COMBINEDDRUGTREATMENTOF TUBERCULOSIS. III. CLINI-CAL APPLICATION OF THE PRINCIPLES OF APPROPRIATEANDADEQUATECHEMOTHERAPYTO THE TREATMENT

OF PULMONARYTUBERCULOSIS

By WILLIAM F. RUSSELL, JR., IRVING KASS, ANGELINE D. HEATON,SIDNEY H. DRESSLERAND GARDNERMIDDLEBROOK

(From the Department of Medicine and Research and Laboratories, National Jewtsh Hospitalat Denver, and the Departments of Medicine and Microbiology, University of

Colorado School of Medicine, Denver, Colo.)

(Submitted for publication September 10, 1958; accepted February 23, 1959)

Microbiologic investigation of the prevention ofemergence of mutant populations of tubercle bacilliresistant to streptomycin and isoniazid emphasizedthe fact that adequate concentrations of each ofthese two appropriate drugs were necessary (1).Delivery of such concentrations of isoniazid tothe multiplying parasites can be estimated bymicrobiologic assay (2-4). Antimicrobially-ac-tive serum streptomycin assayed by a similarmicrobiologic method confirmed that there werealso probably significant variations between in-dividuals in serum levels of this drug as well.These observations suggested that conventionaldosages of these drugs could not be relied upon toinsure the delivery in vivo of sterilizing concen-trations of each agent to physiologically-suscepti-ble tubercle bacilli. The result of consideration ofthese principles was the formulation of a chemo-therapeutic regimen combining high dosage ofstreptomycin with high dosage of isoniazid for allpatients (5-7). The purpose of this report (thethird of a series) is to present the results of clini-cal application of this approach to chemotherapyof pulmonary tuberculosis.

METHODSAND MATERIALS

A. Bacteriology. The bacteriologic studies were per-formed according to methods described elsewhere (8, 9).These routine studies consisted of Ziehl-Neelsen stainedsmears of the concentrated, digested sputum; the re-sults were reported as the number of acid-fast rods seenper oil immersion field. The concentrated sediment of thedigested sputum was then diluted according to the num-ber of acid-fast rods seen on smear. When the smearrevealed less than one acid-fast rod per oil immersionfield, the concentrate was inoculated both undiluted anddiluted 101. When the concentrate smear revealed oneto 10 acid-fast rods per oil immersion field, the sedimentwas diluted 101 and 10'. When the smear revealed more

than 10 acid-fast rods per oil immersion field, the sedi-ment was diluted 10' and 10' prior to inoculation of theculture plates. Every specimen was seeded on solid me-dium (1) containing no antimycobacterial drugs (thecontrol), on the same medium containing streptomycin,2.0 pg. per ml., and on the same medium containing iso-niazid, 0.2 ,ug. per ml. During the period covered in thisstudy, a third direct routine drug-susceptibility test wasperformed. Prior to 1955, this third test was isoniazid,10 Mg. per ml. In 1955, pyrazinamide, 50 Mg. per ml., wasthe test drug for some months. Since that time, para-aminosalicylic acid, 2.0 ,ug. per ml., has been used in thisthird susceptibility test.

The definition of drug-susceptibility employed in thisreport is as follows: A strain or population of tuberclebacilli is susceptible to the action of streptomycin orisoniazid if less than 1 per cent of those members yieldingcolonies on the control are able to grow and produce col-onies on culture medium containing either streptomycin,2.0 pg. per ml. or isoniazid, 0.2 Ag. per ml. Such bac-terial populations were considered significantly drug-re-sistant if more than 1 per cent was recovered on mediumcontaining streptomycin or isoniazid in these concentrations.

Catalase testing, using 15 per cent hydrogen peroxidein 5 per cent Tween® 80, was performed routinely on thecontrol culture (8, 9). As employed, this test providesa crude estimate of the moderate catalase activity oftypical tubercle bacilli, the high activity of the so-called"yellow" bacilli, and the reduced or absent activity ofcatalase-deficient, isoniazid-resistant mutants.

The culture plates were incubated under 2 to 5 percent carbon dioxide in air at 36° C. for 21 days (10).The results of culture, drug-susceptibility testing, andcatalase testing became available for clinical use within25 days following collection of the sputum. This provideda unique opportunity for the selection of the appropriateagents and for close monitoring of the bacteriologic re-sponse of the patient.

Bacteriologic conversion of the sputum to negativewas defined to have occurred when three consecutive spu-tum specimens collected at monthly intervals were found tobe negative both on smear of the concentrate and on culture,and no subsequent positive sputum studies were encountered.The date of conversion was that of the first specimenwhich was negative on both smear and culture. This

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TABLE I

Cumulative bacteriologic conversion(chemotherapy alone)

Negative at Negative at Negative atGroup Total 1-4 mos./total 5-8 mos./total 9-12 mos./total

I (153) 127/153 152/153 141/142*II (39)* 12/39* 15/36* 16/34*

III (117)* 11/117* 13/105* 14/82*

* Losses due to early discharge or conversion con-comitant with surgery or collapse procedures.

has been reported as occurring in the one to four monthperiod (from Day 1 through Day 120 of chemotherapy),the five to eight month period (from Day 121 through Day240 of chemotherapy), the nine to 12 month period, and soforth. When sputum conversion occurred as the result ofcollapse procedures (pneumothorax, pneumoperitoneumor thoracoplasty) or resection surgery, the subject waseliminated from further bacteriologic statistical analysis;this accounts for some of the reduction in actual casesobserved in Table I.

B. Roentgenography. Admission roentgenograms ofthe chest were interpreted according to the DiagnosticStandards of the National Tuberculosis Association, 1955(11), with the following differences: 1) Cavities havebeen reported as single or multiple, and 2) cavity size hasbeen reported as the greatest internal dimension of thelargest single cavity. Thus, in some instances, moderatelyadvanced disease has been reported here associated with alarge cavity or with multiple small cavities the sum of thediameters of which would require designation as far ad-

zanced by N.T.A. Standards. This modification was

necessary because it seemed important to analyze the ex-

tent of disease separate from the cavity component.X-rays made during the one to four month period and

the five to eight month period have been compared withthe admission film in order to assess the response totreatment. Comparison roentgenograms were selectedwhich were made near to the end of the period. How-ever, when collapse procedures or resection surgery were

employed, the film taken just prior to the procedure was

used for the final comparison and no further X-rayevaluation was made for these studies. This accountsfor some of the reduction in total cases observed inTables II and III.

Special roentgenographic studies, including tomography,were performed early during the admission of eachpatient, and were repeated as indicated.

C. Microbiologic assay for serum isoniazid. Estima-tion of the serum concentration of antimicrobially-activeisoniazid has been an important part of the evaluationof our patients during most of the period covered inthis study. Determinations were made by the micro-biologic method in 280 of the patients being reportedhere (12, 13). These patients were divided into three cat-

egories on the basis of their serum levels of active iso-niazid. Thus, 76 had levels less than 0.4 ,ug. per ml.; 94had levels of 0.4 to 0.8 ,ug. per ml.; and 110 had levelsin excess of 0.8 ,Ag. per ml. It has been estimated thata serum level of active isoniazid of 0.4 dg. or more per

ml. at six hours after one-half of the daily dose is ade-quate for optimal chemotherapeutic action (2).

When it was shown that concomitant administration of

BLE II

General roentgenographic response to chemotherapy

None-slight Mod.-markedTotal Worse improvement improvement

1-4 5-8 1-4 5-8 1-4 5-8 1-4 5-8Group mos. mos. mos. mos. mos. mos. mos. mos.

I 153 153 1 1 83 49 69 103II 40 37* 0 2 34 23 6 12

III 123 99* 3 5 116 87 4 7

* Losses due to collapse procedures and/or resection surgery.

TABLE III

Roentgenographic response of cavities to chemotherapy

Cavity status

Present

Total Open Filled Indefinite Lost to view

Group 1-4 5-8 1-4 5-8 1-4 5-8 1-4 5-8 1-4 5-8

I 145 145 117 71 3 12 2 11 23 51II 40 38* 37 30 0 3 2 2 1 3

III 118* 94* 112 79 3 9 1 1 2 5

* Losses due to collapse procedures and/or resection surgery.

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TABLE IV

Criteria for selection of patients*

Total adult pulmonary tuberculosis casesEliminated for following reasons:

1. Sputum negative, consistently2. Sputum positive (minimally), with

unsatisfactory drug-susceptibilitystudies

3. Sputum positive, "atypical yellowbacilli"

4. Sputum positive, typical tuberculebacilli, treated less than 6months-Dead

-Irregular discharge

184

41

16

8t39

288All bacteriologically proven pulmonary tuberculosis

patients excreting typical tubercle bacilli, treatedat least 6 months

* October 1, 1954, to June 1, 1957.t Five nontuberculous deaths; three surgical deaths, all

excreting drug-resistant organisms.

para-aminosalicylic acid (or para-aminobenzoic acid) ele-vated the serum levels of active isoniazid, this drug (inthe dosage of 12 Gm. NaPAS or NaPABA daily) was

added to the basic regimen of those patients known to havelevels less than 0.4 MAg. per ml. (12, 13). The specificpurpose of adding either of these drugs to the basic regi-men in these individuals was to elevate the serum levelof active isoniazid.

D. Microbiologic assay for serum streptomycin. Serumlevels of antimicrobially-active streptomycin have beendetermined in 48 of these patients. There were four whohad levels less than 10 Mug. per ml.; 13 had levels of 10lAg. per ml.; 23 had levels of 20 Ag. per ml.; and eightachieved levels in excess of 20 ug. per ml. The micro-biologic method used was a serial tube dilution techniqueemploying an isoniazid-resistant strain of H37Rv. Thismethod has yielded generally lower values than the filter-paper disc-agar diffusion method using a strain of B.subtilis (3). Limited correlation of chemotherapeuticresponse with streptomycin levels at six hours following

a 20 mg. per Kg. test dose suggested that patienits whofail to achieve a level in excess of 20 ug. per ml. were

receiving what might prove to be insufficient dosage.Thus, in this study, several patients in Group I (see be-low) who had "adequate" isoniazid serum levels and hadnot converted to negative before 10 weeks after initia-tion of chemotherapy and who had streptomycin serum

levels below 20 ,ug. per ml. were given increased dosage ofstreptomycin up to 35 mg. per Kg. per day.

E. Clinical material. Between October 1, 1954, andJune 1, 1957, a total of 606 patients with the clinical diag-nosis of pulmonary tuberculosis were admitted consecu-

tively to the adult service of National Jewish Hospital atDenver (Table IV). Of these, 288 were considered un-

suitable for bacteriologic evaluation of the high dosageregimen. Sputa from 184 of these 288 patients were

consistently negative, and sputa from 41 were so in-frequently and so slightly positive that drug-susceptibilitytests were unsatisfactory.' Sixteen patients were ex-

creting "atypical yellow bacilli," Mycobacterium tubercu-losis luciflavum (14). Of those patients excreting typi-cal tubercle bacilli, eight were lost because of death(five nontuberculous deaths, and three surgical deaths;these latter were excreting drug-resistant tubercle bacilli),and 39 because of irregular discharge, after less than sixmonths of treatment. This study is concerned with theresults of high dosage chemotherapy of 318 adults ad-mitted consecutively with bacteriologically-proven pul-monary tuberculosis, with typical tubercle bacilli recov-

L ered on sputum culture, and with treatment for at leastsix months.

On the basis of the results of drug-susceptibility test-I ing of the populations of tubercle bacilli recovered from

the admission sputa, these 318 patients have been divided

One of these patients became consistently sputum-positive with streptomycin-resistant, isoniazid-resistant,para-aminosalicylic acid-susceptible, catalase-negative tu-bercle bacilli in the sputum four months after admissionand while on chemotherapy with isoniazid/para-amino-salicylic acid.

,BLE V

Background data on 318 patients grouped according to drug-susceptibility studies

Average ageGroup Drug susc. Sex Number (yrs.) Range White Nonwhite

M 94 40.5 (15-68) 80 14I SM-S/INH-S*

F 59 37.6 (17-80) 50 9

SM-R/INH-S M 26 41.5 (21-70) 24 2II

SM-S/INH-Rt F 14 28.8 (17-56) 14 0

M 71 44.6 (20-68) 67 4III SM-R/INH-R

F 54 38.6 (20-69) 47 7

*-S, susceptible to streptomycin (SM), 2 ug. per ml., or isoniazid (INH), 0.2 Mg. per ml.t -R, resistant to SM, 2 ,ug. per ml or INH, 0.2 Mug. per ml.

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TABLE VI

Comparative roentgenographic characteristics

Extent of disease Cavity status Size I II III

Minimal 1 2Moderately advanced, Uni, No cavity 3

Sing. 0.5 4 2 21-2 3 2 42.5-3.5 1 1 54.0-7.5 1

Mult. 1-2 6 1 22.5-3.5 1

Bil, No cavity 3 2Sing. 0.5 2

1-2 17 4 102.5-3.5 10 74.0-7.5 1 1 4

Mult. 0.5 11-2 12 2 22.5-3.5 17 1 84.0-7.5 5 38.0Oor > 1 1

Far advanced, Uni, Sing. 4.o-7.5 1Mult. 1-2 1

Bil, No cavity 1 1Sing. 1-2 4 6 6

2.5-3.5 4 2 44.0-7.5 4 1 68.0Oor > 1

Mult. 1-2 8 2 102.5-3.5 14 5 134.0-7.5 20 5 198.0Oor > 7 4 13Total 153 40 125

into three categories (Table V). Group I patients were

excreting streptomycin-susceptible, isoniazid-susceptiblepopulations of tubercle bacilli. Group II patients were

excreting either streptomycin-resistant, isoniazid-suscepti-ble or streptomycin-susceptible, isoniazid-resistant popu-lations. Group III patients were excreting streptomycin-and isoniazid-resistant populations. All patients ofGroups II and III (except possibly one) who were ex-

creting isoniazid-resistant populations of tubercle bacilligave histories of at least three weeks' previous treatmentwith isoniazid or its chemical derivatives. Similarly, all,except four patients, who were excreting streptomycin-resistant populations gave histories of previous strepto-mycin or dihydrostreptomycin treatment.

These three groups were similar with respect to age,

sex, and race (Table V). Comparison of the membersof these three groups of patients by roentgenographiccharacteristics of disease revealed that they were suffi-ciently comparable for the purposes of this study(Table VI).

F. Basic chemotherapeutic regimen. This consistedof streptomycin sulfate (Squibb), 1 to 2 Gm. daily (20mg. per Kg. per day or more in those patients who hadlow streptomycin serum levels), by the intramuscularroute in a single dose, for at least 90 days. Streptomycinwas not discontinued until the sputum was negative on

smear and culture of the concentrate. Isoniazid (Ny-drazid®, Squibb), 16 or more mg. per Kg. per day, was

administered orally for at least 18 months. Isoniazid

was given in two equal doses each day. Pyridoxine hy-drochloride (Squibb), 50 to 100 mg. per day, was alsogiven orally for 18 months. Pyridoxine was included inthis regimen to reduce the incidence of significant isonia-zid neuropathy (15). It should be emphasized that fol-lowing termination of streptomycin, the isoniazid andpyridoxine were continued alone.

During the period covered in this report, the effect ofshort-term, 21 day chemotherapy with pyrazinamide(2.5 Gm. per day) and cortisone (100 mg. per day), tobe followed at once by the basic regimen, was under in-vestigation in 37 of these patients. So far no significantdifference in results of this strictly randomized studyhas been observed.

Treatment with the basic high dosage regimen was

TABLE VII

Basic chemotherapeutic regimen adaptations

Group Drug-susc. Chemotherapy

I SM-S/INH-S Basic high dosage regimen(SM/INH)*

II SM-R/INH-S INH/PASlSM-S/INH-R SM/PAS plus INH

III SM-R/INH-R VM, PZA, PAS, TTC, SM,and INH*

* Para-aminosalicylic acid (PAS) or para-aminobenzoicacid (PABA) added if serum INH level < 0.4 jg. per ml.

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TABLE VIII

Significant drug reactions

Drug Manifestation Number

Streptomycin Dermatologic reactions* 4t(SM) VIlIth cranial nerve,

vestibular dysfunction* 3Optic neuritis (?) 1Thrombocytopenic purpura,

Isoniazid aggravated by INH 1(INH) Psychosis 4

Neuropathy, incipient 12Dupuytren's contracture 2tEmotional storms 12

Total 39 (12%)

* Severe reactions.t Chemotherapy continued with steroids.

considered to be appropriate for the Group I patients(Table VII). Half of Group II patients were excretingstreptomycin-resistant, isoniazid-susceptible organisms andisoniazid plus para-aminosalicyclic acid (NaPAS, 12Gm. per day) was appropriate. The other half ofGroup II were excreting streptomycin-susceptible, iso-niazid-resistant tubercle bacilli and daily streptomycin, 1to 2 Gm. for at least 90 days with para-aminosalicylic acid(NaPAS, 12 Gm. per day) plus isoniazid, was deemedappropriate chemotherapy.

Patients in Group III were treated with viomycin,pyrazinamide, para-aminosalicylic acid, tetracycline andstreptomycin in a variety of combinations with isoniazid.Most patients in Groups I and III who had active isonia-zid levels less than 0.4 ,ug. per ml. were also given para-aminosalicylic acid or para-aminobenzoic acid to raise theisoniazid level. Isoniazid was included in every regimenbecause it was considered appropriate to prevent back-mutation. Both animal experimentation (16) and invtitro studies (17) have proven that back-mutation toisoniazid-susceptibility can occur.

After initiation of chemotherapy, all patients, exceptthose with severely restricted cardiopulmonary function,were out of bed for 10 to 12 hours daily.

Collapse procedures and/or resection surgery wereperformed on Group II and III patients when deemedappropriate, but not on Group I patients until four monthsafter conversion.

G. Drug reactions. Serious drug reactions with thehigh dosage regimen incorporating pyridoxine have beeninfrequent (Table VIII). Significant reactions to strep-tomycin or isoniazid occurred in 12 per cent of ourpatients. The specific drug was permanently discon-tinued only for those few patients manifesting severevestibular nerve reactions, optic neuritis, aggravation ofthrombocytopenic purpura or psychosis. No apparentchange in clinical response to chemotherapy was ob-served in these patients following termination of theoffending drug.

RESULTS

There was a striking difference in roentgeno-graphic inprovement estimated separately from

changes in cavity status between Group I andGroups II and III after four months of chemo-therapy, and this became more marked duringthe five to eight month period (Table II). Twoof the patients in Group III were subjected tothoracoplasty within 60 days of the start of chemo-therapy, and so do not appear in this analysis.Similarly, three Group II patients and 24 GroupIII patients were eliminated from X-ray evalua-tion by the application of collapse procedures orsurgical resection during the five to eight monthperiod.

In 51 of the Group I patients with demonstra-ble cavitation on admission, no cavities could bedetected on routine X-rays and tomograms by theend of five to eight months of chemotherapy(Table III). As a further evaluation, analysisof the changes in cavity status confirmed the su-perior response to chemotherapy of Group I.

One hundred and fifty-two Group I patientshad sputum conversion to negative by the end ofthe sixth month of chemotherapy (Table I). Nodrug-resistant cultures were obtained from thesepatients prior to conversion. However, there wasone chemotherapeutic failure, a 48 year old whiteman with far-advanced, bilateral cavitary disease.He had been treated with isoniazid, 300 mg. perday, and para-aminosalicylic acid, 12 Gm. per day,three days before admission to this hospital. Thetubercle bacilli proved to be susceptible to strep-tomycin and isoniazid. His serum isoniazid levelat six hours following a 4 mg. per Kg. test dosewas 1.2 ,ug. per ml. His serum streptomycin levelat six hours after a 20 mg. per Kg. test dose was20 ,ug. per ml. Both levels can be consideredadequate for optimal antimicrobial effect. Hissputum became negative on smear and culture inless than 12 weeks of chemotherapy with iso-niazid, 16 mg. per Kg. per day, and streptomycin,20 mg. per Kg. per day. However, six weekslater his sputum suddenly reverted to positive withtubercle bacilli simultaneously resistant to bothstreptomycin and isoniazid; his sputum culturesremained positive.2

2 The possibility that this was the result of exogenousreinfection was considered. Survey of all hospitalizedpatients excreting organisms resistant to streptomycin(SM) and isoniazid revealed only one likely candidate.However, the organisms from that patient were highlyresistant to streptomycin, 16 ug. per ml., while the or-

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TABLE IX

Cumulative bacteriologic conversion(collapse procedures and resection

in addition to chemotherapy)

1-4 5-8 9-12Group Total Mos. Mos. Mos.

1 (153) 127 (153) 152 (142) 141II (40) 13 (40) 19 (39)* 21

III (125) 19 (125) 33 (107)* 39

* None of Group I was subjected to collapse or resectionuntil at least four months after conversion. Losses shownare due to early discharge.

These results with chemotherapy alone forGroup I patients are strikingly different fromthose of both Groups II and III. If the GroupII patients whose sputum became converted tonegative concomitantly with collapse proceduresor resection surgery were considered togetherwith those achieving the converted state by chemo-therapy alone, the total conversions by the endof one year would be only slightly more than halfof the cases (Table IX). Similarly, only aboutone-third of the Group III patients became con-verted to the sputum negative state by the endof one year of treatment.

Follow-up observationts oni Group I patients

Follow-up observations have been completed on90 per cent of the first 113 Group I patients whowere treated for at least six months. At the timeof this study, these patients had been observedfor an average of two years since the start ofchemotherapy, ranging from 18 to 33 months.Fifty-three of these patients were still on chemo-therapy at the time of follow-up study, and 51had terminated all antimicrobial drugs. The aver-age period of time between termination of chemo-therapy and the mailing of the most recent fol-low-up questionnaire was five and four-tenthsmonths, ranging from one to 15 months. Ofthose patients whose chemotherapy was discon-tinued, only one patient has had a relapse, bothbacteriologic and roentgenographic. This 39 yearold white man had partial gastrectomy, cho-ledocoduodenostomy and gastroenterostomy priorto the diagnosis of tuberculosis. He was admittedto hospital with moderately advanced, unilateraldisease and multiple cavities, 1 to 2 cm. in great-ganisms from the failure were fully susceptible to 8 jug.per ml. even though highly resistant to 2 ,ug. SMper ml.

est internal dimension. At that time his electro-cardiogram revealed evidence of old anteriormyocardial infarction. He was treated with thebasic high dosage regimen to which was addedpara-aminosalicylic acid to elevate the serum levelof isoniazid. Early during this hospitalization, hesuffered another myocardial infarction, and wasplaced on strict bed rest. He complained bitterlyof para-aminosalicylic acid gastrointestinal irrita-tion, and was unable to accept this medication regu-larly even though it was needed to elevate theserum isoniazid level. Resection of the cavitarydisease was considered necessary, but surgerywas postponed indefinitely because of the cardiacstatus. The patient left the hospital, discontinuedall drugs in the seventh month of chemotherapy,and six months later he was readmitted to ourhospital excreting streptomycin-susceptible, para-aminosalicylic acid-susceptible, catalase-positive,isoniazid-resistant organisms in his sputum. Todate this is the only known relapse among the153 Group I patients.3

DISCUSSION

The results of serum assays for antimicrobially-active streptomycin and isoniazid have suggestedthat conventional dosage could not be relied uponto insure adequate delivery of sterilizing concen-trations to susceptible tubercle bacilli in chemo-therapy of pulmonary tuberculosis. Therefore, achemotherapeutic regimen was designed combin-ing high dosage of streptomycin with high dosageof isoniazid. This high dosage regimen was ad-ministered to all patients excreting streptomycin-susceptible, isoniazid-susceptible tubercle bacilli onadmission. Six hour serum assays for strepto-mycin and isoniazid were performed on some ofthese patients in order to document the adequacyof these drugs.

3 Since this report was submitted for publication anotherpatient has suffered a bacteriologic relapse. Both re-lapse cases had several points of similarity. Both be-came bacteriologically negative prior to the start ofchemotherapy, even though positive on smear and cultureon admission. Both left the hospital shortly after sixmonths of chemotherapy, and both discontinued chemo-therapy against medical advice in the seventh month.This second patient relapsed 21 months after stoppingmedications. And, finally, these two relapses were theonly patients in Group I zw'ho were treated with druigs forless than nine months,

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A few large groups have carried out extensivestudies on the chemotherapy of pulmonary tuber-culosis (18-21). Retrospective analysis of theresults of these investigations of conventional drugtreatment regimens has revealed that 10 to 20per cent of previously untreated patients fail toachieve the state of bacteriologic negativity withoutcollapse procedures or surgical resection. In vitrodrug-susceptibility tests indicated that most ofthese failures of chemotherapy resulted from theemergence of drug-resistant mutant populations oftubercle bacilli. In spite of the fact that combineddrug regimens were used in these studies, failureof combined chemotherapy employing conventionaldosages frequently was manifested by the earlyemergence of mutants resistant to only one of thetwo drugs administered. Experiments performedin our laboratory have indicated that selection ofmutants resistant to one drug on exposure to twodrugs is a consequence of exposure to effectiveconcentrations of that one drug plus ineffective(sub-inhibitory) concentrations of the companiondrug; in effect, this is equivalent to exposure toonly one drug although the single-drug-resistantmutants recovered are strikingly reduced in num-bers (see Experiment No. 2, in the first report ofthis series).

It seemed reasonable to postulate that increas-ing the dosage of each drug should largely elimi-nate such evidences of inadequate delivery of drugsas the emergence of organisms resistant to one ofthe two drugs. Support for this hypothesis wasfound in the British Medical Research Councilreports (18, 19) which indicated that streptomy-cin, 1.0 Gm. daily, combined with isoniazid, 200mg. daily, gave results superior to streptomycin,1.0 Gm. twice each week, plus the same dosage ofisoniazid (200 mg. per day). Further supportfor this postulate was evident in a study by Biehl(22) who made a clinical comparison betweenstreptomycin, 1.0 Gm. twice each week, combinedwith isoniazid, 5 mg. per Kg. per day, and thesame dosage of streptomycin (1.0 Gm. twice eachweek) plus isoniazid, 20 mg. per Kg. per day.The regimen including high dosage isoniazidcombined with low dosage streptomycin (2.0 Gm.per week) failed to prevent the emergence ofisoniazid-resistant organisms. Viewed in the lightof these studies, the excellent results obtained inGroup I patients could have been anticipated.

The results obtained by application of the highdosage isoniazid-high dosage streptomycin basicregimen to the chemotherapy of Group I patientsare superior to the results reported by other in-vestigators with the exception of Ross, Horne,Grant and Crofton (23, 24). It can be contendedthat this superiority is attributable to factors otherthan chemotherapeutic. The type of patient ma-terial in terms of anatomic extent of disease, num-ber and size of cavities, and duration of activedisease, for example, may be less challengingamong patients accepted for admission to Na-tional Jewish Hospital. There may be other im-portant variables, such as altitude (5,300 ft.), orthe complex advantages of hospitalization per sewhich would favor the obtaining of such results.However, our experience prior to October of1954 with chemotherapeutic regimens which didnot include both high dosage of isoniazid and highdosage of streptomycin provided adequate evi-dence, in our opinion, that such unidentified vari-ables were not operating in these studies. Forexample, between January of 1953 and October of1954, 43 adult patients excreting typical tuberclebacilli susceptible to both streptomycin and iso-niazid were treated with streptomycin, 1.0 Gm.intramuscularly every third day, combined withisoniazid, 8 or more mg. per Kg. per day, for atleast four months before collapse procedures orsurgical resection. Thirty-one (72 per cent) ofthese patients became bacteriologically negativeby Day 120 of chemotherapy, but 11 (26 per cent)were chemotherapeutic failures by virtue of theemergence of isoniazid-resistant organisms (25).Thus, our experiences at this institution prior tothe initiation of the high dosage regimen describedhere were quite comparable to the experiences ofother investigators employing similar chemothera-peutic regimens.

With regard to the results reported by Croftonand his associates, it should be pointed out thattwo uncontrolled variables render their studies in-comparable with the Group I experiences describedhere. First, they have reported that all threedrugs (streptomycin, isoniazid and para-amino-salicylic acid) were often used during the firstsix to eight weeks of chemotherapy, i.e., until theresults of drug-susceptibility studies became avail-able (26). Para-aminosalicylic acid is known todecrease the rate of metabolic inactivation of iso-

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APPROPRIATEAND ADEQUATECHEMOTHERAPYOF TUBERCULOSIS1

niazid (2, 3), and, therefore, can obscure the roleplayed by this phenomenon in determining theeffectiveness of chemotherapy with isoniazid.Second, other therapeutic measures were taken(pneumoperitoneum and so forth) ad lib., in ad-dition to chemotherapy, before four months afterbacteriologic conversion of the sputum to negative.

The poor results of antimicrobial therapy alonein patients of Group III were not surprising(Table I). They point up the need for additionaleffective chemotherapeutic agents for tuberculosis.On the other hand, the 47 per cent conversionrate (16 patients converted out of 34 treated fornine to 12 months without collapse proceduresor surgery) for patients in Group II may seemunexpectedly low. However, many of the pa-tients in this group were excreting populations oftubercle bacilli which were resistant to para-aminosalicylic acid as well as to either strepto-mycin or isoniazid on admission to hospital.Therefore, in effect, several of these patients re-ceived the benefit of only single-drug treatmentduring the period of this study.

It has been customary for some clinicians toreserve "one good drug" for use at the time ofsurgery. These results indicate that it would bebetter to strive for optimal chemotherapeutic re-sults from the initiation of treatment.

The success observed on application of the ap-propriate basic high dosage regimen in patientsexcreting drug-susceptible tubercle bacilli seems toeliminate any doubt as to the clinical importanceof delivering adequate concentrations of at least twoantimicrobial drugs in vivo. As was implied in thefirst report of this series (1), there is a definite,but very low, incidence of mutant forms of tuberclebacilli which are simultaneously resistant to bothstreptomycin and isoniazid. Thus, there will bea definite incidence of chemotherapeutic failureon any two-drug regimen. Just as adequate two-drug regimens markedly decrease the incidence ofchemotherapeutic failure manifested by the emer-gence of one-drug-resistant mutant populations, soshould adequate regimens of three or more drugsdecrease the incidence of chemotherapeutic failuremanifested by the emergence of two-drug-resist-ant mutant populations. It seems fair to assumethat the one instance of double mutation to resist-ance to both isoniazid and streptomycin which oc-curred in these studies could have been prevented

had a third drug, i.e., para-aminosalicylic acid,been added to his therapeutic regimen before thetwelfth week of chemotherapy.

The present study constitutes a relatively short-term evaluation of chemotherapy of pulmonarYtuberculosis in adults. Resting bacterial cellswhich escape sterilization by virtue of being phys-iologically insusceptible to the action of strepto-mycin or isoniazid remain a threat able to initiatean exacerbation of the disease months or evenyears later (27). Therefore, final evaluation ofany approach to the chemotherapy of tuberculosisrequires a long period of follow-up observation.

Among 133 patients in Group I who presentedroentgenographic evidence of cavitary disease oninitiation of chemotherapy and who remained inhospital at least four months after the date ofbacteriologic conversion to negative, 66 (50 percent) had persistence of cavitation ("open cavityhealing" by clinical criteria). This is the highestincidence of "open cavity healing" reported in theliterature. It can fairly be attributed to the avoid-ance of those measures-strict bed rest, and pul-monary collapse procedures-which, in our opinion(28), are in serious conflict with the purposes ofappropriate and effective antimicrobial chemo-therapy.

For patients treated with conventional drugregimens plus bed rest, important unfavorableprognostic significance has been attached to thepersistence of roentgenographically-visible cavita-tion even when it is accompanied by the absenceof acid-fast bacilli from the sputum (29). Onthe other hand, more recently, evidence has beenpresented by some investigators (30, 31) that theassociation of open, air-filled cavities with per-sistently negative sputum bacteriology after moreeffective protocols of chemotherapy does not haveunfavorable significance. It will be interesting todetermine whether patients treated under the highdosage combined drug regimen will show relapserates lower than those which have been observedon previous modes of therapy. Results obtainedfrom bacteriological studies on surgically resectedpulmonary lesions from patients in Group I ofthis study provide corroborative evidence thatthis may, indeed, be anticipated (32).

In retrospect, the observations on the variationsin the metabolic disposition of isoniazid and onthe six hour serum levels after streptomycin, 20

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RUSSELL, KASS, HEATON, DRESSLERAND MIDDLEBROOK

mg. per Kg., indicate that many of the Group Ipatients might have responded as favorably tochemotherapy employing lower dosages of thesedrugs. Indeed, in view of the significant incidenceof drug reactions (12 per cent) in our experiencewith the high dosage regimen for all patients, itseems that routine high dosage imposes an un-necessary risk. Thus, the best approach shouldbe one in which dosages of appropriate drugs areadjusted according to the results of specific phar-macologic assays.

The minimal immediate chemotherapeutic fail-ure and the low early relapse rate in Group Ipatients, on the one hand, and the high failurerate in the Group II and Group III patients, onthe other hand, provide evidence of the clinicalvalue of the drug-susceptibility tests employed inthese studies for predicting response to chemo-therapy with these drugs.

Ninety-nine per cent of the patients who excretedstreptomycin-susceptible, isoniazid-susceptible tu-bercle bacilli at the start of chemotherapy at thishospital achieved sputum conversion on the highdosage regimen without collapse or surgery, orstrict bed rest. Microbiologic serum assay allowedprompt discovery and correction of isoniazid dos-ages in those few individuals who might have hadinadequate drug with the unmodified regimen. Thegeneral principles of primary chemotherapy of tu-berculosis upon which the treatment of the GroupI patients was based have been outlined previously(28). The very low chemotherapeutic failure rateobserved in such patients in this study confirmsour previous opinion (28) that the drug-treat-ment failures of conventional chemotherapy arelargely due to inadequate dosage of drugs or in-discriminate combinations of therapeutic measures.

SUMMARY

Three hundred eighteen adult patients withbacteriologically proved pulmonary tuberculosisdue to typical tubercle bacilli were admitted con-secutively to the National Jewish Hospital atDenver between October 1, 1954, and June 1,1957, and were treated for at least six months.Of these, 153 were excreting organisms whichwere susceptible to both streptomycin and iso-niazid. These patients were subjected to a high(losage streptomycin, highi dosage isoniazid cliemo-

therapeutic regimen. The sputa of 152 of thepatients excreting populations of tubercle bacilliwhich were streptomycin- and isoniazid-susceptiblebecame bacteriologically converted to negative bythe end of the sixth month of chemotherapy.None of these 152 patients excreted drug-resist-ant organisms prior to conversion. There was olneearly chemotherapeutic failure, as manifested bythe failure to achieve conversion and by the emer-gence of double-drug-resistant mutants. Therehas also been one bacteriologic and roentgeno-graphic relapse.

Two other groups of patients, those who werealready excreting drug-resistant organisms on ad-mission to hospital, were studied for comparison:those excreting tubercle bacilli resistant to onedrug, and those with organisms resistant to twodrugs. Neither group demonstrated impressiveresponse to chemotherapy. These results estab-lished the value of the special drug-susceptibilitytesting techniques employed for predicting theclinical response to treatment with streptomycin orisoniazid.

The theoretical and practical consequences ofthese studies were discussed.

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2. Mandel, W., Cohn, M. L., Russell, W. F., Jr., andMiddlebrook, G. Serum isoniazid levels and cata-lase activities of tubercle bacilli from isoniazid-treated patients. Amer. J. med. Sci. 1957, 233, 66.

3. Mandel, W., Heaton, A. D., Russell, W. F., Jr., andMiddlebrook, G. Combined drug treatment of tu-berculosis. II. Studies of antimicrobially-activeisoniazid and streptomycin serum levels in adulttuberculous patients. J. clin. Invest. 1959, 38,1356.

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5. Russell, W. F., Jr., and Dye, W. E. High dosageisoniazid plus daily streptomycin in the chemo-therapy of pulmonary tuberculosis: A pilot studyof 46 highly selected cases of drug-susceptible pul-

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13. Morse, W. C., Curry, F. J., Morse, P. Z., Chambers,J. S., and Lincoln, A. F. Effect of oral PAS onbiologically active isoniazid serum levels int Trans-actions of the 15th Conference on the Chemotherapyof Tuberculosis. St. Louis, Mo., 1956, vol. 15, p.283.

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15. Carlson, H. B., Anthony, E. M., Russell, W. F., Jr.,and Middlebrook, G. Prophylaxis of isoniazidneuropathy with pyridoxine. New Engl. J. AMed.1956, 255, 119.

16. Schmidt, L. H., Grover, A. A., Hoffmann, R., Rehm,J., and Sullivan, R. The emergence of isoniazid-sensitive bacilli in monkeys inoculated with isonia-zid-resistant strains in Transactions of the 17thConference on the Chemotherapy of Tuberculosis.Memphis, Tenn., 1958, vol. 17, p. 264.

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21. The effect of streptomycin on the emergence of bac-terial resistance to isoiliazid; a U. S. Public HealthService cooperative investigationl. Amer. Rev.Tuberc. 1953, 67, 553.

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23. Ross, J. D., Horne, N. W., Grant, I. W. B., andCrofton, J. W. Hospital treatment of pulmonarytuberculosis. A follow-up study of patients ad-mitted to Edinburgh hospitals in 1953. Brit. med.J. 1958, 1, 237.

24. Crofton, J. "Sputum conversion" and the metabolismof isoniazid. Amer. Rev. Tuberc. 1958, 77, 869.

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26. Stewart, S. M. Virulence of tubercle bacilli recoveredfrom patients treated with isoniazid. Amer. Rev.Tuberc. 1954, 69, 641.

27. Schaefer, W. B. The effect of isoniazid on growingand resting tubercle bacilli. Amer. Rev. Tuberc.1954, 69, 125.

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29. Bell, J. W. The problem of resection surgery forpulmonary tuberculosis in the noninfectious pa-tient with persisting cavitary disease. Amer. Rev.Tuberc. 1956, 74, 169.

30. Ryder, J. B. Prognosis of persistent cavitation insputum-negative cases of tuberculosis followinglong-term chemotherapy. Tubercle (Lond.) 1958,39, 113.

31. Wilson, T. M., Doyle, L., and Gardiner, M. P. Openhealing of tuberculous cavities. Results in 40 pa-tients treated conservatively. Brit. med. J. 1958,2, 87.

32. Heaton, A. D., Russell, \W. F., Jr., Denst, J., andMiddlebrook, G. Combined drug treatment of tu-berculosis. IV. Bacteriologic studies on thesputum and resected pulmonary lesions of tuber-culous patients. J. clin. Invest. 1959, 38, 1376.

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