ampicillin levels in sputum, serum, and saliva

Thorax (1970), 25, 304.

Ampicillin levels in sputum, serum, and salivaSHEILA M. STEWART, MARY FISHER, JOY E. YOUNG,

and W. LUTZ

Departments of Bacteriology, Respiratory Diseases and Social Medicinte, Uniiversity of Edinburghanid Wellcome Laboratory, City Hospital, Edinburgh

The ampicillin levels in sputum, serum, and saliva from 40 patients receiving a dose of 250 mg.,26 patients receiving a dose of 500 mg., and 11 patients receiving a dose of I g. were estimated.The ampicillin was given orally four times daily.The 1-2 hour and 2-3 hour sputum levels were similar in individual patients. There was no

difference in the range or mean sputum or saliva levels between specimens from patients receiving250 mg. and 500 mg., but the levels were significantly higher after the 1 g. dose. The mean serum

level sihowed a small increase after 500 mg. ampicillin as compared with the 250 mg. dose and a

big increase after the 1 g. dose: only the latter difference was significant. The sputum levelswere approximately 30 to 40 times lower than the corrzsponding serum levels. There was con-

siderable scatter in the sputum level for any level of ampicillin in the serum: in only two of the1-2 hour sputum specimens was there no detectable ampicillin.There was no correlation between the sputum levels and either the body weight or the dose

in milligrams per kilogram. There was no evidence that corticosteroids or diuretics affected thesputum level.

It was not possible to demonstrate any relationship between the purulence of the sputum andthe level of ampicillin after doses of 250 mg. or 500 mg., but higher levels were found in themore purulent specimens after 1 g. doses.

Previous work (Hafez, Stewart, and Burnet, 1965)showed that there was considerable variation be-tween patients in the level of penicillin in thesputum after intramuscular injection and that thesputum levels were appreciably lower than theserum levels. May and Delves (1965) reportedthe serum, saliva, and sputum levels in a smallgroup of patients with chronic bronchitis treatedorally with ampicillin. They found considerablevariation between patients.The present investigation was carried out to

determine the range of ampicillin sputum levels ina larger series of patients with respiratory diseaseafter treatment with doses of 250 mg., 500 mg. or1 g. orally four times daily. Serum levels were alsoassayed since these are the levels frequently quotedwhen assessing the adequacy of the dosage of adrug. Saliva levels were estimated because con-taminatilon of sputum with saliva containing ap-preciable quantities of th.z drug might give falselyhigh sputuum levels. Various factors which mightaffect the antibiotic levels have been analysed.

Requests for reprints: Dr. S. M. Stewart, Wellcome Labxratory,City Hospital, Greenbank Drive, Edinburgh, EH10 5SB

PATIENTS INVESTIGATED

All patients were in hospital at the time the levelswere investigated. The authors were not respon-sible for their admission, treatment or discharge.This sample of patients can therefore not be con-sidered a truly random sample in the statisticalsense, nor indeed is this a clinical trial withpatients randomly allocated to different treat-ments. Hence the results, and in particular thestatistical analysis, must be interpreted withcaution. Nevertheless since we think that theampicillin levels reached are not strongly relatedto the criteria determining treatment, we feel theanalysis to be helpful in giving some indicationof possible patient response to oral ampicillin.

AMPICILLIN THERAPY

250 mg. Dose Forty patients received 250 mg.ampicillin four times daily. Twenty-five were menand 15 women. Their ages ranged from 22 to85 years with a mean age of 60-6 years.

500 mg. Dose Twenty-six patients received

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Ampicillin levels in sputum, serum, and saliva

500 mg. ampicillin four times daily. Twenty weremen and six women. Their ages ranged from 33to 81 years with a mean age of 61-0 years.I g. Dose Eleven patients received 1 g. ampicillinfour times daily. Nine were men and two women.Their ages ranged from 60 to 76 years, the aver-age being 67-3 years, significantly older than inthe 250 and 500 mg. dose groups.

REPEATED ASSAYS These were carried out on 20patients. In 15 instances the repeated assays weredone on the same dose only. In three casesrepeated assays were done both on the same doseand on a higher dose, and in two cases only ondifferent doses (see details in Table VI). Whererepeated assays were done on the same dose, onlythe first set of readings was used for the mainanalysis.

CLINICAL DIAGNOSIS The clinical diagnosis ondischarge from hospital is shown in Table I.Forty-two of the patients with bronchitis hadexacerbations of chronic bronchitis, one had acutebronchitis, and six had chronic bronchitis withoutan exacerbation. Four of the five patients withasthma also had evidence of an infection.

TABLE ICLINICAL DIAGNOSIS ON DISCHARGE IN PATIENTS

INVESTIGATED

Dose of Ampicillin q.i.d.Clinical Diagnosis

250 mg. 500 mg. I g.

Bronchitis .25 16 8Pneumonia.7 3Bronchiectasis.3 5 2Asthma .. .. .. .. 4 1 0Cavitated bronchial carcinoma I1 0 0Pulmonary infarction with

secondary infection .. 0 1 0

Total No. of patients .. .. 40 26 11

EXCLUSIONS Fourteen further patients were

examined but were excluded from the mainanalysis. Twelve of these patients (4 in the 250 mg.group, 2 in the 500 mg. group, and 6 in the 1 g.

group) were excluded because the antibiotic levelin the saliva was the same as or higher than thesputum level. In these cases an apparent sputumantibiotic level could have been due to contamina-tion of the sputum with antibiotic-containingsaliva. These 12 cases were included in the analysisof the saliva levels. The remaining two cases wereexcluded from all analyses because they hadclinical renal insufficiency that might have affectedthe drug levels (Hoffler, Stegemann, and Scheler,1966).

METHODS

AMPICILLIN THERAPY The dose of ampicillin wasdecided by the clinician in charge. All patients hadbeen receiving ampicillin for at least 18 hours beforethe assay dose. In order to ensure the exact timingand dosage of the drug, the 6 a.m. dose on the dayof assay was omitted, and the last dose was givenat 10 p.m. the previous evening. The assay dose wasgiven by one of us (M. F.) at approximately 9 a.m.No antibacterial drugs other than ampicillin weregiven. The second dose of ampicillin on the day ofassay was withheld until the last specimens for assayhad been collected.

In calculating the duration of therapy the first dayhas been considered as day 1. Only treatment withampicillin in hospital has been taken into account asthe details of other therapy were often not available.

MEALTIMES No attempt was made to regulate or torecord the food ingested by the patients. They hadbreakfast between 7.45 and 8.00 a.m. and coffee ortea (sometimes with biscuits, etc.) at about 10.30 a.m.The final specimens of sputum were invariably col-lected before lunch was served.

CORTICOSTEROIDS AND DIURETICS Ten of the patientson the 250 mg. dose, six of those receiving 500 mg.,and three of those on the 1 g. dose were also receivingprednisolone, 5 to 50 mg. daily. A further patient inthe 250 mg. group was given 25 units of ACTH.and one patient in t;he 500 mg. group was given50 mg. cortisone. both daily.

Five patients in the 250 mg. group and four in the500 mg. group were receiving diuretics at the timeof the ampicillin assay. The diuretics were frusemide,bendrofluazide or mersalyl.

SPECIMENS FOR ASSAY All specimens were collectedby the person administering the ampicillin and deli-vered to the laboratory within two hours of collec-tion.Sputum was collected during the first hour after

administration of the drug and discarded. All thesputum produced between the first and second hourand between the second and third hour was collectedfor assay. In order to reduce as far as possible therisk of contamination of sputum specimens withsaliva, patients were told to collect only materialactually coughed up. At the end of each period ofcollection, the patient was asked to cough forciblyin order to empty the bronchial passages as far aspossible.

Saliva and serum were collected two hours afteradministration of the drug.The degree of purulence of the sputum was esti-

mated by naked-eye examination in the laboratory. Itwas graded as follows: mucoid; less than 30% pus:30 to 70% pus; more than 70% pus.

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Sheila M. Stewart, Mary Fisher, Joy E. Young, and W. Lutz

BACTERIOLOGICAL METHODS

Assays A cup diffusion technique, similar to that-recommended by Heathcote and Nassau (1951), was

used for the ampicillin assay. A peptone yeast extractagar was melted and cooled to 50° C. To 200 ml.agar was added 4 ml. of a 48-hour nutrient brothculture of Sarcina lutea. After thorough mixing,20 ml. quantities were pipetted into sterile 8 5 cm.plastic Petri dishes. The plates were dried at 37° C.Four cups were then cut in each plate using a metal*cork borer of 8 mm. diameter. Of the fluid under test0 06 ml. was placed into each of two cups. Theplates were incubated at 370 C. for 48 hours.

Test Fluids Assays were carried out on neat serumand on 1 in 10 and I in 100 dilutions made in steriledeionized water. Sputum was homogenized by shakingmechanically with an equal volume of sterile waterand four glass beads. The neat homogenate was usedfor the assay. The saliva was assayed without dilu-tion.

Standard Solutions of Ampicillin The followingrange of ampicillin concentrations was used: 1P0, 0-5.0-25, 01l2, and 0-06 Ag. per ml. in sterile water.Each of the standard ampicillin concentrations wasalso put up in duplicate. The standard solutions were

included in each batch of tests.

The diameter of the zones of inhibition wasmeasured by naked eye using dividers. A standardcurve was prepared from the mean of the pairs ofreadings obtained for each of the standard ampicillinconcentrations. From the means of the two readingsfor the test fluids, the ampicillin concentration wasread off the standard graph, using the dilution of thetest fluid giving a zone of inhibition falling withinthe range of those of the standard dilutions. The finalconcentration of the drug in the test fluid was calcu-lated, allowing for any dilution.

EFFECT ON ZONES OF INHIBITION

Sputum Standard solutions of ampicillin were pre-pared over the range 1-0 to 0-06 ,ug./ml. by doublingdilutions in diistilled water anid in sputum which didnot contain any antibiotics. The diameters of thezones of inhibition at the various concentrations withand without sputum were compared after 18 hours'incubation at 370 C.

pH Standard solutions of ampicillin over the range

1.0 to 0-06 Ag./ml. were prepared in phosphate buffersolutions at pH 6-0, 6 6, 7 0, 7-4, 7-8, and 8-0. Similarseries were prepared in specimens of sputum of pH6-0, 6-5, 7-0, 7-5, and 8-0. After incubation at 370 C.for 18 hours, the diameters of the zones of inhibitionwere compared with those obtained with equivalentsolutions in distilled water.

Penicillinase-producing Organism A series of ampi-cillin solutions in sputum containing no other inhi-biting substances were prepared in I ml. quantitiesin duplicate. To one series was added one drop of an18 hours nutrient broth culture of a strain ofEscherichia coli which was known to produce penicil-linase. The diameters of the zones of inhibition werecompared after 18 hours' incubation at 370 C.

ROUTINE BACTERIOLOGICAL INVESTIGATIONS

All specimens of sputum homogenates were cul-tured on blood agar and boiled-blood agar plates andincubated in 10%O COG.

Ampicillin sensitivity tests were carried out usingthe filter paper disc method, serial dilutions in nutrientor boiled-blood agar, and serial dilution in broth.Bactericidal tests were carried out on some strains bysubculturing from the broth tubes, in which growthhad been inhibited, on to nutrient agar plates. Sensi-tivity tests were done on all strains of potentialrespiratory pathogens and on cultures of lactose-fermenting coliforms, Proteus, and Pseudomonaspyocyanea.

RESULTS

EFFECT ON ZONES OF INHIBITION

Sputum The zones of inhibition obtained withthe ampicillin solutions made up in sputum wereidentical with those obtained with ampicillin indistilled water.

pH The diameters of the zones of inhibitionobtained with the ampicillin solutions in phosphatebuffer pH 6-0, 6-6, and 7 0 were the same as thoseobtained with solutions in distilled water. At pH7 4, 7-8, and 8-0 the zones were progressivelysmaller. In sputum, however, the zones weresimilar over the pH range 6-0 to 8-0 and they werethe same as those obtained with equivalent solu-tions of the drug in distilled water.

Penicillinase-producing Organism The presenceof a culture of penicillinase-producing Esch. colicaused a drop of 2 5 mm. in the diameter of thezones of inhibition at all concentrations. Thiswas equivalent to a two-fold drop in level.

PRE-TREATMENT SPECIMENS No pre-treatmentspecimens have been examined in the presentseries because specimens of sputum and serumfrom 18 patients before the start of antibiotictherapy were tested for inhibitors in a previousseries (Hafez et al., 1965) using the same assaymethod as was used in the present investigation.These results showed that 'there was no evidenceof the presence in sputum of substances other thanantibiotics which might cause non-specific inhibi-tion in the cup-diffusi,on assay technique used'.

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EFFECT OF DURATION OF TREATMENT

Figure 2 shows the correlation between theduration of treatment and the 1-2 hour sputumlevel including repeated assays on the same patientin 20 cases. There was no relationship betweenthe duration of therapy and the ampicillin level,but the number of specimens assayed after morethan 8 days' treatment was small.

nil <0-12 0.12 0-25 0 5 1i0 2-02-3 hour sputum level (l,u./ml.)

FIG. 1. Comparison of 1-2 and 2-3 hour sputum levelsafter 250 mg., 500 mg. or I g. ampicillin.

COMPARISON OF 1-2 AND 2-3 HOUR SPUTUM LEVELS

Comparison of the 1-2 and 2-3 hour sputum levelsin patients receiving 250 mg., 500 mg., and 1 g.dos_s of ampicillin is shown in Figure 1. Thelevels were similar in individual patients. Thestatistical analysis for all three doses is shown inthe Appendix (Table A). There was a correlationof approximately 0-8 between the levels of ampi-cillin in the 1-2 and 2-3 hour specimens after the250 mg. dose. The numbSer of estimations afterthe 500 mg. and 1 g. doses was too small toallow of statistical analysis as shown by very wideconfidence limits, although the levels appeared tobe similar in individual patients. Since there was

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FIG. 2. Effect of duration of ampicillin treatment on1-2 hour sputum level. Levels obtained on repeat assaysare shown with an outer ring round the symbol.

EFFECT OF DIFFERENT DOSES OF AMPICILLIN

Sputum Table II shows a comparison of the 1-2hour sputum levels after varying doses of ampi-cillin. There was no statistical difference in thelevels after 250 and 500 mg. doses. The meanlevel of the 11 specimens of sputum collected afterthe 1 g. dose was significantly higher than for thetwo lower doses.

TABLE IICOMPARISON OF 1-2 HOUR SPUTUM LEVELS AFTER

VARYING DOSES OF AMPICILLIN

Dose of Total 1-2 hour Sputum Level (pg./ml.) MeanAmpi- No. of Levelcillin Patients nil <0-12 0-12- 0 25- 0.5+ (ng./ml.)q.id. 0-24 0-49

250 mg.1 40 1 7 13 13 6 0 30500 mg. 26 1 6 11 3 5 0-26lg. 11 10_ 3 7 0-65

Serum Table III shows a comparison of theserum levels after three doses of ampicillin. Theserum levels increased with the dose of ampicillin.The mean serum level in patients on the 1 g. dosewas significantly higher than the mean levels forthe two lower doses which did not in fact differsignificantly from each other.

TABLE IIICOMPARISON OF SERUM LEVELS AFTER VARYING DOSES

OF AMPICILLIN

Dose of Serum Level (pg./ml.)Ampi- Total _ Meancillin No. of 2-0- 4 0- 8-0- 160+ Levelq.i.d. Patients <20 39 79 15 9 (pg./ml.)

250 mg. 40 4 14 9 7 6 7-67500 mg. 26 1 6 9 7 3 8 45Ig. 11 0 00 2 4 5 1662

Saliva The levels of ampicillin in the saliva areshown in Table IV. There was no difference inthe levels after the 250 mg. and 500 mg. doses,but when ampicillin was given in 1 g. doses therewas a significant increase over the levels after the

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smaller doses. A comparison of the sputum andsaliva levels is shown in Figure 3. High salivalevels were not necessarily associated with highsputum levels.

TABLE IVCOMPARISON OF SALIVA LEVELS AFTER VARYING DOSES

OF AMPICILLIN

Dose of Total Saliva Level (,ug./ml.) Mean

cillinNo. of

0-06- 0.25- Levelq...Patients' Nil <006 0-2-905 -

250 mg. 44 9 23 8 2 2 0-10500 mg. 28 2 19 5 1 1 0111g. 17 0 2 7 5 3 0-28

'The number of patients included 4 in the 250 mg. group, 2 in the500 mg. group, and 6 in the I g. group who were excluded from thmain analysis because the saliva levels were the same as or higherthan the sputum levels.

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FIG. 3. Comparison of 1-2 hour sputum and salivalevels after 250 mg., 500 mg. and I g. ampicillin.

Comparison of Sputum and Serum Levels Therelationship between the 1-2 hour sputum andserum levels is shown in Figure 4. The sputumlevels were on the whole 30 to 40 times lowerthan the serum levels, but for any given serumlevel there was a considerable scatter in thesputum levels. There is apparently no close cor-relation between the 1-2 hour sputum level andthe corresponding serum level (see Appendix,Table B).

EFFECT OF CORTICOSTEROIDS ON AMPICILLINSPUTUM LEVELS The number of patients receivingcorticosteroids was small, but the overall impres-sion was that corticosteroids did not affect the 1-2 hour sputum level at any of the three doses ofampicillin studied.

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FIG. 4. Comparison of 1-2 hour sputum level and 2hour serum level after 250 mg., 500 mg. and I g. ampicillin.

EFFECT OF DIURETICS ON AMPICILLIN SPUTUM

LEVELS The number of patients treated withdiuretics was small, but there was no suggestionthat diuretics had any effect on the 1-2 hoursputum levels of ampicillin.

RELATION BETWEEN DEGREE OF PURULENCE AND

SPUTUM LEVEL The mean 1-2 hour ampicillinsputum levels related to the degree of purulenceof the sputum are shown in Table V. There wasno significant correlation between the meansputum level and the degree of purulence aftereither the 250 mg. or 500 mg. dose. In the 11specimens from patients receiving 1 g. ampicillinfour times daily, there was a statistically signi-ficant increase in sputum level with increase inpurulence.

TABLE VMEAN SPUTUM LEVELS OF AMPICILLIN RELATED TO

DEGREE OF PURULENCE OF SPUTUM

Mean 1-2 hour Sputum LevelDose of (jg./ml.)

Ampicillin Total No.q.i.d. < 30% Pus 30 to 70% More than of Specimens

Pus 70% Pus

250 mg. 0-20 0-27 0-38 40500 mg. 0.34 0-26 0-19 261 g. 0-36 0-56 095

RELATION BETWEEN WEIGHT OF PATIENT ANDSPUTUM LEVEL There was no evidence that theweight of the patient influenced the 1-2 hoursputum level.

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REPEATED ASSAYS Table VI shows the 1-2 hoursputum levels in 20 patients in whom assays wererepeated after the same or higher doses ondifferent occasions. There was some variationbetween the assays on the same dose, the tendencybeing for the repeat assay to be lower than thefirst reading. In the few patients tested afterdifferent doses, the level increased with the dose.

TABLE VIREPEATED ASSAYS ON 1-2 HOUR SPUTUM SPECIMENSFROM THE SAME PATIENT ON THE SAME OR A HIGHER

DOSE

Dose of AmpicillinPatient

250 mg. (pg./ml.) 500 mg. (ug./ml.) I g. (pg/ml.)H.P. 0-18 0-12J.McA. 0-2 <0-12R.McG. 0-4 <0 12R.L. 0-18 0-16T.W. 0-12 <0-12D.G. 0-4 0-12M.Han. 012 NilG.S. 0-26 <012T.H. 03 013 1-2M.H. 1-45 0-2 0-36 1-2

0-23J.M. 0-14 0-24H.W. <0 12 0 5 0-8J.McP. 0 54 0-22T.R. <012 025 0-65G.D. 09 0-2M.B. 0 14 0 16G.G. <0 12 0-24W.D. Nil 0 12W.L. <0 12 <0-12 0-6 0-19T.F. <0 12 0-7

ISOLATION OF BACTERIA FROM SPUTUM AND THEIR

SENSITIVITY TO AMPICILLIN Streptococcus pneu-moniae was isolated from eight specimens ofsputum examined before the start of ampicillintreatment, Haemophilus influenzae from five, andStrep. pneumoniae and H. influenzae from one.No pathogens were isolated from 55 pre-treatmentspecimens but many patients had received someantibiotic treatment before the collection of thesputum. No pre-treatment specimens wereexamined from eight patients.From the specimens on which assays were

carried out (i.e., after the start of ampicillin treat-ment) Table VII shows the bacteria isolated:

TABLE VII

AmpicillinOrganism Isolated

250 mg. 500 mg. 1 g.

Staph. pyogenes.5 1 0Kl. pneumoniae.1 2 1Non-haemolytic streptococcus 1 1 0Lactose-fermenting coliform 6 6 2Proteussp. .3 2 2Ps. pyocyanea .. 0 4 2

All strains from specimens taken after the startof treatment were resistant, by all methods tested,to concentrations of ampicillin well above thosefound in the sputum, with the exception of twostrains of Klebsiella pneumoniae; one strain wassensitive in agar but resistant in broth, the otherfailed to grow on subculture and therefore sensi-tivity tests could not be carried out.

DISCUSSION

The estimation of antibiotics in body fluids hasbeen widely employed as a guide to the optimumdosage of the drug to be given: such reports haveusually been based on serum levels. The results ofprevious work with penicillfin (Hafez et al., 1955)and with ampicillin (May and Delves, 1965) sug-gest that sputum levels are appreciably lower thanthe corresponding serum levels and thereforeserum levels may not be a satisfactory indicationof the tissue levels in the lungs or in the walls ofthe bronchi. The current investigation was carriedout to determine the sputum levels obtained afterthe administration of oral ampiCillin in patientswith respiratory disease during courses of 250 mg.,500 mg., or 1 g. 6-hourly. Serum and saliva werealso assayed in parallel with the sputum.The most striking finding in the present in-

vestigation was the difference in ampicillin sputumlevels between patients on the same dose of drug.For example, with a dose of 250 mg. ampicillin,sputum levels varied from nil (in two patients) to1-45 ,ug./ml. This is an even greater range thanin the series reported by May and Delves (1965)in which it was nil to 0-6 ,tg./ml. in 24-hour collec-tions of sputum from patients receiving 1 g. ampi-cillin 6-hourly. One of the possible causes of sucha variation may be experimental error in the assaysystem used. Against this is the comparatively closecorrelation of the 1-2 hour and 2-3 hour sputumlevels found in our patients. Also, when ampicillinsolutions of known concentrations were made upin sputum or serum, and assayed by the standardmethod, the assay level and the known levels werevery similar.A second cause of the variation between levels

in sputum from different patients, and one difficultto control, is the contamination of sputum withsaliva during the collection of the sputum speci-men. If the saliva contains appreciable amountsof the drug, a mixture of sputum and saliva mightgive a falsely high sputum level; this source oferror has been avoided in the present investigationby carrying out assays on saliva in all cases andby excluding from the analysis all patients in

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whom the saliva level was the same as or greaterthan the sputum level. If the saliva contains littleor no antibiotic, as was frequently the case in thecurrent series, contamination of the sputum withsaliva would give a low reading for the sputum.An attempt was made to prevent this contamina-tion by asking the patients to collect only materialactually coughed up, but it cannot be guaranteedthat saliva contamination did not occur. However,the correlation between the 1-2 hour and 2-3 hourreadings suggests that the effect may not be great.Nevertheless the sputum levels must be consideredas minimal readings and salival contaminationmay account for some of the variation. This is avariation inherent in the method and one thatapplies equally to all investigations of drugsputum levels, unless bronchoscopy specimens areused.

It seemed possible that pH or substances in thesputum might contribute to the variable results.However, assays of known concentrations ofampicillin in specimens of sputum of varying pHdid not demonstrate any difference in readingsover the pH range 6 0 to 8-0. Similarly, assays ofsolution's with and without the addition of sputumcontaining no other antibiotics showed no differ-ence in the readings. Therefore it seems unlikelythat sputum pH or factors in 'normal' sputumcontributed to the variations.Maddocks and May (1969) have suggested that

the presence of penicillinase-producing organismsin the sputum may reduce the active level of peni-cillinase-sensitive antibiotics. They found that infive out of six patients with chronic bronchialdisease excreting penicillinase-producing organ-isms in their sputum only a trace of ampicillinwas detectable in the sputum and that ampicillinadded to the sputum was completely inactivatedin all cases. This inactivation was prevented infive of the six cases by the addition of cloxacillinto the sputum ; in the sixth, partial inactivationoccurred and the patient did not respond clinicallyto the combined therapy. In one patient therewas a sputum level of 0-6 ,tg./ml. in spite of thefact that the sputum inactivated the ampicillinin vitro. In a previous report (Hafez et al., 1965)on penicillin levels in sputum, the levels attainedin patients excreting penicillinase-producing organ-isms in the sputum were compared with those notexcreting such organisms. There was no differencein the levels in the two grioups. These tests weretherefore not carried out in the present investiga-tion. However, mixtures of varying ampicillin con-centrations in sputum with and without the additionof a broth culture of a penicillinase-producing

strain of Esch. coli were assayed; there was a two-fold drop in level in the presence of the Esch. coliculture, but in none of the concentrations from1-0 to 0 06 ytg./ml. was the ampicillin activitycompletely neutralized. It seems therefore thatwhile the presence of a penicillinase-producingorganism may cause a decrease in ampicillin level,this may not be much and is unlikely to accountfor the wide variations found. Maddocks andMay's results suggest that any reduction in levelthat occurs may be of clinical significance andtherefore it is the reduced level, as detectable inthe present series, that should be used in assessingthe optimal dosage of ampicillin.

There will also be variation due to differingabsorption rates between individuals. This shouldbe reflected in the serum levels, which also showedbig variations in the present investigation: how-ever, low sputum levels were not necessarily asso-ciated with low serum levels.

Early reports showed that after a single dose theserum level rose in direct proportion to the in-crease in dose from 250 mg. to 1 g. (Knudsen,Rolinson, and Stevens, 1961). May and Delves(1964) found that in sputum from small groups ofpatients the mean level was 0 08 ,ug./rml. six hoursafter a dose of 250 mg. and 0-24 ,ug./ml. six hoursafter 500 mg. A graph showing repeated estima-tions on two patients, one receiving 250 mg. andthe other 500 mg., showed that, while dlifferencesoccurred, these were not consistent and that bythe third to fourth day of treatment the levelswere similar on the two doses. In a later paper(May and Delves, 1965), the mean 24-hour sputumlevel of 20 patients receiving 1 g. six-hourly was0-25 jtg./ml. In our series there was no differencebetween the sputum levels after 250 mg. or 500Ymg. doses, but the levels were statistically higherafter I g.Most of these estimations were carried out after

three or more days of treatment, though therewas no evidence that the duration of therapyaffected the sputum level. The serum levels in-creased with increasing dose, though the differ-ence between the specimens collected after the250 mg. and 500 mg. doses was much less markedthan between the 500 mg. and 1 g. doses. Thesaliva levels were similar after the 250 mg. and500 mg. doses but higher after the 1 g. dose.The most common way of judging if adequate

doses of an antibiotic are being given is to esti-mate the serum level and to compare this withthe minimum inhibitory concentrations for thecommon pathogens. However, the serum levelmay not represent the tissue level. There may be

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a gradient between the blood vessels and thebronchial wall (Crofton, 1969). In chronic bron-chitis the site of infection is on or just below thesurface of the bronchi (Hors and Mulder, 1953).If sputum levels are considered as a reflection ofthe lung tissue level, it is apparent from this in-vestigation and from May and Delves' results(1965) that the levels at the site of infection may

be much lower than those in the serum. Theserum levels obtained after all doses in the presentseries should be adequate to inhibit the growthof Strep. pneumoniae and H. influenzae, since theminimum inhibitory concentrations of thosepathogens are generally given as 0 06 and 0 25,ug. / ml. respectively, although some strains ofH. influenzae are resistant to 10 jug./ml. or above(personal experience). However, sputum ampi-cillin levels of 0 25 Mug./ml. or above were obtainedin only 19 of 40 patients receiving 250 mg. ampi-cillin per dose and in 8 of 26 on 500 mg. All butone of the 1I patients receiving 1 g. ampicillinhad a sputum level of 025 ,ug./ml. and seven ofthose were of 0 5 fig.!/ml. or above. It is thesputum levels rather than the serum levels thatare compatible with May and Delves' (1965),admittedly uncontrolled, clinical findings inchronic bronchitis that the 1 g. dose is more effi-cient than lower doses. Further, in the presentinvestigation all organisms isolated at the time ofthe assay had minimum inhibitory concentrationsabove the sputum levels but not always abovethe serum levels, suggesting that the concentra-tion in the serum was probably not attained at thesite of the organism concerned. However, nostrains of H. influhenzae or Strep. pneumoniaewere isolated, suggesting that these organisms hadbeen eliminated.The results of the present series confirm that

ampicillin is present in the sputum in appreciableamounts after oral administration and that higherlevels are obtained after a dose of 1 g. than after250 mg. or 509 mg. The serum levels were appre-

ciably higher than the sputum levels. Whether theserum or sputum levels are of greater clinicalsignificance in assessing adequacy of drug dosagecould be ascertained only by careful correlationwith the clinical results.

The authors wish to thank Professor J. W. Croftonand Professor B. P. Marmion for their advice. Thepatients investigated were under the care of ProfessorCrofton, Dr. A. C. Douglas. and Dr. G. J. R.McHardy. The authors also thank Miss E. Mercer,Miss M. Davidson, and Mrs. K. Pratt for technicalassistance, Mrs. J. Herson. of the Computer and

Statistics Section, Usher Institute for computationalassistance, and Miss M. White for secretarial assis-tance. Dr. Margaret A. Calder carried out the pre-treatment bacteriological examinations.The research was supported by grants from the

Scottish Hospital Endowments Research Trust, theWellcome Foundation, the Medical ResearchCouncil, and the Chest and Heart Association.

REFERENCESCrofton, J. (1969). Some principles in the chemotherapy of bacteria?

infections. Brit. med. J., 2, 209.Hafez, F. F., Stewart, Sheila M., and Burnet, M. Eileen (1965).

Penicillin levels in sputum. Thorax, 20, 219.Heathcote, A. G. S., and Nassau, E. (1951). Concentrations of

penicillin in the lungs. Lancet, 1, 1255.Hers, J. F. P., and Mulder, J. (1953). The mucosal epithelium of the-

respiratory tract in mucopurulent bronchitis caused by Haemo-philus influenzae. J. Path. Bact., 66, 103.

Hoffler, D., Stegemann, I., and Scheler, F. (1966). Ampicillin levelsin the serum and urine of patients with impaired renal function.Gernm. med. Mth., 11, 138.

Knudsen, E. T., Rolinson, G. N., and Stevens, Shirley (1961). Absorp-tion and excretion of "penbritin". Brit. med. J., 2, 198.

Maddocks, J. L.,and May, J. Robert (1969). "Indirect pathogenicity"of penicillinase-producing enterobacteria in chronic bronchialinfections. Lancet, 1, 793.

May, J. Robert, and Delves, Doreen M. (1964). Ampicillin in thetreatment of Haemophilus influenzae infections of the respiratorytract. Thorax, 19, 298.

(1965). Treatment of chronic bronchitis with ampicillin-Lancet, 1, 929.

APPENDIX TABLE ACOMPARISON OF 1-2 AND 2-3 HOUR SPUTUM LEVELS

Dose of Ampicillin q.i.d.

250 mg. 500 mg. I g

Sample size .40 26 11

Mean level 1-2 hours .. 030 0-26 0-65Mean level 2-3 hours .. 0-36 0 34 0-57SE of mean of 1-2 hour level 0 04 0 04 0-12SE of mean of 2-3 hour level 0-06 0-07 0-11

Correlation between 1-2 and2-3 hour levels 0-82 0 60 0-61

95 ° confidence limits for thecorrelation. 0-69 0-28 0-02

-0 90 -0-80 -0-89

APPENDIX TABLE BCOMPARISON OF 1-2 HOUR SPUTUM LEVEL AND SERUMLEVEL AFTER 250 MG., 500 MG., AND 1 G. DOSES OF

AMPICILLIN

Dose of Ampicillin q.i.d.

250 mg. S00 mg. I g.

Sample size.40 26 |It

Mean sputum level .. 030 0-26 0 65Mean serum level . 7-67 8-45 16 62SE of serummean. 1-26 1-55 3-48

Correlation between serum and sputum 0-38 0-04 0-7795 ° confidence limits for thecorrelation. 0-08 -39 0-31

-0-62 -+039 -0 94

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