Five cultures of differing sensitivity in vitro were injected into groups of guinea pigs which were subsequently treated witll ethionamide. The two least sensitive strains i;; ritro lailed to respond to ethionamide h; vivo and were regarded as primarily resistant.

RI!stJMi~. Des tests de sensibilit6 fi 1"6thionamide sur milieu de LOwenstein-Jensen furent pratiqu6s sur 144 souches de Aid'co. tuberculosis provenant de malades qui n'avaient pas reeu de chimioth6rapie antituberculeuse. La sensibilite rut mesuree en termes de resistance ratio et de concentration minimale inhibitrice fi deu;,: dilutions de I'inoculum, et en termes de proportion de bacilles cultivant sur les milieux contenant 10, 20 et 40 ~.g. d'6thionamide par ml. de milieu. De variations r6elles dans la sensibilit6 fi I'ethionamide furent prouv6es par des tests r6p6t6s de souches probablement r6sistantes, et en testant 2 cultures par malades pour tout un groupe. Cinq cultures dont la sensibilit6 ditTerait in vitro furent inject6es b, des cobayes qui furent ensuite trait6s par 1"6thionamide. L'6thionamide in vi:'o n'eut pas d'action sur les deux souches les moins sensibles in ritro, qui furent consid6r6es comme r6sistantes primaires.

RESUMI-N

Se efectuaron pruebas de sensibilidad a la ethionamida en 144 cepas de :~lj,cobaclerium tuberculosis, en medio de Loewenstein-Jensen, provenientes de enferm~s que no habian recibido tratamiento previo. Se midi6 ia sensibilidad en t6rminos de concentraci6n inhibitoria minima y de relaci6n de resistencia, con dos tipos de in6culo, asi c o m o la proporci6n de cada cultivo en medios con 10. 20 y 40 ~J.g./mi. de ethionamida. Se demostraron reales variaciones en la sensibilidad a la ethionamida repitiendo los tests de las cepas posiblemente resistentes y haciendo 2 cultivos para cada grupo de enfermos. * Present addre.~,,: Trudeau Foundation Research Laboratories. Saranac Lake, New York. t The material in this paper is drawn from an M.D. Thesis submitted to the University of London.

I'THIONAMII)F

DRUG

SI-NSITIVITY

199

Cinco cultivos de diferente sensibilidad in vitro fueron inculados a grupos de cobayos que fueron luego tratados con ethionamida, l.as 2 cepas menos sensibles in vitro no respondieron al tratamiento in vivo con ethionamida y fueron consideradas como resistentes primarias. Introduction

Ethionamide has been used mainly in the treatment of patients with strains of tubercle bacilli which are resistant to isoniazid and streptomycin (British Tuberculosis Association, 1961 and 1963a). Stewart and others (1962), using a resistance ratio method of measuring sensitivity, found a small proportion of such strains which were apparently resistant to ethionamide prior to treatmerit with that drug. The sensitivity of ttle strains was not examined in experimental animals. There have been very few studies of the ethionamide sensitivity of strains of tubercle bacilli isolated from patients who have not received anti-tuberculosis chemotherapy (Le Hir and others. 1964: British Tuberculosis Association/Hong Kong Tuberculosis Services, 1964). A study of British pre-treatment "wild' strains was made in which ethionamide sensitivity was assessed by several methods (Lefford and Mitchison, 1966). One purpose of this study was to find out whether there was variation in the ethionamide sensitivity of wild strains similar to that found with isoniazid, streptomycin and PAS (Lefford, Mitchison and Tall. 1966), thiacetazone (Thomas and others. 1961" East African/ British Medical Research Council. 1963). and 4- 4' di-isoamyloxythioearbanilide (Isoxyl) (Moodie. Aquinas and Foord. 1964). Strains which were found to differ in their ethionarffide sensitivity in vitro were used to infect guinea pigs, and tlae response of the animals to treatment with ethionamidc was evaluated. Methods

Strains o/'M.vco, tuberculosis "l'wo sputum specimens were obtained from each patient with newly diagnosed, untreated pulnlonar.y tuberculosis admitted to the second national survey of the prevalence of primary resistance to isoniazid, streptomycin and PAS. (Millet" and others, 1966). Ethionamide sensitivity te:;ts were performed on randomly :;elected strains from 144 patients: 136 strains were sensitive to isoniazid, streptomycin and PAS, and 8 (5.6'};,) were resistant to one or.more of these drugs. Tile prevalence of drug resistance in the sample and in the completed survey (4.3'~] of 896 strains) was sufficiently similar to indicate that the sample was representative of British wild strains. All cultures were found to produce niacin, denoting that they were ;~lrco. tuberculosis (Britisll Tuberculosis Association. 1963b). Sensitivitv Meditml Pure ethionamide powder was dissolved and diluted in ethylene glycol. Ethionamide solutions were added to L6wenstein-Jensen medium without potato starch (Jensen, 1955)before inspissation in the ratio of I :49 to give tinal concentrations in the medium of 5. 10.20, 40 and 80 lJ.g./ml, ethionamide in "~_"',, ethylene glycol. Dru~-free~ medium containing 2 '~/,,ethylene glycol, and witl'tout ethylene glycol was also prepared. All medium was dispensed in i 5 ml. screw-capped bottle~ in 3"5 ml. amounts. and stored at 4"C for not longer than one month. Sensitivitv Tests Representative growth from cultures on L6wenstein-.lensen medium was dispersed in distilled water to make a suspension containing approximately 4 mg./ml, bacterial moist weight from which four serial tenfold dilutions were made in distilled water. The first dilution was discarded, and the remainder were inoculated on to sensitivity medium as indicated in Table !, placing a 3 ram. Ioopful of suspension on each slope (LetTord and Mitchison, 1966). Using the undiluted suspension the inoculum contained 0.01 rag. moist weight of bacilli, as has been employed in studies under the auspices of the Medical Research Council (Mitchison and Selkon, 1957: Tuberculosis Chemotherapy Centre. Madras, 1959; British Tuberculosis Association, 1963b: Lefford, Mitchison and Tall. 1966). The standard strain H37 Rv was similarly tested with each batch ofcultures.

TI.'B E R ( ' L E

200 T',I~Is

Dilution o f h~:cterttd ~ttspension

L'ndiluted (standard inr I :I0 I :I00 (small inoculum) I :I ,0(JO I :10.000 Viable c o u n t Proportion(";,)

I.--~X-X.MPI.Is (]iF .% SIIN~,ITIVITY TI?.~T O",; A IvVll.l) S'rR..MN

l)rue-]ree slopes* .I B ("

CG+ Disc:irded IC I( 85 92 7 Ii 9.0

99 9 10 a

I00

Concetztration o ] e t h i o n a m i d e (:Lk'. ml. ) 4t) ,~f) 5 I0 20

IC

35

0

I(7 0

17 0

0 0

0

!.7

I0 a 19

0

XlIC ( "~['. tit/. )

40 20

35 0 0.039 < 0 0 0 1

" Drug-free slopes A arid B are plain m e d i u m . Slope C contai'~s 2",, ethylene glycol. t- C G confluent growth" IC i n n u m e r a b l e discrete colonies: o l h e r iigures refer to n u m b e r of colonies up io I(~). and. when u n d e r l i n e d , were t sed for c a l c u l a t i o n of p r o p o r t i o n s .

Sensitivity tests were read after 4 and 6 weeks" incubation at 3 7 C . and the following measure,; of sensitivity were assessed" I. Standard inoculum (undiluted suspension) minimal inhibitory concentration ( M I C ) a n d resistance ratio ( R R ) at four weeks, taking a 20 colony end-point. 2. Small inoculum (! " i 0 0 d i l u t i o n ) M I C and R R at four wt:~ks, taking a 5 colony end-point. 3. The proportions of growth on medium containing 10 and 20 !,tg./ml. e t h i o n a m i d e at four weeks, and I0. 20 and 40 !.J.g.,'ml. e t h i o n a m i d e at six weeks. l h e proportions were estimated as the ratio of the viable count , " drug containing medium to the mean viable count on drug-free medi urn. expressed :i,, ,: . 9 :centage. Initially a sensitivity test x~a,~ perfo. .....:'," rifle culture from each of 144 patients. Those cuiture~; which were found to be provi,;ionall'. ~t. as defined below, were re-tested together with the .,econd culture, when available, fr,~: +;~c - ,t: pai~ent. ()ccasionally. further repeat tests were done in an attempt to classify the ,,tr:t+,; ,n,~, .,ciselv. In addition pairs o f u n e q u i v o c a l l v sensitive cultures from each of 23 r a n d o m l y ,~elc,.:tc,l ?,tt~ents were tested in order to assess the variation of sensitivity of sensitive strains" both culture,, from a giver, patient were tested in the sanle batch of" tests. By these means it was possible to separate the total experimental variation into its c o m p o n e n t parts.

Guinea p(g e.vperiment A total of 70 guinea pigs o f t h e Duncan Hartley strain were divided into five groups of 14 animals. each group being of a p p r o x i m a t e l y equal weight (mean 446 g.. range of 325-650 g.). Each animal was injected intramuscularly with I rag. moist weight of bacilli obtained from a three-week culture on L6wenstein-Jensen medium. Three weeks later each group of animals was further subdivided i n t o (a) five animals which received ethionamide I0 mg./kg, bodyweight/day" (b) five animals which received ethionamide 40 mg./kg, bodyweight/day" and (c) four untreated cont;oi animals. The e t h i o n a m i d e was suspended in 2~ aqueous methyl cellulose gel. and administered by gastric intubation for six days in each week. After six week's treatment (nine weeks after infection ). all the surviving animals were killed and randomised. Postmortem e x a m i n a t i o n s were carried out on these guinea pigs and on any which died during the course of the experiment without knowing to which g r o u p they belonged. The extent of disease was assessed macroscopically in the organs as a score ranging from 0-100 (Mitchison and others. 1961). The a m o u n t of disease was expressed as the 'root index of disease" which was obtained bv dividing the score for each animal by its survival period in days. and taking the square root of this value (Dickii~son and Mitchi.,,on. 1966). This measure is analouous to the root index , f virulence (Mitchison and others, 1961). in addition the

EFIII()NAMII)!!

I) R U ( ;

SI!NSIIIVI

1Y

201

spleen o f each a n i m a l killed :it the end o f the e x p e r i m e n t was c u l t u r e d on l_6~vensiein-Jensen m e d i u m . Sensitivity tests were p e r f o r m e d on the positive cultures, using the s t a n d a r d i n o c u l u m . a n d L f w e n s t e i n - J e n s c n naedium c o n t a i n i n g the following c o n c e n t r a t i o n s of e t h i o n a m i d e : 0, 10. 14-1.20. 28.2, 40, 56.4, 80 a n d 160 :,xg./ml. T h e results vcere read at four weeks and expressed as M IC's, using :! 20 c o l o n y e n d - p o i n t .

Resulls fiutiai.sen.sitivit I" tests A c u l t u r e from each of 144 patients was e x a m i n e d in the initial scsitivity tests, but two patients were excluded from the analyses because their c u l t u r e s were inh:b,iO.-:! hv m e d i u m c o n t a i n i n g e t h y l e n e glycol. T h e results of the initial sensitivity te~i~ ire she,-'-,> +,, I ~t~lcs I I - I V . On the basis ~ ,t ,,~!.. a few of the least o f these d i s t r i b u t i o n s , provisional criteria of resistance ~ 9 cho,.c', sensitive cultures in each d i s t r i b u t i o n w o u l d be clas,, plo~ ~..~c~;~ll,. , .... i,,:~l l h e s e provisional .~ ~l~,,t,' (2) a small inodefinitions wcrc as follows: ( I ) a s t a n d a r d irloculurn MI( 9 ~,f ,',;tl .,tz c u l u m ,'vlIC of 40 F.g./ml. or more, (3) a s t a n d a r d i n o c u l u m R R c~l I,~t, : ,r,. ~lJ.t ,,mall i n o c u l u m RR. 9 t" four or more. (5) 20".,, or m o r e g r o w t h on 10 !,tg./ml. at four ~cct.,~. t~,~ . ' ,~r m o r e g r o w t h on 20 Fg./mi. at four weeks: (7) 20",;, or m o r e g r o w t h on 10 !zg.iml. at six weeks: (8) 5'!.,, or m o r e ..,, or more g r o w t h on 40 Fg./ml. at six weeks. A p p i v i n g g r o w t h on 20 ,:.tg./ml. at six weeks, and (9/ I<"" these nine criteria to the initial sensitivity test results, 19 c u l t u r e s were found to be p r o v i s i o n a l l y

TAIII.F [I.- .... MINIMAl. INItlBITORY (.'ON('I(NTR.XTION OF Wll.l) STRAINS IN ]NIqIAL SI'NSIII%'ITY "FI'STS

MI("

S t a n d a r d inoculum

( !J.,~'., m l . )

A' o.

. . . . . . . . . . . . . . . . . . . . . . . . . . .

.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 I0 20 40 80 > g0

......... 12 63 64 2 I

Total

S m a l l i.uwulum

o/~

1%,,O. .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8.5 44.4 45" l /-4 0-7

~ 40 90

2.1 28.2 63.4

S

5 "6

I

0.7

. . . . .

I00. I

142

,> ."~

i00.0

142

TAIn.E Ill.-- RFslsr,x?,,:ci- R,','FIOS OF \Vlt.D SrR,'~INs IN ].~ITI,xl_ SENSITIVITY TESTS

Resistance ratio .

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!/4

8

5"6

! ,"2

38 82 13 I

26"8 57.7 9-2 0.7

142

i00.0

I 2 4 or m o r e Total

Small

S t a n d u r d iuoculum No. "/,,

inoculum

,\'o. .

.

.

.

5 40 73 19 2 139*

% .

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8.

3"6 28.8 52 "5 13.7 1.4 100.0

* R R ' s o f t h r e e wild strains w e r e not o b t a i n e d b e c a u s e o f c o n t a m i n a t i o n o f H 37R v.

202

TUBFRCLF TABLE IV.--PROPORTION RESULTS Of WILt) STRAINS IN INITIAl. SENSITIVITY "I"I-kSl'S

Ethionamide concentration Proportion

4-week results

6-,'eek results

(%) I

20 ;,tg./ml.

I0 ;zg.:,ml.

No.

o,o ""

NO.

o,' 0

10 10 33 22 23 25 13 5 i

7.0 7.0 2.?.2 15.5 16 "2 17.6 9.2 3"5 0.7

66 37 29 4 5 0 0 ! 0

46 "5 26"! 20"4 2"8 3"5 0'0 O.0 O.7 0"0

I 6 23 22 31 31 i9 8 I

142

99"9

142

142

.< 0.01 0.01 0-1I .025102050-100 Total

p

10 !zg.,'ml. No. 'o'

I00.0

20 ~zg./ml. NO. o,,/o

40 :zg.'ml. NO. o "o

0.7 4.2 16.2 15.5 21.8 21 "8 13.4 5.6 0.7

12 36 53 18 17 3 2 I 0

8"5 25.4 .?7.3 12.7 12.0 2. I 1.4 O. 7 0.0

51 51 33 2 2 I 0 0 0

36.4 364 23.6 1.4 1.4 O. 7 0.0 041 o.0

99.9

142

100.1

140"

99.9

* No result from two cultures because o f c o n t a m i n a t i o n . I'ABt.t- V,---MEAN SE\SITIVIIY l~,IiS,t't,TS OF PROVISIONALLY RI:SlSl"aXT SIRAINS

,~h, astoe o f sensitivity 6- week results

4- week resttlts Strain

No. o f senxitivit.l' Ie SlS

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Stamflu'd Small IO!zg./ml. 20!,H,,./ml. lt'):.t,t,,.;ml. 20!zg. ;ml. 40!z,g.,,ml. inocuhmt inoculum Stan,h~rd S m u l l propn propn propn propn prolm A.IIC .%11C inor inocuhtm (:~g.~ml. (~zg /ml ) RR RR (",O) (%) ("~,) (";;) (";;) .

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I00

80

30

6"3

72"4

38"0

77"6

38"9

7"94

720 372 8O9 1259

50 50 52 46

40 20 30 18

1"3 I "3 J "3 1"5

3"2 i -3 I "5 0.9

5,~?'9 20.0 25. I 19.5

I! .5 3"02 I ",?2 2.09

60..? 30.9 26 "3 2/.4

19.5 12.0 5.89 5"13

0.39 1 "5 i ! .02 0.79

778 903

40 32

23 20

I "3 I "0

I "5 I '0

19.0 4.27

! "02 0"38

23.4 5.62

3.02 ! .82

0.81 ! .12

27

17

0 '8

0"9

1 "8

--*

2"80

0"25

~*

878

Mean results of 142 cultures

3

.

.

* Mean not calculated becatJse of i n c o m p l e t e data due to fililure of m a n y strains to yield growth on nledium c o n t a i n i h g the c o n c e n t r a t i o n o f e t h i o n a m i d e . Underlined values satisfy the a d o p t e d criteria of resistance.

resistant by one or more criteria. Of these cultures, eight satisfied one criterion only: four satisfied two criteria: three satisfied three criteria; 2 satisfied five criteria; 1 satisfied six criteria and one culture satisfied all nine criteria.

I--I'ltlONAMll)E

DRUG

SENSITIVITY

203

Repeat sensilivit l' le.sls Repeat sensitivity tests were carried out on all 19 provisionally resistant strains. For each measure of sensitivity the results of all the sensitivity tests on a given strain were summed up and the geometric mean was calculated. Applying the provisional criteria of resistance to these mean estimates of sensitivity, 12 of the 19 provisionally resistant strains were no longer resistant. The remaining s.wen strains will be considered in detail, and are listed in order of increasing sensitivity in Table V. Of the nine definitions of resistance, strains 778 and 903 satisfied only one, the 10 ,ag./ml. proportion and 40 ,u.g./ml. proportion definitions, respectively, taking the six-weeks reading. In both cases the estimated values barely exceeded the critical levels denoting resistance, and these strains were theretbre regarded as sensitive. Strains 1259, 809. 372. and 720 were of progressively decreasing sensitivity, satisfying three, four, five and five criteria of resistance respectively. Strain 720 appeared to be the least sensitive strain in this group, yielding a high small inoculum MIC and RR; and high proportions of growth on 10 and 20 i.tg./ml, at four and six weeks. This group of four strains was regarded as 'doubtfully resistant'. Finally. strain 878 was evidently the least sensitive strain examined satisf),ing 8 of the 9 critcria of resistance. Even though it failed to produce a standard inoculum RR o,f four, the estimated value of three was appreciably higher than that obtained from any other wild strain. This strain was, accordingly, classified as resistant. Variat,on in senxilirit r r?f.s'ensitive sll'aill.~ Variation in scnsitivity of sensitive strains from one patient to another was evqluated by the method of Lcfford, Mitchison and Tall (1966). Sensitive tests were performed on pairs of sensitive ctllturcs from each of 23 paticnts sct up in eight batches of tests. The results were examined by analysi,; of variance which allowed the total experimental variation to be subdivided into (a) variation bctwccn batches of tests: (b) variation between patients within batches of tests, which is a measure of the dilt'crence in sensitivity from one patient to another: and (c) variation between duplicate cultures from the same patient which largely consists of experimental variation. The measures.of sensitivity assessed were the standard and small inoculum M IC'S and tlae 10 and 20:ag./ ml. proportions. "I"o maintain homogeneity of variance, the proportion results were expressed as Iog,lrithms to the base I0. The MIC's were expressed in working units each of which corresponds to a two-t\~ld difference in drug concentration" zero units represents a standard inoculum MIC ~f 20 :,tg. ,'ml.. and a snlali inoculunl M IC of l0 !xg./ml. The results of the analyses of variance are set out in Table Vi. With only one of the measures of sensitivity (the 20 ,ag./ml. proportion at six weeks) was there signilicant variation between batchcs of tests (Table VI. term a). but therc was significant variation in sensitivity between strains using all measures except the sta::-'9mrd inoculum M IC (Table VI. term b). These results indicate that there was variation in true sensitivity to cthionamide from patient to patient, qhe components of variance for "between patients within batches' and 'between duplicate cultures' were similar (Table VI). These estimates arc subject to considerabic error as they are based on small numbers of tests, but they suggest that the observed variation in the results of tests are approximately equally attributable to technical variation and to variation in true sensitivity from patient to patient. Guinea pig experiment The live strains selected for examination in riro .were" (a) two which were found to be fully sensitive ira the initial sensitivity tests, strains 880 find 884" Col the most sensitive of the doubtfully resistant strains (1259): (c) the least sensitive of the doubtfully resistant strains (720), and (d) the resistant strain (878). Nine guinea pigs died within two weeks of the Commencement of treatment with ethionamide and have been excludcd from the analysis. In all cases death was. caused by~,perforation of the pharynx or oesophagus by the gastric tube. The postmortem scores and mean root-indice's of the remaining 61 guinea-pigs are show in Table VII. The untreated guinea pigs infected witil the sensitive strain 880 gave a mean root index of I. 16, which fell to 0.97 in those on 10 mg./kg, ethionamide, and to 0.66 in those on 40 mgl/kg, ethionamide. The results of the second sensitive strain~

204

TUBERCLE

TABLE VI.-~VARIAT1ON IN SFNSITIVITY OF SENSITI\-'I=. STRAINS: ANAI.YSIS OF VARIANCE .

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Source o] varhttion

of tests

Between patients wit~fin hatches o f tests

Between ~htp/icate cultures Ji'om same patient

(.)

(h)

(c)

Between bat('hes

.'~fi'asure of sensitivity

Standard in(xzulunl M IC

MS F P C

0.380 1.78 N.S. 0-030

0"214 0"98 N.S. Nil

0"217

Small inoculum M IC

MS F i' C

0.260 0.67 N.S. Nil

0"386 3"55 < 0"0 I 0.149

0"109

0.820 0.79 N.S. Nil

I .040 3"61 < 0.01 0"376

0"288

10 ,,Lg./rnl. proptl, at 4 weeks

MS F P C

I

:

20 :.tg ..ml. propn, at 4 weeks

MS F P ("

0.801 0"96 N.S. Nil

0"833 2.12 0.05 0.220

0-393

10 >tg.. ml. propn, at 6 weeks

MS F P C

0.604 1 -69 N.S. 0.044

0-359 2.68 0.02 0"117

0"i34

20 !.~g./ml. propn, at 6 weeks

MS F P C

!'158 2"84 0-04 0-133

0"407 3 '33 0"01 0"142

0.122

DF

7

15

23

M S mean square; F-=variancc ratio; P:-~:probability of occurrence by chance; C~- c o m p o n e n t of variance; DF== degrees of freedom ; NS ==statistically non-significa nt, P :--= > 0.05.

884 were similar, tile corresponding mean root indices being 1.18, 0.85 and 0-65. Tile linear regression of mean root indices on dose o f e t h i o n a m i d e was significant ( P - < 0.001 ). Of the doubtfully resistant strains, strain 1259 was highly virulent with a mean root index of !.27 for untreated guinea pigs. There was a well-marked therapeutic response to the 10 mg./kg. and 40 mg./kg, regimens, the mean root indices falling to i.04 and 0.72 respectively (P== <0-001 ). Strain 720 was of lower virulence (mean root index of 1.06 in untreated animals), but this was not surprising because the strain was isoniaTid resistant. There was a small decrease in tile extent of disease in the treated animals but this was not statistically significant. The resistant strain, 878 was of ~-t rmal virulence and there was no significant response to ethionamide treatment at either dose level. These results confirmed the in vitro sensitivity test findings that strains 880 and 884 were

I'~THIONAMIDE

DRUG

SENSITIVITY

205

TAI~I.E V i i . - - P o s l - M o a r t M SconF~s ^ND R~T-lr~'t)lC~ I.~ GUINEA PIGS TREATED WITIt ETHIONAMIDE f |

No. oJguinea pig, s Strain

Pretreatment sensitivity

U.thionamide (m,e./kg./&O')

ILw'luded 0 0 2

:'

Sensitive

0 I0 40

4 5 3

78, 95, 86, 80 55, 60, 53, 85.48 18, 38, 28

1"!6 0"97 0"66

0

,

4

Sensitive

0 10 40

!

4 4

78.95 (49). 55, 95 (56) 55.46. 48, 36

!"18 0"85 0"65

1259

Doubtfully resistant

0 10 40

0 0 0

720

Doubtfully resistant

0 10 40 0 10 40

tlO.

880

884

~7~

" Resistant i

Score

1 !

Assessed

i

38.28,

26. 18

Mean i root-index

4 5 5

90 (52). 90 (53). 95 (57). 88 70, 7 6 . 9 5 . 7 3 . 3 6 45.28. 26, 28. 33

! "27 1"04 0"72

0 2 2

4

63.83, 9 5 . 4 8 33.43, 90 (50) 73 (62). 48.61

I "06

0

4

1

4

0

5

3 3

48, 100 (58), 100.95 60. 51, 78, 75 88.85, 55, 90. 78

0"96 0"98 1"17 I "02

1"!2

Brackets dcnotc survival period in days of guinea pigs which died of tuberculosis.

sensitive, and strain 878 was resistant. Of the in vitro doubtfully resistant cultures, one was resistant in i'iro (720), and the other was sensitive (1259). Fifty-two guinea pigs survi`.;ed until the 63rd day of the experiment, and spleen cultures wdre performed on all of them. Only two spleens failed to yield growth (from strain 884), and sensitivity test results were available from the remaining 50 cultures. There ,,,,'as no material change in ethionamide sc.~sitivity of the cultures obtained post-treatment, probably because the period of exposure to the drug ,,,,as too short. The mean standard inoculum MIC's were 28.2, 33-5. 46.7, 47.3 and 132 !J.g./ml. for strains 880, 884, 1259, 720 and 878 respectively, These results confirm the original sensitivity test assessments. Of particular interest are the closely similar M IC results of strains 1259 and 720. despite their dilTerent response in vivo. Discussion

Ill this study of the ethionamide sensitivity of British wild strains of Mvco. tuberculosis 2 (I .4%) of 144 were classified as resistant. This designation was based on tile results of nine measures of se.lsitivity derived from multiple sensitivity tests; and also on tile failure, of infected guinea pigs to respond to treatment with ethionamide. Chicou and others (1961), found 1 of 108 and the British Tuberculosis Association/Hong Kong Tuberculosis Treatment Services (1964) found 1 of 148 wild strains which were apparently resistant to ethionamide. The resistance of these strains was not confirmed by repeat testing or animal experiments. Stewart and others. (1962). using Youmans'-serum liquid medium classified 5 of 96 strains as resistant to ethionamide on the basis of a resistance ratio of four or more, confirmed by repeat testing. The population of strains studied had not been in contact with ethionamide, but many were resistant to isoniazid. The high prevalence of resistance may be due to cross-resistance between isoniazid and ethionamide which has been reported by Tan Thiam Hok, (1964), in conflict with the experience of Rist (1960). Since

206

TUBERCLE

the prevalence of primary resistance to isoniazid in this country is 1.7~/(Miller and others. 1966) it is improbable that the true prevalence o1"primary ctl~ionamide resistance exceeds 1 '~,~. Both patients with ethionamide resistant strains were re-interrogated, and it was confirmed that they had not received anti-tuberculosis chemotherapy. Strain 720 was resistant to isoniazid as well as ethionamide, and probably owed its origin to a patient who had remained sputum positivc after treatment with first-line and second-line drugs. The second strain was resistant to ethionamidc alone, and was isolated from a patient whose tuberculous contact had never been treated with ethionamide or thiacetazone. This strain may be an example of 'natural' drug resistance, arising in the absence of previous contact with the drug (Millet" and others, 1966). The heterogeneity of sensitivity of sensitive strains to ethionamide is consistent with analogous findings with respect to other drugs (Thomas and others. 1961 : Aquinas, Moodie and Foord.. ! 964: Lefford, Mitchison and Tall. 1966). The apparent smooth transition from ethionamidc sensitivity to resistance ht vitro, makes it impossible to define the boundary betwecn sensitivity and rcsistance with any precision. By contrast, in the animal experiment, the effect ofctlaionamide on strains ofdiffering sensitivity was clear-cut. Since only a small nttmber of strains were examined in vivo. it is possiblc that in clinical ttlberculosis strains of differing sensitivity, i, vitro, may show gradcd responses to treatment. ! wish to thank Dr. D. A. Mitchison l~+r his helpful criticism and advice, and Miss iris Yee fl+r technical +L~sistancc. Thanks are also due to Mrs. O. Sheldon of May and Baker Ltd., Dagcnhan~ for supplies ofetl'Jionamide.

R EFER ENCES

[JRITISI! TUBERCULOSISAS.',;OCIATION(1961). An invc,~tigation of the value of cthionamide with l~yrazinamide or cycloserine in the treatment of chronic pulmonary tuberculosis. T,l~erch', Loml., 42. 269. Brl'rlslt TUI~I-.RCt!LOSlS ASSOCIA'rlON (1963a). Ethionamide, pyrazinamide and cycloserine in the tree.lien! of drug resistant pulmonary tuberculosis. Tuherch,, Lend., 44. 195. BRIIISU TUI~I-RCULOSIS ASSOCIAIlON (1963b). Acquired drug resistance in patients with l'mlmonary tuberculosis in Great Britail'~a national survey, 1960-61. Tuberch', Lend., 44, I. BRIIiSi! TUIIERCULOSIS AS.'~)CIAIIoN/HON(; KONG TUIIliRCULOSIS TRI!AIMi!NT SI:I~,VICES (1964). A controlled trial of ethionamide with isoniazid in the treatment of puh'rlonary tuberculosis in Hong Kong. l'+dwrch', Lend.. 45. 299. CItlCOU, J., HETRICK, O., LAIILOU, l~'l., MERCIER, A., ~t'.'EL, R., RIST, N., & VINC!!NT, J. (1961). Controlled trial of three types of oral treatrnent of pulmonary tuberculosis in Morocco and -l'angier (isoniazid and ethionamidc: isoniazid and PAS; isoniazid alone). Revue Tuberc., 25. 103 i. DICKINSON, J. M. ~ MITCItlSON. D. A. (1966). In preparation. JENSEN, K. A. (I 955). Towards a standardization of laboratory methods (second report). B, II. int. Un. 7+,here.. 25, 89. LEFFORD, M. J., c~ M ITCtIISON, D. A. (1966). Cort'~parisor~ of methods for testing the ser~sitivity of Alvcohacteriu/J# tuherculosis to r in preparation. LEFFORI), M. J.. MIT('IIISON, D. A.,'L~, TAI_L, R. (1966). Bacteriological aspects of the second national stlrvey of primary drug resistance in pulmonary tuberculosis, 1963. "l',herch,, Loml., 47, 109. I_E HIR. M., ClIICOtJ, J., HiTRICK. G., ~'IERCIER, A., Nliiit,, R.., & R,ISr, N. (1964). Controlled clinical trial of three types of oral trcatnlent in pulmonary tuberculosis. Bull. lt/ld Hlth Org., 30, 701. Mil,I.I;R, A. B., TAI, L, R.. FOX, \V., LI!t'EORD, M. J., & MlrClIISON, D. A. (1966). Primary drug resistance in pulmoilary tuberculosis in Great Britain: second national survey, 1963. T, herch,, Loml,. 47, 92. N|IICFtlSON, D. A., BIIA'rlA, A. L., RAI)IIAKRISIINA. S., SI-I,KON. J. B., SUIIBAIAII, T. V., & \VAI,I.ACI', J. (.~. (1961). The virulence in the guinea pig of tubercle b ~ i l l i isolated before treatrnent from South Indian patients with pulmonary tuberculosis. I. Homogeneity of the investigaiion and a critique o f the virulence test. Bull. It/Id ttlth Ors.. 25, 285. MITCHISO.'<, D. A. & SeLKOX, J. B. (I 957). Bacteriological ~Lspects ofa suryey of the incidence of drug-resistant tubercle bacilli among untreated patients. "l',bercle, Lend., 38.85. Mc.• A. S., AQUINAS. SIS rI-R M., & Ft-x~rD, R. D. (19(~). Controlled clinical trial of 4-4' diisoamyloxythioea,banilidt in the treatment of pulmonary tuberculosis. Tubercle l.ond.. 45, 192. RIs-r, N. ( ! 9(.,0). The anti-tuberculosis action of ethionamide. Adv. Tuberc. Res., 10.69. STJ.:WART, S. M., Hat.t.. E., RII)I)ELL, R. W., & SO.'~t,":I;R. A. R. (I 962). Bacteriological aspects of the use of ethionamide, pyrazinamide and cycloserine in the treatment of chronic pulmonary tuberculosis. Tubereh,, Loml., 43, 417. TAN TJlIAM HOK (19(:,4). A comparative study of the susceptibility to ethionamide, thiosemicarbazone and isoniazid of tubercle bacilli from patients, nevcr treated with ethionamide or thiosemicarbazone. Ant. Rev. resp. Dis.. 90, 468. T|iOMAS. K. L., JosEP|t. S., SUIIBAIAll, T. V., & S[-LKON, J. B. (1961). Identification of tubercle bacilli from Indian patients witi, pulmonary tuberculosis. Bull. WId. HIth. Ork,., 25, 747. TUI~ERCULOSlS CIIEMOTIII-RAPY CENTRE, MADRAS (1959). A concurrent comparison of isoniazid plus PAS with three regimens of isoniazid alone in the domiciliary lreatment of pulmonary tuberculosis in South India. Bull. H/h/ HIth Ors., 21, 51.