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    Identificación de OXAs en Enterobacterias

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    Identificación de OXAs en Enterobacterias

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    © All Rights Reserved
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    7 views5 pages

    Rapid Identification of OXA-48 and OXA-..

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    Aplicaciones Bioquimicas
    Identificación de OXAs en Enterobacterias

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    © All Rights Reserved
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    A sexe Journal of By ees, Clinical Microbiology ®@ CCroseMasle Rapid Identification of OXA-48 and OXA-163 Subfamilies in Carbapenem-Resistant Gram-Negative Bacilli with a Novel Immunochromatographic Lateral Flow Assay Fernando Pasteran,¢ Laurence Denorme, Isabelle Ote.” Sonia Gomez,* Denise De Belde Youri Glupczynski* Pierre Bogaerts,® Barbara Ghiglione,* Pablo Power,¢ Pascal Mertens,” Alejandra Corso" Ministero de Salus de a Naién Clad Auténoms de Buenos res, Age Monsonng of animicotil Resistance GrarNegativeBacter 3, DnantGeinne, Ul sumac Bioguimis, Unease Buenos Ares, 8 : We assessed a novel immunochromatographic lateral flow assay for direct identification of OXA-48-Ui “nen Ann obianes, ste Nacional nat Cots BioConcep, Gembou,Bekium Natonal Aeferece Lavoro for sades nfeccosas INE, ANLIS De Caos toro de Resistencia Bact i Baku’ Labor carbapenemases and accurate differentiation of allele variants with distinct substrate profiles (OXA-48 or OXA-163 subfamilies). The assay allowed rapid (less than 4 min) and reliable direct confirmation of OXA-163- and/or OXA-48-like enzymes (with 100% sensitivity and 100% specificity) from cultured colonies that were recovered from both solid medium and spiked blood culture bottles. The emergence and spread of Enterobacteriacea that produce plasmids encoding class A (KPC, GES, Sme, NMC-A),B (IMP, VIM, NDM), and D (OXA-48-like) carbapenemases are world- wide public health threats. To prevent the spread of carbapen- emase producers and define an appropriate empirical antimicro- bial therapy, the rapid detection of carbapenemase-producing organisms has become imperative (1). One of the major concerns for controlling the spread of OXA-48 of reliable phenotypical teats that might contribute to their easy recognition. This is due in part to relatively low carbapenem MICs, alack of suitable inhibitor compounds for use in confirme- tory tess, and the very low expression of earbapenemase activity ‘which cannot be detected readily by recently developed biochem- ical methods (1-5). Recently, aovel means of detecting OXA-48- like enzymes via an antbody-mediated approach was developed (6). The OXA-48 K-SeT test (Coris BioConcept, Belgium) relies ‘on immunological capture of two epitopes specifi to the OXA-48 variants OXA~48, OXA-I81, OXA-204, OXA-252, and OXA-244 Using colloidal gold nanoparticles bound to nitrocellulose mem- brane within a lateral low device (6). The reported sensitivity and specificity were both 100%, with the result obtained in ess than 10| rin (6-10), Noteworthy is that new allele variants of OXA-48 hhave emerged, namely, OXA-163 and the related variants OXA- 247, OXA-405, and OXA-438 (11-14), which are not recognized by the OXA-48 K-SeT test (6, 8,10), OXA-1683, which isthe only variant of this subfamily that has spread to several hospitals in Argentina and Egypt (15, 16), differs from OXA-48 by a single amino acid substitution anda four-amino-acid deletion, In carer studies, OXA-163 exhibited almost undetectable carbapenemase activity with a substrate profle that included broad-spectrum cephalosporins (11). However, recent reports indicate that this allele might produce enhanced carbapenemase activity in the presence of carbonates (13, 17), suggesting that its activity is strongly influenced by the rate of carboxylation ofthe active ste, asobserved for other carbapenen-hydrolyzing class D B-lactama- ses (18, 19). Additionally, in vivo data suggested that OXA-162 right cause carbapenem treatment failures critically patients (12, 15,20) or favor intrapaient selection ofnew OXA-48 variants roducers isthe absence Joaral of Cnc Macrobiology (12), which highlights the urgent need for efficient identification tests Definitive confirmation of OXA-163- and/or OXA-48-Like producers currently relies on molecular assays and gene sequenc- ing fr the assignment of allelic variants. However, these methods are of limited practical use for deily application in most clinical laboratories due tothe high cost and requirement for significant expertise to interpret test results. We recently developed new immunochromatographic lateral flow assay, the OXA-163/48 Duo K-SeT test (Coris BioConcept), for direct identification and accurate differentiation of OXA-48- and OXA-163-Iike subfami- lies, in which two monoclonal anti-OXA-48 antibodies are se- lected as specific capture reagents on two lines for the test (21), ‘The test included (i) a first antibody directed against all current (OXA-48-like variants (OXA~48, OXA-162, OXA-181, OXA-204, ‘OXA.232, and OXA-244) but not the OXA-163 subfamily (first line, labeled 48), and (ii) a second antibody directed against an- other epitope present in all OXA~48-like enzymes, which also binds the OXA-163 subfamily (second line labeled 163) (Fig. 1)-1F the sample contains OXA-48, it will remain bound to the first capture antibody (anti-OXA-48). If the sample contains OXA- 163, it will not react with the first antibody but will bind to the second capture antibody (anti-OXA-163 andanti-OXA48). Ifthe sample contains a large amount of OXA~48, the second line may give a faint signal from a portion of OXA~48 that is not captured by the fast ine, Finally, a third antibody directed against a thied Feceved 5 ay 20 Retumetfor medication 5 Je 2015 Accepted 9 gst 2016 Accepted manuscript pasted online 17 Aug egeer clone Powet> Mertens, Cao 2076 Rad deviation of ‘ete and Ont ures in caapenem-estat Grar-negaive ‘sth novelimmungetromsteranhic tao shay Cn Merl 2082-7886 doi TO1 BICMOS 16 Feito 2 PteL Mayo Chic zs orerpondencee Ferman Pasta, fosterangrallcom. Copy © 201, arvarcanSocty for Microbiology. Ags Reserved, November 2016 Velume 54. Number 11 ysenB Aq 9102 “yz 48q0}90 Uo /Buowse wWol//:dyY Woy papeo|juMog cokes esses ose ae OXA-48, es ce («A and 0X14 Detection by Inmunaehromategaphy aa _oueaer omens, nem "control OxA-163 Sea HIG 1 OXA-163/48 Duo K-SeT assays forthe detection of OXA-48-type (a and OXA165-type (b) enzymes. For positive results. in addition to areédish-purple band at the contre ne (©), a visible hand attest ne postion 48 corresponds toa sample containing OXA-A8 (or closely related variants, bat not OXA-163), regardless ofthe presence or absence af aint signal at the 163 testine, Ube only positive test lines ine 165, te sample contains the OXA-163 variant or doely ‘elated variant) For negative results, a single line appeats atthe position ofthe conta line (C) only. The vera azow indiates the dzection af lateral low epitope present in all OXA-48-Ike variants (including the OXA- 163 subfamily) was chosen as detection reagent. In this study, we challenged the performance ofthe Duo K-SeT assay fr the detec- tion of OXA-48-like and OXA-163-Lke enzymes against acolle- tion of Gram-negative bacill ‘he evaluation of the test was performed with a panel of 75 clinical isolates. PCR and DNA sequencing of entire bla alleles ‘were considered the gold standard for B-lactamase characteriza- tion (4). The selected isolates comprised 64 carbapenemase pro- ducers, among which 50 were confirmed to belong to the OXA-48 family (Table 1). The isolates included were found to be nonclonal by pulsed-field gel electrophoresis (PFGE) (data not shown). Spe- cies identification was confirmed using matrix-asisted laser de- sorption ionization-time of flight mass spectrometry (MALDI- "TOK [BD Biotyper 3; Bruker, Germany]). MICs were obtained using an Fest (bioMérieux; Matcy l'Rtoile, France) or by agar

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