Download Full protocol V9 06082013

Version 9
August 6, 2013
WP 1
D1.1
Multicenter open-label RCT to compare colistin alone vs.
colistin plus meropenem
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BACKGROUND
Colistin has resurged in the last decade for the treatment of multidrug-resistant Gram-negative
bacteria due to lack of other antibiotics. This antibiotic, firstly discovered in 1947, belongs to the
polymyxin family and is a mixture of polymyxin E1 and polymyxin E2. The polymyxins act by
disrupting the cell membrane. They have a strong positive charge and a hydrophobic acyl chain
that confer them a high binding affinity to lipopolysaccharide (LPS) molecules. They interact
electrostatically with these molecules and competitively displace divalent cations from them,
causing disruption of the membrane. [1, 2] Electron microscopy studies show protrusions and
bleb formation of the cell membrane with leakage of cell contents. [3-5] Colisitn is bacteriocidal;
whether interaction with membranes is the cause of bacterial cell death is unknown. [1]
Polymyxins also bind to the lipid A portion of the LPS and, in animal studies, block many of the
biologic effects of endotoxin. [6] Colistin has broad in-vitro activity against Gram-negative
bacteria, with the exception of Proteus spp., Providencia spp., Serratia spp., and rarer bacteria
(Brucella spp., Edwardsiella spp., Pseudomonas mallei and Burkholderia cepacia). Breakpoints
for susceptibility are defined for enterobacteriaceae, Acinetobacter spp. and Pseudmonas spp.
The CLSI define 2 mg/L for all and EUCAST defines 2 mg/L for Acinetobacter sp. and
enterobacteriaceae and 4 mg/L for Pseudomonas sp. [7]
Colistimethate sodium (CMS) is the preparation currently used for systemic treatment. CMS is a
prodrug that undergoes spontaneous hydrolysis in-vivo or in aqueous solutions to the active drug,
colistin. The existence of these two forms has complicated PK/PD studies, since old bioessays
did not differentiate between the two forms, which have different half lives and modes of
excretion. [8] To further complicate matters, colistin is measured using different units. One mg
of CMS is equivalent to 12,500 international units (IU, where 1 IU is defined as the minimal
concentration which inhibits the growth of E. coli 95 I.S.M in 1 ml broth at pH 7.2 [9]). One mg
of colistin base activity (CBA, the unit of measurement used in the US formulation) is equivalent
to 33,250 IU. Considering a 70kg adult, the classically maximal recommended daily dose of
colistin in the US, 5 mg/kg CBA would translate to 11.5 mill IU, while in Europe 9 mill IU per
day would translate to 3.9 mg/kg CBA.
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Since its resurgence, observational studies have tried to examine the effectiveness of colistin.
Although individual studies reported favourable results regarding both effectiveness and safety, a
compilation of these studies shows higher mortality among patients treated with colistin or
polymyxin B compared to patients given other antibiotics, mostly beta-lactams (Figure 1). [2] In
most studies colistin was used in combination with other antibiotics, mainly carbapenems, and
colistin was probably underdosed. In the largest study, conducted in Israel, colistin was given
almost always as monotherapy at a mean dose of 6.1 ±2.3 MU/day and mortality was
significantly higher with colistin when compared to carbapenems or ampicillin-sulbactam. [10]
Pooling of adjusted results from multivariable analyses or matched studies shows similar results
(Figure 2).
In the same comparative studies rates of nephrotoxicity were higher with colistin compared to
other antibiotics (Figure 3). Rates of nephrotoxicity in recent studies designed to assess this
outcome have ranged from 6-14% in some [11-15] to 32-55% in others[16-20]. The wide range
of nephrotoxicity rates is explained at least partially by different definitions of renal failure. Both
the daily dose [17, 20] and the total cumulative dose [15, 16, 21] have been associated with
increased risk of nephrotoxicity. Among patients with colistin-induced nephrotoxicity between
0-1.5% [16, 20] to ~20% [14, 18, 19] required short-term renal replacement therapy. Studies
monitoring patients up to 1-3 months after colistin last dose demonstrated reversibility of renal
failure in at least 88% of patients [12, 16, 18]. The other feared toxicity of colistin is
neurological. Manifestation range from dizziness, muscle weakness, paresthesias, hearing loss,
visual disturbances and vertigo to confusion, hallucinations, seizures, ataxia, and neuromuscular
blockade with apnea[22]. The latter manifestations are rare in clinical practice.
Studies currently focus on improving the efficacy and safety profile of colistin. A first step is the
optimization of dosing and schedule of administration. Recent PK studies demonstrate that it
takes about 36-48 hours for colistin (rather than CMS) to reach therapeutic concentrations in
plasma (≥2 mg/L) using classical dosing in patients with normal renal function [23, 24]. Thus, a
loading dose, equaling to about the total daily dose is currently recommended. Furthermore,
these studies demonstrate that once or twice daily dosing is probably sufficient[8]. For example,
targeting a colistin steady state level of 2.5 mg/liter for a patient with a creatinine clearance of 70
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ml/min/1.73, requires 337.5 mg CBA per day (11.2 mill IU). [23] With higher creatinine
clearance rates the dose increases further. A recent study reported on the clinical experience of
treating critically ill patients with colistin using a 9 mill IU loading dose followed by 4.5 mill IU
q12h for normal renal function. [25] A response rate of 82.1% (23/28) and nephrotoxicity of
17.8% (5/28) was reported.
The suboptimal efficacy of colistin and the nephrotoxicity associated with high dosing regimens
has led to the search for combination therapies that might improve clinical success via better
killing or inhibition of the pathogen, more rapid killing, killing or inhibition at lower drug
concentrations, thus avoiding toxicity, and prevention of resistance selection or emergence.
Combinations suggested with colistin include various beta-lactams, azithromycin, cotrimoxazole, rifampin, doxycycline, minocycline, tigecyclin, vancomycin, aminoglycosides,
quinolones, fosfomycin and sulbactam[26]. Most of the clinical experience exists with
carbapenems that are sometimes used alone or in addition to colistin for carbapenem-resistant
infections when MICs are relatively above the susceptibility breakpoint, mainly for
Acinetobacter baumannii, in the assumption that high dosing might overcome resistance, but few
data support this practice. In a mouse model, intratracheal meropenem was significantly more
effective than colistin for carbapenem-resistant Acinetobacter baumannii pneumonia with an
MIC for meropenem of 32 μg/ml[27]. The main rationale for combination therapy lies in the
existence of in-vitro synergy. Synergistic interaction between antibiotics is usually defined as a
>2-log10-lower number of CFU/ml for the combination than for its most active component in
time-kill studies. Antagonism is defined as >2-log10 increase in CFU/ml between the
combination and the most active single agent and additivity is defined as a 1 to <2-log10-lower
number of CFU per milliliter for the combination. Other interactions are considered indifferent.
[28-30] In the checkerboard and Etest methods, synergy is defined using the fractional inhibitory
concentrations index (FICI), where FICI is the sum of the FICs of individual antibiotics in a
combination and the FIC of an antibiotic is defined as the combination’s MIC divided by the
MIC of the antibiotic alone. The common convention is that FICIs of <0.5, >0.5–4, and >4
represent synergy, no interaction and antagonism, respectively, [31-33] although variations exist
and older studies considered FICIs>1 as antagonistic. [34]
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For in-vitro data, a systematic review and meta-analysis of the literature was performed as part of
the background for the clinical trial. The following search string was used to locate all studies
published in PubMed:
(colistin OR colisti* OR colistimethate OR polymyxin) AND (imipenem OR meropenem OR
doripenem OR ertapenem OR carbapenem) AND (pharmacokinetic OR pharmacodynamic OR
synergy OR synerg* OR antagonis* OR additive) AND (in-vitro OR checkerboard OR time-kill
OR Etest OR E-test OR microdilution OR agar dilution OR susceptibility). A search was run also
in Google scholar and the ICAAC, IDSA and ECCMID conference proceedings for the years
2007-2012. References of all included studies were reviewed for more eligible studies.
(colistin OR polymyxin) AND (imipenem OR meropenem OR doripenem OR carbapenem)
AND (combination[ti] OR synergy[ti] OR synerg*[ti] OR combin*[ti]). In addition, the
references of all included studies were searched for additional studies.
For each study, we sought to extract the method of in-vitro synergy testing, bacterial species, the
type of carbapenem and polymyxin used, and number of isolates tested. Reported MICs of study
isolates for the carbapenem and polymyxin tested were also extracted and susceptibility was
assessed according to the European Committee on Antimicrobial Susceptibility Testing
(EUCAST) published breakpoints. [35] We calculated synergy rates, where synergy was counted
as an event and the sample size was the number of isolates tested. We used mixed-effects
analysis in order to provide a pooled rate. The I2 statistic was used to test heterogeneity.
Comprehensive Meta-Analysis V2.2 (Biostat, Englewood NJ, 2005) was used for analysis.
Thirty-eight published studies and 15 conference proceeding were included, reporting on 244
different tests on 1050 bacterial isolates. A summary of selected studies are presented in Table 1.
In time-kill studies, combination therapy showed synergy rates of 77% (95% CI 64-87) for A.
baumannii, 44% (95% CI 23-51%) for Klebsiella pneumoniae and 50% (95% CI 30-69%) for
Pseudomonas aeruginosa with low antagonism rates for all. For A. baumannii, meropenem was
more synergistic than imipenem, whereas for P. aeruginosa the opposite was true. Checkerboard
and Etest studies generally reported lower synergy rates than time-kill. Comparisons of
resistance development between monotherapy and combination therapy were found in one study
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on 3 A. baumannii isolates and four studies on 14 P. aeruginosa isolates, all recent studies. Use
of combination therapy led to less resistance development in-vitro.
Thus, in-vitro studies show variable results, but overall synergy is substantial. Carbapenempolymyxin synergy is probably more likely when isolates are more susceptible to one or both of
the drugs in the combination. It was observed more frequently with A. baumannii than with K.
pneumonia or P. aeruginosa strains and this could be related to lower MICs for A. baumannii to
carbapenems in general. Difference between carbapenems is less clear and depended on bacteria
type, with doripenem having some advantage.
Learning from in-vitro studies on clinical effects is difficult because the bacterial inocula differ,
drug levels may be affected by practical constraints of antibiotic administration and clinical
effects are confounded by underlying conditions and adverse effects. Furthermore, poor
correlation has been shown between different in-vitro methods for synergy testing. [34] Indeed,
despite strong in-vitro proof of synergy and prevention of resistance induction for beta-lactamaminoglycoside combinations for various Gram-negative and Gram-positive bacteria,
randomized controlled trials do not show a clinical benefit for the same combinations compared
with beta-lactams alone in the treatment of sepsis by the same bacteria[36]. Detriments of
combination therapy may comprise of further resistance induction, increased toxicity and
antagonistic interactions between antibiotics. Thus, the effects of combination therapy must be
tested in clinical studies
Data from in-vivo and human studies on combination therapy is weak. Three in-vivo studies
examined the role of carbapenem-polymyxin combination (Table 2), all examining the effect of
combining imipenem and colistin. While two studies P. aeruginosa studies found improved
outcome with combination, the third tested on A. baumannii showed no benefit with this
combination. Three studies were found reporting on the clinical effects of combination therapy
(Table 3) [37-39]. Two were retrospective comparative studies, comparing carbapenem-colistin
combination therapy to colistin monotherapy. One showed worst survival with combination
therapy [37], but there was an inherent difference between patient groups in that patients with P.
aeruginosa were treated with colistin monotherapy while combination therapy was given mostly
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to patients infected by A. baumannii. The second very small study showed improved survival in
five patients receiving combination therapy compared to seven patients treated with polymyxin
monotherapy among patients with K. pneumoniae bacteremia[38]. The last study compared any
combination therapy (the most common combination was tigecycline and colistin) to any
monotherapy (the most common was tygecycline) and found an overall advantage to
combination therapy[39]. Colistin monotherapy was given to 22 patients and colistin-meropenem
combination therapy to 6 patients in this study.
The objective of the current trial is to examine the clinical effects of colistin-carbapenem
combination therapy in the optimal trial design. Basing on the review of PK studies we will
select the currently optimal dosing regimen for colistin, including a loading dose. Given no
difference in the expected interactions, we will select meropenem as the carbapenem tested since
high doses can be given to critically-ill patients and is the carbapenem of choice in the trial
centers. To avoid bias we will conduct a randomized controlled trial, but given the expected
difficulties in obtaining informed consent we will prospectively collect data from all eligible
patients, documenting their treatment regimen if not recruited into the randomized controlled
trial.
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Table 1: Studies examining colistin-carbapenem combination therapy
First author
Year
published
Polymyxin
tested
Carbapenem
tested
Bacteria type
no. of Carbapene Polymyxin
Synergy
isolates
m
Resistance
methods
resistance
33
R,S
R
checkerboard,
time-kill
2
S
S
time-kill
Chan[34]
1987
colistin
imipenem
Rynn[40]
1999
colistin
meropenem
P. aeruginosa, S.
maltophilia
P. aeruginosa
Yoon[41]
2003
polymyxin B
imipenem
A.baumannii
8
R
R
Landman[42]
2005
polymyxin B
imipenem
P. aeruginosa
10
R
Bratu[43]
2005
polymyxin B
imipenem
K. pneumoniae
16
Timurkaynak[44]
2006
colistin
meropenem
A. baumannii, P.
aeruginosa
Wareham[45]
2006
polymyxin B
imipenem
Tateda[46]
2006
polymyxin B
Biancofiore[47]
2007
Cirioni[48]
Outcome reported
FICI
AUKBC
S
checkerboard,
time-kill
time-kill
FICI, time-kill synergy,
bactericidality
bactericidality
R
R,S
time-kill
bactericidality, time-kill synergy
10
R,S
S
checkerboard
FICI
A. baumannii
5
R
S
FICI
imipenem
P. aeruginosa
12
R
R
Etest
checkerboard
breakpoint
colistin
meropenem
A. baumannii
1
R
S
2007
colistin
imipenem
P. aeruginosa
2
R,S
R
Tripodi[49]
2007
colistin
imipenem
9
R
S
Pankuch[50]
2008
colistin
meropenem
A. baumannii
P. aeruginosa, A.
baumannii
102
R,S
R,S
time-kill
time-kill synergy
Tascini[51]
2008
colistin
imipenem
E. cloaca
1
S
S
checkerboard
FICI
Guzel[52]
2008
colistin
meropenem
50
S
S
checkerboard
FICI
Guelfi[53]
polymyxin B
meropenem
20
R,S
S
checkerboard
FICI
colistin
meropenem
A. baumannii
5
R
S
time-kill
bactericidality, time-kill synergy
Ullman[55]
2008
2008
ICAAC
2008
ICAAC
P. aeruginosa
P. aeruginosa, A.
baumannii
colistin
meropenem
A. baumannii
3
R,S
S
PK/PD time-kill
Pankey[56]
2009
polymyxin B
meropenem
A. baumannii
8
R
S
Etest, time-kill
bactericidality
FICI, bactericidality, time-kill
synergy
Souli[57]
2009
2009
ICAAC
2009
ICAAC
colistin
imipenem
K. pneumonia
42
R,S
R,S
time-kill
time-kill synergy
colistin
imipenem
A. baumannii
5
R
S
time-kill
bactericidality, time-kill synergy
colistin
doripenem
P. aeruginosa
2
S
S
checkerboard
FICI
Burgess[54]
Burgess[58]
Hilliard[59]
8
FICI
checkerboard
FICI
checkerboard,
time-kill
FICI, time-kill synergy
time-kill
bactericidality, time-kill synergy
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Milne[60]
2010
colistin
Pongpech[61]
2010
colistin
meropenem,
imipenem
meropenem,
imipenem
Rodriguez[62]
2010
colistin
Elemam[63]
2010
Lin [64]
P. aeruginosa
144
R,S
R,S
A. baumannii
30
R
imipenem
A. baumannii
14
polymyxin B
imipenem
K. pneumoniae
2010
colistin
Shields[65]
2010
colistin
imipenem
imipenem,
doripenem
Sopirala[66]
2010
colistin
imipenem
Urban[67]
2010
polymyxin B
doripenem
Pankuch[68]
2010
2010
ECCMID
2010
ECCMID
colistin
doripenem
colistin
colistin
imipenem
meropenem,
ertapenem
imipenem,
meropenem,
doripenem
colistin
Steed[69]
Souli[70]
FICI, SBPI
S
Etest, SBPI
checkerboard,
time-kill
R,S
R,S
time-kill
bactericidality, synergy
12
R
R
checkerboard
FICI
E. cloaca
1
S
S
time-kill
bactericidality, synergy
A. baumannii
17
R
S
FICI, bactericidality, synergy
A. baumannii
K. pneumoniae, A.
baumannii, P.
aeruginosa, E. coli
A. baumannii, P.
aeruginosa
8
R
S
Etest, time-kill
checkerboard,
Etest, time-kill
20
R,S
R,S
time-kill
bactericidality
50
R,S
R,S
time-kill
time-kill synergy
A. baumannii
8
R
S
time-kill
bactericidality, time-kill synergy
K. pneumoniae
55
R,S
R,S
time-kill
time-kill synergy
P. aeruginosa
57
R
-
FICI
doripenem
Acinetobacter
6
R
R
checkerboard
checkerboard,
time-kill
FICI, bactericidality
colistin
meropenem
A. baumannii
3
R
S
PK/PD time-kill
bactericidality, time-kill synergy
colistin
doripenem
P. aeruginosa
3
S
R,S
PK/PD time-kill
bactericidality
colistin
FICI
FICI, time-kill synergy
Ly[74]
2010
ICAAC
2010
ICAAC
2010
ICAAC
2011
ICAAC
Liang[75]
2011
colistin
meropenem
A. baumannii
4
R
S
time-kill
Pankey[76]
2011
polymyxin B
meropenem
K. pneumoniae
14
R,S
R,S
bactericidality, synergy
FICI, bactericidality, time-kill
synergy
FICI, bactericidality, time-kill
synergy
Khuntayaporn[71]
Dorobisz[72]
Srispha-Olarn[73]
Sheng[77]
2011
colistin
imipenem
A. baumannii
18
R
S
Etest, time-kill
checkerboard,
time-kill
Bergen[78]
2011
colistin
imipenem
P. aeruginosa
6
R,S
R,S
time-kill
bactericidality, time-kill synergy
colistin
doripenem
P. aeruginosa
2
R,S
R,S
PK/PD time-kill
bactericidality, time-kill synergy
Bergen[79]
2011
Santimaleeworagun[8
0]
2011
colistin
imipenem
A. baumannii
8
R
S
checkerboard
FICI
Lim[81]
2011
polymyxin B
meropenem
P. aeruginosa
22
R
S,R
time-kill
bactericidality
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Mohamed[86]
2011
ECCMID
2011
ECCMID
2011
ICAAC
2011
ICAAC
2011
ICAAC
Peck[87]
2012
colistin
imipenem
A. baumannii
6
Jernigan[88]
2012
colistin
doripenem
K. pneumoniae
12
R
S,R
time-kill
bactericidality, synergy
bactericidality, time-kill synergy,
AUBKC
Deris[89]
2012
colistin
K. pneumoniae
4
R,S
R,S
PK/PD time-kill
bactericidality, time-kill synergy
Ozseven[90]
2012
polymyxin B
doripenem
imipenem,
meropenem
A. baumannii
34
R
S
checkerboard
FICI
He[91]
2012
colistin
doripenem
P. aeruginosa
100
R
S
Etest, time-kill
FICI
Morosini[82]
Poudyal[83]
Teo[84]
Principe[85]
colistin
meropenem
K. pneumoniae
1
S
S
time-kill
bactericidality, FICI
colistin
doripenem
A. baumannii
3
R,S
S
PK/PD time-kill
bactericidality, time-kill synergy
polymyxin B
doripenem
P. aeruginosa
16
R
-
time-kill
bactericidality, time-kill synergy
colistin
doripenem
A. baumannii
24
R,S
-
checkerboard
synergy
colistin
meropenem
P. aeruginosa
2
R,S
S
PK/PD time-kill
bactericidality, time-kill synergy
R
R,S
time-kill
R - resistant, S - sensitive, MDR – multidrug resistant, XDR – extremely drug resistant, AUBKC – area under the bacterial killing
curve
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Table 2 – in-vivo studies
Study
Methods
Type of
Bacteria (MIC
Outcome
carbapenem 1 in mg/L)
Cirioni
In-vivo randomized
2007 [31]
Imipenem
Effect on defined
Effect summary
outcome
P. aeruginosa, 1
Deaths
Control strain: 8/20
significant
(BALB/c male mice
quality control
(colistin vs.
vs. 2/20
effects in-vivo on
with bacteremia
strain: imipenem combi)
Clinical strain: 6/20
survival and
following IV injection of
MIC 0.5, colistin
vs. 3/20
bacteremia
P. aeruginosa)
MIC 4)
Control strain: 8/20
clearance
One CR MDR
Positive
vs. 2/20
clinical isolate:
blood culture
Clinical strain: 13/20
imipenem MIC
at 24h
vs. 3/20 (p<0.05 for
32 colistin MIC
all)
8
Aoki
In-vivo - BALB/c
2008[92]
imipenem
P. aeruginosa, 1
Survival,
female mice pneumonia
PAO1 strain and
lung bacterial combination vs 10%
effects on
model (intranasal and
6 clinical strains
burden
in monotherapy,
survival and
reduced bacterial
bacterial burden
burden
with colistin
subcutaneous)
Song
In-vivo randomized
2009 [93]
(neutropenic mice with
Imipenem
90% survival in
Significant
A. baumannii, 1
Lung
Combi 7.15 ± 3.56
In-vivo bacterial
clinical CR
bacterial
vs.
load, bacteremia
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pneumonia following
isolate OXA-51
tracheal A. baumannii
positive.
0.98
inoculation)
Imipenem MIC
Combi 0/3 vs.
64, colistin ≤0.5
loads at 48h
Bacteremia
Colistin alone 0/3
eradication at
Combi 1/3 vs.
48h
Colistin alone 1/3
Mortality at
48h
12
Colistin alone 6.35 ±
and mortality
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Table 3- observational studies
Study
Methods
Type of
Bacteria (MIC
Outcome
carbapenem 1 in mg/L)
Effect on
Effect summary
defined
outcome
Falagas
Clinical,
2006 [37]
Meropenem
Colistin alone
In-hospital death
0/14 vs.
_
retrospective
(14 patients)
(colistin vs. combi)
21⁄57
More deaths with
(most pneumonia,
mostly P.
Response
(36.8%),
combi
24% bacteremia)
aeruginosa
p=0.007
Combi (57
12/14
patients (mostly
Nephrotoxicity
(85.7%) vs.
39⁄57
A. baumannii)
(68.4%),
NS
0/14 vs.
4⁄57 (7%),
NS
Qureshi
Clinical,
NS (probably
CR K.
Death (colistin vs.
4/7 (57.1%)
+
2012 [38]
retrospective (all
mostly
Pneumoniae
combi)
vs. 1/5
Less deaths with
bacteremia)
imipenem)
MIC50 colistin
(20%)
combi
MIC ≤0.25,
imipenem MIC 4
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Tumbarello
Clinical,
Meropenem
CR K.
Death (colistin alone vs. 11/22 vs.
Overall advantage to
2012 [39]
retrospective (all
(definitive
Pneumoniae
2 or 3-drug
12/37
combination.
bacteremia)
therapy)
combinations including
(32%)
Colistin-carbapenem
colistin-meropenem
not specifically
combination)
examined.
1
Unless otherwise stated, data the carbapenem was combined with colistin
2
Data obtained from Landman 2008 [94]
CI – continuous infusion
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Figure 1: Synergy rates for polymyxin and carbapenem combination by type of bacteria
Study names are comprised of first author and either publication year or convention name and
year accordingly. Subgroups within studies (according to resistance profile, antibiotic used, etc –
see Methods section) were listed separately and denoted by continuous numbering in parenthesis
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Figure 2: All-cause mortality in observational studies comparing colistin or polymyxin B vs.
comparator antibiotics for sepsis 1
Colistin
Study or Subgroup
Comparator
Events Total Events
Odds Ratio
Total Weight
M-H, Fixed, 95% CI
Qureshi 2012 AAC
5
14
4
14
2.2%
1.39 [0.28, 6.84]
Durakovic 2011 Intern Med
3
26
3
26
2.3%
1.00 [0.18, 5.48]
Betrosian 2008 J Infect
5
15
4
13
2.4%
1.13 [0.23, 5.54]
Garnacho-Montero 2003 CID
13
21
9
14
3.5%
0.90 [0.22, 3.68]
Gounden 2009 BMC Infect
16
32
9
32
3.8%
2.56 [0.91, 7.20]
Kvirko 2011 (polyB) JAC
30
45
25
88
4.8%
5.04 [2.32, 10.93]
Rios 2007 Eur Resp
16
31
14
40
5.1%
1.98 [0.76, 5.16]
Hachem 2007 AAC
19
31
30
64
6.5%
1.79 [0.75, 4.30]
Kallel 2007 Int CM
21
60
15
60
8.3%
1.62 [0.73, 3.56]
Oliveira 2008 (polyB) JAC
63
82
54
85
10.5%
1.90 [0.97, 3.75]
Reina 2005 Int CM
16
66
34
130
14.8%
0.90 [0.46, 1.79]
Paul 2011 JAC
78
200
85
295
35.8%
1.58 [1.08, 2.31]
861
100.0%
1.70 [1.36, 2.13]
Total (95% CI)
Total events
623
285
286
Heterogeneity: Chi² = 13.29, df = 11 (P = 0.27); I² = 17%
0.01
0.1
1
10
100
Favours colistin Favours comparator
Test for overall effect: Z = 4.67 (P < 0.00001)
1
Odds Ratio
M-H, Fixed, 95% CI
Betrosian 2008 was a quasi-randomized study, using alternation for patient allocation
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Figure 3: Adjusted all-cause mortality in observational studies comparing colistin or polymyxin
B vs. comparator antibiotics for sepsis
Odds Ratio
Study or Subgroup
Betrosian 2008 J Infect
log[Odds Ratio]
SE Weight
Odds Ratio
IV, Fixed, 95% CI
0.1178 0.8131
3.2%
1.13 [0.23, 5.54]
0 0.8681
2.8%
1.00 [0.18, 5.48]
Kallel 2007 Int CM
0.4796 0.4027
13.2%
1.62 [0.73, 3.56]
Kvirko 2011 (polyB) JAC
0.6471 0.3017
23.5%
1.91 [1.06, 3.45]
Oliveira 2008 (polyB) JAC
0.7275 0.3561
16.9%
2.07 [1.03, 4.16]
Paul 2011 JAC
0.3646
40.4%
1.44 [0.92, 2.26]
100.0%
1.63 [1.22, 2.17]
Durakovic 2011 Intern Med
0.23
Total (95% CI)
Heterogeneity: Chi² = 1.54, df = 5 (P = 0.91); I² = 0%
IV, Fixed, 95% CI
0.05
0.2
1
5
20
Favours experimental Favours control
Test for overall effect: Z = 3.34 (P = 0.0008)
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Figure 4: Nephrotoxicity for patients treated with colistin vs. comparator antibiotics in
observational studies
Colistin
Study or Subgroup
Comparator
Events Total Events
Odds Ratio
Total Weight
Betrosian 2008 J Infect
5
15
2
13
Durakovic 2011 Intern Med
3
26
0
Garnacho-Montero 2003 CID
5
21
6
Hachem 2007 AAC
7
31
Kallel 2007 Int CM
0
60
Kvirko 2011 (polyB) JAC
5
Lim 2011 J Korean med
10
Oliveira 2008 (polyB) JAC
Paul 2011 JAC
Odds Ratio
M-H, Fixed, 95% CI
M-H, Fixed, 95% CI
3.0%
2.75 [0.43, 17.49]
26
0.9%
7.89 [0.39, 160.91]
14
11.5%
0.42 [0.10, 1.79]
14
64
14.8%
0
60
45
6
88
7.6%
1.71 [0.49, 5.94]
20
10
35
7.6%
2.50 [0.80, 7.84]
18
69
21
81
30.0%
1.01 [0.49, 2.10]
26
168
17
244
24.6%
2.44 [1.28, 4.67]
Reina 2005 Int CM
0
55
0
130
Not estimable
Rios 2007 Eur Resp
0
31
0
20
Not estimable
Total (95% CI)
Total events
541
79
775
1.04 [0.37, 2.92]
Not estimable
100.0%
1.58 [1.11, 2.26]
76
Heterogeneity: Chi² = 9.11, df = 7 (P = 0.25); I² = 23%
0.002
0.1
1
10
500
Favours colistin Favours comparator
Test for overall effect: Z = 2.51 (P = 0.01)
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METHODS
Trial design: Randomized, open-label, controlled clinical trial (RCT).
Setting: Multicenter study including the following sites/ departments:
1. Italy, Rome: Universita Cattolica del Sacro Cuore, Agostino Gemelli Hospital. Four
departments: ICU, Infectious Diseases, Internal Medicine and Pneumology
2. Greece, Athens: Laikon Hospital, all departments
3. Greece, Athens: Attikon Hospital, all departments
4. Israel, Tel-Aviv: Tel Aviv Sourasky Medical Center, all departments
5. Israel, Petah-Tikva: Rabin Medical Center, Beilinson hospital and Hasharon Hospital. All
departments.
6. Israel, Haifa: Rambam Medical Center, all departments
Inclusion/ exclusion criteria
We will include adult inpatients ≥18 years with clinically-significant infections as defined below
caused by carbapenem- non-susceptible and colistin-susceptible Gram-negative bacteria:
Acinetobacter sp., P. aeruginosa or any Enterobacteriaceae (including but not limited to K.
pneumoniae, E. coli and Enterobacter sp.). Patient recruitment will occur only after
microbiological documentation and susceptibility testing.
Types of infections and definitions:

Bloodstream infection (BSI): growth of the relevant bacteria in one or more blood culture
bottles accompanied by the systemic inflammatory response syndrome (SIRS) within 48h of
blood culture taken time. BSIs can be either primary or secondary to any other source of
infection.

Ventilator-associated pneumonia (VAP) or healthcare-associated pneumonia (HAP):
pneumonia fulfilling CDC/NHSN surveillance definition of health care-associated infection
for pneumonia with specific laboratory findings (PNU2) with modifications to the laboratory
criteria. [95] Ventilator-associated pneumonia will be defined in persons who had a device to
assist or control respiration continuously through a tracheostomy or by endotracheal
intubation within the 48-hour period before the onset of infection. BAL will not be
performed routinely for the purposes of the trial. The specific criteria required for diagnosis
of pneumonia will be all of the following:
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1. Chest radiograph with new or progressive and persistent infiltrate, consolidation or
cavitation.
2. At least 1 of the following signs of sepsis: Fever >38ºC with no other recognized cause;
Leukopenia <4000 WBC/mm3 or leukocytosis >12,000 WBC/mm3; For adults >70 years
old, altered mental status with no other recognized cause
3. At least 1 of the following respiratory signs/symptoms: New onset of purulent sputum or
change in character of sputum or increased respiratory secretions or increased suctioning
requirements; New onset or worsening cough or dyspnea or tachypnea >25 breaths per
minute; Rales or bronchial breath sounds; Worsening gas exchange, including O2
desaturations, PaO2/FiO2 <240, or increased oxygen requirements
4. Laboratory criterion: Growth of the relevant bacteria in culture of sputum, tracheal
aspirate, bronchoalveolar lavage or protected specimen brushing. For any lower
respiratory secretion other than bronchoalveolar lavage (BAL) or protected specimen
brush (PSB), the respiratory sample has to contain >25 neutrophils and <10 squamous
epithelial cells per low power field, identified by Gram stain

Probable ventilator-associated pneumonia (VAP): pneumonia fulfilling CDC/NHSN 2013
revised surveillance definition, omitting the criterion of antimicrobial treatment before
randomization and modifying the microbiological criteria: [96]
1. Mechanical ventilation for ≥3 calendar days
2. Worsening oxygenation, following ≥ 2 calendar days of stable or decreasing FiO2 or
PEEP, presenting as:
o Minimum daily FiO2 values increase ≥ 0.20 (20 points) over baseline and remain
at or above that increased level for ≥ 2 calendar days OR
o Minimum daily PEEP values increase ≥ 3 cmH2O over baseline and remain at or
above that increased level for ≥ 2 calendar days.
3. Temperature > 38 °C or < 36°C, OR white blood cell count ≥ 12,000 cells/mm3 or ≤
4,000 cells/mm3
4. Purulent respiratory secretions AND positive respiratory culture; OR positive culture of
pleural fluid. For any lower respiratory secretion other than bronchoalveolar lavage (BAL)
or protected specimen brush (PSB), the respiratory sample has to contain >25 neutrophils
and <10 squamous epithelial cells per low power field, identified by Gram stain.
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
Urinary tract infection: positive urine culture with relevant bactera ≥105 CFU/ml with pyuria,
accompanied by the systemic inflammatory response syndrome (SIRS) with 48h of taken
time and no other explanation for SIRS
Microbiological criteria:
We will include patients with infections caused by carbapenem non-susceptible bacteria (using
EUCAST breakpoints for disc testing or MIC >2) that are sensitive to colistin (by disc testing or
MIC≤ 2 mg/L for Acinetobacter sp. and enterobacteriaceae and ≤4 mg/L for Pseudomonas sp.)
We will exclude infections when the carbapenem-resistant isolate is sensitive to quinolones or
any beta-lactam, but include those sensitive to tetracyclines, tigecycline cotrimoxazole or
aminoglycosides since these are not established treatments for such infections. We will exclude
patients with polymicrobial infections where one or more of the clinically-significant gramnegative bacteria are susceptible to any beta-lactam. We will permit the inclusion of patients
with polymicrobial infections where the non-trial isolate/s are carbepenem-resistant Gramnegative bacteria, Gram-positive bacteria or anaerobes (see permitted additional antibiotics
below).
Inclusion will be based on the testing performed in individual study hospitals (disc diffusion
essays, E-test, Vitek or other automated systems) with the breakpoints defined above. Isolate
identification and carbapenem MICs will be confirmed in a central laboratory.
Exclusion criteria

Previous inclusion in the trial. Patients will be included in the RCT only once for the first
identified episode of infection

Colistin administered >96 hours prior to randomization. Although prior treatment is allowed
by protocol for 4 days, all efforts should be made to recruit patients as soon as possible after
isolate identification.

Pregnant women

Epilepsy or prior seizures

Known allergy to colistin or a carbapenem
Interventions
Colistin arm: Patients will be given a loading dose of 9 mill IU, regardless of renal function. For
patients with normal renal function (CrCl ≥50 ml/min), the loading dose will be followed by 4.5
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mill IU q12hr. [24, 97] Colistin will be administered as a 30 min intravenous infusion. Patients
treated with colistin before randomization will be given a loading dose if treated for <48 hours
and not given a loading dose at start of treatment. Patients already given a loading dose or who
have been treated for 48 hour or more will continue colistin without a loading dose, using the
trial schedule.
Dose adjustment for patients with renal failure will be based on the study by Garoznik et al.
aiming to achieve a colistin steady state average level of 2-2.5 mg/L [23]

Patients with CrCl <50 ml/min, without renal replacement therapy: Total daily dose in mill
IU = [2*(1.5*CrCl + 30)]/30. CrCl should be expressed in ml/min/1.73 m2, using the MDRD
formula, Cockcroft and Gault equation or other means.

Continuous renal replacement therapy: fixed dose of 6 mill IU q12h

Intermittent hemodialysis: 1 mill IU q12h, with a 1 mill IU supplement dose after dialysis.
Combination arm: Colistin will be administered as above and combined with IV meropenem 2gr
q8hr for patients with normal renal function (CrCl>50 ml/min). Meropenem will be administered
as prolonged infusion over 3 hr. For patients with renal function the following algorithm will be
used: [98]
CrCl 26-50 ml/min
2gr q12hr
CrCl 10-25 ml/min and continuous renal replacement therapy
1gr q12hr
CrCl <10 ml/min. Supplemental dose given after intermittent
1gr q24hr
hemodialysis
No dosage adjustments will be performed for hepatic insufficiency for both antibiotics. Duration
of antibiotic treatment will be 10 days for all listed indications. If infectious complications
mandate longer treatment, duration will be prolonged as appropriate. The day of randomization
will be defined as day 1. Modifications of antibiotic treatment will be determined by the patients’
physicians, although we will request physicians to refrain from antibiotic changes in the first 72
hrs. unless a severe adverse event is observed. We will permit the concomitant administration of
the following antibiotics for polymicrobial infections in both study arms: vancomycin, oxacillin
derivatives, cefazolin, ampicillin, penicillin or metronidazole. We will not permit the routine
addition of: rifampin, tigecycline, minocycline, aminoglycosides or colistin inhalations. In case
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of clinical deterioration, any treatment modification will be permitted and this will be counted as
treatment failure (see secondary outcomes).
Outcomes
Primary outcome: Clinical success, defined as a composite of all of the following, all measured
at 14 days:

Patient alive

Systolic blood pressure >90 mmHg without need for vasopressor support

Stable or improved SOFA score, define as:
o for baseline SOFA ≥ 3: a decrease of at least 30%;
o for baseline SOFA <3: stable or decreased SOFA score

For patients with HAP/ VAP, PaO2/FiO2 ratio stable or improved

For patients with bacteremia, no growth of the initial isolate in blood cultures taken on day
14 if patient still febrile
Secondary outcomes:

14 and 28-day all-cause mortality

Clinical success, as defined above, but any modification to the antibiotic treatment not
permitted by protocol will also be considered as failure. This will include any change or
addition of antibiotics not permitted by study protocol during the first 10 days after
randomization. Early discontinuation of antibiotic treatment will not be considered as failure.

Time to defervescence, defined as time to reach a temperature of <38°C with no recurrence
for 3 days

Time to weaning from mechanical ventilation in VAP for patients weaned alive

Time to hospital discharge for patient discharged alive

Change in functional capacity from baseline before infection onset to discharge from hospital.
Function capacity will be classified into 3 grades: I. Independent II. Need for assistance for
activities of daily living III. Bedridden

Microbiological failure, defined as isolation of the initial isolate (phenotypically identical) in
a clinical sample (blood or other) 7 days or more after start of treatment or its identification
in respiratory samples.
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o For all patients with VAP/ HAP sputum or tracheal aspirates will be obtained on day
7, regardless of clinical response
o For all patients with UTI, a repeat urine culture will be obtained on day 7, regardless
of clinical response
o For patients with bacteremia, blood cultures will be repeated on day 7 and 14, only if
the patient is febrile at that time

Superinfections, defined as a new clinically or microbiologically-documented infections by
CDC criteria within 28 days

Colonization or infection by newly-acquired (other species than the initial infection)
carbapenem-resistant or colistin-resistant Gram-negative bacteria. Colonization will be
assessed by rectal surveillance (see surveillance protocol below)

Clostridium-difficile-associated diarrhea, defined by diarrhea with a positive C. difficile toxin
test
Adverse events

Renal failure using the RIFLE GFR criteria [99] at day 14 and day 28
RIFLE category
GFR criteria
Risk
Serum creatinine increased 1.5 times
Injury
Serum creatinine increased 2.0 times
Failure
Serum creatinine increased 3.0 times or creatinine = 4 mg//dl (355
μmol/L) when there was an acute rise of >0.5 mg/dl (44 μmol/L)
Loss
Persistent acute renal failure; complete loss of kidney function
requiring renal replacement therapy for longer than 4 weeks
End-stage renal disease
End-stage renal disease requiring renal replacement therapy for
longer than 3 months

Seizures or other neurological adverse events including critical illness neuropathy

Other adverse event requiring treatment discontinuation
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If patients are discharged or death occurs before end of follow-up (day 28), we will end data
collection at that date. We will attempt to determine survival status at day 28 for all patients
(central registry in Israel; re-admissions, rehabilitation centers, hospital transfers in Greece and
Italy).
Randomization
Blocked randomization will be performed in each participating center with random block sizes.
[100]. A computer generated random code, stratified by site using blocked randomization with
random block size between 4-8 patients will be accessed through a central web-based
randomization page only after patients’ recruitment to ensure adequate allocation concealment.
No blinding will be used after randomization.
Sample size
To show an improvement in clinical success (primary outcome) from 55% with colistin alone to
70% with combination therapy with a 1:1 randomization ratio, a sample of 324 patients (162 per
group) is needed (uncorrected chi-squared test, alpha=0.05, power=0.8, PS Power and Sample
Size Calculations). Assuming a non-evaluability rate of about 10%, we plan to recruit 360
patients. The graph below plots the change in number of patients in one arm (y axis) if the failure
rate in the combination arm is lower that 30% (x axis).
200
160
120
80
40
0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Probability of the event in experimental group
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0.9
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For a 3-year trial duration we will have to recruit 120 patients/ year. Basing on the current
prevalence of carbapenem-resistant colistin-susceptible isolates in the trial sites, the plan should
be to recruit 60 patients in 3 Israeli hospitals, 50 in 2 Greek hospitals and 10-20 in Italy.
Colistin level monitoring
Blood samples for CMS and colistin concentration determination will be drawn from all patients
to evaluate PKPD-relationships and potential covariate relationships. A sparse sampling schedule
with two samples drawn at 45 minutes after start of the first infusion (i.e. 15 minutes after the
end of the loading dose infusion), and at 22 hours (i.e. 22 hours after the start of the first infusion
= 10 hours after the start of the second infusion = 2 hours before the start of the third infusion)
was determined to be suitable based on optimal design theory. Sampling at 23-24 h may risk that
CMS concentrations are not quantifiable in the assay; therefore it is important to take the sample
no later than 22-23 h after the start of the first infusion. For patients treated with colistin prior to
randomization, the first sample will be taken 15 minutes after the end of the first infusion postrandomization and the second 2 hours before the start of the third infusion. To enable
interpretation of the measured colistin levels, we will record colistin start and end administration,
the dose and timing of colistin administration prior to randomization and the exact time of blood
sampling. Both CMS and colistin will be measured in the two samples. 70 Developed population
PK models will be used to obtain each individual’s PK parameters in the NONMEM 7 software.
Handling of PK samples

Draw 5 ml venous blood in EDTA (or heparin) tubes. The sampling site has to be
different from the vein used for CMS infusion.

VERY IMPORTANT: Place on ice bath immediately after sampling.

Note the exact time of the sampling (clock time and time in relation to start and stop of
infusion(s).

Within 20 min after sampling: Centrifuge in chilled centrifuge for 10 minutes at 20002500 g.

Transfer the supernatant (1.5-2 ml plasma) to polypropylene Eppendorf tubes. The tube
should be marked. Please include the following information on the labels:
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AIDA STUDY
Study center
Patient randomization number:
Sample #: (1st or 2nd)
Date:
Time of sampling: HH:MM
Please also add Randomization number and sample number on the cap with a waterproof
marking pen.

Freeze at -70 degrees (or cooler). It is acceptable to store the sample at -20 degrees for a
few days.

Ship batch of samples to Uppsala University (approximately every 3-6 months) on dried
ice by USPS, DHL or similar service. Before shipping, it is important to confirm with
Uppsala that there will be personnel to take care of the samples upon arrival
([email protected]; [email protected])
Microbiological methods
Index culture
The index culture is defined as the pathogen/s causing the infection for which the patient was
included in the trial. The index culture will be documented in the electronic CRF and must be
kept and frozen at -70ºC.
Clinical samples
Blood cultures will be repeated every 48 hours as long as fever >38 or signs of SIRS are present.
Other cultures will be taken as deemed appropriate by attending physicians. Document in
electronic CRF all positive samples associated with clinically-significant new infections ("New
infection" day 28, only pathogen name). Enter detailed pathogen information into new culture
page for all repeat (index pathogen) or new carbapenem-resistant Gram-negative bacteria. Freeze
and store all repeat samples of index pathogen (same bacterium, carbapenem-resistant, similar
susceptibility pattern).
Surveillance samples
Sputum cultures for patients with HAP/ VAP and urine samples for patients with UTI will be
repeated at day 7 routinely, regardless of clinical signs/ symptoms. Document and store isolates
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according to the criteira mentioned under clinical samples. Growth of bacteria different from the
index culture will be disregarded unless associated with a clinically significant infection.
Rectal surveillance cultures will be obtained using rectal swabbing at randomization and once
weekly thereafter until day 28 (or death/ discharge from hospital). Rectal swabs content will be
frozen (after extraction into glycerol containing media) using a validated protocol and shipped to
the study central laboratory (TASMC) every 4-6 months. Exact MIC of the clinical isolates to the
study drug will be determined using agar based methods. All rectal swabs will be evaluated to
determine carriage of carbapenem and/or colistin resistant organisms. Using methodology
developed in FP7 SATURN project, quantitative analysis will be performed to examine the
effect of treatment regimen on density of resistant strains, and the co-carriage of various
carbapenem-resistant strains. Co-carried resistant strains belonging to different species and
newly acquired-resistant strains will be studied for the mechanisms of resistance. When cocarriage or a new acquisition event of a carbapenem resistant strain will be detected, strains will
be analyzed to examine between-species transfer of genetic elements encoding for resistance
(plasmids, transposomes and genes) and to determine the relationship of transfer events to the
treatment protocol. Clonality of clinical and surveillance resistant isolates will be determined
using a combination of PFGE, PCR and sequencing based (MLST) genotyping methods, as
appropriate. Mechanisms of resistance in selected clinical isolates, and in surveillance isolates in
which resistance has emerged, will be determined. These will include carbapenemase activity
assays, beta-lactamase identification, porin loss determination, and efflux pump expression.
Population analysis and modified population analysis profile (PAP) to detect colistin
heteroresistant subpopulations, analysis for stable vs. unstable colistin resistance among resistant
subpopulations and molecular analysis of the mechanism of resistance will be performed to
detect and quantify carbapenamases and OMP changes.
Synergy tests for the combination of meropenem and colistin will be conducted using time-kill
studies. For synergy testing, we will select 10 isolates each of A. baumannii, K. pneumoniae, P.
aeruginosa and E. coli. All will be carbapenem-resistant and we will try to select from each
species 5 isolates with MIC<32 to meropenem and 5 isolates with higher MICs. Correlation
between carbapenem MICs, colistin MICs, molecular typing, PAP, mechanisms of resistance and
synergy studies will be determined and correlated with the following treatment outcomes: a.
clinical success b. microbiological failure, c. emergence of resistance.
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Data collection
We will collect data using the electronic interface/ database platform EPI-INFO
(http://wwwn.cdc.gov/epiinfo/). The data collected will include:

Patient demographics

Background conditions, including the revised Charlson comorbidity index [92] and McCabe
score

Source of infection and diagnostic criteria for VAP and HAP including type of respiratory
specimen used for patient classification

Devices present at infection onset and risk factors for MDR colonization and infection

Antibiotic treatment prior to onset of the infectious episode, empirical antibiotic treatment
and all antibiotics used from randomization until day 28. We will document colistin
administration times.

Concomitant nephrotoxic agents: aminoglycosides, IV contrast material, cyclosporine

Therapeutic procedures throughout the infectious episode (surgery, catheter extraction, etc.)

Use of colistin inhalation therapy

All outcomes as defined above
Study visits/ trial flow (grey actions not mandating a study visit):
 Notification from laboratory for isolation of CR-GNB.
 Application of inclusion criteria: relevant pathogen (as defined above) and relevant clinical
syndrome (BSI, VAP, HAP, probable VAP or UTI) define a potentially eligible patient
 For all potentially eligible patients, enter patient into Epi-Info (first and second page). Follow
trial flow according to inclusion/ exclusion criteria – patient not fit for study, observational
study or RCT. Complete EpiInfo for all patients classified for the observational study or RCT.
For RCT only:
 Document last 5 digits of Epi-info unique identifier above.
 Perform randomization at: http://ozeuss.pythonanywhere.com
(username:[email protected], password: projectaida)
 Document randomization number given above and enter to EpiInfo.
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 Take clinical cultures as appropriate + rectal surveillance sample. Ensure storing of index
culture in the lab.
 Time 0 – colistin loading dose 9MIU in 30min, followed by meropenem 2gr in 3hrs.
Document start/ stop timing
 45min – colistin level testing, take on ice to centrifugation with 15 min of sampling,
centrifuge using cold centrifuge and freeze (-70˚C). Document timing of sample and
centrifugation.
 Make sure that the 8, 12 and 16 hr. doses are documented and implemented
 8hr – IV meropenem 2gr in 3 hrs
 12hr – colistin 4.5 MIU in 30 min
 16hr – IV meropenem 2gr in 3 hrs
 22hr – document start/ stop timing of the 2nd dose of colistin and 2nd/ 3rd doses of meropenem.
Colistin level testing, carry on ice and centrifuge within 15 min and freeze immediately
(-70˚C). Instruct on continued treatment and repeat cultures as clinically appropriate.
 24hr - colistin 4.5 MIU (30min) followed by meropenem 2gr (3hr)
 48hr– clinical follow-up, adherence monitoring (avoid treatment modifications until 72 hrs),
blood cultures if febrile
 Day 5 - clinical follow-up, clinical cultures as appropriate, blood cultures if febrile
 Day 7 – rectal surveillance sample, sputum culture for HAP/VAP, urine culture for UTI,
blood cultures if febrile, outcome data collection
 Day 9 - clinical follow-up, clinical cultures as appropriate, blood cultures if febrile
 Day 10 – clinical follow-up, clinical cultures as appropriate, blood cultures if febrile
 Day 14 –rectal surveillance sample, clinical cultures as appropriate, blood cultures if febrile,
outcome data collection
 21 days – rectal surveillance sample
 28 days –rectal surveillance sample, outcome data collection
If patient discharged at any time before day 28, complete case report form at the time of
discharge, except for death. If death before day 28, complete case report form at the time of
death. Complete as many fields as possible given known information.
Store/ freeze
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 Index culture
 All repeat isolates of index culture following randomization until day 28
 4 X rectal surveillance incubated in BHI
 2 X colistin/ meropenem level sampling
Ethical considerations
The study will be approved by the ethics committees at each participating center. We will request
to defer informed consent among critically-ill patients. The study is planned to include
mechanically ventilated patients, other severely ill patients in ICU and patients during the acute
stage of sepsis who will not be able to provide informed consent at the time of randomization.
The interventions examined are both accepted and used in clinical practice; and there is no better
treatment known for the targeted infections. Patients who are able to provide informed consent at
the time of randomization, will be included only if providing informed consent. A data and
safety monitoring committee will be appointed and will review the study data quarterly.
Statistical analysis
An intention to treat analysis will be performed. Baseline characteristics and outcome of study
groups will be compared. Significance will be set at p=0.05 and all tests will be 2-sided. Time to
event outcomes will be assessed using survival analysis.
Predefined subgroup analyses

Patients who did not receiving covering antibiotic treatment for more than 48 hours prior to
randomization

Study patients, excluding those recruited for the indication of UTI or probable VAP

Patients in whom the infecting bacteria has an MIC to meropenem <32 mg/dl
We will conduct a multivariable analysis of the randomized cohort and the randomized +
observational cohorts (see below), to examine the independent effect of the study regimen on
28-day mortality.
Concomitant observational study
We will collect all clinical data and treatment regimens from patients not included in the RCT for
the reason detailed below, but otherwise fulfilling clinical and microbiological inclusion criteria.
31
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
Unable to provide informed consent or otherwise no informed consent

Identified later than 96h after start of treatment

Second and subsequent episodes of infection for patients included in the RCT. A separate
episode of infection will be defined as an infection occurring at least 28 days after the index
episode of infection and separated by at least 7 days off antibiotics.
Treatment in this arm will be based on attending physicians’ decisions. Clinical and
microbiological samples for these patients will be collected only for routine purposes and will
not be kept or analysed as for the main trial. Data will be kept anonymously. Informed consent
for data collection will not be required, as no intervention isplanned. This arm will serve for
comparison of randomized and non-randomized patients to examine the external validity of the
trial and for an observational comparison between the trial treatment regimens in the overall
cohort.
Pooled analysis with the NIH trial
A concurrent NIH-funded randomized controlled trial will be conducted in the US, assessing
similar interventions and using comparable microbiological methods. An agreement has been
reached between the current and the NIH trial PIs to examine possible collaboration. We will try
to ensure comparability between the current and the NIH trial, in particular with respect to the
outcomes assessed to allow for comparison and compilation of results after analysis of the
current trial. Heterogeneity, if existent, will be explained by differences in patient and infection
characteristics. If non-heterogeneous, we will pool results using methods of individual patient
level meta-analysis. The combined sample size will allow for subgroup analyses by types of
infections and microbiological characteristics (MICs and synergy).
The differences that currently exist between the trial protocols are the following:
Inclusion criteria, types of
NIH
AIDA
BSI and/or pneumonia (VAP)
BSI, VAP, HAP, UTI
Include preliminary result of
Only documented
infection
Inclusion criteria,
32
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microbiological
gram-negative non-lactose
carbapenem-resistant GNs
fermenter that is oxidase
included
negative
Include prior history (within
Only current isolate
last 6 months) of XDR-GNB
considered
and is susceptible to colistin
Permit polymicrobial
Exclude susceptible GNs
infections with susceptible
GNs, but do not allow
treatment with carabepenems
in the mono arm
Exclusion criteria, prior
Colistin for more than 96
Exclude patients if more than
treatment
hours and imipenem,
96 hours elapsed since culture
doripenem or meropenem in
with the study pathogen taken.
the 36 hours prior to
Patients may received up to 96
enrollment
hours of colistin or other
antibiotics before
randomization
Exclusion criteria, other
Neutropenics and those
Neutropenics included
recently treated with GCSF
excluded
Complicated infections
Complicated infections
excluded: endocarditis,
included (treatment
osteomyelitis, prosthetic joint
prolongation and intrathecal
infections, meningitis and/or
treatment allowed)
other central nervous system
infections.
Hemodialysis
Any type of renal replacement
therapy included
Interventions
Colistin vs. colistin +
33
Colistin vs. colistin +
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imipenem
meropenem
Colistin dose?
Colistin dose: 9 MIU loding
dose followed by 4.5 MIU
q12h
Primary outcome
Other
Duration: 14 days
Duration: 10 days
Mortality
Composite treatment success
Outcome time: 14 and 28 days
Outcome time: 14 and 28 days
None
Concomitant observational
study
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