a Departments of Paediatrics and Child Health, Pharmacology, Medical Biochemistry and Internal Medicine, University of Stellenbosch, Cape Town; b Medical Research Council, Tygerberg, South Africa; c St George's Hospital Medical School, London SW17 0RE, UK
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Abstract |
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Introduction |
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Materials and methods |
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All patients were of mixed race (previously called Cape coloured) living in the Western Cape district, where there is a serious epidemic of tuberculosis. They were admitted for the study into Tygerberg Hospital, Cape Town, South Africa. Permission for all studies was obtained from local ethics committees and the patients gave written informed consent. Normal renal function of all patients was confirmed as a criterion for admission. Studies were carried out before and after the test period on each patient of haematology, urinalysis and serum chemistry, including liver function tests, electrolytes, cholesterol, triglycerides and glucose. No estimates of concentrations of total amikacin in blood were made, as these had already been estimated in healthy volunteers, nor were concentrations of amikacin in sputum measured.
Estimation of the EBA
Details of the procedures have been described elsewhere.9 In all patients, a 16 h collection of sputum was made on the night of the first day (S1 collection). Soon after the completion of this collection the first dose of liposomal amikacin 30 mg/kg body weight was given by slow iv drip over a 2 h period. A further 16 h sputum collection (S2 collection) was made on the second day followed by another iv dose of liposomal amikacin 30 mg/kg. This procedure was repeated once more on the third day (S3 collection). Finally one further 16 h collection was made (S4 collection) and the patient was then given standard multidrug chemotherapy. Thus, each patient was treated for 3 days and had four sputum collections.
Microbiological methods
Sputum in the S1 and S4 collections was examined conventionally by direct smear, culture and susceptibility testing. The procedure for sputum processing was similar to that described previously.9 Briefly, sputum collections were homogenized on a magnetic stirrer by adding a teflon-coated follower bar to the entire specimen which was then stirred for 2030 min. Ten millilitres of the homogenate was added to an equal volume of 1:10 dithiothreitol (Sputasol, Oxoid, Basingstoke, UK) in a 50 mL screw-capped tube containing three to six glass beads (10 mm) and vortex mixed for 20 s. The specimen was then mechanically shaken for c. 30 min until well digested. Sets of dilutions were prepared for each collection in sterile distilled water by making 10-fold dilutions from neat to 10-2 and thereafter by serial five-fold steps. From each of the dilutions, aliquots of 100 µL were spread in duplicate on to half selective 7H11 oleic acid albumin agar plates. These were placed into polyethylene bags together with a plate inoculated with Mycobacterium phlei to provide CO2 and were incubated for 3 weeks at 37°C before colonies were counted at that dilution which permitted counting between 20 and 200 colonies.
Statistical methods
The results of the cfu counts were entered on Microsoft Excel worksheets and the corresponding log10 counts/mL sputum, Y1, Y2, Y3 and Y4, were calculated from the cfu counts on the S1, S2, S3 and S4 collections, respectively. Three EBAs were then calculated: S1S2 EBA = Y1Y2; S1S3 EBA = (Y13)/2; and S1S4 EBA = (Y1Y4)/3. Shapiro-Francia tests for normality of distribution, ANOVA and regression analyses were done on these estimates using Stata release 6 (Stata Corp., College Station, TX, USA)
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Results |
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Discussion |
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The failure to detect EBA indicates a failure of liberation of the amikacin from the liposomes in liposomal amikacin. It seems probable that liberation happens only when the liposomes are phagocytosed by macrophages. This accounts for the high activity of liposomal amikacin in acute murine tuberculosis, where bacilli are mainly intracellular within macrophages. As there are few macrophages close to tubercle bacilli in cavities,12,15 this mechanism may not produce an adequate concentration in those parts of the caseum where the great majority of the bacilli exist. More amikacin might, however, be released in areas of pneumonic infiltration where macrophages abound and there is a closer resemblance to murine histology. Even so, the mouse experiments clearly demonstrate that amikacin had little bactericidal activity once immunity has developed,6 which is certain to have happened in human lesions. In HIV-seropositive patients, macrophages are less likely to cause bacillary stasis, so that it is still possible that liposomal amikacin might be of value in their treatment. The very low EBA of amikacin whether administered in the free form or in liposomes may result from an acid micro-environment of the bacilli due to the presence of acute inflammation, as argued elsewhere.9 It is necessary to postulate such an acid micro-environment to account for the sterilizing activity of pyrazinamide.16 Thus, it appears that amikacin in any form has low initial bactericidal activity, mainly because of the lesional pH. While the great majority of the bacilli at the start of treatment are extracellular, it has been postulated that the persisting bacilli in the later stages of treatment have often been phagocytosed.15 Drugs incorporated in liposomes would then be liberated close to bacilli after they had been phagocytosed by macrophages containing bacilli. This mechanism might allow liposomal amikacin to be more bactericidal in the later stages of treatment. Unfortunately, because all aminoglycosides are poor sterilizing drugs, as shown in the treatment of chronic murine tuberculosis6,17 and of pulmonary tuberculosis,18,19 they are also ineffective in killing bacilli during the later stages of treatment and do not shorten the duration of treatment. Nevertheless, whereas aminoglycosides and amikacin in particular may be less bactericidal in the treatment of human tuberculosis than other drugs, they serve a purpose in preventing the emergence of drug resistance. Although the role of liposomal amikacin appears to be strictly limited in the treatment of pulmonary tuberculosis, enclosure of drugs with higher sterilizing activity, particularly rifampicin, in liposomes or by micro-encapsulation20 could offer a mechanism for drug delivery that has advantages over conventional oral administration.
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Acknowledgements |
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Notes |
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References |
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Received 19 February 2001; returned 21 June 2001; revised 3 August 2001; accepted 10 September 2001