a Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand; b Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK
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Abstract |
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Introduction |
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We have evaluated retrospectively the prognostic value of serum bactericidal and inhibitory titres in determining treatment outcome in severe melioidosis.
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Patients and methods |
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Adult patients (over 14 years old), admitted to Sappasitprasong Hospital with suspected severe melioidosis, were enrolled into one of a series of treatment trials, as described previously, if they or their relatives gave informed consent.24 Patients were randomized to either ceftazidime or one of intravenous conventional; four-drug therapy, co-amoxiclav or imipenem. The drug regimens consisted of: ceftazidime 120 mg/kg/day in three divided doses by slow iv injection; chloramphenicol succinate 100 mg/kg/ day, doxycycline 4 mg/kg/day, trimethoprim 10 mg/kg/day and sulphamethoxazole 50 mg/kg/day in divided doses; co-amoxiclav 160 mg/kg/day in six divided doses by slow iv injection; or imipenem 50 mg/kg/day in three divided doses by iv infusion over 90 min. Pre-dose (immediately before) and post-dose (1 h after slow iv injection dose or end of the infusion) samples were collected, 4872 h after starting therapy, by venepuncture. Specimens were centrifuged as soon as possible at 3000 rpm at 4°C. Serum was stored at 30°C until assays could be performed, if these could not be performed immediately.
Laboratory procedures
B. pseudomallei was isolated as described previously.5 Paired serum specimens from each patient were serially double-diluted in tryptic soy broth (TSB) (Unipath, Basingstoke, UK) in U-well microtitre plates to give a range of titres of 1:21:128. The patient's original isolate of B. pseudomallei was incubated overnight in TSB and then diluted to achieve a final inoculum per well of approximately 1 x 105 cfu/mL. Precise inocula were determined by dilution and sub-culture. Plates were incubated overnight at 3537°C in air, unshaken. The highest dilution without visible growth was recorded as the serum inhibitory titre (SIT) and 20 µL volumes from each well were subcultured on to Columbia agar. After overnight incubation at 37°C, plate colony counts were performed. The highest dilution at which 99.9% kill of the original inoculum was achieved was recorded as the serum bactericidal titre (SBT). Titres from some of these patients have been published previously.3,5
Serum creatinine concentrations in samples collected from patients on admission were determined in the Biochemistry Laboratory of Sappasitprasong Hospital using an automated alkaline picrate method. Data analysis was performed using the statistical computing package SPSS 8.0 for Windows (SPSS Inc., Chicago, IL, USA).
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Results |
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Titre results for the four main drug regimens are shown in Table I. There were no significant differences overall between sexes for SIT or SBT and no significant associations with age. There were strong positive correlations overall between pre- and post-dose titres for both inhibitory and bactericidal determinations overall, as well as between inhibitory and bactericidal titres either pre- or post-dose (all correlations, Spearman rank correlation coefficient r > 0.74, P < 0.001).
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The post-dose bactericidal titres for all patients, by outcome, are shown in Figure 1. Overall, higher inhibitory or bactericidal titres were not associated with improved survival, either pre- or post-dose. However, for ceftazidime, higher post-dose SBTs were associated significantly with mortality (survivors, median = 16, interquartile (IQ) range 832; non-survivors, median = 32, IQ range 1664, P = 0.015), as were higher pre-dose SBTs (median 4 versus 12, respectively, P = 0.003) and SITs (median 8 versus 24, P = 0.022). For imipenem, pre-dose bactericidal titres only were associated with higher mortality (P = 0.03). Higher titres for co-amoxiclav and the four-drug regimen were not significantly associated with death.
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SBT/SIT ratios
Both overall, and for the individual drug regimens, higher pre- and post-dose SBT/SIT ratios were not associated with improved outcome.
Post-dose/pre-dose SIT and SBT ratios
The ratios of post- to pre-dose titres (both inhibitory and bactericidal) are shown in Figure 2. Overall there was an association between SIT post-/pre-dose ratios and outcome (survivors, median ratio 2, IQ range 24; non-survivors, median ratio 2, IQ range 14, P = 0.032), but not between SBT ratios and outcome (P = 0.525). There were no associations between outcome and ratios for any of the individual drug regimens, or the three ß-lactam agents combined.
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Admission renal function was correlated positively with mortality overall, i.e. patients with impaired renal function were more likely to die. Admission serum creatinine concentrations and related mortality are shown in Table II.
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Overall, there were positive correlations between serum creatinine concentrations and pre-dose titres (SIT, r = 0.194, P = 0.009; SBT, r = 0.20, P = 0.006), but not post-dose titres. Creatinine concentration was correlated with pre-dose titres (SIT, r = 0.38, P = < 0.001; SBT, r = 0.37, P = < 0.001) for ceftazidime-treated patients, and with pre-dose SBT for imipenem (r = 0.54, P = 0.009), but not with titres for either co-amoxiclav or the four-drug regimen.
Those patients who had a pre-dose bactericidal titre 1:4 had a mean creatinine of 2.47 mg/dL (95% CI 2.102.90) but for patients with a titre < 1:4 the mean creatinine was 1.85 mg/dL (1.632.09), P = 0.005. This was also true of pre-dose inhibitory titres (means 2.32 versus 1.87 mg/dL, respectively, P = 0.04). Post-dose titres of > 1:4 were not significantly associated with renal function.
Logistic regression analyses were performed to examine the relative influence on mortality of renal function and both SITs and SBTs. These multivariate analyses compared age, gender, drug regimen, sGOT, serum creatinine concentration, plasma lactate, the presence of positive blood cultures and either SIT or SBT (pre-dose titres > 1:4). Serum creatinine (SBT, P = 0.006, OR 1.32, 95% CI 1.081.60) and plasma lactate (SBT, P = 0.11, OR 1.18, 95% CI 0.961.46) concentrations were the most important predictors of mortality. Similar results were found for inhibitory titres.
There were significant inverse correlations between serum creatinine concentration and either bactericidal (r = 0.29, P = 0.006) or inhibitory (r = -0.31, P = <0.001) post-/pre-dose ratios. In a multiple logistic regression analysis as described above, renal function was the most important predictor of mortality (P = 0.004, OR 1.37, 95% CI 1.111.71) and neither SIT nor SBT ratios were independent risk factors.
Duration of hospital stay and fever clearance times
The median duration of hospital stay in survivors was 22 days (range 466). Both overall, and for patients with normal renal function, there were no significant correlations between SIT, SBT or post-/pre-dose ratios, and either duration of hospital stay or fever clearance. There was no evidence to suggest that those surviving patients with the lowest SBTs or SITS ( 1:2) had longer stays in hospital or prolonged fever.
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Discussion |
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Our results demonstrate that SBT and SIT determinations are not useful in the individual management of severe melioidosis. Outcome, duration of stay in hospital and fever clearance times were all unrelated to these titres. The three ß-lactam antibiotics all achieved reasonable inhibitory and bactericidal activity in serum, although the four-drug regimen had relatively poor inhibitory activity and very little bactericidal action. This is reflected in the considerably higher mortality in this group of patients.
In the present study, significantly higher inhibitory and bactericidal titres, particularly pre-dose, were seen in patients who subsequently died. These higher pre-dose titres were a result of the greater degree of renal impairment present in those patients who died; such impairment of renal function was the most important determinant of mortality. Although high ratios between post- and pre-dose titres appeared to be beneficial, this effect was lost when renal function was taken into account. This relationship between renal impairment and higher SITs and SBTs is unsurprising, as such functional impairment reduces the urinary excretion of antibiotics, the main route of elimination of the ß-lactam agents. Furthermore, the principal determinant of therapeutic outcome when severe bacterial infections are treated with ß-lactam antibiotics is the time (T) for which plasma concentrations remain above the MIC for the infecting organism. The steep concentrationeffect relationship with these drugs and their usually rapid elimination result in an abrupt transition from maximum activity (Emax) to no activity, once levels have fallen to the MIC. The peak and trough titres measured here are an imprecise measure of T. The patients with renal impairment had extremely high levels, probably achieving Emax throughout the dosing interval. Relationships between SIT or SBT and therapeutic effect would therefore not have been expected in this group, but nor were we able to demonstrate any such relationship in patients with normal renal function.
In conclusion, determination of serum bactericidal activity does not provide useful prognostic information and is of no value in the management of acute severe melioidosis.
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Acknowledgments |
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Notes |
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Present address. Public Health Laboratory, Derriford Hospital, Plymouth, UK
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References |
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Received 22 February 1999; returned 12 May 1999; revised 24 June 1999; accepted 31 August 1999