La diagnosi microbiologica delle infezioni batterichenel2019 · Fondazione Policlinico...

La diagnosi microbiologica delle infezioni batteriche nel 2019

Teresa Spanu

Fondazione Policlinico Universitario A. Gemelli, IRCSSUniversità Cattolica del Sacro CuoreIstituto di Microbiologia

Fondazione Policlinico Universitario A. Gemelli, IRCCSUniversità Cattolica del Sacro CuoreIstituto di Microbiologia

Microbiological diagnostic stewardship “coordinated guidance and interventions to improve appropriate use of microbiological

diagnostics to guide therapeutic decisions. It should promote appropriate, timely diagnostic testing, including specimen

collection, and pathogen identification and accurate, timely reporting of results to guide patient treatment

Clinical bacteriology is a discipline that focuses on rapidly characterizing pathogen samples to

direct the management of individual infected patients (diagnostic microbiology) and to

monitor the epidemiology of infectious disease (public health microbiology)

HOT TOPIC/ Diagnosis of bacterial infections

Time toresults

3

• Non lab time• Ordering time

• Collection time

Significant delays between collection of samples and lab results. Delays can occur both at the patient's bedside and in the laboratory

To the lab AS SOON AS POSSIBLE for entry in the blood culture system

• Trasportation time

• Lab time: hours of opening

The median time from blood culture collection to growth detection was reduced by 10.1 h in the Bactec ON arm

(12/7 vs 24/7)

Murray PR, Masur H. Crit Care Med. 2012 Dec;40(12):3277-82.

•Time to growth



Banerjee et al . Clin Infect Dis. 2016 15;63:1332-1339



P Morency-Potvin et al. CMR 2016

.

Blue arrows represent the conventional microbiology pathway, orange arrows represent the RDT pathway, and green arrows represent opportunities for the laboratory and antimicrobial stewardship teams to improve communication of results. AST, antimicrobial susceptibility testing.

There are a number of changes underway in modern clinical microbiology laboratories

SIR - new definitions 2019

Susceptible

Normal exposureNormal

exposureIncreasedexposureIncreasedexposure

Resistant



Gunnar Kahlmeter and the EUCAST Steering Committee



HE = High exposure for agent (see table of dosages, last tab in breakpoint table)

HE= high exposure

Pseudomonas spp.


HE= high exposure




All clinical breakpoints are related to the achievable level of exposure* of the microorganism.

The achievable level of exposure* depends on many factors. Individual differences in pharmacokinetics are allowed for in the calculations leading up to pharmacodynamic indices following population simulation. Others factors as follows are determined by the the site of infection or can be varied during therapy:

1. Site of infection – concentration in certain tissues and body fluids may be high (urine, bile, lymphatic

tissues).2. Dose and dosing frequency3. Mode of administration (Oral, Intravenous, IV infusion etc)

*Exposure is a function of how the mode of administration, dose, dosing interval, infusion time, as well as distribution,

metabolism and excretion of the antimicrobial agent will influence the infecting organism at the site of infection.

Gunnar Kahlmeter and the EUCAST Steering Committee

Acceleration of identification of positive blood cultures by MALDI-TOF MS

Short term incubation cultures: The incubation time needed to achieve species-level identification was 5.9 and 2.0 h for Gram-positive aerobic cocci (GPC, n = 86) and Gram-negative aerobic rods (GNR, n = 42), respectively. Idelevich EA. CMI. 2014 Apr 3.

Detection of microbial growth

Lab resultsPLoS One.2009; 4:e8041S


Bruker's MALDI Biotyper ; Vitek MS Funke DJ, Kumar A. Critical Care Clinics 2011 27: 53-76; May 2011 Clinical Laboratory News: Bloodstream Pathogens Fournier et al. Nat Rev Microbiol. 2013; 11:574-585Bauer et al. Clin Infect Dis. 2014.59 : S134Advice E, Carroll KC. Infect Dis Clin North Am. 2014 Jun;28(2):215-35 Clinical Infectious Diseases 2009; 49:543–51 AST, antimicrobial susceptibility testing; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry


Rapid AST directly from blood culture bottlesEUCAST has published recommendations for short incubation (4, 6 and 8 hours) AST directly from positive blood culture bottles:


Riedel and Carroll, 2016 Clin Chest Med

Acceleration of identification of positive blood cultures by molecular methods



Ramanan et al. CMR 2018Riedel and Carroll, 2016 Clin Chest Med

Although multiplex assays have potential benefits over routine testing by reducing turnaround time, there are certain disadvantages to consider. These tests do not overcome the technical and sensitivity issues of blood cultures (BCs), detect only a limited spectrum of microorganisms, may not be capable of identifying all pathogens in mixed infections, even if the organisms are included in the panel and are largely “add-on” tests. False-positive results may also occur. To realize maximal benefits, these assays should be performed 24 h a day/7 days a week, adding logistical hurdles for both the laboratory and the stewardship system

BSI, bloodstream infection

HOT TOPIC/ Microbiological diagnosis of bacterial infections

Acceleration of identification of positive blood cultures by molecular methods

In order to enable the rapid escalation or deescalationof antimicrobial therapy, the results of these assays should be reported to providers as rapidly and directly as possible and should also ideally be communicated to an expert in antimicrobial stewardship who can work with the providers to optimize therapy

Studies evaluating the effects of panel-based molecular assays for detection of select microorganisms and resistance genes in positive blood culture bottles on clinical and economic outcomes for patients with bacteremia


Ramanan et al. CMR 2017



Opota et al . CMI 2015

Blood-culture (BC) independent nucleic acid methods for the microbial diagnosis of bacteremia



BC-independent nucleic acid amplification-based detection of pathogen-associated molecular patterns applied on whole blood does not only circumvent the limiting BC step but also provides a much lower turnaround time

Banerjee et al. CID. 2016 15;63:1332-1339Opota et al . CMI 2015Riedel and Carroll, 2016 Clin Chest Med

BC-based nucleic acid methods


1-2.5 h



Mwaigwisya et al. 2015; Expert Rev. Mol. Diagn. 15(5), 681–69

Because of current limitations, these assays are used to complement blood culture rather than replace it



CSF PCR demonstrates sensitivity of 87-100 % and specificity of 98-100%, respectively. It is especially useful if antibiotics have been given prior to lumbar puncture

ABM, acute bacterial meningitis; CSF, cerebrospinal fluid; PCR, polymerase chain reaction

Workflow pathways for diagnosing ABM: CSF PCR



ESCMID guidelines, 2016IDSA guidelines, 2004UK guidelines, 2016

Multiplex respiratory panel


Allplex

√

√√

√

√Legionella pneumophila

√√√

√√

√

√

Allplex





Echaverria et al Journal of Clinical Virology 2018

Diagnostic yield was significantly higher by FilmArray-RP (81%) than by immunofluorescence assay (IFA; 31%)(p < 0.001)

Improved clinical sensitivity and range of pathogens detected by FilmArray™ compared to immunofluorescence assay



Echaverria et al Journal of Clinical Virology 2018

Impact on Clinical Decision-making

R Patel. Mayo Clin Proc. 2016





FilmArray detected pathogens in 35.3% of specimens, compared to 6.0% for culture. Median time from collection to result was 18 hours for FilmArray and 47 hours for culture.

Cybulski et al. CID 2018

Improved clinical sensitivity and range of pathogens detected by FilmArray™ compared to traditional stool culture



Patients with an infectious cause of acute gastroenteritis detected by FilmArray (B) were more likely (P = .0148) to receive targeted rather than empirical therapy when compared to those detected by culture (A)

Cybulski et al. CID 2018

Impact on Clinical Decision-making

(A) (B)



Test ordering guidance for infectious diarrhea

R Patel. Mayo Clin Proc. 2016

aWarning signs and risk factors for severe disease include fever, bloody diarrhea, dysentery, severe abdominal pain, dehydration, hospitalization, and immunocompromised state. bGastrointestinal Pathogen Molecular Panels test for common bacterial, viral and parasitic causes of diarrhea.cSubmit 3 stool collected on separate days for maximum sensitivity.

Multiplex syndromic panels are more sensitive than conventional methods, although questions remain regarding clinical utility and cost effectiveness.

Blood cultures grew clinically significant bacteria for 12 out of the 75 patients (16%)

In comparison, 16S metagenomics detected clinically significant bacteria in 18 patients (24%), including 8 of the 12 blood culture positive patients and 10 patients with a negative blood culture. In total, 22 patients (29.3%) were classified as confirmed bBSI by blood culture and/or 16S metagenomics

bBSI, bacterial bloodstream infection



Paisse et al. Transfusion 2016

Comprehensive description of blood microbiome from healthy donors assessed by 16S targeted metagenomic sequencing

Most of the blood bacterial DNA is located in the BC (93.74%), and RBCs contain more bacterial DNA (6.23%) than the plasma (0.03%). The distribution of 16S DNA is different for each fraction and spreads over a relatively broad range among donors. BC, buffy coatRBC, red blood cells



Caliendo et al. Clin Infect Dis 2013;57:S139–5175Murdoch D R et al. JCM 2009; 47: 3405-08Banerjee et al . Clin Infect Dis. 2016 15;63:1332-1339

We engage an educational campaign to teach clinician teams appropriate indications for ordering, collection, transportation and placement of BC bottles

Guidance in the preanalytic stage at the Fondazione Policlinico UniversitarioA. Gemelli IRCCS

BC, blood culture

Check-in

Sample entry (24 X 7)

Transportation time

Collection time

Ordering time




Menchinelli et al. Sumitted


Efficient Inactivation of Clinically Relevant Antimicrobial Drug Concentrations by BACT/ALERT® or BACTEC™ Resin-Containing Media in Simulated Adult Blood Cultures

At concentrations almostequivalent to the lowest possiblelevels of antibiotics in the plasma, both BACT/ALERT® and BACTECTM

Plus media were efficient against13 antibiotics, with the exceptionof meropenem (but only in combination with E. coli or K.

pneumoniae).


Fiori B et al JCM 2016; Bruker's MALDI Biotyper ; Vitek MS Funke DJ, Kumar A. Critical Care Clinics 2011 27: 53-76; May 2011 Clinical Laboratory News: Bloodstream Pathogens Fournier et al. Nat Rev Microbiol. 2013; 11:574-585Bauer et al. Clin Infect Dis. 2014.59 : S134Advice E, Carroll KC. Infect Dis Clin North Am. 2014 Jun;28(2):215-35 Clinical Infectious Diseases 2009; 49:543–51AST, antimicrobial susceptibility testing; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry


Acceleration of identification of positive blood culturesHOT TOPIC/ Diagnosis of bacterial infections

Rapid AST directly from blood culture bottles

Lab results

Workflow for rapid identification of BSIs at the clinical microbiology laboratoryof Fondazione Policlinico Universitario Gemelli

FilmArray BCID


MRSA POSITIVE

Parameter Result

A1 S.aureus positive

A2 mecA positive

A3 mecC negative

A4 S.epidermidis negative

A5 Sample control valid

A6 Inhibition control valid

A7

A8

Detection of resistance genes

Infectious diseaseconsultant AST

Lab results

MALDI-TOF MS


BSI, bloodstream infection; AST, antimicrobial susceptibility testing; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, BC, blood cultureGenes detected: mecA, mecC, CTX-M-1, CTX-M-9; KPC, VIM, NDM-1, OXA-48, OXA-23, OXA-181




The appropriateness of antibiotic treatments was assessed at the time of BC collection (empirical therapy) and after notification of the procedure (targeted therapy) or AST (definitive therapy) results

Appropriateness of antimicrobial treatments in subgroups defined by the infecting pathogen's resistance status

Abbreviations: BSI, bloodstream infections; none refers to strains not carrying carbapenemase, extended-spectrum and plasmid-mediated AmpC β-lactamase genes

Fiori et al. submitted


Development of a multiplex PCR platform for rapid, culture-independent detection of bacteria, antibiotic resistance, and Candida in human blood samples

Liotti et al. submitted

The sensitivity and specificity were 86.6% and 97.0% among patients with positive BCs for the organisms targeted by the assay, or fulfilling criteria for infection. The mean times to positive or negative assay’s results were 5.3 ± 0.2 h and 5.1 ± 0.1 h, respectively.



Results

Work-flow for diagnosis of ABM at the microbiology laboratory of Fondazione Policlinico Universitario Gemelli IRCCS

ABM, acute bacterial meningitis; ID, identification AST, antimicrobial susceptibility testing;

Culture-based method Microscopic examination Molecular methods

16SrRNA PCR



Results for 48 patients with bacterial meningitis by method

Results of

Microscopic examination

Culture testing

Rapid molecular method

PCR and sequencing

L. monocytogenes 3 6 6 6

N. meningitidis 4 3 9 9

S. agalactiae 1 1 1 1

S. pneumoniae 15 14 20 20

E. coli 2 3 3 3

C. koseri 1 1 0 1

F. nucleatum 1 0 0 1

E. cloacae 0 1 0 1

S.marcescens 0 1 0 1

S. pyogenes 2 2 0 2

Total 29 32 39 45

From 117 samples, bacterial DNA was detected in 45 (38.5%) and virus in 14 (12%). The rapid molecular method detected 1 pathogen in 39 (33.3%) specimens, compared to 32 (27.3%) detected by culture and to 29 (24.7) by microscopic examination

Multiplex respiratory panel


√





Microbial pathogens identified in 63 samples collected from 67 patients with pneumonia

Sample result Bacteria MoldsBacteria+

moldsBacteria+

virus

Bacteria+ molds+ virus

Virus

Positive 441 22 1 9 23 5

1 Mycobacterium tuberculosis (1)2 Pneumocystis jirovecii(1)3 Pneumocystis jirovecii (1)

A total of 82 microorganisms were detected in 54 samples. Of those, 79 (9%) weredetected by FilmArray pneumonia and 64 (81.4%) by the culture based method.

Additional resultsby Culture

Additional results by FilmArray

Concordantresults

Acinetobacter baumannii 0 1 9Aspergillus fumigatus* 2 0 0Escherichia coli 0 1 7Haemophilus influenzae 0 6 9Klebsiella aerogenes 0 0 2Klebsiella pneumoniae 0 0 2Legionella pneumophila 1 0 3Moraxella catharralis 0 1 4Mycoplasma pneumoniae 0 1 0Pseudomonas aeruginosa 0 2 6Staphylococcus aureus 0 3 13Streptococus pneumoniae 0 3 5Streptococus pyogenes 0 0 1Total 3 18 61

Results of the clinical evaluation of Film Array Pneumonia panel HOT TOPIC/ Diagnosis of bacterial infections


*Species not detectable by the FilmArray pneumonia panel


Antibiogram data

Clinical microbiology laboratories also contribute to the success of stewardship programs through compilation of aggregate antimicrobial susceptibility data

Batteri Gram Negativi N° isolati

Am

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lavu

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Pip

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cill

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bact

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fota

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Ce

ftazi

dim

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Ce

fep

ime

Ert

ap

ene

m

Me

rop

en

em

Imip

en

em

am

ikaci

na

Ge

nta

mic

ina

Cip

rofl

oxaci

na

Co

trim

oxazo

lo

Co

list

ina

Tig

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clin

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Acinetobacter baumanni 5 R R 0% 0% 0% 0% 0% 100%

Citrobacter freundii 0

Enterobacter aerogenes 1 R R 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%

Enterobacter cloacae 2 R R 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%

Escherichia coli 3 33% 100% 100% 100% 100% 100% 100% 100% 100% 67% 67% 100% 100%

Klebsiella pneumoniae 4 R 100% 100% 75% 75% 75% 100% 100% 100% 100% 100% 75% 75% 100%

Morganella morganii 1 R R 100% 0% 0% 0% 100% 100% 100% 0% 0% 0% R R

Proteus mirabilis 2 100% 100% 100% 100% 100% 100% 100% 100% 50% 50% 100% R R

Pseudomonas aeruginosa 2 R R 0% 0% 0% R 100% 100% 100% 100% 100% R R

Serratia marcescens 7 R R 100% 100% 100% 100% 100% 100% 100% R 71% 100% 100% R

Stenotrophomonas maltophilia 0

>=90% Sensibili

70-89 % Sensibili

< 70% Sensibili

Intrinsecamente resistenti

Antibiogramma cumulativo-Materiali Invasivi

Periodo 01/01/2016 - 30/09/2018

Percentuali di sensibilità


• Molecular methods based on seeking specific targets for pathogen identification and resistance genes are likely to be phased out in the coming years and replaced by more comprehensive methods, such as MALDI-TOF MS and genomic sequences method.

• These techniques are expected to contribute substantially to improving the etiological diagnosis and precision medicine.

• The tremendous promise of these new methods will require the successful development of clinical microbiologists capable of interpreting and evaluating microbiological data and placing these data in the appropriate clinical context.

• Appropriate budgets are need to make available these activities.

Concluding remarks

MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry



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