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The use of pyrazinamide plus isoniazid in the treatment of pulmonary tuberculosis
Tubercle, Lond., (I959) , 4 o, 14
14
The Use of Pyrazinamide Plus Isoniazid in the Treatment of Pulmonary Tuberculosis By H. M. M A C L E O D , D. H A Y and S H E I L A hi. S T E W A R T * from the Department of Tuberculosis and Diseases of the Respiratory System, University of Edinburgh Reports in the literature have indicated that pyrazinamide plus isoniazid is a highly effective antituberculous combination but fear of the toxic effects of pyrazinamide has restricted its use. T h e work of Perry and Morse (I955) suggested that pyrazinamide plus isoniazid might prove to be effective clinically even in the presence of isoniazid resistant bacilli, as organisms resistant to 5 to 15 lag. of isoniazid per ml. appeared in vitro to be more susceptible to pyrazinamide than organisms which were isoniazid sensitive. T h e clinical study of Donnerberg and others (I957) did not suggest that these bz vitro findings had an hz vivo application. In this present paper a further small but detailed study is reported of the use of pyrazinamide and isoniazid in patients whose organisms showed a varied range of resistance to isoniazid. Material Eleven patients were included in the trial, 9 males and 2 females. Their ages ranged from 22 to 73 years and the known duration of illness was from two to fourteen years; in 8 it was four years or more. All had extensive bilateral cavitated disease except one who had already had a pneumonectomy. Four were in good general condition; 2 were in fairly good condition and 5 were in poor condition. Owing to respiratory insufficiency only 3 patients were considered possible candidates for surgery. T w o patients (Cases I and 2) had never received treatment with isoniazid and their organisms were sensitive to this drug. T h e other 9 had all received isoniazid therapy in drug combinations now known to be unsatisfactory in preventing the emergence of resistance. O f these, 5 patients were excreting bacilli resistant to 0.2 to I-o lag. isoniazid per ml. L6wenstein-Jensen medium and 3 resistant to 5 to 5 ~ lag. per ml. These results were confirmed on at least two cultures. In the remaining patient, bacilli of a high degree of isoniazid resistance were isolated four months before treatment, but a month before treatment organisms of only a low degree (0.2 lag. per ml.) were isolated and a filrther two cultures, one three days before treatment and the other three days after the start of treatment, were fully sensitive. None of the patients had previously been treated with pyrazinamide. In all of the patients the bacilli were known to be resistant to streptomycin and to p-aminosalicylic acid. Methods
Treatment Pyrazinamide was given in a dose o f 4 o mg. per kg. of body weight per day in two divided doses. Isoniazid was given in a dose of Ioo rag. twice daily to patients with organisms sensitive to isoniazid (Cases I and 2) and to 3 of the patients (Cases 4, 6 and xo) whose organisms were resistant. T h e remaining 6 patients (Cases 3, 5, 7, *Royal Victoria ttospital Tuberculosis Trust Research Fellow in BacterlologT.
P Y R A Z I N A M I D E PLUS 1SONIAZID
I5
8, 9 and 1I) were given 300 mg. twice daily. Patients on the higherisoniazid dosage received pyrldoxine 4 ~ rag. three times daily. The period of treatment varied from two to twenty-four months. In the one patient (Case 4) who received less than three months' treatment, chemotherapy was discontinued because of possible toxicity to pyrazinamide. O f the remaining xo patients, 4 (Cases 7, 9, IO and t I) were treated for three to six months, 2 (Cases 5 and 6) for seven months and 4 (Cases I, 2, 3 and 8) for seventeen, twenty-four, thirteen and twenty months respectively. All the treatment was given in hospital except in 3 patients who were treated as out-patients for the last five (Case 3), eleven (Case I), and twelve (Case 2) months of their total course. Bacteriological Methods Sputum Positivity.- All patients in the trial had had intensive bacteriological studies and had been regularly sputum positive for at least three months - in most cases for several y e a r s - b e f o r e the start of treatment. The positivity of the sputum was assessed by comparing a direct smear of the test sputum with standard smears. Details of the method are given elsewhere (Stewart and others, I957). The results were expressed as follows: + +-Jr--~ More than five bacilli per I/12 in. oil immersion field. + + =Less than five bacilli per field but more than one bacillus per five fields. + = L e s s than one bacillus per five fields. A change in positivity was considered to have occurred if there was a rise or fall of at least one grade in each of two or more consecutive sputa. The assessments were carried out on two specimens of sputum during the week immediately preceding the start of treatment with pyrazinamide and isoniazid and weekly during treatment. All specimens were cultured on L6wenstein-Jensen slopes. Sputum conversion was considered to have occurred if there had been at least three consecutive cultures negative after six weeks' incubation. The term 'maintained conversion' was used where cultures had remained negative to the end of observation (a minimum of fourteen months) and 'transient conversion' where conversion was followed by subsequent reversion to positivity. Resistance Tests. - Tests for resistance to pyrazinamide and isoniazid were made from the cultures. Pyrazinamide: These were carried out in Davis and Dubos medium without 'Tween 80' at pH 5.8 (Stewart and others, I957). Final concentrations of 2,ooo lag. per ml. by two-fold dilutions to 15 lag. per ml. were used. The results were expressed as resistance ratios to a standard strain, Z762 , that is, the ratio of the minimum inhibitory concentration for the test strain to that for the standard strain. A ratio of eight or above was considered to indicate the development of resistance. Isoniazid: Isoniazid resistance tests were carried out by the method recommended by the Medical Research Council of Great Britain (1953) using L6wensteinJensen medium. The isoniazid concentrations used were 5 o, IO, 5, I .o and 0.2 lag. per ml. The results were expressed as the highest concentration allowing growth of twenty or more colonies. Growth on slopes containing o'2 lag. per ml. or above was considered indicative of resistance. Population Studies. - Prior to the start of treatment an assessment of the percentages of the total viable population resistant to varying concentrations ofisoniazid was made on two specimens of sputum from each of xo of the i i patients by the method already described (Stewart, x956). Counts were made on L6wenstein-Jensen plates containing 64, I6, 4, I .o, 0.25 and o.o6 lag. isoniazid per ml. medium. The results were expressed as a percentage of the total viable count. Clinical Studies Monthly records of the general condition, weight, erythrocyte sedimentation rate (E.S.R.) and temperature were made. Chest radiographs were taken monthly and
x6
TUBERCLE
TABLE I.
- BACTERIOLOGICAL RESULTS
Sputum positivity Isouiazid group
Maintained* conversion
Transient t conversion
Positive throughout
First appearance of p.)'razinamide resistance (months)
2
o
o
Nil
I
O
I
O
12
4~
I
I
2
N i l , 5, 2, 3
3
O
I
2
3~ 27 1
~ . of Cases
Sensitive Sensitivepreviously resistant Resistant O"2--1 "O
/ag. per ml. Resistant 5-5o /~g. per ml.
[
*Cultures remained negative to tile end of observation (minimum of fourteen months). "]'Cultures became negative but reverted to positive. ,+The patient whose treatment was stopped after two months is excluded from the table. assessed by a panel unaware of the treatment being given. Each series was assessed independently on two separate occasions. Weekly tests of the urine for urobilonogen, and fortnightly estimates of the serum bilirubin, alkaline phosphatase and thymol turbidity were done as a check on liver function. Results
Bacteriological Results A summary of the bacteriological results is given in Table I. The I patient (Case 4) in whom treatment had to be stopped after two months because of toxicity has been omitted. His sputum remained positive throughout treatment, but no pyrazinamide resistance was detected. I n the 2 patients with isoniazid sensitive organisms (Cases I and 2) who had not been previouly treated with isoniazid, conversion to sputum negativity occurred by the third month and has been maintained to the present, a total of seventeen and twenty-four months' treatment respectively; treatment is still continuing. One other patient (Case 8) whose organisms showed a low degree of resistance to isoniazid before treatment became negative within a month and the negative state has been maintained throughout twenty months' treatment and seven 9months" observation thereafter. This patient's sputum was positive only on culture at the start of treatment in contrast to all others in the trial whose sputa were positive on smear as well as on culture. Three patients became sputum negative but this state was not subsequently maintained. One of these (Case 3) was the patient whose bacilli had apparently reverted to isoniazid sensitivity at the start of treatment though it was known that they had been previously isoniazid resistant. This patient's sputum converted within one month but became positive again at twelve months with the emergence of bacilli resistant to both pyrazinamide and isoniazid. The last five months of his treatment were given at home and it is at least possible that he failed to adhere faithfully to the drug regime prescribed. The other 2 patients (Cases 5 and 9) became sputum negative during the third and first month respectively, but reverted to sputum positive with pyrazinamide resistant organisms during the fifth and third month respectively. In one of these patients (Case 5) the organisms were of a low degree of resistance to isoniazid at the start of treatment, and in the second (Case 9) they were of a high degree. Four patients (Cases 6, 7, xo and II) remained positive on smear and culture
PYRAZINAMIDE PLUS ISONIAZID
I7
throughout treatment. Pyrazinamide resistant organisms were detected in all of these cases by the third month of treatment. Isoniazid Population Studies. - No resistant bacilli were detected by the pre-treatment population studies in the two patients (Cases I and 2) who had never received treatment with isoniazid, nor in the case (Case 3) in which reversion to full sensitivity appeared to have occurred before the start of treatment with pyrazinamide p h u isoniazid. Nevertheless, in the last case sputum conversion was only temporary. But the total viable count was low (3 X Io '~ per ml.) and therefore the population studies may have failed to detect small numbers of resistant bacilli. Population studies were only done in I of the other 2 cases in which a fall in sputum positivity occurred at the start of treatment. In that case (Case 5) 2o per cent of the bacilli were sensitive to o-o6 lag. per ml. and 9 2 per cent to o.2 5 lag. per ml. I n only one other case (Case 4) were any sensitive organisms detected by the population study. This was the patient in whom treatment had to be stopped at the end of the second month because of toxicity. In the remaining 6 cases Ioo per cent of the bacilli were resistant to o.2 5 lag. per ml. or above. In only I of these patients (Case 8) was the sputum converted to negative by treatment. Clinical Results Radiographic State. - The 2 patients who had never had isoniazid before were radio-
graphically improved. In I of these (Case 2), who had gross cavitation, the improvement was considerable and included closure of all cavities by the end of the fifth month of treatment. In the other (Case I), who had moderate cavitation, improvement was slight; cavitation remains though the sputum has converted to negative. Two patients (Cases 9 and Io) in the high isoniazid resistance group showed moderate deterioration following the emergence ofpyrazinamide resistant organisms. The remaining patients were radiographically unchanged throughout treatment. Clinical State. - O f the 3 patients who showed persistent sputum conversion, I (Case 2) gained 14 lb. in weight, I (Case i) showed no change and the third (Case 8) lost 14 lb. Only I of the 3 patients showing transient sputum conversion (Case 5) had a significant weight gain. Only i patient (Case I o) was febrile at the start of treatment; he remained so throughout and his sputum remained positive. There was no significant clinical change in the remaining patients. Toxicity
Toxicity ascribed to pyrazinamide was noted in 2 cases (Cases 2 and 4). In each case the serum bilirubin rose and in addition I of the patients showed excessive urobilinogen in the urine. Neither patient was clinically jaundiced. In I case the serum bilirubin rose from his pre-treatment level o f o . 5 rag. per IOO ml. to I. 5 rag. per zoo ml., and his thymol turbidity from three units to ten units after two months' treatment and it was decided to stop treatment. Within five weeks his serum bilirubin returned to normal. In the other patient, his serum bilirubin rose from an average of 0. 4 mg. per Ioo ml. to I "5 mg. per zoo ml. during the eighth months of treatment, but there was no other biochemical or clinical manifestation of liver disorder. His treatment was stopped for one month by which time his serum bilirubin was normal. He then resumed pyrazinamide and isoniazid in the same dosage for a further sixteen months without incident. None of the remaining IO patients showed any symptoms of toxicity. Although the incidence of toxicity in this trial was low and its effects slight, pyrazinamide can produce a fatal toxic necrosis of the liver. We are indebted to Dr I. W. B. Grant for permission to publish the following short account of a case showing this fatal outcome, and to Dr J. Davidson for the pathology" report. 'A m a n aged 2 3 who had pulmonary tuberculosis for two years was treated on a m b u l a n t chemotherapy with p-aminosalicylic acid to g. daily and isoniazid 2oo rag. daily for one year but failed
18
TUBERCLE
to take his drugs properly and developed slight radiographic extension of a lesion in his left lung while still on chemotherapy. Despite the radiographic deterioration he was in good clinical condition and had no significant chest symptoms. It was considered probable that he had resistant organisms though they could not be isolated for study. As his disease was mainly confined to the left apicoposterior segment, resection of the affected area under cover ofstreptomycin t g. daily, pyrazinamide 3 g. daily and isoniazid 200 rag. daily was undertaken. The operation, which was performed eleven days after starting his drugs, was uneventful. The total period of drug administration was sixty-four days. On the sixty-fourth day he complained of nausea and vomited on three occasions. His urine was found to contain excess urobillnogen and the drugs were discontinued forthwith. On the third day after stopping pyrazinamide he became obviously jaundiced and liver function tests showed a serum bilirubln value of 4-z mg. per too ml., and a cephalin-eholesterol floceulation test of 3+. On the fifth day he became confused and irrational and lapsed into coma, and the following day he developed convulsions, tie died on the seventh day after stopping pyrazinamide despite treatment along the usual lines for hepatic failure. A post-mortem examination was carried out within twentyfour hours of death and the following is an extract from the report: The liver wlfich weighed 800 g. was much reduced in size and its capsule showed slight wrinkling. On section the parenehyma was markedly congested and haemorrhagie, so much so that the outline of tile liver lobules was not distinguishable except in the subeapsular portion of the left lobe. Various parts of the organ were examined mlcroseopically and these showed extreme necrosis of the parenchyma. The few remaining cells chiefly in relation to the capsule showed profound fatty changes. Amongst the necrotic tissue there was intense haemorrhage. A few plugs of bile pigment were noted; for the most part tile bile duets were intact. The kidneys were of average size and weight. The capsules stripped easily leaving smooth, slightly congested surfaces. The cortex was broad and well defined from the medulla. Microscopically there were marked degenerative changes in the lining cells of tile secretory tubules varying from advanced cloudy swelling to complete necrosis. Fatty changes were present in the lining cells of the loops of Henle and also in some of the convoluted tubules. The glomeruli and interstitial vessels were congested. The stomach was filled with altered blood material. The mucosa was atrophic and a smaU area of congestion and ulceration, 0. 5 cm. in diameter, was present in the middle of the greater curvature and there were also a few small areas of congestion near the pylorus. Black tar-like material was present in the duodenum and jejunum.'
Discussion T h r e e of the I I patients in this trial d e r iv e d a p p a r e n t l y lasting benefit from treatm e n t with p y r a z i n a m i d e and isoniazid. O f these, 2 had bacilli sensitive to isoniazid and I had bacilli o f low resistance to isonlazid. It m a y be significant t h a t the latter pat i en t had s p u t u m positive only on c u l tu r e at the start o f t r e a t m e n t a n d could therefore be p r e s u m e d to h a v e a low bacillary population. O n e o t h er patient, whose organisms were k n o w n to have been resistant to isoniazid but in whose s p u t u m no resistant bacilli w e r e detected i m m e d i a t e l y before the start o f t r e a t m e n t , m a i n t a i n e d s p u t u m conversion for twelve months, b u t then reverted to positive with p y r a z i n a m l d e and isoniazid resistant organisms. I n 2 o t h e r patients transient s p u t u m conversion occurred, b u t the s p u t u m later b e c a m e positive with the e m e r g e n c e o f p y r a z i n a m i d e resistant organisms. T h e r e f o r e there is no e v i d e n c e from the present series that p y r a z i n a m i d e plus isoniazid is likely to be effective in p r e v e n t i n g the e m e r g e n c e o f p y r a z i n a m i d e resistant bacilli in a d v a n c e d eases o f p u l m o n a r y tuberculosis in w h o m the organisms are resistant to isoniazid before t r e a t m e n t . Th ese results are in accord with those o f D o n n e r b e r g and others (I957). T h e failure o f p y r a z i n a m i d e plus isoniazid in the p at i en t in w h o m reversion to isoniazid sensitivity had a p p a r e n t l y o c c u r r e d before the start o f t r e a t m e n t , c a n n o t be a c c u r a t e l y assessed. It m a y h a v e been d u e to small n u m b e r s o f isoniazid resistant bacilli wh i ch w e r e not detected by the resistance tests and p o p u l a t i o n studies a n d from w h i c h bacilli resistant to p y r a z i n a m i d e developed. T h i s p a t i e n t was, however, treated at h o m e for the last five m o n t h s o f t r e a t m e n t and it is possible t h a t he did not take the drugs as prescribed. A n assessment o f p y r a z i n a m i d e - i s o n i a z i d t h e r a p y as initial t r e a t m e n t for tuberculous patients encounters difficulties because o f the k n o w n risk o f toxicity, sometimes fatal. I n view o f t h e well-proved efficacy o f the s t a n d a r d drugs, streptomycin, PA S a n d isoniazid, the physician m a y r i g h t l y hesitate to s u b m i t his patients to a controlled clinical investigation. T h e risk o f toxicity m a y be j u d g e d from a short review o f the literature d e a l i n g with this point.
PYRAZINAMIDE
PLUS ISONIAZID
I9
In a series of 66 patients reported by McDermott and others (1954) the incidence of hepatitis was io per cent. This included I fatal case, 3 severe cases and 2 mild. These authors concluded that the high incidence of hepatitis in m a n during treatment with pyrazinamide and isoniazid makes the use of this combination inadvisable as a treatment for tuberculosis. T h e dosage of pyrazinamide used was 5 ~ rag. per kg. of body weight with isoniazid in a dosage of 5 mg. kg. of body weight. T h e fatal case of hepatitis occurred about the eighth week of treatment and 4 of the non-fatal cases on the twenty-fourth week. These authors remind us that, although isoniazid is normally non-toxic to the liver in a dose of 3 to 5 mg. per kg. of body weight, it could become toxic to a liver simultaneously damaged by pyrazinamide. In an attempt to reduce toxicity Allison (i 956) treated 69 patients on about half the dosage used by previous workers. With a dosage of 1.5 g. of pyrazinamide and I5O mg. of isoniazid per day they found abnormal liver function tests in only 4 patients and stopped treatment on that account. Although there was reduced toxicity they found a high incidence of isoniazid resistance- I8 per cent of patients at eight mouths. Other authors using standard amounts of 4o to 5 ~ mg. pyrazinamide per kg. report much less toxicity than McDermott and others (1954). In view of the accepted liver toxicity of pyrazinamide its place in the modern treatment of tuberculosis is limited. I t should not be used alone except to provide short-term cover, e.g. for operation, when a bactericidal effect m a y be expected for about four to six weeks before resistance develops. It is unlikely to be of benefit for more than this time in combination with isoniazid in patients whose bacilli are resistant to isoniazid: pyrazinamide resistance m a y occur in a high percentage of such cases. Its use in these circumstances is therefore not recommended. In patients whose organisms are sensitive to streptomycin, PAS and isoniazid, or a combination o f a n y 2 ofthese, there is no justification for the inclusion ofpyrazinamide. T h e r e may, however, be a place for pyrazinamide plus isoniazid in the treatment of patients whose organisms are resistant to streptomycin and PAS, but sensitive to isoniazid and who are unsuitable for surgical treatment, provided there is no pre-treatment evidence of liver disease. T h e slight risk of fatal toxicity should not weigh heavily against the use of pyrazinamide in this strictly defined group whose outlook without effective chemotherapy is very poor. Although no direct comparisons have been made it seems possible that a combination of pyrazinamide with isoniazid would be more effective as long-term chemotherapy in this group than the alternative combination of isoniazid with oxytetracycline (Stewart and others, i954). Summary Eleven patients with advanced pulmonary tuberculosis whose bacilli were either sensitive to isoniazid or of a low or high degree of isoniazid resistance were treated with a combination of pyrazinamide and isoniazid for periods ranging from two to twenty-four months. T w o patients whose bacilli were sensitive to isoniazid showed sputum conversion and radiographic improvement. Sputum conversion without radiographic improvement occurred in I patient whose bacilli were of a low degree ofisoniazid resistance: sputum conversion lasting for one year without radiographic improvement occurred in I other patient whose bacilli had apparently reverted to isoniazid sensitivity at the start of treatment though it was known that they had previously been resistant. T w o patients whose bacilli were resistant to isoniazid became sputum negative for periods of three and five months but then reverted to sputum positive with pyrazinamide resistant organisms. O f the remaining 5 patients whose bacilli were isoniazid resistant, 4 remained positive on smear and culture throughout treatment and pyrazinamide resistant organisms were demonstrated in all. One patient whose treatment was stopped after two months because of abnormal liver function tests was omitted from further bacteriological studies.
20
TUBERCLE
A b n o r m a l liver function tests were o b t a i n e d i n 2 patients necessitating the disc o n t i n u a n c e of t r e a t m e n t , t h o u g h in I this was only temporary. O n e fatal case of liver toxicity, not in the series studied, is recorded a n d reference is m a d e to the incidence of hepato-toxlcity from reported series. A l t h o u g h liver toxicity is a hazard, p y r a z i n a m l d e m a y ]lave a place as short-term t h e r a p y to cover surgery, or as l o n g - t e r m t h e r a p y in c o m b i n a t i o n with isoniazid for patients whose bacilli are sensitive to the latter d r u g b u t resistant to streptomycin a n d PALS. Its use with isoniazid in the presence of resistance to the latter d r u g appears unprofitable. This investigation was carried out on behalf of Professor J. W. Crofion and Dr J. D. Ross. We are indebted to the nursing and laboratory staffs of the City, Southfield, and the Royal Victoria Hospitals, Edinburgh, the East Fortune Hospital, Drem, and the Department of Pathology, University of Edinburgh, and to Mrs M. Robertson for secretarial assistance. The work was supported by a generous grant from the Royal Victoria Hospital Tuberculosis Trust. References
Allison, S. T. (x956) Amer. Rev. Tuberc., 74, 4~ Donnerberg, R. L., Atwell, R. J., and Browning, R. H. (x957) Amer. Rev. Tuberc., 75, 846. McDermott, W., Ormond, L., Muschenheim, C., Deuschle, K., McCune, R. M., Jr., and Tompsett, R. (1954) Amer. Rev. Tuberc., 69, 319. Medical Research Council (x953) Lancet, ii, 2x3. Perry, C. R., and Morse, W. C. (1955) Amer. Rev. Tuberc., 72, 84o. Stewart, Sheila M. (1956) Amer. Rev. Tuberc., 73, 39o. Stewart, Sheila M., Murdoeh, J. McC., Crofton, J, W., and Hay, D. (1957) Brit. 07. Tuberc., 5x, x58. Stewart, Sheila M., Turnbull, F. W. A., and Crofton, J. W. (i954) Brit. reed. 07., 2, x5o8.
14
The Use of Pyrazinamide Plus Isoniazid in the Treatment of Pulmonary Tuberculosis By H. M. M A C L E O D , D. H A Y and S H E I L A hi. S T E W A R T * from the Department of Tuberculosis and Diseases of the Respiratory System, University of Edinburgh Reports in the literature have indicated that pyrazinamide plus isoniazid is a highly effective antituberculous combination but fear of the toxic effects of pyrazinamide has restricted its use. T h e work of Perry and Morse (I955) suggested that pyrazinamide plus isoniazid might prove to be effective clinically even in the presence of isoniazid resistant bacilli, as organisms resistant to 5 to 15 lag. of isoniazid per ml. appeared in vitro to be more susceptible to pyrazinamide than organisms which were isoniazid sensitive. T h e clinical study of Donnerberg and others (I957) did not suggest that these bz vitro findings had an hz vivo application. In this present paper a further small but detailed study is reported of the use of pyrazinamide and isoniazid in patients whose organisms showed a varied range of resistance to isoniazid. Material Eleven patients were included in the trial, 9 males and 2 females. Their ages ranged from 22 to 73 years and the known duration of illness was from two to fourteen years; in 8 it was four years or more. All had extensive bilateral cavitated disease except one who had already had a pneumonectomy. Four were in good general condition; 2 were in fairly good condition and 5 were in poor condition. Owing to respiratory insufficiency only 3 patients were considered possible candidates for surgery. T w o patients (Cases I and 2) had never received treatment with isoniazid and their organisms were sensitive to this drug. T h e other 9 had all received isoniazid therapy in drug combinations now known to be unsatisfactory in preventing the emergence of resistance. O f these, 5 patients were excreting bacilli resistant to 0.2 to I-o lag. isoniazid per ml. L6wenstein-Jensen medium and 3 resistant to 5 to 5 ~ lag. per ml. These results were confirmed on at least two cultures. In the remaining patient, bacilli of a high degree of isoniazid resistance were isolated four months before treatment, but a month before treatment organisms of only a low degree (0.2 lag. per ml.) were isolated and a filrther two cultures, one three days before treatment and the other three days after the start of treatment, were fully sensitive. None of the patients had previously been treated with pyrazinamide. In all of the patients the bacilli were known to be resistant to streptomycin and to p-aminosalicylic acid. Methods
Treatment Pyrazinamide was given in a dose o f 4 o mg. per kg. of body weight per day in two divided doses. Isoniazid was given in a dose of Ioo rag. twice daily to patients with organisms sensitive to isoniazid (Cases I and 2) and to 3 of the patients (Cases 4, 6 and xo) whose organisms were resistant. T h e remaining 6 patients (Cases 3, 5, 7, *Royal Victoria ttospital Tuberculosis Trust Research Fellow in BacterlologT.
P Y R A Z I N A M I D E PLUS 1SONIAZID
I5
8, 9 and 1I) were given 300 mg. twice daily. Patients on the higherisoniazid dosage received pyrldoxine 4 ~ rag. three times daily. The period of treatment varied from two to twenty-four months. In the one patient (Case 4) who received less than three months' treatment, chemotherapy was discontinued because of possible toxicity to pyrazinamide. O f the remaining xo patients, 4 (Cases 7, 9, IO and t I) were treated for three to six months, 2 (Cases 5 and 6) for seven months and 4 (Cases I, 2, 3 and 8) for seventeen, twenty-four, thirteen and twenty months respectively. All the treatment was given in hospital except in 3 patients who were treated as out-patients for the last five (Case 3), eleven (Case I), and twelve (Case 2) months of their total course. Bacteriological Methods Sputum Positivity.- All patients in the trial had had intensive bacteriological studies and had been regularly sputum positive for at least three months - in most cases for several y e a r s - b e f o r e the start of treatment. The positivity of the sputum was assessed by comparing a direct smear of the test sputum with standard smears. Details of the method are given elsewhere (Stewart and others, I957). The results were expressed as follows: + +-Jr--~ More than five bacilli per I/12 in. oil immersion field. + + =Less than five bacilli per field but more than one bacillus per five fields. + = L e s s than one bacillus per five fields. A change in positivity was considered to have occurred if there was a rise or fall of at least one grade in each of two or more consecutive sputa. The assessments were carried out on two specimens of sputum during the week immediately preceding the start of treatment with pyrazinamide and isoniazid and weekly during treatment. All specimens were cultured on L6wenstein-Jensen slopes. Sputum conversion was considered to have occurred if there had been at least three consecutive cultures negative after six weeks' incubation. The term 'maintained conversion' was used where cultures had remained negative to the end of observation (a minimum of fourteen months) and 'transient conversion' where conversion was followed by subsequent reversion to positivity. Resistance Tests. - Tests for resistance to pyrazinamide and isoniazid were made from the cultures. Pyrazinamide: These were carried out in Davis and Dubos medium without 'Tween 80' at pH 5.8 (Stewart and others, I957). Final concentrations of 2,ooo lag. per ml. by two-fold dilutions to 15 lag. per ml. were used. The results were expressed as resistance ratios to a standard strain, Z762 , that is, the ratio of the minimum inhibitory concentration for the test strain to that for the standard strain. A ratio of eight or above was considered to indicate the development of resistance. Isoniazid: Isoniazid resistance tests were carried out by the method recommended by the Medical Research Council of Great Britain (1953) using L6wensteinJensen medium. The isoniazid concentrations used were 5 o, IO, 5, I .o and 0.2 lag. per ml. The results were expressed as the highest concentration allowing growth of twenty or more colonies. Growth on slopes containing o'2 lag. per ml. or above was considered indicative of resistance. Population Studies. - Prior to the start of treatment an assessment of the percentages of the total viable population resistant to varying concentrations ofisoniazid was made on two specimens of sputum from each of xo of the i i patients by the method already described (Stewart, x956). Counts were made on L6wenstein-Jensen plates containing 64, I6, 4, I .o, 0.25 and o.o6 lag. isoniazid per ml. medium. The results were expressed as a percentage of the total viable count. Clinical Studies Monthly records of the general condition, weight, erythrocyte sedimentation rate (E.S.R.) and temperature were made. Chest radiographs were taken monthly and
x6
TUBERCLE
TABLE I.
- BACTERIOLOGICAL RESULTS
Sputum positivity Isouiazid group
Maintained* conversion
Transient t conversion
Positive throughout
First appearance of p.)'razinamide resistance (months)
2
o
o
Nil
I
O
I
O
12
4~
I
I
2
N i l , 5, 2, 3
3
O
I
2
3~ 27 1
~ . of Cases
Sensitive Sensitivepreviously resistant Resistant O"2--1 "O
/ag. per ml. Resistant 5-5o /~g. per ml.
[
*Cultures remained negative to tile end of observation (minimum of fourteen months). "]'Cultures became negative but reverted to positive. ,+The patient whose treatment was stopped after two months is excluded from the table. assessed by a panel unaware of the treatment being given. Each series was assessed independently on two separate occasions. Weekly tests of the urine for urobilonogen, and fortnightly estimates of the serum bilirubin, alkaline phosphatase and thymol turbidity were done as a check on liver function. Results
Bacteriological Results A summary of the bacteriological results is given in Table I. The I patient (Case 4) in whom treatment had to be stopped after two months because of toxicity has been omitted. His sputum remained positive throughout treatment, but no pyrazinamide resistance was detected. I n the 2 patients with isoniazid sensitive organisms (Cases I and 2) who had not been previouly treated with isoniazid, conversion to sputum negativity occurred by the third month and has been maintained to the present, a total of seventeen and twenty-four months' treatment respectively; treatment is still continuing. One other patient (Case 8) whose organisms showed a low degree of resistance to isoniazid before treatment became negative within a month and the negative state has been maintained throughout twenty months' treatment and seven 9months" observation thereafter. This patient's sputum was positive only on culture at the start of treatment in contrast to all others in the trial whose sputa were positive on smear as well as on culture. Three patients became sputum negative but this state was not subsequently maintained. One of these (Case 3) was the patient whose bacilli had apparently reverted to isoniazid sensitivity at the start of treatment though it was known that they had been previously isoniazid resistant. This patient's sputum converted within one month but became positive again at twelve months with the emergence of bacilli resistant to both pyrazinamide and isoniazid. The last five months of his treatment were given at home and it is at least possible that he failed to adhere faithfully to the drug regime prescribed. The other 2 patients (Cases 5 and 9) became sputum negative during the third and first month respectively, but reverted to sputum positive with pyrazinamide resistant organisms during the fifth and third month respectively. In one of these patients (Case 5) the organisms were of a low degree of resistance to isoniazid at the start of treatment, and in the second (Case 9) they were of a high degree. Four patients (Cases 6, 7, xo and II) remained positive on smear and culture
PYRAZINAMIDE PLUS ISONIAZID
I7
throughout treatment. Pyrazinamide resistant organisms were detected in all of these cases by the third month of treatment. Isoniazid Population Studies. - No resistant bacilli were detected by the pre-treatment population studies in the two patients (Cases I and 2) who had never received treatment with isoniazid, nor in the case (Case 3) in which reversion to full sensitivity appeared to have occurred before the start of treatment with pyrazinamide p h u isoniazid. Nevertheless, in the last case sputum conversion was only temporary. But the total viable count was low (3 X Io '~ per ml.) and therefore the population studies may have failed to detect small numbers of resistant bacilli. Population studies were only done in I of the other 2 cases in which a fall in sputum positivity occurred at the start of treatment. In that case (Case 5) 2o per cent of the bacilli were sensitive to o-o6 lag. per ml. and 9 2 per cent to o.2 5 lag. per ml. I n only one other case (Case 4) were any sensitive organisms detected by the population study. This was the patient in whom treatment had to be stopped at the end of the second month because of toxicity. In the remaining 6 cases Ioo per cent of the bacilli were resistant to o.2 5 lag. per ml. or above. In only I of these patients (Case 8) was the sputum converted to negative by treatment. Clinical Results Radiographic State. - The 2 patients who had never had isoniazid before were radio-
graphically improved. In I of these (Case 2), who had gross cavitation, the improvement was considerable and included closure of all cavities by the end of the fifth month of treatment. In the other (Case I), who had moderate cavitation, improvement was slight; cavitation remains though the sputum has converted to negative. Two patients (Cases 9 and Io) in the high isoniazid resistance group showed moderate deterioration following the emergence ofpyrazinamide resistant organisms. The remaining patients were radiographically unchanged throughout treatment. Clinical State. - O f the 3 patients who showed persistent sputum conversion, I (Case 2) gained 14 lb. in weight, I (Case i) showed no change and the third (Case 8) lost 14 lb. Only I of the 3 patients showing transient sputum conversion (Case 5) had a significant weight gain. Only i patient (Case I o) was febrile at the start of treatment; he remained so throughout and his sputum remained positive. There was no significant clinical change in the remaining patients. Toxicity
Toxicity ascribed to pyrazinamide was noted in 2 cases (Cases 2 and 4). In each case the serum bilirubin rose and in addition I of the patients showed excessive urobilinogen in the urine. Neither patient was clinically jaundiced. In I case the serum bilirubin rose from his pre-treatment level o f o . 5 rag. per IOO ml. to I. 5 rag. per zoo ml., and his thymol turbidity from three units to ten units after two months' treatment and it was decided to stop treatment. Within five weeks his serum bilirubin returned to normal. In the other patient, his serum bilirubin rose from an average of 0. 4 mg. per Ioo ml. to I "5 mg. per zoo ml. during the eighth months of treatment, but there was no other biochemical or clinical manifestation of liver disorder. His treatment was stopped for one month by which time his serum bilirubin was normal. He then resumed pyrazinamide and isoniazid in the same dosage for a further sixteen months without incident. None of the remaining IO patients showed any symptoms of toxicity. Although the incidence of toxicity in this trial was low and its effects slight, pyrazinamide can produce a fatal toxic necrosis of the liver. We are indebted to Dr I. W. B. Grant for permission to publish the following short account of a case showing this fatal outcome, and to Dr J. Davidson for the pathology" report. 'A m a n aged 2 3 who had pulmonary tuberculosis for two years was treated on a m b u l a n t chemotherapy with p-aminosalicylic acid to g. daily and isoniazid 2oo rag. daily for one year but failed
18
TUBERCLE
to take his drugs properly and developed slight radiographic extension of a lesion in his left lung while still on chemotherapy. Despite the radiographic deterioration he was in good clinical condition and had no significant chest symptoms. It was considered probable that he had resistant organisms though they could not be isolated for study. As his disease was mainly confined to the left apicoposterior segment, resection of the affected area under cover ofstreptomycin t g. daily, pyrazinamide 3 g. daily and isoniazid 200 rag. daily was undertaken. The operation, which was performed eleven days after starting his drugs, was uneventful. The total period of drug administration was sixty-four days. On the sixty-fourth day he complained of nausea and vomited on three occasions. His urine was found to contain excess urobillnogen and the drugs were discontinued forthwith. On the third day after stopping pyrazinamide he became obviously jaundiced and liver function tests showed a serum bilirubln value of 4-z mg. per too ml., and a cephalin-eholesterol floceulation test of 3+. On the fifth day he became confused and irrational and lapsed into coma, and the following day he developed convulsions, tie died on the seventh day after stopping pyrazinamide despite treatment along the usual lines for hepatic failure. A post-mortem examination was carried out within twentyfour hours of death and the following is an extract from the report: The liver wlfich weighed 800 g. was much reduced in size and its capsule showed slight wrinkling. On section the parenehyma was markedly congested and haemorrhagie, so much so that the outline of tile liver lobules was not distinguishable except in the subeapsular portion of the left lobe. Various parts of the organ were examined mlcroseopically and these showed extreme necrosis of the parenchyma. The few remaining cells chiefly in relation to the capsule showed profound fatty changes. Amongst the necrotic tissue there was intense haemorrhage. A few plugs of bile pigment were noted; for the most part tile bile duets were intact. The kidneys were of average size and weight. The capsules stripped easily leaving smooth, slightly congested surfaces. The cortex was broad and well defined from the medulla. Microscopically there were marked degenerative changes in the lining cells of tile secretory tubules varying from advanced cloudy swelling to complete necrosis. Fatty changes were present in the lining cells of the loops of Henle and also in some of the convoluted tubules. The glomeruli and interstitial vessels were congested. The stomach was filled with altered blood material. The mucosa was atrophic and a smaU area of congestion and ulceration, 0. 5 cm. in diameter, was present in the middle of the greater curvature and there were also a few small areas of congestion near the pylorus. Black tar-like material was present in the duodenum and jejunum.'
Discussion T h r e e of the I I patients in this trial d e r iv e d a p p a r e n t l y lasting benefit from treatm e n t with p y r a z i n a m i d e and isoniazid. O f these, 2 had bacilli sensitive to isoniazid and I had bacilli o f low resistance to isonlazid. It m a y be significant t h a t the latter pat i en t had s p u t u m positive only on c u l tu r e at the start o f t r e a t m e n t a n d could therefore be p r e s u m e d to h a v e a low bacillary population. O n e o t h er patient, whose organisms were k n o w n to have been resistant to isoniazid but in whose s p u t u m no resistant bacilli w e r e detected i m m e d i a t e l y before the start o f t r e a t m e n t , m a i n t a i n e d s p u t u m conversion for twelve months, b u t then reverted to positive with p y r a z i n a m l d e and isoniazid resistant organisms. I n 2 o t h e r patients transient s p u t u m conversion occurred, b u t the s p u t u m later b e c a m e positive with the e m e r g e n c e o f p y r a z i n a m i d e resistant organisms. T h e r e f o r e there is no e v i d e n c e from the present series that p y r a z i n a m i d e plus isoniazid is likely to be effective in p r e v e n t i n g the e m e r g e n c e o f p y r a z i n a m i d e resistant bacilli in a d v a n c e d eases o f p u l m o n a r y tuberculosis in w h o m the organisms are resistant to isoniazid before t r e a t m e n t . Th ese results are in accord with those o f D o n n e r b e r g and others (I957). T h e failure o f p y r a z i n a m i d e plus isoniazid in the p at i en t in w h o m reversion to isoniazid sensitivity had a p p a r e n t l y o c c u r r e d before the start o f t r e a t m e n t , c a n n o t be a c c u r a t e l y assessed. It m a y h a v e been d u e to small n u m b e r s o f isoniazid resistant bacilli wh i ch w e r e not detected by the resistance tests and p o p u l a t i o n studies a n d from w h i c h bacilli resistant to p y r a z i n a m i d e developed. T h i s p a t i e n t was, however, treated at h o m e for the last five m o n t h s o f t r e a t m e n t and it is possible t h a t he did not take the drugs as prescribed. A n assessment o f p y r a z i n a m i d e - i s o n i a z i d t h e r a p y as initial t r e a t m e n t for tuberculous patients encounters difficulties because o f the k n o w n risk o f toxicity, sometimes fatal. I n view o f t h e well-proved efficacy o f the s t a n d a r d drugs, streptomycin, PA S a n d isoniazid, the physician m a y r i g h t l y hesitate to s u b m i t his patients to a controlled clinical investigation. T h e risk o f toxicity m a y be j u d g e d from a short review o f the literature d e a l i n g with this point.
PYRAZINAMIDE
PLUS ISONIAZID
I9
In a series of 66 patients reported by McDermott and others (1954) the incidence of hepatitis was io per cent. This included I fatal case, 3 severe cases and 2 mild. These authors concluded that the high incidence of hepatitis in m a n during treatment with pyrazinamide and isoniazid makes the use of this combination inadvisable as a treatment for tuberculosis. T h e dosage of pyrazinamide used was 5 ~ rag. per kg. of body weight with isoniazid in a dosage of 5 mg. kg. of body weight. T h e fatal case of hepatitis occurred about the eighth week of treatment and 4 of the non-fatal cases on the twenty-fourth week. These authors remind us that, although isoniazid is normally non-toxic to the liver in a dose of 3 to 5 mg. per kg. of body weight, it could become toxic to a liver simultaneously damaged by pyrazinamide. In an attempt to reduce toxicity Allison (i 956) treated 69 patients on about half the dosage used by previous workers. With a dosage of 1.5 g. of pyrazinamide and I5O mg. of isoniazid per day they found abnormal liver function tests in only 4 patients and stopped treatment on that account. Although there was reduced toxicity they found a high incidence of isoniazid resistance- I8 per cent of patients at eight mouths. Other authors using standard amounts of 4o to 5 ~ mg. pyrazinamide per kg. report much less toxicity than McDermott and others (1954). In view of the accepted liver toxicity of pyrazinamide its place in the modern treatment of tuberculosis is limited. I t should not be used alone except to provide short-term cover, e.g. for operation, when a bactericidal effect m a y be expected for about four to six weeks before resistance develops. It is unlikely to be of benefit for more than this time in combination with isoniazid in patients whose bacilli are resistant to isoniazid: pyrazinamide resistance m a y occur in a high percentage of such cases. Its use in these circumstances is therefore not recommended. In patients whose organisms are sensitive to streptomycin, PAS and isoniazid, or a combination o f a n y 2 ofthese, there is no justification for the inclusion ofpyrazinamide. T h e r e may, however, be a place for pyrazinamide plus isoniazid in the treatment of patients whose organisms are resistant to streptomycin and PAS, but sensitive to isoniazid and who are unsuitable for surgical treatment, provided there is no pre-treatment evidence of liver disease. T h e slight risk of fatal toxicity should not weigh heavily against the use of pyrazinamide in this strictly defined group whose outlook without effective chemotherapy is very poor. Although no direct comparisons have been made it seems possible that a combination of pyrazinamide with isoniazid would be more effective as long-term chemotherapy in this group than the alternative combination of isoniazid with oxytetracycline (Stewart and others, i954). Summary Eleven patients with advanced pulmonary tuberculosis whose bacilli were either sensitive to isoniazid or of a low or high degree of isoniazid resistance were treated with a combination of pyrazinamide and isoniazid for periods ranging from two to twenty-four months. T w o patients whose bacilli were sensitive to isoniazid showed sputum conversion and radiographic improvement. Sputum conversion without radiographic improvement occurred in I patient whose bacilli were of a low degree ofisoniazid resistance: sputum conversion lasting for one year without radiographic improvement occurred in I other patient whose bacilli had apparently reverted to isoniazid sensitivity at the start of treatment though it was known that they had previously been resistant. T w o patients whose bacilli were resistant to isoniazid became sputum negative for periods of three and five months but then reverted to sputum positive with pyrazinamide resistant organisms. O f the remaining 5 patients whose bacilli were isoniazid resistant, 4 remained positive on smear and culture throughout treatment and pyrazinamide resistant organisms were demonstrated in all. One patient whose treatment was stopped after two months because of abnormal liver function tests was omitted from further bacteriological studies.
20
TUBERCLE
A b n o r m a l liver function tests were o b t a i n e d i n 2 patients necessitating the disc o n t i n u a n c e of t r e a t m e n t , t h o u g h in I this was only temporary. O n e fatal case of liver toxicity, not in the series studied, is recorded a n d reference is m a d e to the incidence of hepato-toxlcity from reported series. A l t h o u g h liver toxicity is a hazard, p y r a z i n a m l d e m a y ]lave a place as short-term t h e r a p y to cover surgery, or as l o n g - t e r m t h e r a p y in c o m b i n a t i o n with isoniazid for patients whose bacilli are sensitive to the latter d r u g b u t resistant to streptomycin a n d PALS. Its use with isoniazid in the presence of resistance to the latter d r u g appears unprofitable. This investigation was carried out on behalf of Professor J. W. Crofion and Dr J. D. Ross. We are indebted to the nursing and laboratory staffs of the City, Southfield, and the Royal Victoria Hospitals, Edinburgh, the East Fortune Hospital, Drem, and the Department of Pathology, University of Edinburgh, and to Mrs M. Robertson for secretarial assistance. The work was supported by a generous grant from the Royal Victoria Hospital Tuberculosis Trust. References
Allison, S. T. (x956) Amer. Rev. Tuberc., 74, 4~ Donnerberg, R. L., Atwell, R. J., and Browning, R. H. (x957) Amer. Rev. Tuberc., 75, 846. McDermott, W., Ormond, L., Muschenheim, C., Deuschle, K., McCune, R. M., Jr., and Tompsett, R. (1954) Amer. Rev. Tuberc., 69, 319. Medical Research Council (x953) Lancet, ii, 2x3. Perry, C. R., and Morse, W. C. (1955) Amer. Rev. Tuberc., 72, 84o. Stewart, Sheila M. (1956) Amer. Rev. Tuberc., 73, 39o. Stewart, Sheila M., Murdoeh, J. McC., Crofton, J, W., and Hay, D. (1957) Brit. 07. Tuberc., 5x, x58. Stewart, Sheila M., Turnbull, F. W. A., and Crofton, J. W. (i954) Brit. reed. 07., 2, x5o8.