TheH9252-Lacta Test for Direct Detection of Extended-Spectrum- [616081]

The/H9252-Lacta Test for Direct Detection of Extended-Spectrum-
/H9252-Lactamase-Producing Enterobacteriaceae in Urine
Salah Gallah,aDominique Decré,b,cNathalie Genel,bGuillaume Arleta,b,c
Assistance Publique-Hôpitaux de Paris, HUEP, Hôpital Tenon, Department of Bacteriology-Hygiene, Paris, Francea; Université Pierre et Marie Curie-Paris 6, Medical School,
Department of Bacteriology, Paris, Franceb; Assistance Publique-Hôpitaux de Paris, HUEP, Hôpital Saint Antoine, Department of Bacteriology, Paris, Francec
With the /H9252-Lacta test, production of extended-spectrum /H9252-lactamases (ESBLs) was assayed in 200 urine samples showing Gram-
negative bacilli during direct microscopic examination. While 168 samples tested negative, all samples yielding ESBL-producing
Enterobacteriaceae after culture gave positive (n /H1154930) or uninterpretable (n /H115492) results. The sensitivity and specificity of ESBL
detection were 94% and 100%, respectively.
Asurvey conducted from 2002 to 2010 by the French national
infection control program demonstrated an increase of
282% in the incidence of extended-spectrum /H9252-lactamase-pro-
ducing Enterobacteriaceae (ESBLE), particularly ESBL-producing
Escherichia coli (1). The increasing importance of multiresistant
ESBL-producing E.
colistrains in the community should make
clinicians aware of potential treatment failures associated with
serious and potentially life-threatening infections ( 2). A recent
study
by Peralta et al., conducted in 19 Spanish hospitals, found
that the ESBLE causing bacteremia were mainly from the urinary(55.3%) and biliary (12.7%) tracts. E. coli accounted for 89% of all
ESBLE strains, and 45.7% of these were multidrug resistant (e.g.,resistant to /H9252-lactam–/H9252-lactamase inhibitor combinations, ceph-
alosporins, quinolones, and aminoglycosides) ( 3). Furthermore,
empirical
antibiotic treatment was adequate in only 48.8% of the
cases, and the in-hospital mortality was 20.9% ( 3). ESBL-produc-
ingE.
coliisolates, particularly those producing CTX-M enzymes,
account for a significant number of cases of bacteremia in hospi-
talized and nonhospitalized patients ( 4). Moreover, Livermore
has
reported that, since 2003, 90% of ESBL-producing E. coli iso-
lates in the United Kingdom produce CTX-M-15 ( 5).
Urinary
tract infections (UTI) are among the most frequent
bacterial infections in the community and in the health caresetting ( 6). Broad-spectrum cephalosporins, /H9252-lactam–/H9252 -lac-
tamase
inhibitor combinations, or fluoroquinolones are rec-
ommended for first-line empirical therapy of sepsis arisingfrom the urinary tract in community-acquired and nosocomi-ally acquired cases ( 4,7). These recommendations might be
difficult
to apply if a significant proportion of such infections is
caused by ESBLE ( 4).
A
new chromogenic test (/H9252 -Lacta test [BLT]; Bio-Rad, Marnes-
La-Coquette, France) was developed for the rapid detection (in lessthan 15 min) of strains of Enterobacteriaceae with decreased suscep-
tibility or resistance to third-generation cephalosporins (3GC)conferred by enzymes such as ESBLs and carbapenemases. Thistest provides useful information to guide antibiotic treatment be-fore full results of antimicrobiotic susceptibility testing are avail-able ( 8). The aim of this study was to highlight the time saved in
cases
of UTI when the BLT is performed directly with urine rather
than bacterial colonies.
Urine samples. All urine samples received over a 3-month pe-
riod and showing Gram-negative bacilli (GNB) upon direct mi-croscopic examination and on Gram stains of uncentrifuged urinewere prospectively included. Urine samples with hematuria which
would interfere with the chromogenic BLT were excluded.
/H9252-Lacta test. The BLT was performed directly on urine sedi-
ments according to the manufacturer’s recommendations ( 8).
Two
collection periods were scheduled to test for possible varia-
tions in BLT efficiency; during the first 5 weeks, 1-ml urine sam-ples collected from 100 patients were centrifuged for 2 min at3,000/H11003gin Eppendorf tubes, and during the following 7 weeks,
1.5-ml samples collected from an additional 100 patients were
Received 9 June 2014 Returned for modification 3 July 2014
Accepted 22 July 2014
Published ahead of print 30 July 2014
Editor: A. B. Onderdonk
Address correspondence to Guillaume Arlet, guillaume.arlet@tnn.aphp.fr.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.01629-14TABLE 1 Phenotypes of Gram-negative bacilli isolated from urine
samples subjected to the /H9252-Lacta test
Organism(s) or test resultNo. of strains with indicated phenotype
Total Wild BSBLaESBLAmpC-type
/H9252-lactamasesb
Escherichia coli 79 48 16 4 147
Klebsiella pneumoniae 9 0 14 0 23
Enterobacter spp. 13 0 3 5 21
Proteus mirabilis 34 0 1 8
Citrobacter koseri 30 0 0 3
Morganella morganii 31 0 0 4
Proteus vulgaris 10 0 0 1
Obligate aerobesc90 0 5 1 4
Total 120 53 33 15 221No./H9252-Lacta test positive 0 0 31 0 31
No./H9252-Lacta test
uninterpretable00 2 0 0
aBSBL, broad-spectrum /H9252-lactamases, including TEM-1, TEM-2, OXA-1, and IRT-2.
bAmpC-type /H9252-lactamases conferring resistance to ceftazidime or cefotaxime
(chromosome or plasmid mediated).
cTen Pseudomonas aeruginosa strains, one Acinetobacter baumannii strain, and three
Stenotrophomonas maltophilia strains.
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centrifuged for 5 min, also at 3,000 /H11003g. After elimination of the
supernatant, the BLT was performed in the sediment-containing
tube. The sediment was completely homogenized in one drop (ca.50/H9262l) of each reagent, and after incubation for 15 min at room
temperature, the test was interpreted visually as follows: nochange in color indicated a negative result, a color change to red orpurple indicated a positive result, and a color change to orangewas uninterpretable. In some cases, an immediate color changewas observed (in less than 1 min).
Antibiotic susceptibility testing. Standard disk diffusion re-
sults were interpreted 48 h after the BLT, and 3GC (i.e., cefotaximeor ceftazidime)-resistant isolates were screened for ESBL produc-tion using the double-disk synergy test by following the recom-mendations of the Comité de l’Antibiogramme of the SociétéFrançaise de Microbiologie (CA-SFM) ( 9).Molecular
characterization of /H9252-lactamase genes. All strains
suspected of producing one or more acquired broad-spectrum/H9252-lactamases (BSBLs) or extended-spectrum /H9252-lactamases (ESBLs)
and strains resistant to ceftazidime or cefotaxime were screened usingPCR and sequencing as described previously ( 10,11).
Culture
characteristics of GNB. In total, 200 urine samples
containing GNB, as seen using direct microscopy and Gram stain-ing of uncentrifuged urine, were included in this study. Fromthese, 221 strains grew on Uriselect 4 agar (Bio-Rad) after 16 to 24h of incubation at a threshold of detection of /H1135010
4CFU/ml. They
were mainly Enterobacteriaceae (n/H11005207; 94%), including 147 E.
coli strains (71%), 23 Klebsiella pneumoniae strains (11%), 21
Enterobacter species strains (10%), 9 Proteus species strains (4%),
4Morganella morganii strains, and 3 Citrobacter koseri strains (Ta-
ble
1). Fourteen obligate aerobes (6%) were recovered on the sameTABLE 2 /H9252-Lacta test results during the first and second collection periods for extended-spectrum /H9252-lactamase-producing Enterobacteriaceae
/H9252-Lacta
samplecollectionperiod ESBLEAmt of GNBatmicroscopicexamination CFU/ml by cultureInitialcolorImmediatecolorchange(/H110211 min)Color at15 min Result blagene
1st Klebsiella pneumoniae Several /H1102210
5and 105P.
aeruginosaYellow Yes Purple Positive CTX-M-15
Klebsiella pneumoniae Plenty 105Yellow No Orange Uninterpretable CTX-M-15
Escherichia coli Abundant 105Yellow No Red Positive CTX-M-15
Klebsiella pneumoniae Abundant /H11022105and/H11022105
E. coliYellow No Red Positive CTX-M-15
Escherichia coli Few /H11022105Yellow No Red Positive CTX-M-64*
Klebsiella pneumoniae Several /H11022105Yellow No Red Positive CTX-M-15
Klebsiella pneumoniae Plenty 105Yellow Yes Purple Positive CTX-M-15
Escherichia coli Abundant 105Yellow No Red Positive CTX-M-15
Escherichia coli Plenty /H11022105Yellow No Red Positive CTX-M-55
Klebsiella pneumoniae Several /H11022105Yellow No Purple Positive CTX-M-15
Escherichia coli Few 104Yellow Yes Orange Uninterpretable CTX-M-1
Klebsiella pneumoniae Plenty /H11022105Yellow No Purple Positive CTX-M-15
2nd Enterobacter cloacae Abundant /H11022105Orange Yes Purple Positive CTX-M-15
Escherichia coli Abundant 105Yellow Yes Purple Positive CTX-M-55
Enterobacter cloacae Few 105Yellow No Purple Positive CTX-M-15
Klebsiella pneumoniae Abundant /H11022105Yellow Yes Purple Positive CTX-M-15
Klebsiella pneumoniae Several /H11022105Yellow No Purple Positive CTX-M-15
Klebsiella pneumoniae Few /H11022105Yellow No Purple Positive CTX-M-14
Escherichia coli Several /H11022105and/H11022105
EnterococcusspeciesYellow No Purple Positive CTX-M-15
Escherichia coli Abundant /H1102210
5Yellow No Purple Positive CTX-M-15
Escherichia coli Several 105Yellow No Purple Positive CTX-M-55
Escherichia coli Several 105Yellow Yes Purple Positive CTX-M-15
Escherichia coli Few /H11022105and/H11022105
E. cloacaeYellow No Red Positive CTX-M-64**a
Escherichia coli Abundant 105Yellow No Purple Positive CTX-M-15
Escherichia coli Several 105Yellow No Red Positive CTX-M-14
Escherichia coli,
KlebsiellapneumoniaeFew 10
5,1 05, and 105
A. baumanniiYellow No Red Positive CTX-M-15
Klebsiella pneumoniae Abundant 105Yellow No Red Positive CTX-M-27
Escherichia coli Plenty /H11022105Yellow No Red Positive CTX-M-1
Escherichia coli Several /H11022105Yellow Yes Purple Positive CTX-M-55
Klebsiella pneumoniae Plenty /H11022105Yellow No Purple Positive CTX-M-15
Enterobacter cloacae Several 105Yellow Yes Purple Positive CTX-M-15
Klebsiella pneumoniae Several /H11022105Yellow No Purple Positive CTX-M-15
aCTX-M-64** was found twice in the same patient in two independently collected urine samples.Direct ESBL Detection in Urine with the /H9252-Lacta Test
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medium, including 10 Pseudomonas species strains, 3 Stenotroph-
omonas maltophilia strains, and 1 Acinetobacter baumannii strain
(Table 1).
Performance
of the BLT. In total, 30 (15%) of the 200 urine
samples were found to be BLT positive, and two (1%) gave a unin-
terpretable result. During the first collection period, 10 out of 100samples tested positive with the BLT and two samples gave uninter-pretable results (Table 2). During the second collection period, BLT
seemed
to be somewhat more efficient, with 20 samples out of 100
testing positive and all results being interpretable ( Table 2).
Phenotypic
characterization of all GNB isolates from urine
(Table 1) confirmed that samples giving a positive or an uninter-
pretable
BLT result were ESBL producers, and no negative BLT
results were obtained for ESBL producers. All GNB strains pro-ducing /H9252-lactamases other than ESBLs, such as broad-spectrum
/H9252-lactamases (TEM-1, TEM-2, IRT-2, and OXA-1) and AmpC-type enzymes, gave negative BLT results. Thirty-three ESBLs wereisolated from the 32 BLT-positive urine samples, including 16 E.
coli,1 4K. pneumoniae, and 3 Enterobacter species strains (Table
1). BLT has shown sensitivities of 87% in the first collection periodand
of 100% in the second and a specificity of 100% in both peri-
ods. The presence of other bacterial morphotypes in the polymor-phic flora seen at direct microscopic examination did not interferewith the BLT results.
The molecular results confirmed the phenotypic enzyme char-
acterizations and revealed that all ESBLs belonged to the CTX-Mgroup (Table 2), in apparent keeping with the ongoing CTX-M
/H9252-lactamase
pandemic (5, 12).
The
test yielded very high values for both sensitivity (94%) and
specificity (100%). Recently, Renvoisé et al., analyzing isolatesgrown for 16 to 24 h, demonstrated that the sensitivity and spec-ificity of BLT were 99.6% and 87.7%, respectively, for Enterobac-
teriaceae overexpressing ampC and that both were 100% for ESBL
producers (8). This demonstrates that the /H9252-lactam
ring of the
BLT chromogenic substrate (HMRZ-86) is very efficiently hydro-lyzed by ESBL but not AmpC-type activities.
All screening tests for rapid detection of ESBL-producing GNB
require at least 16 to 24 h, including those that use specific agarmedia, e.g., ESBL agar (AES Chemunex), ChromID ESBL agar(bioMérieux), or Brilliance ESBL agar (Oxoid). They have sensi-tivities and specificities of 81.3 to 87.5% and 60.8 to 82.1%, re-spectively (13). Recently, a biochemical test, ESBL NDP, was pro-
posed
by Nordmann et al. (14). Preliminary culturing of the
isolate
was required, but the test took less than 1 h; it was found to
have a specificity of 100% and a sensitivity of 92.6%.
In conclusion, BLT may be considered an efficient test for the
detection of ESBL in urine, and due to the rapidity and ease withwhich it is performed, it is a valuable adjunct in specifying empir-ical UTI management, including measures to limit cross-trans-mission. The BLT may be an adequate tool for efficient detectionof carbapenemase-producing Enterobacteriaceae in countries with
an elevated prevalence of theses enzymes (e.g., Klebsiella pneu-
moniae carbapenemase and class B carbapenemases) ( 15).
ACKNOWLEDGMENTS
We thank Manette Juvin and Caroline Dallenne for support during this
study.
This study was conducted as part of our routine work, and we received
no extra funding.
We have no conflicts of interest to declare.REFERENCES
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