Tubercle , Lond. , (1963), 44, 417

T H E R E S P O N S E TO TREATMENI" WITH T H I A C E T A Z O N E OF G U I N E A - P I G S

A N D M I C E INFECTED W I T H T U B E R C L E BACII]~I O B T A I N E D F R O M UNTREATEI) A F R I C A N P A T I E N T S

By MAIIGAP, I-T I]&P, NETT. S. R. M. BuslmY. fi'Om IVellcome Rl'se~trlh Laborator)', Bel'kenhrinl, Kent

JIAN ~4. DICKINSON alld O. A. MITCItlSON .fi'om Ml'lh'cwl Research Council'.~ Unit /~lr Research on Drltg Sel;silivl O, ill Tuberculosis

Po.llgralltmte Medical School o] London

SUMMARY

Four strains of Al) 'co, tuberculosis , chosdn as being highly resistant to thiacetazone, with rcsistal/ec i'atios of 4. 16. 16 alld 64 or nlorc, and t}lrcc sensitive strains, with resistance ratios of Olle, were obtained froln pretreatmcnt spLItUlll cultures of East African patients participating ill co-operative daemotherapy studies�9 All of the seven patients denied, having received any previous antitugerculosis chemotherapy.

Guinea-pigs were infected with I rag. of the slraills alad, after three weeks, were treated for four weeks with 5, 20 or 80 mg./kg, body weight thiacetazone given by oral iotubation. Response to treatnlent was ineasured by thc extent of tile disease find by spleen culture at seven weeks, in comparison with untreated animals. With tile sensitive strains, responsewas absent witb the 5 mg./kg, dose. was slight with the 20 mg./kg, dose and was considerable wlth the 80 mg./kg, dose. No definilc response was obtained at any dose level in the animals infected with the fol.lr resistant strains, although thc results with olle attenuated strain could not be assesscd.

Mice were infected with 2 and 0.5 rag. or I and 0.25 rag. o f the strains: treatment with 12.5. 25, 50, 100. 200 or 250 mg./kg, thiacetazone, given by oral iotubation, was startcd immediately after infection and was cc3ntlnued until death or for 80 days. Compared with untreated mice, tile sLirvival of groups of treated naicc infected .with the sensitive straios was prolonged, but only with a dosage of 100-250 mg./kg. Tile survival times of mice infected with the resistant strains was only slightly prolonged by dosages of 200 and 250 mg./kg. These findings demonstrate that differ_cafes of in vitro sensitivity of pretrcatment stl'ains ale reflected ill corresponding responses of [nl'ected animals to treatment with tldaeetazone. Tile guinea-pig test was irtol-e satisfactory thai] tile mouse test ill demonstrat- ing these (tiffe[ences.

There is evidence (Rist, Cals, & Jullien, 1951; East African/British Medical Research Council, 1960) that strains of tubercle bacilli obtained from patients before treatment with thiacetazone differ ill their sensitivity to tbis drug. The response of Afiican patients with pulmonary tuberculcsis to treatment with various regimens of isoniazid plus thiacetazone, and of isoniazid plus para- aminosalicylic acid (PAS) has recently been studied in cooperative controlled trials (East African/ British Medical Research Council, 1963a), All of tile strains obtained fiom the patients before tile start of their allocated chemotherapy in these trials were tested for their sensitivity to isonlazid, but thiacetazone sensitivity tests were done Oll!y o11 pretreatlnellt strains from those who �9

41~ I"U I}I~R('LI

treated ',~itfi isonlazid plus tt/iacctazone. A g roup of ['our strains chosen as Imvlng /lie higl~cst degrees of I'r to IIliacctazolle fl-onl those available, atld another group of three slrahls %\ iIIl a Iligh degree of sensitivity were further studied. The response of gulnea-pigs and mice. inl'ec/ed with these seven Sll-ains. and then Ires/ted wilh tbiacelazonc, is reporled here.

Ori~hls of Strains o | Tubercle Bacilli

The four tfiiacetazone-leSistaut stl-aim; and tile three sensitive strains used [!l this s tudy were selected floln the prctreatnlent CLll!tll'eS 0[" spu[tllll frolll pat[elliS who WCFC aOnlitlcd to l \ \o COOl)era- tire chemotherapy studies in East Africa (East AfricaniBrifisfi Medical Research Council. 1963al and wile salisfled tile following cr i tmia:

(I) They had acute, extensi'~c puhnonary ttiberccl[osis of leeenl or ig in : (2) they had had less than one mollLh's known antitubercuk~sis chemotherapy : (3) they yielded cuhures which were sensitive to isoniazid oil admiss ion.

O f the 363 patients satisfying these criteria, who were allocated to t reatment w h h various reghnens of thiacetazonc, and whose strains were tllerefore tested Ii)[ lhiacetazonc SCilSifivity. 18 were found by Ihe cooperat ing East African laboratories to have a resistance ratio of cigfit or nloFe to tfilaceta- zone [n sensitivity tests 6n tit least one of the two prelreatnlent stl-ains which were tested as a rouline for each patient. Only seven of tile 18 slrahls were available for the present study and these were sent to London where further sensitivity tests were done. The three slralns wflh the highesl degree of resislance were selected for an imal tests. A fburth strain was obtained {'1"Oill a patient who was allocated to treatnlent with PAS plus isoniazld alld was found tO l/ave a pretreatnlcllt sit,tin resistant to lhiacetazone io a snbsidiary laboratory investigation. The three sensitive strains ~xet-e chosen as Itaving resistance ratios e l 'one or less in similarly repeated sensitivity tests.

As far as could be discovered, none of file seven patients who yielded tim strains that were used ill tile present study ]lad received any previous aniittlbercLllOSiS c]lemothcrapy. Six of the seven patients were in tile in-patient study (East AflicanlBritisl~. Medical Research Cotlneil, 1963a) in wfiicll the patients were repeatedly qncst ioaed on admission and dur ing IIeahllent as to previous chemotbcrapy. The seventh patient (strain 209L who was in tile octl-patlent study, was only ques- tioned at tile start of t realmenl . As the strains prodLlced i/]acill \rhea tested by tile metl lod of tile British Tuberculosis Associat ion (t963). riley were all considered as likely to be lhc human variety of Ml co. luber~zUosis.

Metllods Thiacetazone Scnsitiri O, Tests Sensitivily tests for tile prel iminary selection ,of the strains were carried out by inoeula l ing

approximately 0.0t rag. (moist weight) of bacilli oil to a series of g/Swenstein-.lenscn mediunl slopes containing serial two-fold concentrat ions of thiacetazone and reading tile minima] concefltration that inkibited <growth (defined as 20 or more colonies) a l ler four weeks' incubation. The results were expressed as a resistance ratio, namely, tile ratio of tile mininla] inhibi tory concentrat ion for the test strain to the minimal inhibi tory concentrat ion for strain H37Rv, set up ill each batch of tests. "rite method has been described in detail elsewhere (East African/Britisl'~ Medical Research Council . 1960).

'Popula t ion study' sensitivity tests were carried out on the strains sfiorlly before animal inocu- lation and on tile bacilli isolated from the spleens of the infected guinea-pigs. The inoculum was pre- pared by shaking a weighed amoun t of bacterial growth from a three-week old culture on L6wenstein- Jensen medium in a -} oz. screw-capped bottle cmlta ining glass beads and 0 '2 ml. sterile water, and then adding water to yield a final concentrat ion of 1 rag. (moist weight) bacilli per ml. A loopful from the suspension was inoculated oil to each of a series of slopes of LirwensleJn-Jensen medium containing serial two-fold di lut ions fro'm 32 to 0-06 ~.g./ml. thiacctazone and on to a drug-free slope: tire final concentrat ion of triethylene glycol necessary for solution of tile tbiacetazone was 0.5 '}<; in all slopes.

] I t lACI2TAZONE ]'RI~A1 M I~NT d,]9

A corresponding series o f slopes were also inoculated f lonl I :<40 and 1:1600 di lut ions in water o f the suspension. The slopes were read after Ibur weeks' hletlbadol~ at 3 7 C. and an estimate of the viable count oil each eonceot radon of thhieetazone was obtained. Strain l137Rv was tested in the same mailner in each batch of tests.

Gidlwa-iU~ lJx[iori#ll~'lllf Guinea-pigs of tile Duncan Hartley strain (Mhchison and others, 1960) of average weight 367 g.

(range 235-550 e.) were injected in t ramuseukuiy i n the ' r ight thigh with I rag. moist weight of tile strain of tubercle baeillL prepared as described above for the popula t ion study thiaeetazone scnsilh, ity tests. At till-CO weeks, [loall]]ellt with thiacetazolle was started al~d was cont inued for a ft{rther four weeks. The thiaeetazone was given onec daily oil six days a week in doses of 5, 20 or 80 m g . / k g body wr ight : it wa~ administered in 2".. aqueous metbylcellulose by gastric hl tubat lon. The guinea-pigs x~ere weighed weekly and tile dose adjusted accordingly. Surviving animals were killed at file end o[" tile period o{" treatment, seven weeks after infection. U ntreated guinea-plgs were killed at three weeks (pletreatnlent controls) and at 7 weeks.

A pos tqnor tem examinat lon was carried out on guinea-pigs thai died dur ing treatment with thiacetazonc or that were killed al scroll weeks. The examiuat ion of tile latter g roup was carried out in a r ao dom order alld the identity of the strain or t reatment was not knowIt to the observer. The extent of tuberculotis disease visible to the naked eye in file spleen, liver, lUllgS aild local lynlpll nodes was assessed as a score raitging from 0 to 100, as described by Mitchlson and others (1960). For reasons given elsewhere (Mitchison and others, 196l ), the squate root of the ratio of the score to the su rvlval 0me in days was determined for each animal and was called tile root-index. The root- index was employed as a measure of tile rate of progression of the disease in tile guinea-pigs, ks it takes ilttO LICCOLlUL both tile post-nlortcfu score and tile survival period of tile animals.

The ~plecns of tile gtdnea-pigs killed at seven weeks were eulltlred by removillg due quar te r of the spleen with aseptic precautions, ]tonlogenis]ilg with 5 ml. of water and illocula[d~g slopes of kSwen- stein-Jonson medium with two s tandard Ioopfuts of tile result ing suspension and of a 1:20 di lut ion of tile suspensiou. The nunlber of colonies growing on the slopes after four weeks' i l lcabation was eOUllted. Populutiou study thkicetazollc sensitivity tests were set up on spleen Ctllttlres fI'OI]] tWO guinea-plgs in each tl-eatl]len{ group infected with each strain.

I'LJO ii ~l? [~\pCl'illlcllI5 Groups of'Swiss mice o f the Scholield strain, weighing ]5- ]7 g., each were injected intravenously

will] suspensions of bacilli in Dubos mediuln, prepared in a s imilar nlanner to those used in the popula t ion study thktcetazone sensitivity tests. Each strain was given at two dose levels, which were elthcr 0.25 rag. and I mg., or 0'5 rag. and 2"0 rag. per mouse, according to tile results of previous virtden,r tests hi mice, Trea tment with thiacetazone ill doses ranging from 0.[ rag. to 2.0 nlg. per mouse was started immediately after infection and was given by gastric intubatiou twice daily on five days a week. once daily oil tile sixth day and uot oil tile seventh day. Treatulent was continued uutil there were lid stlrvivors, or for 80 days.

AI~.RANGI MI NT OF EXPEI{IMI!NTS E~pr ]

One tlliacetazone-sensitive strain alld {wo tl l iacetazone-reslstant strains were each injected into 21 guinea-plgs, o f whlcll three were killed at three weeks as pretreatment cotMrolsl the remaining animals , in g roups of six, were itot treated or were treated with 5 or with 20 mg./kg, th iace tazone . Each of tile same three strains was injected into 100 mice, 50 at the high dose level and 50 at the low dose level. The mice infected at each of rite two dose levels were divided into five groups of 10 animals . One g roup was not treated, and the other four groups were treated with 0- l , 0"2, 0.4 or 0"8 mg. thiacetazone per nloLlsg, respec0vely.

Exl)erimenl 2 The results of experiment 1 were not in fol-matlve because one of the resistallt s trains was attenu-

420 TUBI~R CL]~

ated in tile guinea-plgs, and because tile dose of thiacetazone was too low. In experiment 2, the same strains were used. except that tile attentuated resistant strain was replaced by a third resistant strain. Each strain was injected into 24 guinea-pigs, which were divided inlo two pre{reatment control animals, six unlreated animals and two groups oF eight anhnals , treated xdth 20 or 80 mg./kg. thiaeetnzouc, respeetlvely. Tile arrangemel l l o f the mouse experiment was the same as hi experiment I, except that the gl-oups of treated mice were given 0 I4 , *) 18 , 1.6 and 2-0 111 g I thiacetazone per mouse,

E.rperiment 3 Three addit ional strtlhls, t ~ o sensitive arid one resistant, were tested enlployh/g tile same arrange-

menls :is in experiment 2.

TIIIACEIAZONE ~ENSITIYITY TES]S Results

Tile results oF tile poptHation study thiacetazone sensitivity tests on tile Stl'aills used in thc: animal experhnems are shown in Fig. ~. ] he majori ty of the bacterial poptdal lons ill the three sensitive strair~s and in slrah'~ H37Rv (l::ig. 1, upper row) were each inhibited by l[zg./ml, lhlacetazone. Consider ing tile four resistant strains (Fig. t, lower row). tile ma,iority of tile bacterial popula t ion of strain 269 grew at 32 y.g./ml, thiacetazoae, tile highest concentrat ion tested, and strains 316, 209 and 236 were inhibited, ill the main. by 4, 16 and 16 {,g,/nll. thiacetazonc, respectively. With tile s train 236, and per[laps wilh strain 316. there was a tendency for tile viable counts to decrease with increasing colleelllraliolls O[ tbiacetazone over tile range o/col lcentr , l l ions lbat allowed the growth of tile major i ty oF tile bacterial [~opukttion, probably indicat ing tile presence of organisms whh varying degrees of sensitivity.

Tile results of tile sensitivity tests carried out on the spleen cultures from Ibe untreated guinea-pigs were s imilar to those sho~vn in Fig, I. except that most of tire strains ~ere inhibited by halt" tile

2

N 37 Rv

~N,N

012 05 ? 2 32 0 01Z 0~ 2 8 22 D ~2 ~'S ? B 22 0 t~12 25 2 2 ]2 Thi~c~laz~ne ~oncefl~ration IjJg/m[.}

FIG, 1 Popula io sl i h, sensilivi v e s o I1 lace azo e Upper } ne se s ve slrziJns Lo~er

�9 tine: ]'esistant strains. RR~esismnce ratio.

THIACI!I~AZONE TREATMI~NT 421

concentration of thiacetazollr Thus, the m~ljorhy of Ibe organisms in the sensitivc s t ra ins including strtlin H37Rv, were inhibited by 0"5 ?.g./ml,, strain 236 by 8 ~• and strain 209 by 8 i~g,/ml. thiacetaz(me. Strain 269 grew heavily oll the slope containing 32 :zg./ml. fldacctazone. No spleen cultures were done with s~ruin 316. Tl',ere appeared, therefore, to be, a~ advantage in expressing the results of the tests as resistance ratios, since these remained constant despite variation in tile minimal inhibitory concentratioll o[" the strains. The resistaltce ratio for each of tile three sensitive strains was one. Amollg the resistant strains, tile resistance ratios were 64 or more for strain 269, I~oLir f~./r slrain 316 and 16 lbr strains 209 and 23fi.

3"he results of fl~e single population study sensitivity tests shown in Fig. 1 are approxinmfions. since growth on [hc uoncel~trations of thiacetazone near the end-point was sometimes not pro- portional in tile slopes inoculated wifll lhe dill'erent dihltions of the bacillary suspensions. However, tile resistance ratios quoted above tire reasonably ~lccurale since alh except strain 316. arc based on at least three tests.

GUINEA-PiG EXPIiRIMEN1 S Po,~7-tt~ortotl ,~c'ores attd rool-#ldir162

The post-mortem scores and the corresponding root-indices obtained in the guinea-pig experi- ments are set out ic~ Table I, As indicated by the post-ntol-~enl scores On the pretrealment control guinea-pigs, a moderate amount Of disease was present when treatolent was started at 21 days after infection. A proportion of the animals, particularly high ill e• I. died before 40 days as a result of perforation of the oesophagus during intnbation and their results have been excluded from the calcukttion of tile mean scores and the mean root-indk.-cs. Ill experiment I. the //lean root-indices obtained with the sensiGve stalin 175 were similar ti"~r tile tmtreated guinea-pigs (I.28) and for ttle guinea-pigs treated with 5 mg./kg, thiacetazone (I "34), but there was a significanl (P <0.01) fall from the mean for these two groups ( [ ' 3 [ ) to 1.15 ill those treated with 20, mr. /ks , thiacctazone. In experiment 2, the mean root-indices with strain t75 decreased fiom 1.26 in the untreated guinea- pigs to 1.03 in flwsc treated with 20 mr. /ks, thiacetazm/e, and to 0.84 in those treated with 80 mg,/kg, flliacetazone. The corresponding mean root-indices for the remaining two sensitive strains. 26 NI and 29 NI in experiment 3 w e n I .[g, 0, '95.0.7g and 1.24, 1,05 and 0,78 respectively. The association between the mean root-indlces and the dose of thiacetazone (for tile purpose, ao treatment, 20 mg./kg, and 80 reg./ks, were scored as 0, 1 and 2 respectively) attained statistical significance at the 0,1 ~'/,, level for each of the lhree strains.

In contrast to the se21sitive strains, the resistanl strains (lower half of Table I) showed little response to thiacetazone. [n experiment I. Ihc mean root-indices obtained with strain 269 were similar in untreated guinea-pigs ([ "02) and in guinea-plgs treated with 5 mgjkg , thiaeetazane (0.98) and widl 20, mg./kg, thiacctazone (~ "05), In experiment 2. the same strain yielded mean rool-indices of 1.06, 1,14 and 1"12 in untreated guinea-pigs and in those treated with 20 mr. /ks , and witl3 80 mg./kg, thiaeefazone, respectively. Strain 316 w~as attenuated, with little progression of tlle disease between 21 and 49 days and low scores at 49 days, so that tile results of tl 'eatment are difficult to interpret. The mean root-indices obtained with this strain in untreated and treated guinea-pigs were similar. In experiment 2. strah~ 20,9 was substituted [or strain 316 and the mean root-indices de- creased slightly from t '22 in untreated guinea-pigs to 1.19 in guillea-pigs treated with 20 mr . /ks . thiacetazone, and to 1.13 in those treated with 80 mg,/kg, thiacetazone. A similar result was oblaincd wifll strain 236 ill experiment 3, the corresponding mean root-indices being 1.04, 1.0 and 0"99. None of tile differences between the mean root-indices of untreated and treated guinea-pigs infecled with a:,y of tlle res{stant strains attained stabstical significance.

The response to treatment with thiaeetazone of the guinea-plgs infected with the sensitive and tlle resistant strains is sumtualized in Fig. 2. Tile sells]live strains show a clear decrease hi the mean root-lndices as tile dose of thiacetazone was increased. The mean root-indices obtained with the resistant ~trains were hardly affected by treatment with thlacetazone.

422 -I u IH ]~c I .

B

/

E .

=~ ~ _ 4 =

I ' l l IA CI~TA Z O N I T R I~A Ti',I [~ N1 423

o ~ c . . . . .

E

7~

424 T U [1 [iU, C L I~

Spleen Cultures The spleens of the guinea-pigs in experiments 2 and 3 were cultured by a quanti tat ive medlod, Only

one spleen cuhure (strain 29 N[, treated with 80 mg,/kg, thiacetazone) was negative. The geometric means of the viable cotlnts OI1 the spleens of tile gtlinca-pigs ill the three tlcatnlel3t groups ial'eeted wi th each strain arc shown in Fig. 3. With the sensitive strain 175 the counts liell From Iog~. 6.10 viable units per spleen in untreated guinea-pigs to 4.77 in the guinea-pigs trealed with 20 mg,/kg. thiaeetazonc and to 3.47 in those treated with 80 m g , / k g thiacetazone. Similar results were obtained with the remaining two sensitive strains 26 NI and 29 NI, Each of the ditlErcnccs between the mean count Oll untreated guinea-pigs and oi1 those treated with 20 and 80 nlg./kg, thiacctazone atlained statistical significance at llle 0, t ~,~; level.

1.5

~3

g

i

0.R

0.1

SENSITIVE STRAINS

. ..,.

1

I I 10 20 t.0 0i0

RESISTANT STRAINS

6

- - o . . . . . . . . . ~ 5

Oose ol thiacetazone (mg./kg.}

Fio. 2 Roo~-iIldices obtainc~t in Guinea-pigs treated ~ith nnacclazone.

I : Strain 26N1, Expt. 3; 2: Slrain 29N1. Exp,. 3: 3: Strain 175. Uxpt. 2: 4: Strain 175, Expt. l; 5: Strain 31~, Expt. 1: 6: Strain P236, Expt. 3; 7: Straiq 269, Expt. I ; 8: Strain 269. Expt. 2: 9: Strain 209, Expt. 2.

The spleen counts on the three resistant strains were s imilar in the untreated and treated guinea- pigs. The counts on the treated guinea-pigs were slightly higher than those on the untreated guJnca- p~gs with strain 269 and slightly lower with strains 209 and 236. None of the differences between the mean counts on treated and untreated guinea-pigs infected wi[h each of tlle resistant strains at tained statistical significance

Thiocetazone Sensitivity Tests on SpleeJ~ Cultures Thiacetazone sensitivity tests by the 'popula t ion study' nlet]lod were do[le on Ctl]tUl'eS froln two

spleens in each t reatment g roup from each strain in experintents 2 and 3. O f the 24 spleens fl'om guinea-pigs treated with 20 or 80 mg./kg, thiacetazone, 23 yielded strains of similar sensitivity to those used for infection, or obta ined from untreated guinea-pigs. One strain From a gulnea-pig infected with the sensitive strain 26 NI and treated with 80 mg./kg, thiacetazone, contained about 3 ~ of organisms four to eight t imes more resistant than the parent strain,

1 HIACI2TAZONE ]REATMI NT 425

SENSITIVE STRA(NS RES(STANT STRAINS

6.;0 175

5

Iili!ii;}ii!ii;il iiiiiiiiii iT{iiiiiiiiiiii))

_ 20NI

555

- ~ C81

269 5 72

5 2o 209

o s l ; 29 N1 236

3 0 20 20 O 20 00

Bose of thiacetazone (mg./kg.]

Fm. 3 aesuhs of cullurr oF spleens of Guinea-pigs trealcd wilh thiace[azonr

M OLISI] EX PERI~,I L'N3 S The survival periods of the mice infected with the sensitive strains are set out in Table II and of

those infected witll the resistanl strains, ill Table II I. The mean survival periods relaled to the dose of thlacetazone, expressed as mg./kg, body weight/day, are shown in Fig. 4. Although Ihe maximum dose o f thiacctazone/kg, body weight was higher in the mice than in the guinea-pigs, the survival of the mice infected with the sensitive strains was less inflt/enced by treatmeat with thiacetazone titan would be expected from the guinea-pig ivsults. The mean survival periods of the untreated mice ranged fl'om 5-0 to 22.4 days in the groups infected with the two dose levels of the three sensitive strains and similarly from 2"9 to 26.2 days in the groups infected with the two dose levels of the

426

g L~ f 30

2g -

SENSITIVE SIffAINS

..8

12 5 25 511 100 2DO

RESISTANT STRAINS

I ~00

, /

/

15 , J '* 16

. . . . . �9 - . , ~ /

i 2J5 ~ p i _ _ _ u 12 5 R 100 200 t, DO

Dose of thiacetazone ( mg./kg )

FJ(;. 4 Survival periods oR mlce trealed ~ith thiaceltlzonc.

I: Strilil3 175, I~xpl, 1. 2'0 rag. inoc~lum: 2: Stlnin 26NI. Expt. 3, 2 O t~3g. h]octl[~kl]l: 31 Str[lin 175, Expt, 2 20 rag. inocutul~l: 4: Strain 29NI. ~ixpt. 3, 20 illg. itlocllluill: 5: Strain 175, Expl. I. 0.5 Rig. iflOCLIJtHlli 6: Stlain 2(~NI, Expl. 3, (1.5 illg, Jr~ocu]klm: 7: Slri/i~l 175, EXI3I. 2, 05 n]g inoculum: S: Str;lin 2~)N'I, 15XDI. 3, 0"5 rag. il)OC[IILI~]3: 9: S[lilifl [)236. EXI)i. 3, 2 0 Illg. iROCU~[II]I: I(); S{rain 209. Exl~l. 2, 2 n rag. inocu)tlm:

I I; Straill 269, Expt. 2. 1.0 nlg, ilIOCLllLII]I: 12: StCaill 269. Expl. I, 10 n]g incct~lum: 13: Straill 316, Expl. 1, 2.0 rag, il]OCtl~ll]l: 14: S~(ai{~ 209, Fxpt. 2. 05 rag. illog'LlltHIl: 15: Strain 26t1, EXD I. ], 0"25 ing. [[]OCLlltl[l/: [6: Str'a/n 2(,9. Iixpt. 2, 025 Ill~. i~lOCU[tl[ll: 17: Str~lin P236. Fxl)t, 3, 0"5 rag. i[]ectt]ttll]: 18: Strait] 316, i~xpl, i, 05 Illg. inoctl[trn/.

four resistant strains. In the groups of treated mice infected with the three sensitive strains the sarvival period was longer as the dose or tlaiacetazol~e was increased, particularly ~,ith the doses o['0-8, 1-6 and 2'0 reg./mouse (100. 200 and 250 mg./kg/day), whereas even with the higher doses of thiacetuzone there was only a slight increase in the sarvival times of the trea{ed grotlps of mice infected with the four resistant strains. The mean survival periods in the mice receiving the 2'0 rag. dose of thiacctazonc ranged from 14.9 to 42-7 days with the sensitive strains, but only from 4.4 to 26"0 days with the resistant strains. The mice which received 0-5 rag. bacilli of the resistant strain 316 (Fig. 3. ]lumber 18) appeared to show a greater respm]se to treatment, bat tile range of the survival periods of these animals was exceptionally large and no statistically significant diJYerences were found between the mean survival periods ill the five treatment grotlps.

D i s c u s s i o n

The results oF the thiacetazone sensitivity tests on the sever Stlai1~s of tubele}e bacilli used in the study provide good evidence of real di/Terences in the h7 vitro sensitivity of organisms isolated fi'om untreated African patients. The four resistant cultures were selected as being the most resistant from a large 17Ollaber of pretreatment cultures and it is probable ttmt such strains occur only infreq uently.

In the guinea-pigs infected witlt the three sensitive strains, the extent of tuberculosls ~tt post- mortem examination and the number of viable bacilli in the spleen were lower in animals treated with 20 mg./kg, thiacetazone than in untreated animals, and even Iowo- in those treated with 80

T H / A C E T A Z O N E T R I ? A T M E N T

TAIILE II.--SUaVlVAI O1 MI( ' I INrEC31D \ ' q lH TIIIACIIAZONE'SENSNIVI TU/]IIRCI.I }3AC/LLI AND TREAIID \Ir T}IJAt EqAZONE

( )d/ t i le no. and I I SCqllilll'#)' tO ' Dose oy i : Dose o / I Per cent Survival period //u~wetazone bacilli I JL~pt. ! thiacetazotle [ .lulviving o f mi{e (dav~) (le~i,llttltcl' (rag.) I10. ] (n(~,]t)sOtt.~e) IIIl'ce at

roll'o) [ J 2] th(l',l RanRe Mean

0 i 0 I - 2 7'1 0 ' I j 0 1-10 7"1

2"0 I : 0 '2 I fl 6-14 ' 8 '4 0 ' 4 0 - 3 6"3

i o .} z2-42 1 . . . . . . . . . . . . 1 . . . . . . . . . . . . . 18"5

0-1 I 30 13 36 i 19-3 0 '5 (12 30 ! 12-26 I 183

0"4 4{1 ] i 12-44 ! 19"8 175 0"8 ] 100 ' ! 2 t - 40 ! 27"1

. . . . . . . . . . . > i 0 0 ] 1-7 , 5 0 {t-4 0 3- l I 7 '8

] ' ~ R I 2 ' 0 2 0'8 10 1-21 10"5

I 6 10 0-24 I 1"2 2"0 60 ! 1 4 - 4 3 26"0

. . . . . . . . . . . . . . . . . . . . . . . . . t - - - I

' i 0 2 0 '] I i -29 ! 18 4 0'4 h'O I 20-37 , 27"5

0"5 2 0 8 [00 i 23-57 3 7 0 I ( I 90 19-52 34-3 2 0 LO(} 27-60 4 0 2

0 0 1-12 i 5"8 0 4 0 2-11 6"9

26NI 2 0 3 0"8 0 I 16 7 '9 1'6 I0 I 29 12"9 2 0 10 1-31 1 4 9

. . . . . . . . . . . . . . . . . . . . . . i . . . . 0 30 12-38 21 '5 0'4 30 12"54 i 24"8

I { [ { : I 0 '5 3 0 8 30 13-58 i 25"5 1'6 40 22-62 ! 3 I ' 2 2 0 60 21-66 34'8

. . . . - - . . . . . . . . I . . . . . . . I . . . .

0 I0 I0-21 12"4 0"4 30 I 1-27 15'8

2"0 3 0 8 30 12-33 18'5 29NI I "6 40 I 1-38 21 ' I

2"0 80 12-55 26"9

R R : I 0 50 12-46 22"4 0 "4 i 80 18-40 29 4

i

0"5 3 0"8 100 2 t -48 30"3 1"6 l 100 27-53 39"5 2 0 100 23-72 42"7

4 2 7

428 TUBERCLI2

TaiJLe I11. SURVIVAL OI •41('1 [NIIC]ID ~XlII{ TI4IACEqaZt)NI:-REsI~FANI TUBI R( LE BACILI I AND TREAq 11) 3~tl [i TItIAf'F]AZCINI

CH/tllpr all d ~ , Slltl,[vcllp[,tiod senfft irilv to Dote o/ D o s e o[" Fer tc,m o['mt'ce (fktls d~iaceluzoae 6acilh" L:~pr dtiacetttzonr ~urviving ] tl't,,~istt~t1(e (Hig,) no. (mg.. l , louse) mice ell I . . . . . . . .

ratio) 21 /k/l's [ Rallgt, /'~lr

0 0 8-15 108 01 0 I0-18 13'3

1 0 1 0 2 : 0 10-15 11'3

0"4 0 ~ 7-15 10'4 0"8 0 8-14 t I 0

0 ' O 14-20 176 OJ 20 15~23 ~8'6

269 0'25 I 0 2 I0 15-21 180 0'4 20 7-28 17 0 0'8 40 14-32 20'7

RR :64 0 0 7-13 9 9 or more 0 4 0 6-13 9,7

I '0 2 0.8 0 7-12 %5 1.6 0 8-L3 10'4 2"0 0 8-16 106

316

RR~=4

209

R R = I 6

0 20 16-23 18"2 0",1 20 t 5-24 18 -6

025 2 0"8 10 12-42 1%5 I "6 40 t8-38 22-5 2"0 50 16-37 23-2

!

0 i 20 9-25 )4"1 0'1 0 8-14 117

2'0 I 0"2 30 10-44 19"5 0"4 0 9-14 l l - I 0 8 20 10-34 [5-9

0"5 I

2"0 2

0'5 2

0 0'1 0-2 0'4 0 8

0 0 4 0'8 1'6 2'0

0 0'4 0'8 1'6 2'0

70 15-43 26'2 50 14-73 37"7 80 18-80* 47 "4 90 17-80t 49"6

I00 21 -80 t 51 "5

0 6-8 7"0 0 1-9 6'6 0 I-[I 7"6 0 7-13 8"6 0 3-12 8"7

0 t2-~4 132 0 1]-15 13.3 0 11-16 13-5 0 [I-18 i4"7

I0 14-22 16.2

(cont iuued on nex t pc~ge

] IIIACI~TAZONI! TRI A'FMENT 429

TA ml. 11 I. (colttitttwd)

C*dmre no. emd Szo vival period sens#h'#y to Dose o/ Dose o/ Per cent ol mhe Oho's) Ihhl,'erozotre I,aci/h" KvW. fllt~gcelasolw ~ttlv/l'/l~g

I'e.~islat:~e (otG) I~0. (l~g,,/nlo~rae} iit&e at . . . . . . . . . *at#a) 21 laoa Ratlge Mett~z

0 0 1-6 2 '9 % 4 0 1-7 3 '6

2"0 3 0'8 0 0=7 2"7 P 236 1"6 0 I-7 4 O

2'0 0 0-9 4'4

0 0 10-19 14"5 R R 16 0 '4 I 0 13-27 I 7 '0

(}'5 3 0'8 20 / 3-3f~ 19 0 I '6 6 0 )6-44 249 2 0 i 60 I ?-49 26"0

* "FJn'ee mice survived al 80 daft. i T\~o nlice survived at 80 days,

mg./kg, thiacetazooe. There >,,as, however, little response 'to IrefiulaeiM iIl the guinea-pigs h'fccted with tim four resistant straius, The results with one of these resistant strains (316) were d[mcult to interpret since the disease was i'~ot progressive ill the untreated allJmals, With tile remaining fllree vh'uletll resistant slFahls, the extent of the disease alld the spleen counts were slightly lower" in the trealed (han it1 tile untreated guhlea-pigs infected with the two strahls lhat had resistance ratios of 16, whereas they were sfightly higher in tile trcated guinea-pigs infected with the strain I[lat had a resistance ratio of 64 or f0orc, Thlls , guinea-pigs hlfccted with (he sensitive strains i 'esponded much better to [realnlent 01aii those in~'ccled with the i-esistallt Stl-ai[is alld, amoi lg the resistant strains, there is a suggest ion that the response might have been associated with the degree of resistance,

All of the mice infected with *l~e, three sensitive strains died. even when treated with 250 reg./ks. tltiaeetazone, tile highest dose used. The mean stn'vival t imes were prolonged by treatment, btlt only to a noticeable extent when the dose of thiacetazone was I00-250 m&/kg, In the mice infected with the Iour resistant strains, only a very slight prolongat ion ill the nlean survival t imes occtnTed its *! result of t reatment with 200 and 250 ms . /ks , thiacetazone, These results indicate that the response to treatmetlt of mice hlfectcd with sensitive slrahls was greater than the response of mice infected with resistant strains, but the difFelvnce is less convincing than in the experiments with guinea-pigs.

The dJfl'erence between the responses to treatnlent o(" guinea-pigs and mice infected with the sensitive strains might have been dtie to the natm-c of filch- disease dur ing tile period of treatment. Trea tment of the guinea-pigs wlts star[ed three weeks after inlkction, when allergy would have developed and the tubercle bacilli would be lying mainly outside tile host cells. However, t rea tment of the mice was started immediately after infection when tile bacilli would be mainly h~tracellular. If" thiaceta/_of/e does ilot penetrate inlo cells oz is inactive wilhit~ then1, the pool- response of the mice might have been due to the intracellular location of their bacilli. Streptomycin is known to be less active against intraceltular than against extraee|lular tubercle bacilli (Maekaness & Smith, 1953). Steenken & Pratt (1949) and Jensen (1949) have shown that the progress of experimental tttberctl- losis in the guinea-pig was Iittle affected by treatment with s t reptomycin dur ing the first fortnight after inl~clion, perhaps ana logous to the pool- response of our mice to thiacetazone. However. there was a good response to tl-eatment with s t reptomycin dur ing tile fol lowing weeks, posslb]y corles- ponding to o u r experience with guinea-pigs.

4 3 ( ) T U I~ E I /C L E

In experillaenls Io be reported elsewhere, guinea-pigs were found (o buve sertlm concelltl'tdiolls averaging 2.6 [~,g./ml. thiaectazone at three hours after the last of several daily oral doses of thiai:;ela- zone 20 ms. /ks , body weight (one of the doses used in the present study) and concentrations of 4 ' l lJg,/ml, at six hours and 0-8 !ag./ml. at 24 hours were obtained. In man, Heilmeycr & Heilmeyer (1949) found peak serum concentrations g-6 [zg./ml. :It four hours after a dose of 300 nlg. thiaceta- zone. Irrom the data of Geks (1950). it can be calculated tha~ serum concentrations ot"0.5-2 1).g/ml. were present 3 hour~ after a dose o[" 100 ms.. and 2-5 :~.g/ml. after doses of 300-500 mS. Simonds and others (1950) obtained serum concentrations of 0-5-1.0 b,.g,/ml, at 3-6 hours. 0"5-0'68 !*g./ml, at 12 Iltltlrs and 0"25-0"5 [*g./ml, at 24 hOtlrs after a single dose of 200 nlg. thiaceta- zone. but, after repeated doses of 200 I l lg, , ill a total dosage of 800 nlg. t3(21" day, file SelUnl GOllCell- tratioi/s in paticllts wcrc 6-17 ,].g./ml, Estinlatcs o1" the serum concelltraliOllS atlained whetl thiaceta- zone is given in a single daily dose of 150 ms., as reconlmcllded in the main rcport of the thiacetazone study least Afriean/l}ritish Medical I~.esearcb Council, 1963a), are not yet available. However. it can be concluded thai the concenlralions o[" Ihiacetazone attained in the blood o1" the guinea-pigs in the present study were not greatly dilI'crent.from those in patients given the usual dosage o1" thiacetazone, even though the dose exprcssed per ks. body weight was considerably h i g h e r in the gk l i l lea-p igs t han i l l m a n .

All aecompal/ying rcporl (East Af/ican/lh-itlsh Medical I~.esearch Cocmcil. 1963b) exttmhlcs the inlItlCllCe of pretreatment drug-resistance ell tile response to chemotherapy of tile patients partici- pating in the two chemotherapy sttidies 0-on1 which the strains tested in anlmfds were obtained. A weak association was Found bctwcen the initial sensitivity o[" the organisms to thiacelazone and the pl'OglesS 01" the patiellts during treatnlent with thiacetazorle and isoniazid. The eollelusiorl that pretreatmenl (hlaeelazone sensitivity probably allEcted the results of treatment is strengthened by tile findings of the present study. However, it should bc emphaslzcd that resistant cultures with as high a degree of resistance to thiacetazone as those used in the animal experiments wine rarely isolated from the paticnts~ Only 10 of the 228 patients assessed had pre-trealment cultures with a mean m i n i n l a l i n h i b i t o r y c o n e e n t r n t i o n s o f inore t h a n 4 ,,zg./nlt. t h i ace tazone ( f o u r o r more t imes Ihe median sensitivity of all pretreatnlelll -drains). Thus, pretreatnlent lesistanee to thiacetazone is likely to p]ay only a minor role in 111e treatment o[" East African patients with thiaeetazone piLlS ison iazid.

We are gralel"tll Io Miss Jant21 Ll{~yd for techllical {D, sislanci2,

I~. E FI'ZR ENCES I ] 1 / t I istl ~[/]~t R( ULI)NJS ANS[)( [A I ION { ] 963). 7)dwrc/e. LomL, 44. I. EASI AIRICAN/I]RI HSll b,41DI('AL I~I-%IS~RCII COUNCIL ThiacclaZOllC/Diphcnyhhhxwea Investigation (19(10). 7)lberd<'.

L(md,, 41, 399. �9 EAS[ AFRIUAN]BRI IISII MII)If. AI R[SIAI/L'II C()UNCII. (1963a). 7)tbe~de, Lemd. [11 press. LzASr AJIIICAN/I]RIIISlt MII)ICAI. RfSE.%RCII COUNCIL 11963b). Tuberch', gotld, In pl'eSs. Gigs, F J. (1950).Z.H.!7,'. lt!/~,r'l.-Kr.. 131, 345. th II ~.11 yl!14. I, & HEILMEyI R, I,. (1949). Kiln. [t'ischr.. 27, 790. JENSI N. K. A. (1949). lit'let, ndwtc., sc~md,. SuppL 21, 42. MM'KANI:';:;. G. [L & SMIlII. N. (1953). Anwr. Rev. 7)there.. 67. 322. MIICIIIS;f)N. D, A.. BIIAIIA. A. L., RAI)IIAKR(SIINA. S., SILKEN, J, 8., SUllIIAIAII4 T. V.. & WAI LACI, J. G. (1961).

Btdl, IVh/. II/Ih. 0r 25, 285. MII('IIISON, D, A., WAI IA(:[. J, G., BIIA[IA, A, L,, SH.KON. J. B,. SUIll]AIAII, -r, v., & LANCASIER, M- C. (1960).

1)IbeJch'. l.ond,> 41, I. Rlsr, N., CAI.S, M., & JLI t l lN , W. {]951). Aim. ht.w. Pavte~w, 81, 324, SIMMONS. O,, HOIISON, [.. 1]., RISNICK. A.. DI NI('OLA, R,, & [3ENNEI-I. R, I-I. (1950). Amer. Rev. 7)/hl,l&. 62, 128. SIll NKI N, W, & PI/A I'T, P. C. { 1949). Anwr. Rev. 7)thl,rc., 59, 664.