Detection of Carbapenemase and CTX-M Encoding Genes Directly from Bronchoalveolar Lavage Using the CRE and ESBL ELITe MGB Assays: Toward Early and Optimal Antibiotic Therapy Management of Critically Ill Patients with Pneumonia

Abstract

The detection of carbapenemase extended-spectrum β-lactamase (ESBL)-producing Enterobacterales (EB) has become a major issue among critically ill patients, especially due to their impact on appropriate antimicrobial therapy. This study aimed at evaluating the potential contribution of molecular assays to early optimization of empirical antibiotic therapy among critically ill patients with carbapenemase- and/or CTX-M-producing EB pneumonia. The CRE and ESBL ELITe MGB^® assays were evaluated directly on 197 bronchoalveolar lavage (BAL) samples obtained from 120 patients. Molecular results were then compared to routine culture-based diagnostic results, and a retrospective analysis of the therapeutic antimicrobial management was performed. Among the 197 clinical specimens, bla_KPC-like and bla_CTX-M-like were detected in 20 (10.2%) and 12 (6.1%) specimens belonging to 15 and 11 patients, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of the CRE ELITe MGB Kit were 85% [95% confidence interval [CI]: 64.9–94.6] and 100%, respectively. PPV and NPV of the ESBL ELITe MGB Kit were 75% [95% CI: 49.4–90.2] and 100%, respectively. Retrospective analysis of the therapeutic antimicrobial management at the time of BAL collection showed that in ∼50% of patients with carbapenemase- and CTX-M-producing EB pneumonia empirical antibiotic therapy could have been optimized at least 48–72 hr earlier if positive molecular data had been used. The CRE and ESBL ELITe MGB assays might be an interesting tool for expediting optimization of empirical antibiotic therapy in critically ill patients with pneumonia, depending on local epidemiology of antibiotic resistance, patient risk stratification for EB infection, and availability of an antimicrobial stewardship team.

Introduction

Pneumonia requiring care in an Intensive Care Unit (ICU) is associated with considerable morbidity, mortality, length of stay, and high hospital costs, especially in patients with significant comorbidities.^1–6 Multidrug resistant (MDR) Gram-negative bacteria are responsible for most bacterial cases of pneumonia among critically ill patients.^7,8 Adequate empirical therapy should be based on local epidemiology and patient's risk stratification for MDR pathogens. However, the growing threat of antimicrobial resistance hampers the choice of empirical treatment demanding a rapid turnover of microbiological results. In this regard, the detection of carbapenemase- and extended-spectrum β-lactamase (ESBL)-producing Enterobacterales (EB) has become a major issue among ICU patients, due to their impact on infection control measures and prompt and appropriate antimicrobial therapy.

Conventional phenotypic diagnostics for respiratory samples typically takes 48–72 hr, leading to an inconvenient delay in pathogen identification and load quantification, which compromises targeted therapy and risk reduction for antibiotic resistance selection.

Molecular tests targeting carbapenemase and ESBL encoding genes have been successfully used directly from blood culture^9,10 and respiratory samples^11–15 but there is limited evidence about their potential impact on early optimization of empirical antimicrobial management among critically ill patients with pneumonia.

The platform ELITe InGenius^® (ELITechGroup Molecular Diagnostics, Turin, Italy) is an integrated system that automatically performs nucleic acid extraction, real-time PCR, and interpretation of results in less than 3 hr. The CRE and ESBL ELITe MGB^® Kits are two qualitative multiplex real-time PCR assays for the detection of the most prevalent carbapenemase and ESBL encoding genes in EB, respectively. The CRE ELITe MGB Kit can detect bla_KPC-like, metallo β-lactamase (i.e., bla_NDM-like, bla_VIM-like, bla_IMP-like), and bla_OXA-48-like genes, whereas the ESBL ELITe MGB Kit can identify bla_CTX-Ms genes belonging to group 1 (including CTX-M-15) and group 9 (including CTX-M-14).

This study aimed at evaluating the performance of CRE and ESBL ELITe MGB assays on bronchoalveolar lavage (BAL) of critically ill patients. This study also intends to present how molecular assays detecting antibiotic resistance genes would shorten delays to optimal therapy and impact on early optimization of empirical antibiotic therapy among critically ill patients with pneumonia.

Materials and Methods

Routine culture-based microbiological diagnostics

At the Microbiology and Virology Unit of “Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino” (Turin, Italy) BAL samples were subjected to Gram staining and subculture on appropriate solid medium at the time of arrival to the laboratory. Matrix assisted laser desorption ionization time-of-flight mass spectrometry analysis was used for bacterial identification, and antimicrobial susceptibility testing was carried out on overnight subcultures using MicroScan WalkAway plus System (Beckman Coulter, Brea, CA) according to the manufacturer's instructions. Antimicrobial susceptibilities were interpreted according to EUCAST breakpoints as updated in 2019.¹⁶ Furthermore, the Total ESBL Confirm Kit (Rosco, Taastrup, Denmark) was used to identify ESBL production if cefotaxime (CTX) and/or ceftazidime (CAZ) minimal inhibitory concentrations (MICs) were >1 mg/L. MASTDISCS^® combi Carba plus disc system (Mast Group Ltd, Bootle, United Kingdom) was used to characterize carbapenemase producers when meropenem (MEM) MIC was >0.125 mg/L. Detection of carbapenem resistance genes was carried out using the Xpert Carba-R assay (Cepheid, Sunnyvale, CA).

Specimen collection and study design

BAL samples included in the study were obtained from critically ill patients and consisted of samples submitted for standard of care bacterial culture, which were prospectively collected for a 6-month period.

The CRE and ESBL ELITe MGB Kits were assayed using InGenius system directly on 197 BAL samples obtained from 120 patients, starting from 200 μL of a 1:2 dilution in dithiothreitol solution (Sputasol, Oxoid Ltd, Basingstoke, United Kingdom) previously heated in a thermoblock at 90°C for 5 min. The ELITe MGB Kits' internal control, as well as positive and negative controls, was used as previously described.¹⁷

Molecular results were then compared to routine culture-based microbiological diagnostic results to estimate the accuracy of genotypic analysis. Molecular results for the CRE and ESBL targets were interpreted as shown in Table 1. All cycles with a Ct value >35 were considered negative for detectable signal. Routine culture-based microbiological diagnostic result was considered positive when carbapenemase- and/or ESBL-producing EB was detected.

Table 1.

Interpretation of Molecular Results on Bronchoalveolar Lavage Samples for the Detection of Carbapenemase and ESBL Encoding Genes

CRE ELITe MGB^® Kit	ESBL ELITe MGB Kit	Interpretation	Report
+	−	Carbapenemase encoding gene	KPC, NDM-IMP-VIM, OXA-48 DNA detected
+	+	Carbapenemase and ESBL encoding genes	Both KPC, NDM-IMP-VIM, OXA-48 DNA detected and CTX-M_s DNA detected
−	+	ESBL encoding gene	CTX-M DNA detected
−	−	Neither carbapenemase nor ESBL encoding genes	Neither KPC, NDM-IMP-VIM, OXA-48 nor CTX-M_s DNA detected

All cycles with a cycle threshold (Ct) value >35 were considered negative.

ESBL, extended-spectrum β-lactamase.

Accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the CRE and ESBL ELITe MGB Kits with 95% confidence interval (95% CI) were computed using the free software MedCalc website.

Subsequently, a retrospective analysis of the therapeutic antimicrobial management of the patients with positive molecular results at the time of the first BAL collection was performed to evaluate the potential contribution of genotypic data to early optimization of empirical antibiotic therapy. Empirical therapy was deemed optimal when causative bacterial strains were susceptible in vitro to at least one prescribed drug and the antibiotics prescribed were in accordance with current evidence and recommendations regarding the treatment of carbapenemase- and/or ESBL-producing EB infections.^18–22

Finally, according to the obtained results, an algorithm for the application of molecular assays detecting the main carbapenemases and ESBL encoding genes in critically ill patients with pneumonia was presented, providing a comprehensive microbiological response to clinicians in case of discordance between molecular and conventional phenotypic results.

Results

Detection of carbapenemase and CTX-M encoding genes

Table 2 shows the comparison between molecular and conventional phenotypic results.

Table 2.

Performance of the CRE and ESBL ELITe MGB Kits on 197 Bronchoalveolar Lavage Samples Compared with Conventional Phenotypic Results

CRE, ESBL ELITe MGB^® Kits	Conventional phenotypic results		Accuracy	Sensitivity [95% CI]	Specificity [95% CI]	PPV [95% CI]	NPV [95% CI]
CRE, ESBL ELITe MGB^® Kits	Positive	Negative	Accuracy	Sensitivity [95% CI]	Specificity [95% CI]	PPV [95% CI]	NPV [95% CI]
bla _KPC-like
Positive	17	3	100%	100% [81.6–100]	98.3% [95.2–99.4]	85% [64.9–94.6]	100%
Negative	0	177	100%	100% [81.6–100]	98.3% [95.2–99.4]	85% [64.9–94.6]	100%
bla _CTX-M-like
Positive	9	3	100%	100% [70.1–100]	98.4% [95.4–99.5]	75% [49.4–90.2]	100%
Negative	0	185	100%	100% [70.1–100]	98.4% [95.4–99.5]	75% [49.4–90.2]	100%

NPV, negative predictive value; PPV, positive predictive value.

Accuracy, sensitivity, specificity, PPV, and NPV of the CRE ELITe MGB Kit were 100%, 100% [95% CI: 81.6–100], 98.3% [95% CI: 95.2–99.4], 85% [95% CI: 64.9–94.6], and 100%, respectively.

Among the 197 clinical specimens, the CRE ELITe MGB Kit detected bla_KPC-like in 20 (10.2%) specimens collected from 15 patients. Seventeen (85%) bla_KPC-like positive specimens had concomitant positive culture results with bacterial load >10⁴ CFU/mL. bla_KPC-like was not detected in 177 specimens showing 100% accuracy. The three presumed false positive samples were collected from patients who presented culture positive to KPC-producing Klebsiella pneumoniae in the following 7 days (blood culture = 1, tracheal aspirate = 1, bronchoaspirate = 1). Neither bla_{NDM-IMP-VIM-like} nor bla_OXA-like genes were detected by the CRE ELITe MGB Kit, and no samples yielded positive culture results.

Accuracy, sensitivity, specificity, PPV, and NPV of the ESBL ELITe MGB Kit were 100%, 100% [95% CI: 70.1–100], 98.4% [95% CI: 95.4–99.5], 75% [95% CI: 49.4–90.2], and 100%, respectively.

Among the 197 clinical specimens, bla_CTX-M-like was detected in 12 (6.1%) specimens belonging to 11 patients by ESBL ELITe MGB Kit. bla_CTX_-M-like was found in four (2%) positive bla_KPC-like positive specimens that were accounted for the bla_KPC-like group. Nine (75%) bla_CTX-M-like positive specimens also had concordant positive culture results with bacterial load >10⁴ CFU/mL. bla_CTX-M-like was not detected in 185 specimens, and all these samples (100%) yielded negative culture results for ESBL-producing EB. The three presumed false positive samples belonged to patients which were on empirical antibiotic therapy theoretically active against ESBL-producing EB at the time of BAL sample collection (piperacillin/tazobactam = 2; ceftolozane/tazobactam = 1).

Potential impact of molecular assays detecting the main carbapenemase and CTX-M encoding genes on early optimization of empirical antibiotic therapy of critically ill patients with pneumonia

Analysis of the empirical antibiotic therapy at the time of the first BAL collection among critically ill patients with carbapenemase- and/or CTX-M-producing EB pneumonia is reported in Table 3.

Table 3.

Analysis of the Empirical Antibiotic Therapy at the Time of the First Bronchoalveolar Lavage Collection Among Critically Ill Patients with Carbapenemase- and/or ESBL-Producing EB Pneumonia

	Empirical therapy
KPC-producing EB pneumonia (n = 15)^a	Optimal (n = 3) CZA+VAN (n = 1) MEM+TGC+CST (n = 1) MEM+ETP+GEN (n = 1)	Nonoptimal (n = 10) MEM+VAN (n = 3) PTZ+LVX (n = 2) PTZ+SXT (n = 1) GEN+FOS (n = 1) CRO+LZD+GEN (n = 1) LVX+VAN (n = 1) DAP (n = 1)
CTX-M-producing EB pneumonia (n = 11)^b	Optimal (n = 5) PTZ+LVX (n = 2) PTZ (n = 1) CZT+FOS (n = 1) MEM+VAN (n = 1)	Nonoptimal (n = 3) PTZ (n = 2) FEP (n = 1)

Two patients with negative culture-based results were on empirical antibiotic therapy with PTZ+LVX+LZD (n = 1) and PTZ (n = 1).

Three patients with negative culture-based results were on empirical antibiotic therapy with PTZ (n = 2) and CZT (n = 1).

CRO, ceftriaxone; CST, colistin; CZA, ceftazidime/avibactam; CZT, ceftolozane/tazobactam; DAP, daptomycin; EB, Enterobacterales; ETP, ertapenem; FEP, cefepime; FOS, fosfomycin; GEN, gentamycin; LVX, levofloxacin; LZD, linezolid; MEM, meropenem; PTZ, piperacillin/tazobactam; SXT, trimethoprim–sulfamethoxazole; TGC, tigecycline; VAN, vancomycin.

Among patients with KPC-producing EB pneumonia (n = 15), 3 and 12 patients were on optimal and nonoptimal empirical antibiotic therapy, respectively, while in 2 patients the culture-based approach yielded negative results.

Among patients with CTX-M-producing EB pneumonia (n = 11), five and three patients were on optimal and nonoptimal empirical antibiotic therapy, respectively, while in three patients the culture-based approach yielded negative results.

Overall, in ∼50% of patients (13/26 or 13/21 if excluding patients with negative culture-based results) empirical antibiotic therapy could have been optimized at least 48–72 hr earlier if positive molecular data had been used.

Algorithm for the application of molecular assays detecting the main carbapenemase and CTX-M encoding genes in critically ill patients with pneumonia

The use of molecular assays detecting the main carbapenemase and CTX-M encoding genes on BAL could be implemented to early optimize empirical antibiotic therapy and infection control practices in critically ill patients with pneumonia, as shown in Fig. 1.

FIG. 1.

Algorithm for the application of molecular assays detecting the main carbapenemase and ESBL encoding genes directly from bronchoalveolar lavage of critically ill patients with pneumonia. *Targeted therapy toward isolated pathogens expressing resistance genes undetectable by ELITe MGB^® assays or without Targeted therapy toward isolated pathogens expressing resistance genes undetectable by ELITe MGB assays or without β-lactam resistance mechanisms. EB, Enterobacterales; ESBL, extended-spectrum β-lactamase.

In case of a positive result on either CRE or ESBL ELITe MGB Kits, infection control measures should be strengthened and optimal therapy considered. If a positive molecular result is confirmed by the conventional phenotypic approach, targeted therapy would then be recommended. Conversely, if the positive molecular result is not confirmed by conventional phenotypic methods, extremely low bacterial load, previous antibiotic therapy, and inactivation or downregulation of antibiotic resistance genes should be borne in mind and optimal therapy considered.

In case of a negative result on CRE and ESBL ELITe MGB Kits together with a positive conventional phenotypic result, other resistance genes or mechanisms, as well as mutations affecting annealing of primers, should be considered. However, infection control measures should be strengthened and targeted therapy considered. Conversely, if the molecular negative result was confirmed by conventional phenotypic approach, targeted therapy toward isolated pathogens expressing resistance genes undetectable by ELITe MGB assays or without β-lactam resistance mechanisms would then be recommended.

Discussion

Current guidelines for the management of MDR Gram-negative pneumonia^23,24 provide highly sensitive and insufficiently specific clinical recommendations, while empirical antibiotic therapy is to be mostly guided by the patient's hemodynamic status, leading to excessive broad spectrum empirical therapy.^25,26 Although genotypic methods are not currently recommended by international guidelines for the microbiological diagnosis of pneumonia in critically ill patients and evidence regarding their impact on therapeutic management is still undefined, the role of molecular assays in clinical practice is broadly shared,^27–29 especially in high MDR endemicity settings and in solid organ transplant recipients.²⁰

In the European scenario where carbapenemases and CTX-M variants are the major drivers of carbapenem resistance and ESBL production,³⁰ respectively, the use of molecular assays detecting the most widespread antibiotic resistance genes among EB directly from BAL sample seems to be reasonable and highly warranted.

In this study, the CRE and ESBL ELITe MGB Kits showed high NPV and more variable PPV.

Despite NPV suggesting a strong correlation with negative conventional culture, the antibiotic resistance genes, which were tested, are limited and do not include other less common mechanisms that could play a role in such resistance. For this reason, negative CRE and ESBL ELITe MGB assay results do not allow definite exclusion of resistance toward main beta-lactam classes. Conversely, PPV showed wide confidence intervals because the ELITe MGB Kits provided in six samples a positive molecular result despite negative conventional culture. However, among these presumed false positive samples, three were collected from patients who eventually became culture positive in the next 7 days and the other three were collected from patients who were on appropriate empirical therapy before BAL collection. All these findings may allow us to presume that confidence intervals of sensitivity and PPV were probably narrower and that molecular data could, in some cases, reveal the positive conventional culture result a few days in advance.

This study also provides an interesting view on the potential contribution of molecular results in the early optimization of empirical therapy and/or prevents dissemination at least 48 hr prior to the availability of conventional culture-based results. Indeed, in ∼50% of patients empirical antimicrobial therapy could have been optimized on the same day of bronchofibroscopy execution, contributing theoretically to reduce time of inappropriate therapy, longer antibiotic courses, use of second-line antibiotic regimens, risk of antibiotic resistance selection, and poor clinical outcomes. Given that molecular results together with knowledge of local epidemiology susceptibility patterns and antibiotic stewardship programs could be used to expedite optimization of empirical antibiotic therapy and infection control practices,^11,31 the algorithm proposed in this study showed the potential for the use of CRE, ESBL ELITe MGB Kits in clinical practice when managing critically ill patients with higher risk for MDR EB pneumonia. However, conventional culture-based antimicrobial susceptibility testing continues to be required to confirm molecular results and to detect other antibiotic resistance mechanisms.

One of the limitations of this study was the lack of prospective assessment of direct implications of molecular results on antimicrobial stewardship, clinical outcomes, and cost effectiveness. Furthermore, it was not known how molecular data could have impacted on clinicians' confidence to change antibiotic therapy, especially to de-escalation since the main resistance mechanisms of Pseudomonas aeruginosa and Acinetobacter baumannii cannot be detected by the ELITe MGB Kits. Unfortunately, we did not evaluate the CRE and ESBL ELITe MGB assays on BAL samples with metallo β-lactamase- and OXA-48-like-producing EB, as they were not present during the study period.

In conclusion, the ELITe MGB assays showed high accuracy for the detection of the main carbapenemases and ESBL encoding genes from BAL of critically ill patients with pneumonia caused by EB. These assays might be an interesting tool for expediting optimization of empirical antibiotic therapy and infection control practices, depending on local epidemiology of antibiotic resistance, patient risk factors associated with MDR EB infection, and availability of an antimicrobial stewardship team with well-established fast track diagnostics. However, further studies are required to confirm these results, assessing the clinical impact of the ELITe MGB assays on antimicrobial stewardship, patient outcome, and infection control programs and determining their cost effectiveness.

Ethical Approval

This study was conducted in accordance with the Declaration of Helsinki. Formal ethical approval was obtained by our Center's institutional review board (Protocol No. 0029345).

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this study.

References

Waterer

G.W.

, Self

W.H

, Courtney

D.M

, Grijalva

C.G

, Balk

R.A

, Girard

T.D

, Fakhran

S.S

, Trabue

, Mc Nabb

, Anderson

E.J

, Williams

D.J

, Bramley

A.M

, Jain

, Edwards

K.M

, and Wunderink

R.G.

. 2018. In-hospital deaths among adults with community-acquired pneumonia. Chest. 154:628–635.

Hortal

, Giannella

, Pérez

M.J

, Barrio

J.M

, Desco

, Bouza

, and Muñoz

. 2009. Incidence and risk factors for ventilator-associated pneumonia after major heart surgery. Intensive Care Med. 35:1518–1525.

Tejerina

, Frutos-Vivar

, Restrepo

M.I

, Anzueto

, Abroug

, Palizas

, González

, D'Empaire

, Apezteguía

, and Esteban

. 2006. Incidence, risk factors, and outcome of ventilator-associated pneumonia. J Crit Care. 21:56–65.

Ranjan

, Chaudhary

, Chaudhry

, and Ranjan

K.P.

. 2014. Ventilator-associated pneumonia in a tertiary care intensive care unit: analysis of incidence, risk factors and mortality. Indian J Crit Care Med. 18:200–204.

, Wu

, Zhang

, and Zhong

. 2019. Risk factors of ventilator-associated pneumonia in critically III patients. Front Pharmacol. 10:482.

Wałaszek

, Kosiarska

, Gniadek

, Kołpa

, Wolak

, Dobroś

, and Siadek

. 2016. The risk factors for hospital-acquired pneumonia in the intensive care unit. Przegl Epidemiol. 70:15–20.

Bassetti

, Righi

, Vena

, Graziano

, Russo

, and Peghin

. 2018. Risk stratification and treatment of ICU-acquired pneumonia caused by multidrug-resistant/extensively drug-resistant/pandrug-resistant bacteria. Curr Opin Crit Care. 24:385–393.

Rhodes

N.J.

, Cruce

C.E

, O'Donnell

J.N

, Wunderink

R.G

, and Hauser

A.R.

. 2018. Resistance trends and treatment options in gram-negative ventilator-associated pneumonia. Curr Infect Dis Rep. 20:3.

Bianco

, Boattini

, Iannaccone

, Sidoti

, Cavallo

, and Costa

. 2019. Detection of antibiotic resistance genes from blood cultures: performance assessment and potential impact on antibiotic therapy management. J Hosp Infect. 102:465–469.

10.

Wang

, Hecht

, Kline

, and Leber

A.L.

. 2019. Staphylococcus aureus and methicillin resistance detection directly from pediatric samples using PCR assays with differential cycle threshold values for corroboration of methicillin resistance. J Microbiol Methods. 159:167–173.

11.

Paonessa

J.R.

, Shah

R.D

, Pickens

C.I

, Lizza

B.D

, Donnelly

H.K

, Malczynski

, Qi

, and Wunderink

R.G.

. 2019. Rapid detection of methicillin-resistant Staphylococcus aureus in BAL: a pilot randomized controlled trial. Chest. 155:999–1007.

12.

Burillo

, Marín

, Cercenado

, Ruiz-Carrascoso

, Pérez-Granda

M.J

, Oteo

, and Bouza

. 2016. Evaluation of the Xpert Carba-R (Cepheid) assay using contrived bronchial specimens from patients with suspicion of ventilator-associated pneumonia for the detection of prevalent carbapenemases. PLoS One. 11:e0168473.

13.

Cercenado

, Marín

, Burillo

, Martín-Rabadán

, Rivera

, and Bouza

. 2012. Rapid detection of Staphylococcus aureus in lower respiratory tract secretions from patients with suspected ventilator-associated pneumonia: evaluation of the Cepheid Xpert MRSA/SA SSTI assay. J Clin Microbiol. 50:4095–4097.

14.

Ullberg

, Lüthje

, Mölling

, Strålin

, and V. Özenci. 2017. Broad-range detection of microorganisms directly from bronchoalveolar lavage specimens by PCR/electrospray ionization-mass spectrometry. PLoS One. 12:e0170033.

15.

Boattini

, Bianco

, Iannaccone

, Charrier

, Almeida

, De Intinis

, Cavallo

, and Costa

. 2020. Accuracy of the ELITe MGB^® assays for the detection of carbapenemases, CTX-M, Staphylococcus aureus and mecA/C genes directly from respiratory samples. J Hosp Infect. 105:306–310.

16.

EUCAST 2020. Available at www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_10.0_Breakpoint_Tables.pdf

17.

Girlich

, Bernabeu

, Fortineau

, Dortet

, and Naas

. 2018. Evaluation of the CRE and ESBL ELITe MGB^® kits for the accurate detection of carbapenemase- or CTX-M-producing bacteria. Diagn Microbiol Infect Dis. 92:1–7.

18.

Bassetti

, Giacobbe

D.R

, Giamarellou

, Viscoli

, Daikos

G.L

, Dimopoulos

, and Management of KPC-producing Klebsiella pneumoniae infections. 2018. Management of KPC-producing Klebsiella pneumoniae infections. Clin Microbiol Infect. 24:133–144.

19.

Rodríguez-Baño

, Gutiérrez-Gutiérrez

, Machuca

, and Pascual

. 2018. Treatment of infections caused by extended-spectrum-beta-lactamase-, ampC-, and carbapenemase-producing Enterobacteriaceae. Clin Microbiol Rev. 31:pii:e00079-17.

20.

Pouch

S.M.

, Patel

, and Infectious Diseases Community of Practice

AST

. 2019. Multidrug-resistant Gram-negative bacterial infections in solid organ transplant recipients-guidelines from the American Society of Transplantation Infectious Diseases Community of Practice. Clin Transplant. 33:e13594.

21.

Pana

Z.D.

, and Zaoutis

. 2018. Treatment of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBLs) infections: what have we learned until now?. F1000Res. 7:pii: F1000 Faculty Rev-1347.

22.

Corcione

, Lupia

, Maraolo

A.E

, Mornese Pinna

, Gentile

, and De Rosa

F.G.

. 2019. Carbapenem-sparing strategy: carbapenemase, treatment, and stewardship. Curr Opin Infect Dis. 32:663–673.

23.

Kalil

A.C.

, Metersky

M.L

, Klompas

, Muscedere

, Sweeney

D.A

, Palmer

L.B

, Napolitano

L.M

, O'Grady

N.P

, Bartlett

J.G

, Carratalà

, El Solh

A.A

, Ewig

, Fey

P.D

, File Jr

T.M

, Restrepo

M.I

, Roberts

J.A

, Waterer

G.W

, Cruse

, Knight

S.L

, and Brozek

J.L.

. 2016. Management of adults with hospital-acquired and ventilator-associated pneumonia: 2016 Clinical Practice Guidelines by the Infectious Diseases Society of America and the American Thoracic Society. Clin Infect Dis. 63:e61–e111.

24.

Torres

, Niederman

M.S

, Chastre

, Ewig

, Fernandez-Vandellos

, Hanberger

, Kollef

, Li Bassi

, Luna

C.M

, Martin-Loeches

, Paiva

J.A

, Read

R.C

, Rigau

, Timsit

J.F

, Welte

, and Wunderink

. 2017. International ERS/ESICM/ESCMID/ALAT guidelines for the management of hospital-acquired pneumonia and ventilator-associated pneumonia: guidelines for the management of hospital-acquired pneumonia (HAP)/ventilator-associated pneumonia (VAP) of the European Respiratory Society (ERS), European Society of Intensive Care Medicine (ESICM), European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and Asociación Latinoamericana del Tórax (ALAT). Eur Respir J. 50:1700582.

25.

Ekren

P.K.

, Ranzani

O.T

, Ceccato

, Li Bassi

, Muñoz Conejero

, Ferrer

, Niederman

M.S

, and Torres

. 2018. Evaluation of the 2016 Infectious Diseases Society of America/American Thoracic Society Guideline Criteria for risk of multidrug-resistant pathogens in patients with hospital-acquired and ventilator-associated pneumonia in the ICU. Am J Respir Crit Care Med. 197:826–830.

26.

Cillóniz

, Dominedò

, and Torres

. 2019. An overview of guidelines for the management of hospital-acquired and ventilator-associated pneumonia caused by multidrug-resistant Gram-negative bacteria. Curr Opin Infect Dis. 32:656–662.

27.

Torres

, Lee

, Cilloniz

, Vila

, and Van der Eerden

. 2016. Laboratory diagnosis of pneumonia in the molecular age. Eur Respir J. 48:1764–1778.

28.

Douglas, I.S. New diagnostic methods for pneumonia in the ICU. Curr Opin Infect Dis 2016;29:197–204.

29.

Giacobbe

D.R.

, Giani

, Bassetti

, Marchese

, Viscoli

, and Rossolini

G.M.

. 2019. Rapid microbiological tests for bloodstream infections due to multidrug resistant Gram-negative bacteria: therapeutic implications. Clin Microbiol Infect. pii:S1198–743X(19)30524–30525.

30.

Bianco

, Boattini

, Iannaccone

, Cavallo

, and Costa

. 2020. Evaluation of the NG-Test CTX-M MULTI immunochromatographic assay for the rapid detection of CTX-M extended-spectrum-β-lactamase producers from positive blood cultures. J Hosp Infect. 105:341–343.

31.

Saliba

, Aho-Glélé

L.S

, Karam-Sarkis

, and Zahar

J.R.

. 2019. Evaluation of polymerase chain reaction assays for direct screening of carbapenemase-producing Enterobacteriaceae from rectal swabs: a diagnostic meta-analysis. J Hosp Infect. https://doi.org/10.1016/j.jhin.2019.11.017.