BACTERICIDAL ACTIVITY OF DIFFERENT BRANDS OF CEFEPIME ... · Bano and Arsalan, 2018). The...
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Pakistan Journal of Pharmacology ORIGINAL ARTICLE
Vol.36, No.1 & 2, January-July,2019, pp.53-67
BACTERICIDAL ACTIVITY OF DIFFERENT BRANDS OF
CEFEPIME MARKETED IN PAKISTAN AGAINST GRAM-
POSITIVE AND GRAM-NEGATIVE CLINICAL ISOLATES
ADEEL ARSALAN1*, MIRZA TASAWER BAIG
1, SYED ABID ALI
2,
AND MUDASSAR HUSSAIN3
1Faculty of Pharmacy, Ziauddin University, 4/B, Shahrah-e-Ghalib, Clifton, Karachi, Pakistan 2HEJ Research Institute of Chemistry, ICCBS, University of Karachi, Karachi, Pakistan
3Faculty of Pharmacy, University of Karachi, Karachi, Pakistan
ABSTRACT
An important issue is the hesitation of patients or physicians receiving antibiotics about the
quality of a generic medicine is equal to, greater than or less than its innovator one. This
present study has been carried out to evaluate the bactericidal activity of parenteral cefepime
(500 mg) against clinical isolates. A number of 62 clinical isolates of Gram-positive
Staphylococcus aureus (n=33) and Gram-negative Escherichia coli (n=29) were collected
from different hospitals in Karachi, Pakistan. The minimum inhibitory concentration 90%
(MIC90) of the pathogens was determined by broth dilution method, according to clinical and
laboratory standards institute (CLSI) guidelines. Cefepime (standard) was tested against E.
coli standard (ATCC=25922) with (MIC90; 0.015-0.25µg/ml) and compared with different
seven different brand of cefepime injection 500mg with (MIC90; 0.015-64.0µg/ml). While in
the case with Gram-positive S. aureus standard (ATCC=25923) sensitivity against cefepime
(standard) was (MIC90; 1.0-4.0µg/ml) and similarly when it was compared with same
parentral cefepime (MIC90; 1.0-64.0µg/ml). It has been revealed that in vitro antimicrobial
analysis had shown that both the innovator ones and me-too (generics) products have not
any significant statistical variability. Now it is very important to conclude by evidence that
all commercialized drugs were appropriate for therapeutic use.
Keywords: Antimicobial resistance, cephalosporin, in-vitro, bactericidal activity, clinical
isolates.
INTRODUCTION
Microorganisms are found everywhere in
nature (Arsalan et al., 2010, 2013a, 2013b;
2014a). It has been found there is increase in
resistance of microorganisms with the span of
time (Ventola, 2015; Banerjee et al., 2018;
Bano and Arsalan, 2018). The resistance of
antibioticis one of the most critical problems
encountered by health-associated professionals
(Arsalan et al., 2014b; Sommer et al., 2017). It
has been observed a number of root cause for
the resistance among them lack of information
about antibiotics, inadequate diagnosis of
infections among prescribing doctors
(Ayukekbong et al., 2017). Some of the
prescribed medicines by practitioners are of
incorrect antibiotics or dose, route and/or
duration of treatment (Rezal et al., 2015;
Davidoff et al., 2015). This may often occur in
response to pressure from companies or
patients and the desire for profit (WHO, 1999).
It has been known that medical representatives
or commercially oriented publications were
the main sources of information about
medicines (Silva et al., 2010; Arsalan et al.,
2015).
Moreover, the quality of raw material has
been also play an significant role in resistance
that‟s why some physicians consider that
generic drugs are not as valuable as innovator
*Corresponding author: e-mail: [email protected]
Bactericidal activity of different brands of cefepime 54
ones, so it is very important to show that all
commercialized drugs are suitable for
therapeutic use (Goff et al., 2017). Antibiotics
are measured by their potency or biological
activity compared against an international
standard. Therefore, the commercial products
should meet the biological assays and other
analytical procedures according to
international reference standard (Russel, 1996;
Diaz et al., 2011).
It has been the prime objective of
pharmaceutical industry to comply with
standards of quality, efficacy, and consistency,
aspects as predetermined by various regulatory
authorities (ICH, 2005; Diaz et al., 2011;
Pezzola and Sweet, 2016; Lamanna et al.,
2018). In recent years, discussions about the
quality and efficacy of generic antibiotics has
taken place in conferences and workshops and
a number of workers have been concluded that
some generics do not meet regulatory
standards and even no similar effect of drugs
has been observed in animal model (Jones et
al., 2008; Zuluaga et al., 2009; Giovagnoli et
al., 2017). It has been mystified by some
workers that some innovator ones are “gold
standard” which are recognized for purposes
of bioequivalence and bioavailability studies
while in the case of intravenous antibiotics, the
bioavailability is 100%, and therefore,
pharmacodynamic studies must be supported
with validated analytical results (Diaz et al.,
2011).
Globally infectious diseases are the
second foremost reason of death after
cardiovascular disease and one of the top
causes of death by the World Health
Organization in 2008. Under developed
countries are severely affected by infectious
diseases because of low-income, high cost of
antibiotics, irrational use of antibiotics, lacking
of follow-up, self-medication (Shi et al., 2017;
Ayukekbong et al., 2017; Bano and Arsalan,
2018). It has been found low level of antibiotic
concentration in tissue and plasma the chances
of resistance is increased many folds (Tangden
et al., 2017). Due to more bioavailability and
rapid onset of action, the parenteral antibiotics
are currently commonly used in severe
infections (Fauci, 2001; Bryant and Katz,
2018). Since last two decades, the use of broad
spectrum antibiotic has been doubled because
of wide-spectra and less chances of
antimicrobial resistance (Craig, 1998; Martens
and Demain, 2017) adverse reactions, drug-
drug interactions and cost (Stanley et al.,
1996; Martens and Demain, 2017). Thus,
broad spectrum has permitted the option of
monotherapy to over-come the hazards to
decrease the risk for toxicity and drug
interactions, reduced drug expenditures, and
other pharmacoeconomic benefits (Badaro et
al., 2002; Singh and Yeh, 2017).
The most favorable efficacy of beta-
lactams against microbes and adequate plasma
levels for as long as possible through the
dosing interval for prolong span (Davies et al.,
2000; Grupper et al., 2016). It has not being
surprising that dosing regimens for beta-
lactam antibiotics has been reevaluated to
maintain plasma levels above MICs for longer
periods of the dosing period and even in
continuous infusions (Turnidge, 1998;
Crokaert, 2001; Veiga and Paiva, 2018).
Cefepime is a zwitterionic, anti-
pseudomonal fourth generation cephalosporin,
having broad-spectrum activity even against
resistant strains (Marwa et al., 2015; Lee et al.,
2017). Cefepime have a low propensity toward
the development of resistance and low affinity
for most beta-lactamases. Due to good safety
profile and wide spectrum of activity, it is
commonly used as empirical therapy of
critically-ill patients (Lipman et al., 2003; Lee
et al., 2017). Since late 1990s, cefepime has
been introduced into clinical practice and
approved for the treatment of moderate-to-
severe infections, such as pneumonia,
uncomplicated and complicated urinary tract
infections (UTIs), skin and soft tissue
infections, intra-abdominal infections and
febrile neutropenia (Endimiani, 2008; Kalil,
2011; Nakane et al., 2015). Konstantinou with
his colleagues (2004) have been observed
cefepime has lower resistance as compared to
other extended spectrum cephalosporins.
Adeel Arsalan et al 55
Infectious Disease Society of America (IDSA)
has been included cefepime as one of the anti-
pseudomonal antibiotics. It has also been used
against extended spectrum beta-lactamase
producing Escherichia coli (Nguyen et al.,
2009; Nakane et al., 2015; Wang et al., 2016).
Since the last two decades, resistant Gram-
positive and Gram-negative microbes are
mainly involved in the community, long-term
care, and hospital settings, posing serious
problems in the choice of an appropriate
antibiotic for treatment (Exner et al., 2017;
Cerceo et al., 2017). Gram-positive
Staphylococcus aureus, is resistant to many
antibiotics. While Gram-negative Escherichia
coli and Klebsiella species are resistance to
beta-lactam antibiotic by producing class A
extended-spectrum β-lactamases (ESBLs)
(Maglio et al., 2004; Diaz et al., 2011).
Globally it has been observed Gram-positive
Staphylococcus aureus and its resistant sera
has been one of the most commonly isolated
bacteria, Gram-negative bacilli especially E.
coli is major causes of infection (Isais-
Agdeppa and Bravo 2005; Diaz et al., 2011).
MATERIALS AND METHODS
Materials and reagents
Reference cefepime was a kind gift
sample from M/s Barrett Hodgson
Pakistan (Pvt) Ltd. Seven different brands of
cefepime were purchased from retail
pharmacies of Karachi (Pakistan) market.
Representative Gram positive (S. aureus,
ATCC=25923) and Gram-negative (E. coli,
ATCC=25922) standard organism has been
obtained. The clinical isolates were obtained
from July 2018 to March 2019 from different
sources and tertiary care hospitals at Karachi.
Mueller Hilton broth (Merck Germany),
distilled water were prepared freshly to
prepare different dilution (Wiegand et al.,
2008).
Instrumentation
For the determination of MICs of
cefepime content in raw material and their
dosage form a Shimadzu UV-1601
spectrophotometer was utilized for quantitative
calculation, where as for visual observation
laminar hood with light box was used.
Broth dilution method
A Complete protocol for performing these
tests is found in the Clinical and Laboratory
Standards Institute (CLSI) publication, for
broth dilution methods, decreasing
concentrations of the antimicrobial agents(s) to
be tested, usually prepared in serial two fold
dilution, are place and tubes of a broth
medium that has been supported the growth of
the test microorganism (Wiegand et al., 2008).
Preparation of macfarland standard
First we prepared 1% sulfuric acid in
1.175% aqueous solution of barium chloride.
Slowly, and with constant agitation, add the
designated amounts of the two solutions to the
tubes make a total of 10ml per tube. The
suspended barium sulfate precipitate
corresponds approximately to homogenous E.
coli cell densities per ml throughout the range
of standard (Wiegand et al., 2008).
Preparation of inoculums
These suspensions were prepared by using
the top of the colonies of the standard and
isolated microorganisms. Standard E. coli
(ATCC 25922) and S aureus (ATCC 25923)
and clinical isolates of E. coli, and S. aureus
were incubated in test tubes at 37oC for 2-8
hours until the turbidity exceeds that of 0.5
McFarland standards (Wiegand et al., 2008).
Preparation of antibiotic stock solution
Accurately weighed for a required amount
of standard antibiotic powder (standard
powder of cefepime and its different brands).
Prepare stock solution using the formula
(Wiegand et al., 2008).
P
1000CVW
Where P is potency of the antibiotic base,
V is volume in ml required,
C is final concentration of solution and
W is weight of the antimicrobial to be dissolved in V.
Bactericidal activity of different brands of cefepime 56
Fig. 1: Resistance pattern of gram-negative clinical isolates against different brands of cefepime
injection (500 mg)
Fig. 2: Resistance pattern of gram-positive clinical isolates against different brands of cefepime
injection (500 mg).
Adeel Arsalan et al 57
Preparation of antibiotic dilution range
Usually prepared a series of varying
concentrations two fold serial dilutions (0.015,
0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64
g/ml). Equal volume of inoculums has been
added in test tubes (Wiegand et al., 2008).
Procedure
Prepared Mueller Hilton broth, arranged
sufficient sterile test tubes for each antibiotic
to cover the range of antibiotic dilutions.
Transferred 9 ml of broth and 1 ml of each
antimicrobial agents dilution in separate broth
tubes. Now inoculums have been added to
each anti-microbial containing tube in the
dilution series. The tubes have been incubated
at 37oC for 12 to 18 hours. Examined the tubes
with visually and with spectrophotometer (546
nm) for the presence or absence of growth and
compared the result with the growth in the
control tube (Wiegand et al., 2008).
RESULTS AND DISCUSSION
During the present study, antibacterial
activity of seven different brands of cefepime
has been evaluated against Gram-positive and
Gram-negative pathogens. The study has
revealed that among Gram-positive and Gram-
negative, S. aureus and E. coli are the most
frequent clinical isolates in various infections.
A total 62 clinical isolates (S. aureus and E.
coli) have been collected from July 2017 till
March 2018 from different tertiary care
Fig. 3: Overall resistance pattern of clinical isolates against marketed cefepime
Table 1: Summary of clinical isolates
Clinical Isolates Source of isolates Number of
isolates
Gender
Male Female
Staphylococcus
aureus
Surgical, Burn and Accidental
wound pus (Skin and soft tissue
infections)
33 19 (57.57%) 14 (42.42%)
Escherichia coli Stool and Urine (Intra-abdominal
and urinary tract infections) 29 23 (79.31%) 06 (20.68%)
Bactericidal activity of different brands of cefepime 58
Table 2: Comparison of Standard Cefepime and Different Brands of Cefepime against Standard
Escherichia coli & Different Clinical isolates of Escherichia coli
STD CF CF 01 CF 02 CF 03 CF 04 CF 05 CF 06 CF 07
STD E. coli
ATCC 25922 0.015 0.12 0.015 0.03 0.03 0.015 0.12 0.03
Ec 01 1 1 1 2 2 1 2 4
Ec 02 4 16 32 4 2 64 16 32
Ec 03 2 2 8 16 16 16 64 16
Ec 04 1 1 2 2 8 1 4 2
Ec 05 2 4 2 64 2 2 64 1
Ec 06 4 16 64 16 64 1 64 64
Ec 07 2 16 2 4 64 4 2 32
Ec 08 1 1 64 2 1 2 64 2
Ec 09 1 4 8 8 2 16 2 1
Ec 10 4 16 64 32 16 64 32 64
Ec 11 1 1 16 2 2 2 8 4
Ec 12 1 2 16 64 4 4 32 16
Ec 13 4 32 32 64 16 64 8 16
Ec 14 2 4 32 16 2 8 32 1
Ec 15 1 1 64 4 64 4 4 4
Ec 16 1 4 4 2 1 64 8 16
Ec 17 1 16 2 16 1 2 4 8
Ec 18 2 2 8 2 2 4 4 4
Ec 19 4 64 16 64 32 16 32 16
Ec 20 2 16 4 4 2 2 4 2
Ec 21 4 64 64 16 32 16 64 16
Ec 21 2 4 64 2 16 8 2 64
Ec 23 1 4 2 2 64 2 2 2
Ec 24 8 64 64 32 64 64 32 64
Ec 25 2 2 32 16 2 1 64 2
Ec 26 1 4 16 2 64 2 1 2
Ec 27 2 2 2 16 1 16 2 8
Ec 28 4 16 64 16 64 1 64 64
Ec 29 1 1 4 1 4 4 4 1
Adeel Arsalan et al 59
Table 3: Comparison of standard cefepime and different brands of cefepime against standard
Staphylococcus aureus and different clinical isolates of Staphylococcus aureus
STD CF CF 01 CF 02 CF 03 CF 04 CF 05 CF 06 CF 07
STD. S. aureus
ATCC 25923 1 1 4 1 2 2 4 1
Sa 01 1 4 2 1 4 4 32 2
Sa 02 1 1 4 64 64 4 1 16
Sa 03 1 4 8 2 1 1 16 1
Sa 04 2 32 8 1 8 4 32 64
Sa 05 1 32 16 64 1 1 1 64
Sa 06 2 8 1 1 8 2 64 1
Sa 07 4 16 64 4 16 32 2 16
Sa 08 1 1 16 64 64 1 1 1
Sa 09 1 2 2 1 2 32 64 1
Sa 10 2 16 1 2 64 1 64 32
Sa 11 1 1 64 32 8 4 4 16
Sa 12 1 1 1 64 16 8 1 8
Sa 13 4 64 32 1 16 64 8 1
Sa 14 2 16 16 32 64 32 1 64
Sa 15 4 64 64 16 32 64 64 2
Sa 16 4 32 64 16 64 64 32 64
Sa 17 1 2 16 16 4 2 8 1
Sa 18 1 4 2 64 1 1 64 64
Sa 19 2 16 32 1 4 64 4 16
Sa 20 4 64 64 16 64 32 64 64
Sa 21 1 8 16 64 8 64 1 1
Sa 21 1 16 32 4 2 1 2 1
Sa 23 2 1 4 64 8 16 64 4
Sa 24 2 32 16 8 16 64 1 16
Sa 25 1 4 1 2 2 2 64 1
Sa 26 1 16 4 64 1 16 1 2
Sa 27 2 16 16 8 16 4 32 1
Sa 28 4 64 64 64 64 1 64 32
Sa 29 1 64 16 1 1 32 1 1
Sa 30 1 1 2 16 64 1 64 16
Sa 31 8 64 64 64 32 16 1 1
Sa 32 2 32 1 8 8 1 2 64
Sa 33 4 16 64 64 64 64 64 1
Bactericidal activity of different brands of cefepime 60
Table 4: Resistance pattern of clinical isolates of Escherichia coli against Different Brands of
Cefepime injection (500mg)
Marketed cefepime code Susceptible Intermediate Resistant
CF 01 18 (62.06%) 8 (27.58%) 3 (10.34%)
CF 02 13 (44.82%) 8 (27.58%) 8 (27.58%)
CF 03 15 (51.72%) 10 (34.48%) 4 (13.79%)
CF 04 16 (55.17%) 6 (20.69%) 7 (24.13%)
CF 05 19 (65.51%) 5 (17.24%) 5 (17.24%)
CF 06 16 (55.17%) 6 (20.69%) 7 (24.13%)
CF 07 16 (55.17%) 8 (27.58%) 5 (17.24%)
Table 5: Resistance pattern of clinical isolates of Staphylococcus aureus against different brands
of cefepime injection (500 mg)
Marketed cefepime code Susceptible Intermediate Resistant
CF 01 14 (42.42%) 13 (39.39%) 6 (18.18%)
CF 02 14 (42.42%) 11 (33.33%) 8 (24.24%)
CF 03 15 (45.45%) 7 (21.21%) 11 (33.33%)
CF 04 17 (51.51%) 7 (21.21%) 9 (27.27%)
CF 05 18 (54.54%) 8 (24.24%) 7 (21.21%)
CF 06 17 (51.51%) 5 (15.15%) 11 (33.33%)
CF 07 18 (54.54%) 8 (24.24%) 7 (21.21%)
Table 6: Resistance pattern of clinical isolates against cefepime
S.
No Country Year
% Resistance of
Staphylococcus aureus
% Resistance of
Escherichia coli Reference
1 Canada 1997 21.0% – Halpern et al., 1997
2 USA 1996-1997 – 0.4% Jones et al., 1998
3 Brazil 1997-1999 44% 4% Sader et al., 2001
4 Romania 2005 – 22% Vlad, 2005
5 North
America 2005 – 6% Sader et al., 2005
6 Taiwan 2006 – 33% Liao et al., 2006
7 Pakistan 2002 – 19% Iqbal et al., 2002
8 Pakistan 2010 15% 7% Ale-Zehra et al., 2010
9 Pakistan 2013 30% 18% Nasiri et al., 2013
10 Pakistan 2013 8% 22% Siddiqui et al., 2013
11 Pakistan 2012-2013 37% 26% Arsalan et al., 2015
12 Pakistan 2015 38% 27% Arsalan et al., 2016
13 Hungary 2004-2015 – 1-8% Magyar et al., 2017
14 Ethiopia 2016 1% – Deyno et al. 2017
15 United
Kingdom 2013-2014 0% – Xu et al., 2018
Adeel Arsalan et al 61
hospitals at Karachi, Pakistan as shown in
table 1. The anti-bacterial sensitivity of
reference and different brands of cefepime
against clinical isolates has been shown in
tables 2-3. The resistance pattern of E. coli
clinical isolates has been revealed in table 4
and fig. 1 while Gram-positive S. aureus
susceptibility has been shown in table 5 and
fig. 2 against cefepime marketed in Pakistan.
The overall resistance pattern of clinical
isolates has been shown in fig. 3. Due to
irrational use of cefepime and change in
serotype and source of microbes, there is a
change in resistance pattern of clinical isolates
as shown in various studies table 6.
Cefepime is fourth generation
cephalosporin, possessing good activity
against Gram-positive and Gram-negative.
Because of its wide spectra even against
resistant pathogens, it has been recommended
in severe infections (Nguyen et al., 2009; Lin
et al., 2018). The susceptibility test has
indicated that the MIC90 of reference cefepime
(gifted by M/s. Barrett Hodgson Pakistan
(Pvt.), Ltd.) against standard E. coli (ATCC®
25922) with range of (MIC90 = 0.015–
0.25µg/ml). Moreover, for standard S. aureus
(ATCC® 25923) MIC90 with range of
(MIC90=1-4µg/ml) According to CLSI (2011),
for both pathogens MIC90less than 8μg/ml is
considered as susceptible and 16μg/ml and
more than 32μg/ml is considered as
intermediate and resistance respectively.
Seven different brand of cefepime injection
500mg have been compared with clinical
isolates of Gram-negative E. coli and Gram-
positive S. aureus with (MIC90; 0.015-
64.0µg/ml) and (MIC90; 1.0-64.0µg/ml)
respectively.
The generics should be identical to
innovator ones in purity, quality, and strength
while in the case with parenteral preparations
sterility of medicines along with dissolution of
drug have become the prime object (WHO,
1998; Vega et al., 2015). Any considerable
dissimilarity, in the dissolution profiles among
same generics show lacking in the entire drug
formulations. Solubility and dispersibility of
drug in parenteral preparation plays an
important role as a quality control tool to
monitor batch to batch consistency of drug
release (Kumar and Palmieri, 2010). Taylor et
al. (2009) have been found that some
medicines do not meet the quality
specifications as claimed by manufacturers.
Several workers have reported treatment
failure cases and economic burden due to
substandard medicines (Petralanda, 1995;
Johnston and Holt, 2014; Sammons and,
Choonara, 2017; Ozawa et al., 2018). The
evaluation of the antibacterial activity of an
antibiotic, the inhibition of the multiplication
of pathogenic bacteria by recognized
concentrations of antibiotic with a reference
standard, bring into being meaningful results
by well illustrated procedures (Mendez et al.,
2005).
It has been observed that urinary tract
infections were common in female patients as
supported by Kodner and Gupton (2010),
while S. aureus has been mainly isolated from
pus secretions and male patients (Kaleem et
al., 2010). It has been proposed by several
workers that the bactericidal activity of many
generic antibiotics have showed lesser extent
as compared to innovator against pathogenic
microorganisms (Rodriguez et al., 2004;
Zuluaga et al., 2009; Venkatesh et al., 2011;
Silver 2011; Exner et al., 2017). It can be
probably possible that certain generics have
not fulfilled the quality standards for that
pharmaceutical product (purity, content, etc.)
especially in active pharmaceutical ingredients
due to low in cost (Mallu et al., 2015;
Valverde and Pisani, 2016). These active
pharmaceutical ingredients may be
contaminated which may interfere antibacterial
activities of antibiotics (Zuluaga et al., 2009;
Han et al., 2016; Richardson, 2017).
In the last two decades, there has been a
constant increased in the use of cefepime for
the treatment of severe bacterial infections.
The continuous use and often abuse of
cefepime have led to increase in resistance
against cefepime (Endimiani et al., 2008;
Chong et al., 2010). A number of factors have
been involved in the quality by design of
medicine like manufacturing technology,
Bactericidal activity of different brands of cefepime 62
quality of excipients, production processes,
overall quality, and release proficiency of
medicament. To ensure the requisite quality
certain tests have been performed during and
after manufacturing even during the shelf life
(Chow, 1997; Pifferi et al., 1999; Newton et
al., 2010; Xu et al., 2017).
The results has obtained after application of
ANOVA has been confirmed that the brands
of cefepime purchased from the local market
produce same antibacterial effect in
comparison to the standard cefepime. It has
been noticed in the present study that there
were no significant variations found in MIC90
values of different brands of cefepime. It has
been indicated that cefepime has been good
choice of drug against S. aureus and E. coli
associated infections.
CONCLUSIONS
Nearly all the pharmaceutical industries
fulfill the requirements of drug regulatory
authorities of their respective countries. It
should be considered that all generics are
pharmaceutical equivalent to innovator ones.
The MIC90 values of the cefepime against each
strain have tested behave approximately same
bactericidal activity. It has been revealed from
the present study that there is no significant
variations have been observed between the
me-too (generics) and trademarks (innovator
ones). The faith should be discouraged by
doctors, pharmacist, and microbiologist to
patients that expensive or innovator ones are
better as compared to generics.
ACKNOWLEDGEMENT
We would like to thank Mr. Shamim
Mumtaz for his keen interest and valuable
suggestions during the preparation of
manuscript.
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