Metodologie molecolari per la identificazione dei batteri multiresistenti
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Metodologie molecolari per la
identificazione dei batteri multiresistenti
Vittorio Sambri, MD, PhD
Unit of Microbiology
the Greater Romagna Area Hub Laboratory
DIMES – University of Bologna
Pievesestina, Cesena (Italy)
Vittorio.sambri@auslromagna.it – vittorio.sambri@unibo.it
A “syndromic” approach
• Classic Microbiology • Molecular Microbiology
– Culture based – Specific gene(s) ID
– Phenotypic ID – Growth is not necessary
– Phenotypic AST (sometime!)
– Immunocomplex ID – Multiple techniques
– Immune response – Very low LOD
detection – Fast and quick
– Time is an issue – More germs “who is the
– First come First got bad guy”
vittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdr
Antibiogramma Molecolare
• Determina la presenza di geni di resistenza
– Non serve il batterio vitale
• Bassi LOD (dipendente da numero di target e da reazione)
• Non influenzato da on going therapy
• TAT molto rapido
– Determina ciò che “noi vogliamo, non quello che c’è”
• Singolo target
• Pannelli (quanto completi)
• Sensibilità della reazione
• Mutazioni
vittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdr
vittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdr
ISSN: 1473-7159 (Print ) 1744-8352 (Online) Journal hom epage: ht t p:/ / www.t andfonline.com / loi/ iero20
Em erging m et hodologies for pat hogen
ident ificat ion in posit ive blood cult ure t est ing
Grégory Dubourg & Didier Raoult
To cit e t his art icle: Grégory Dubourg & Didier Raoult (2015): Emerging methodologies
for pathogen identification in positive blood culture testing, Expert Review of Molecular
Diagnostics, DOI: 10.1586/14737159.2016.1112274
To link t o t his art icle: http://dx.doi.org/10.1586/14737159.2016.1112274
Published online: 11 Nov 2015.
Submit your article to this journal
Expert Review of Molecular Diagnost ics
Article views: 18
View related articles
ISSN: 1473-7159 (Print ) 1744-8352 (Online) Journal hom epage: ht t p:/ / www.t andfonline.com /loi/iero20
View Crossmark data
Emerging met hodologies for pat hogen
vittorio sambri CUEB 27 novembre 2019
04/12/2019
ident ificat ion in posit ive blood cult ure tpcr est ing
mdr
Available online at www.sciencedirect.com
Recent and emerging technologies for the rapid
ScienceDirect
diagnosis of infection and antimicrobial resistance
Alexander J Trotter1,2, Alp Aydin1,2, Michael J Strinden1,2 and
O’Grady1,2
Justin Recent and emerging technologies for the rapid
diagnosis of infection and antimicrobial resistance
Alexander J Trotter1,2, Alp Aydin1,2, Michael J Strinden1,2 and
The rise in antimicrobial resistance (AMR) is predicted to cause appropriate antibiotics (i.e. improved antibiotic steward-
10 million deaths per year by 2050 unless 1,2 steps are taken to ship) [2]. T he final O’N eill report states that by 2020 all
Justin O’Grady
prevent this looming crisis. Microbiologic al culture is the gold antibiotic prescriptions should be supported by a rapid
standard for the diagnosis of bacterial/fungal pathogens and diagnostic test where available [1].
antimicrobial resistance and takes 48 hours or longer. Hence,
antibiotic prescriptions are rarely based
The rise in antimicrobial on a definitive
resistance Current standard
(AMR) is predicted to cause methods
appropriate for diagnosing
antibiotics bacterial
(i.e. improved infec- steward-
antibiotic
diagnosis and patients often receive inappropriate treatment.
10 million deaths per year by 2050 unless steps are taken to tion are ship) [2]. T he final O’Neill report states that
based on microbiological culture and have longby 2020 all
Rapid diagnostic tools are urgently required to guide turn-around
prevent this looming crisis. Microbiological culture is the gold times, offer poor clinical sensitivity
antibiotic prescriptions should be supported and areby a rapid
appropriate standard
antimicrobial therapy, thereby improving patient not
for the diagnosis of bacterial/fungal pathogens and fit-for-purpose for acute serious infection
diagnostic test where available [1]. such as
outcomes and slowing AMR development. We discuss new
antimicrobial resistance and takes 48 hours or longer. Hence,sepsis, pneumonia and meningitis. Acute infections force
antibiotic prescriptions are rarely based on a definitive clinicians into early broad-spectrum treatment, before
technologies for rapid infection diagnosis including: sample-in-
Current standard methods for diagnosing bacterial infec-
culture results become available, highlighting the
tion are based on microbiological cultureneed
answer-out PCR-based tests, BioFire FilmArray and Curetis
diagnosis and patients often receive inappropriate treatment. and have long
Unyvero; rapid susceptibility tests, Accelerate Pheno and for rapid diagnostics [3–6]. A paradigm shift in diagnostic
Rapid diagnostic tools are urgently required to guide
microfluidic tests; and sequencing-based approaches,
turn-around times, offer poor clinical sensitivity and are
microbiology is urgently required, with the ultimate goal
appropriate antimicrobial therapy, thereby improving patient
focusing on targeted and clinical metagenomic nanopore not fit-for-purpose for acute serious infection such as
of providing pathogen identification and resistance/sus-
sequencing.
outcomes and slowing AMR development. We discuss new sepsis, pneumonia and meningitis. Acute
ceptibility information to clinicians before antibiotics are infections force
technologies for rapid infection diagnosis including: sample-in-
administered.clinicians into early broad-spectrum treatment, before
Addresses answer-out PCR-based tests, BioFire FilmArray and Curetis culture results become available, highlighting the need
1
University ofUnyvero; rapid
East Anglia, susceptibility
Norwich Research tests, Accelerate
Park, Norwich, Pheno and
Norfolk, for rapid
I n this review, diagnostics
we highlight [3–6].
recent andAemerging
paradigmtests
shiftfor
in diagnostic
NR4 7TJ, UKmicrofluidic tests; and sequencing-based approaches, microbiology is urgently required, with the ultimate goal
2
Quadram Institute Bioscience, Norwich Research Park, Norwich,
the rapid diagnosis of pathogens, antimicrobial resistance
focusing on targeted and clinical metagenomic nanopore of providing
and antimicrobial pathogenand
susceptibility identification and resistance/sus-
their current/future
Norfolk, NR4 7UQ, UK
sequencing. ceptibility information
clinical applications. We describetosome
clinicians before
of the antibiotics are
current
Corresponding author: O’Grady, Justin (justin.ogrady@quadram.ac.u k) tests that administered.
utilise genotypic methods such as PCR for
Addresses pathogen identification and antibiotic resistance testing.
1
University of East Anglia, Norwich Research Park, Norwich, Norfolk,
We also describe technologies
In this review, and techniques
we highlight that
recent and com- tests for
emerging
Current Opinion in Microbiology 2019, 51:39–45
NR4 7TJ, UK
2 bine pathogen
the identification
rapid diagnosiswith
of rapid phenotypic
pathogens, anti- resistance
antimicrobial
Quadram
This review comes fromInstitute
a themedBioscience, Norwich Research Park, Norwich,
issue on Antimicrobials
Norfolk, NR4 7UQ, UK biotic susceptibility
and testing
antimicrobial (AST ). Finally,
susceptibility andwe outline
their current/future
Edited by Matt Hutchings, Andrew Truman and Barrie Wilkinson
key advances in the
clinical application We
applications. of Ddescribe
N A sequencing
some offorthe current
the rapid
Corresponding author: O’Grady, Justin (justin.ogrady@quadram.ac.uk) diagnosis
tests thatofutilise
infection and AMmethods
genotypic R that could
suchbe as PCR for
implemented clinically
pathogen in the nearand
identification future.
antibiotic resistance testing.
Current Opinion in Microbiology 2019, 51:39–45 We also describe technologies and techniques that com-
https://doi.org/10.1016/j.mib .2019.03.001
bine pathogen identification with rapid phenotypic anti-
1369-5274/ã This
2018review comes
Elsevier from
Inc. All a themed
rights issue on Antimicrobials Rapid PCR-based pathogen and antimicrobial
reserved.
resistance biotic susceptibility testing (AST ). Finally, we outline
detection
Edited by Matt Hutchings, Andrew Truman and Barrie Wilkinson
vittorio key advances
D iscerning bacterial 27
sambri CUEB inviral
the infections
novembre
from application
2019isofthe
DNA sequencing for
simplest
04/12/2019 the rapid diagnosis of infection and AMR that could be
pcr that
level of diagnosis mdrcan be clinically useful to guide
implemented clinically in the near future.
antimicrobial therapy, reducing unnecessary antibiotic
vittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdr
04/12/2019 vittorio sambri CUEB 27 novembre 2019 pcr mdr
04/12/2019 vittorio sambri CUEB 27 novembre 2019 pcr mdr
vittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdr25 negative bacteria (GNB).
28 Data sources: PubMed, Scopus and ISI Web of Knowledge.
Rapid molecular tests for detection of antimicrobial resistance determinants in Gram-
26 Objectives: To review and meta-analyse the evidence for using commercially available molecular
29 Study eligibility criteria: Clinical studies evaluating the performance of two major commercial
negative organisms from positive blood cultures: a systematic review and meta-analysis◊
27 tests for the direct detection of AMR determinants in GNB-positive blood cultures (PBCs).
1 30 systems, namely the Verigeneâ and FilmArrayâ systems, for rapid testing of GNB-PBCs, in
G. De Angelis , A. Grossi 2, G. Menchinelli 1, S. Boccia 2, 3, M. Sanguinetti 1, 4, *, B. Posteraro 5, 6
28 Data sources: PubMed, Scopus and ISI Web of Knowledge.
31 comparison with the phenotypic or genotypic methods performed on GNB-PBC isolates.
29 Study eligibility criteria: Clinical studies evaluating the performance of two major commercial
1) 32 Methods: Literature search according to the Preferred Reporting Items for Systematic Reviews and
Istituto di Microbiologia, Università Cattolica del Sacro Cuore, Rome, Italy
30 systems, namely the Verigeneâ and FilmArrayâ systems, for rapid testing of GNB-PBCs, in
2) 33 Meta-Analyses criteria and, for meta-analysis of sensitivity and specificity of both systems,
Sezione di Igiene, Istituto di Sanità Pubblica, Università Cattolica del Sacro Cuore, Rome, Italy
31 comparison with the phenotypic or genotypic methods performed on GNB-PBC isolates.
3) 34 bivariate random-effects model.
Dipartimento di Scienze della Salute della Donna e del Bambino e di Sanità Pubblica, Fondazione
32 Methods: Literature search according to the Preferred Reporting Items for Systematic Reviews and
35 Results: Twenty studies were identified (3310 isolates) from 2006 to 2019. Nine studies were
Policlinico Agostino Gemelli IRCCS, Rome, Italy
33 Meta-Analyses criteria and, for meta-analysis of sensitivity and specificity of both systems,
4) 36 conducted in East Asia. In 15 studies using phenotypic comparators (1930 isolates), 1014 (52.5%)
Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario
34 bivariate
37 isolates random-effects
were Escherichia model.
coli, and 287 (14.9%) of all the isolates displayed AMR phenotypes. In 5
A. Gemelli IRCCS, Rome, Italy
3538 Results: Twenty
studies using studies comparators
genotypic were identified (3310
(1380 isolates)
isolates), 585 from 2006
(42.4%) to E.
were 2019.
coli,Nine studies
and 100 wereof
(7.2%)
5)
Istituto di Patologia Medica e Semeiotica Medica, Università Cattolica del Sacro Cuore, Rome,
3639 conducted in East
all the isolates Asia. InAMR
displayed 15 studies using Pooled
genotypes. phenotypic comparators
sensitivity (1930 isolates),
and specificity estimates1014
for (52.5%)
detection
Italy
37
40
isolates
of AMR were Escherichiabycoli,
determinants the and 287 (14.9%)
Verigeneâ of all theIMP,
(i.e. CTX-M, isolates displayed
KPC, NDM, AMROXA phenotypes. In 5
and VIM) and/or
6)
Dipartimento di Scienze Gastroenterologiche, Endocrino-Metaboliche e Nefro-Urologiche,
38 studies using genotypic comparators (1380 isolates), 585 (42.4%) were E. coli, and 100 (7.2%) of
41
Fondazione Policlinico FilmArrayâ
Universitario(i.e.
A. KPC)
Gemellisystems
IRCCS, were 85.3%
Rome, Italy(95% CI 79.9%–89.4%) and 99.1% (95% CI 98.2%–
39 all the isolates displayed AMR genotypes. Pooled sensitivity and specificity estimates for detection
*Corresponding 42author. 99.5%),
M. respectively,
Sanguinetti, across the di
Istituto 15 studies, and 95.5%
Microbiologia, (95% CI 89.2%–98.2%)
Fondazione Policlinico and 99.7% (95% CI
40 of AMR determinants by the Verigeneâ (i.e. CTX-M, IMP, KPC, NDM, OXA and VIM) and/or
Universitario A. 43 99.1%–99.9%),
Gemelli respectively,
IRCCS, Università across
Cattolica del the 5 studies.
Sacro Cuore, Largo A. Gemelli 8, 00168
4144 FilmArrayâ
Conclusions:(i.e.
OurKPC) systems
findings were
show that85.3% (95% CI 79.9%–89.4%)
the Verigeneâ and 99.1%
and FilmArrayâ systems(95%
mayCIbe98.2%–
a valid
Rome, Italy.
42 99.5%),
adjunctrespectively, across the 15 studies, and 95.5% (95% CI 89.2%–98.2%) and 99.7% (95% CI
E-mail address: 45 to the conventional
maurizio.sanguinetti@unicatt.it (M.microbiology
Sanguinetti)(phenotypic or genotypic) methods used to identify AMR
vittorio sambri CUEB 27 novembre 2019
04/12/2019
◊ 43 99.1%–99.9%), respectively, acrossdetects pcr mdr
the 5 studies.
Presented in part46at thein28th
GNBs. FilmArrayâ
European Congresssystem
of Clinical only
Microbiologyone and
AMR genotype,
Infectious namely KPC, limiting its
Diseases,40 of AMR determinants by the Verigeneâ (i.e. CTX-M, IMP, KPC, NDM, OXA and VIM) and/or
1 Rapid molecular tests for detection of antimicrobial resistance determinants in Gram-
41 FilmArrayâ (i.e. KPC) systems were 85.3% (95% CI 79.9%–89.4%) and 99.1% (95% CI 98.2%–
2 negative organisms from positive blood cultures: a systematic review and meta-analysis◊
42 99.5%), respectively, across the 15 studies, and 95.5% (95% CI 89.2%–98.2%) and 99.7% (95% CI
3 1
G. De Angelis , A. Grossi 2, G. Menchinelli 1, S. Boccia 2, 3, M. Sanguinetti 1, 4, *, B. Posteraro 5, 6
43 99.1%–99.9%), respectively, across the 5 studies.
4
44 Conclusions: Our findings show that the Verigeneâ and FilmArrayâ systems may be a valid
1)
5 Istituto di Microbiologia, Università Cattolica del Sacro Cuore, Rome, Italy
45 adjunct to the conventional microbiology (phenotypic or genotypic) methods used to identify AMR
2)
6 Sezione di Igiene, Istituto di Sanità Pubblica, Università Cattolica del Sacro Cuore, Rome, Italy
46 in GNBs. FilmArrayâ system detects only one AMR genotype, namely KPC, limiting its
3)
7 Dipartimento di Scienze della Salute della Donna e del Bambino e di Sanità Pubblica, Fondazione 2
47 utilization. Both Verigeneâ and FilmArrayâ systems can miss important
8 Policlinico Agostino Gemelli IRCCS, Rome, Italy
48 cephalosporin/carbapenem resistance phenotypes in a minority of cases. However, sensitivity and
4)
9 Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario
49 specificity of both systems render them valuable clinical tools in timely identification of resistant
0 A. Gemelli IRCCS, Rome, Italy
50 isolates. Further studies will establish the prominence of such rapid diagnostics as standard of care
5)
1 Istituto di Patologia Medica e Semeiotica Medica, Università Cattolica del Sacro Cuore, Rome,
51 in patients with bloodstream infections.
2 Italy
52 Keywords: Antimicrobial resistance, Blood cultures, Molecular diagnostics, Performance
6)
3 Dipartimento di Scienze Gastroenterologiche, Endocrino-Metaboliche e Nefro-Urologiche,
53 characteristics, Gram-negative bloodstream infections
4 Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
54
5 *Corresponding author. M. Sanguinetti, Istituto di Microbiologia, Fondazione Policlinico
6 Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168
7 Rome, Italy.
8 E-mail address: maurizio.sanguinetti@unicatt.it (M. Sanguinetti)
vittorio sambri CUEB 27 novembre 2019
04/12/2019
◊
pcr mdr
9 Presented in part at the 28th European Congress of Clinical Microbiology and Infectious Diseases,N. papers
250
200
150
N. papers
100
50
0
2018 2017 2016 2015
vittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrAnalisi dei Dati
Maggio – Giugno 2015
• 84 campioni per 78 pazienti
• 60% dei risultati TAT >2 giorni
Day 0 Day 1 Day 2 Day 3 Day 4
13% ricevuti Risultati
1pm (L-G) Day 2-3
19% campioni ricevuti Risultati
Venerdì, Sabato o festivi Day 3-4CPE Screening algorithm: from image analysis
to sample reporting
Negative Direct reporting on LIS
24h: Negative
Automatic streaking Automatic
on Chromogenic reading after 16
plates h of incubation Direct reporting on
LIS 24h:negative
Positive Unknown Patient for
CPE Id(Malditof) +
rapid molecular test
Direct reporting on
LIS 24h: positive
(target bacteria+
resistant gene)
Known patient for CPE:
Id(Malditof)+ confirmation
with sinergic test
Direct reporting Direct reporting on LIS 48h:
on LIS 24h: positive
Negative (target bacteria + phenotypic)
vittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdr04/12/2019 vittorio sambri CUEB 27 novembre 2019 pcr mdr
vittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdr1 Article
2 Comparison of four commercial screening assays for
3 detection of blaKPC, blaNDM, blaIMP, blaVIM and blaOXA48
4 from rectal secretion collected by swabs.
5 Francesca Del Bianco 1,*, Manuela Morotti 1, Silvia Zannoli 1, Giorgio Dirani 1, Michela Fantini 1,
6 Maria F Pedna 1, Patrizia Farabegoli 1 and Vittorio Sambri 1,2
7 1 Unit of M icrobiology, The Great Romagna H ub Laboratory, 47822 Pievesestina (FC), Italy
8 2 Department of Experimental, Diagnostic and Specialty M edicine, University of Bologna, 40126 Bologna,
9 Italy
10 * Correspondence: francesca.delbianco@auslromagna.com
11 Received: date; A ccepted: date; Published: date
12
13 A total of 1015 non-duplicated rectal swab specimens were prospectively collected
Abstract: The spread of carbapenem-resistant Enterobacteriaceae (CRE) has been enabled by the
lack of control measures directed at carriers of multidrug-resistant organisms in healthcare
14 settings. Screening patients for asymptomatic colonization and implementation of contact
15
16
using ESwab™ (COPAN Italia S.p.A., Brescia, Italy). The samples were transported to
precautions, on the other hand, reduce patient-to-patient transmission. Screening plates represents
a relatively low -cost method for isolating CRE from rectal sw abs, how ever molecular assays have
17 become w idely available. This study compared the performances of four commercial molecular
18
19
the Laboratory upon collection, processed within 24 hours and reported in 48 hours.
platforms in detecting clinically significant carbapenemase genes versus routine CRE screening
protocol. A total of 1015 non-duplicated rectal sw abs w ere cultured on chromogenic
20 carbapenem-resistant selective medium. A ll grow ing Enterobacteriaceae strains w ere tested for
21 carbapenemase related genes confirmation. The same specimens w ere processed using the
22 follow ing molecular assays: A llplex™ Entero-DR, A mplidiag® CarbaR+M CR, AusDiagnostics M T
23 CRE EU and EasyScreen™ ESBL/CPO Detection. The KPC-producing Enterobacteriaceae
24 prevalence detected by sw ab culture w as 2.2%, w hile the OXA -48 and M BLs-producing organisms
25 w ere infrequent. The cost of CRE related infection-control precautions, w hich must be kept in place
26 w hile w aiting for screening results, are significant, so the molecular tests could become
27 cost-competitive, especially w hen the turnaround time is decreased dramatically. M olecular assays
28 represent a pow erful diagnostic tool as they allow the rapid detection of the most
29 clinically-relevant carbapenemases.
30 Keywords: enterobacteriaceae; multidrug-resistant organisms; asymptomatic colonization;
31 screening assays.
32
33 1. Introduction
34 A ntimicrobial resistance is one of the most complex global health challenges [1]. The World
35 H ealth Organization puts development of new antibacterial agents to treat carbapenem-resistant
36 Enterobacteriaceae (CRE) among the most critical priorities [2]. Because CRE are resistant to the
37 majority of beta-lactams, carbapenem resistance has minimized the usefulness of many
38 commercially available drugs [3]. In addition to that, CRE frequently carry mechanisms conferring
39 resistance to other antimicrobial classes, thus further limiting the available therapeutic options [4-6].
40 Resistance to carbapenems is typically based on tw o main mechanisms. The first one is related to
41 structural mutations combined w ith the activity of other β-lactamases, such as A mpC
42 cephalosporinase (A mpC) and Extended spectrum beta-lactamases (ESβL). The second, and likely
43 the most important mechanism is the carbapenemase enzyme production. These versatile
44 beta-lactamases are able to hydrolize carbapenems and other beta-lactam antibiotics [7-10].
Microorganisms 2019, 7, x; doi: FOR PEER REVIEW w w w .mdpi.com/journal/microorganisms
vittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrvittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrWhen I say “we”………. I mean THEM
vittorio sambri CUEB 27 novembre 2019
04/12/2019
pcr mdrYou can also read
