Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT01725841 |
| Other study ID # |
200809012R |
| Secondary ID |
|
| Status |
Completed |
| Phase |
N/A
|
| First received |
October 8, 2009 |
| Last updated |
November 8, 2012 |
| Start date |
April 2009 |
| Est. completion date |
September 2009 |
Study information
| Verified date |
November 2012 |
| Source |
National Taiwan University Hospital |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
Taiwan: Department of Health |
| Study type |
Observational
|
Clinical Trial Summary
Resistance to broad-spectrum cephalosporins through the acquisition and expression of
Extended-Spectrum β-lactamase (ESBL) among Enterobacteriacea is increasing. The clinical
implications of ESBLs are extremely serious and sensitive diagnostic methods are urgently
needed to guide therapy, monitor resistance development and implement intervention
strategies. Conventionally, detection of expression of ESBLs was based on reduction of
ceftazidime of cefotaxime MICs by ≥ 3 two fold dilutions in the presence of clavulanic acid.
However, the use of the above method was limited to cover only some of the bacterial
species, including predominantly E. coli and Klebsiella spp., or tested strains which were
all transconjugants generated in vitro. ESBLs are now reported in a growing number of genera
other than E. coli or Klebsiella spp., and Serratia marcescens.
Carbapenems, including ertapenem, imipenem and meropenem, are the drugs of choice used for
severe ESBL-producing bacterial infections. Failure to detect ESBL at the presence of AmpC
β-lactamase might result in an important clinical concern because 4th generation
cephalosporins, which are stable to AmpC β-lactamase, is not a drug of choice for severe
infections caused by ESBLs-producing isolates. Fluoroquinolone-resistance in ESBL-producing
Enterobacteriacea is common. In this study, the investigators will use isolates of
Enterobacteriacea collected from different hospitals (isolates offered by the Taiwan
Surveillance of Antimicrobial Resistance [TSAR] program) to investigate the susceptibility
of ertapenem and five other antimicrobial agents against ESBLs-producing Enterobacteriacea.
Description:
Materials and Methods
1. Collection of bacterial isolates
The staffs in National Health Research Institute have established a long term
surveillance of antimicrobial resistance from 22 to 44 hospitals distributed in all
parts of Taiwan-Taiwan Surveillance of Antimicrobial Resistance (TSAR) since 1998. It
has been conducted five times in year 1998, 2000, 2002, 2004, and 2006 and were named
as TSAR-I, TSAR-II, TSAR-III, TSAR-IV and TSAR-V. In this study, isolates of
Enterobacteriaceas from TSAR-IV will be recruited. Each 50 isolates of Escherichia
coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Serratia
marcescens, and Morganella morganii will be enrolled (about 10% of the TSAR IV
collected isolates) for the following microbiological studies.
Isolates will be divided to ESBL-producing or non-producing groups by screening tests.
All the available isolates were identified to the species or genus level by means of
conventional methods. Isolates with same species recovered from the same an individual
patient, are considered one isolate. The isolates were all stored at -70℃ in trypticase
soy broth (Difco Laboratories, Detroit, MI) supplemented with 15% glycerol before being
tested.
2. Data Collection
Information collected for each isolate included: collection date, test date,
identification, site of infection, hospital unit of the patient, and whether the
isolate was acquired in the community (i.e., specimen obtained <48 hours after
admission), the length of patient's hospital stay. The susceptibility of all the
isolated against targeted antimicrobial agents will be determined by their Minimum
Inhibitory Concentration (MIC values) follow the CLSI breakpoints for respective
antimicrobial agent.
3. Antimicrobial susceptibility testing
- Inoculums preparation Sterile saline (0.85% NaCl), suitable buffer or broth for
inoculums preparation, depending on the organism. The CLSI inoculums procedure for
aerobes and other microorganisms should be used. 0.5 McFarland turbidity will be
used as the standard for aerobes.
- Inoculation Use sterile loops/swabs to obtain optimal amount of inoculums
suspension. Swab the entire agar surface three times; rotating the plate
approximately 90 degrees each time to ensure an even distribution of inoculums.
Allow excess moisture to be absorbed for about 10-15 minutes so that the surface
is completely dry before applying E test strips.
- Application of E test strips Ensure that the inoculated agar surface is completely
dry before applying E test strips. Use a Etest applicator or use a forceps to grip
the handle of the strip (are labeled E) and place them on to the inoculated agar
surface, ensuring that the MIC scale is facing upwards and that the concentration
maximum is nearest to the rim of the plate. Make sure the whole strip is in
complete contact with the agar surface. Once applied, the strip cannot be moved
because of the instantaneous release of antibiotic into the agar. It is
recommended to apply 4-6 different E test strips onto a 150 m agar plate. In this
study, the E test strips of six antimicrobial agents, including cefepime, cefepime
plus clavulanic acid (for the identification of ESBL producing isolates),
ciprofloxacin, cefmetazole, amikacin, tigecycline will be applied in addition to
ertapenem.
- Incubation The incubation temperature and atmosphere used must be optimal for
growth of particular bacterial species and the antibiotic being tested. CLSI
recommend 35℃/16-18 hours/ambient atmosphere for non-fastidious aerobes and
facultative anaerobes.
- Reading of the MIC After complete the period of incubation and when bacterial
growth become distinctly visible, read the MIC value at the point of intersection
between the inhibition ellipse edge and the E test strip. Use the E test reading
Guide and other technical guides to correctly read different patterns. Etest strip
(AB Biodisk, Solna, Sweden) containing cefepime (MIC test range, 0.25-16 mg/L) and
cefepime (MIC test range, 0.064-4 mg/L) plus 4 mg/L clavulanic acid will be used
to detect the expression of ESBL according to the manufacturer's
instruction.reduction of MIC by ≥3 two-fold dilutions in the presence of
clavulanic acid is indicator of ESBL production. Deformation of the ellipses or
the presence of "phantom" zone is also indicator of ESBL production even if the
MIC ratio is <8 or can not be read.
- Quality control In order to check the system with respect to correct handing,
quality and consistency of materials and methods, test quality control strains
with the antibiotics concerned. S. pneumoniae and H. influenzae can be determined
by broth microdilution under ambient incubation. The following organisms are
included as control strains: S. aureus ATCC 29213, E. faecalis ATCC 29212, S.
pneumoniae ATCC 49619, E. coli ATCC 25922, E. coli ATCC 35218, K. pneumoniae ATCC
700603, and P. aeruginosa ATCC 27853. For every 500 Etests performed, a control
test including the above 5 control strains will be done.
- Molecular studies Isolates with expression of ESBLs will be further typed using
Pulsed field gel electrophoresis (PFGE) method. The detailed procedures of PFGE
and the interpretations of PFGE banding patterns are as the description in our
previous report (13).
Data Analysis
Isolates are categorized into susceptible, intermediate, and resistant according to the
guidelines provided by the CLSI. Isolates intermediate or resistant to antimicrobial agents
are also categorized as nonsusceptible to the agents. The rates of nonsusceptibility among
major bacterial pathogens will be analyzed according to the different sites of infection and
origin of the isolates. The associations between geographic area, hospital types from whom
these isolates were obtained against the antimicrobial susceptibility will be analyzed.
