UNYVERO HPN HOSPITALIZED PNEUMONIA APPLICATION …€¦ · 3.2 unyvero hpn hospitalized pneumonia cartridge 18 3.3 workflow 19 3.4 unyvero software 20 3.5 facts – pneumonia 23 3.6

© Curetis GmbH, 2018 | Unyvero HPN Application Manual | Item no. 00255 Rev. 7.0 – EN – G2

UNYVERO

HPN HOSPITALIZED PNEUMONIA

APPLICATION MANUAL

2 © Curetis GmbH, 2018 | Unyvero HPN Application Manual | Item no. 00255 Rev. 7.0

0483

No part of this manual may be reproduced in any form or translated into other languages without prior permission

and written consent of Curetis GmbH.

Curetis GmbH

Max-Eyth-Str. 42

71088 Holzgerlingen

Germany

Phone: +49 (0) 7031 49195 10

Fax: +49 (0) 7031 49195 19

(hereinafter referred to “Curetis“)

Last updated: 12 / 2018

Please contact Curetis (if assistance is provided directly by Curetis)

or your local distributor regarding the current print edition.

Unyvero is a registered trademark of Curetis.

This is an operating manual for the Unyvero HPN Application and should only be used by qualified health

professionals.

© Curetis GmbH, 2018 | Unyvero HPN Application Manual | Item no. 00255 Rev. 7.0 3

TABLE OF CONTENTS

INTRODUCTION

1.1 PATENTS 5

1.2 SYMBOLS USED 5

1.3 CONVENTIONS USED 6

1.4 LIABILITY 7

1.5 SERVICE AND SUPPORT 8

1.6 E-LABELING 8

SAFETY

2.1 INTENDED USE 10

2.2 RESPONSIBILITY OF THE USER 11

2.3 STORAGE AND USE 12

2.4 BIOLOGICAL SAFETY 12

2.5 CHEMICAL SAFETY 13

BASIC PRINCIPLE

3.1 UNYVERO COMPONENTS 16

3.2 UNYVERO HPN HOSPITALIZED PNEUMONIA

CARTRIDGE 18

3.3 WORKFLOW 19

3.4 UNYVERO SOFTWARE 20

3.5 FACTS – PNEUMONIA 23

3.6 UNYVERO HPN PANEL 24

3.7 ANALYSIS PRINCIPLE 27

PERFORMING A TEST

4.1 PREPARATION 29

4.2 SAMPLE STORAGE 32

4.3 SAMPLE TRANSFER 32

4.4 STARTING THE TEST 37

4.5 THAWING THE UNYVERO MASTER MIX 38

4.6 PERFORMING THE LYSIS 39

4.7 LYSIS STATUS 41

4.8 REMOVING THE SAMPLE 41

4.9 ASSEMBLING THE CARTRIDGE 43

4.10 MEASURING IN THE ANALYZER 46

4.11 ANALYSIS STATUS 47

4.12 REMOVING THE CARTRIDGE 48

TEST RESULTS

5.1 VIEWING RESULTS 50

5.2 READING THE RESULTS 51

5.3 INVALID RESULTS 57

PERFORMANCE EVALUATION STUDY

6.1 ANALYTICAL TESTING

(ANALYTICAL PERFORMANCE DATA) 59

6.2 INTERFERING SUBSTANCES 62

6.3 CLINICAL TRIAL (DIAGNOSTIC

PERFORMANCE TEST) 63

INTRODUCTION

5© Curetis GmbH, 2018 | Unyvero HPN Application Manual | Item no. 00255 Rev. 7.0

1.1 PATENTS

The Unyvero System is protected by the following patents: EP10005128.3 EP 2388312; PCT/EP/2011/02303 WO 2011/

144304 EP 11 718 666.8 EP 2571976 A1; US 9,598,721 SG 185466 JP6100162 (Div) JP 2015-214794 HK 13108207.7 AU

2011254936 ES 2637589; EP10005237.2; PCT/EP/2011/002507 WO 2011/144345 EP 11 720 402.4 EP 2571617 IN: 9813/

DELNP/ 2012 US 9,592,511 ZL 201180024484.8 HK Nr 1183460A AU 2011254887 SG 185467 JP 5992904 CA 2,799,676; EP

2737294 WO2013/013687A1 AU 201137373961 US 9,963,670 US 15/942,702 CA 2,839,951 ZL 201180072624,9 JP 6130831;

EU 001 197 560; JP 1456898; EU 001 966 433; US D683,044; CH 138 957; EU 002 070 441; US 29/433,060 D723,180; CH

139 083; EU002210401-0001; EU 002210401-0002; EU002210401-0003; EU002210401-0004; DE 30 2008 010 146.6/44,

CH 603 237; US 77/930,558; EU 007 160 955; DE 30 2010 032 715.4/01; CH 613 203; US 85/068,577; EU 009 532 029.

1.2 SYMBOLS USED

SAFETY SYMBOLS

WARNING

A warning contains information related to tasks that, if not performed correctly, could result in injury to the user or even death.

Follow instructions that include a warning only when you have understood the risk, and can therefore carry out the task

without risk of injury.

CAUTION

A caution contains information related to tasks that, if not performed correctly, could result in damage to the product or

invalidate the results of the analysis. Please do not carry out any tasks until you have understood the potential risks involved

and can perform the task correctly.

NOTE

Notes are pieces of information that make it easier to work with the product more efficiently and effectively.

Interactive soft keys are shown in bold.


The Unyvero System consists of the Unyvero Analyzer (item no. 60001), the Unyvero Lysator (item no. 60002), the Unyvero

Cockpit (item no. 60003) incl. Unyvero OS and the Unyvero Sample Tube Holder (item no. 60010).

The Unyvero HPN Application uses the following components: Unyvero HPN Cartridge Set (item no. 10047, contains 12 x

Unyvero HPN Cartridges item no. 10046), Unyvero T1 Sample Tube & T1 Sample Tube Cap Set (item no. 10069, contains 12

x 10066), Unyvero T1 Sample Transfer Tool (item no. 10014, contains 12 x item no. 10005), Unyvero M1 Master Mix (item no.

10011, contains 12 x item no. 10002) as well as the Unyvero HPN Plug-In. The Unyvero T1 Sample Transfer Tool can also be

used apart from the Unyvero HPN Application for the transfer of viscous patient samples.

1.3 CONVENTIONS USED

SHORT NAMES

FULL NAMES SHORT NAMES

Unyvero A50 Analyzer Unyvero Analyzer

Unyvero L4 Lysator Unyvero Lysator

Unyvero C8 Cockpit Unyvero Cockpit

Unyvero M1 Master Mix Tube Unyvero Master Mix

Unyvero Operating Software Unyvero OS

Unyvero HPN Hospitalized Pneumonia Cartridge Unyvero HPN Cartridge

Unyvero HPN Plug-In Unyvero HPN Plug-In

Unyvero Sample Tube Holder Sample Tube Holder

Unyvero T1 Sample Tube T1 Sample Tube

Unyvero T1 Sample Tube Cap T1 Sample Tube Cap

Unyvero T1 Transport Cap T1 Transport Cap

Unyvero Sample Transfer Tool Sample Transfer Tool

Table 1

Unyvero components


1.4 LIABILITY

The content of this manual is subject to change by Curetis at any time without prior notice.

All data and information contained in this manual were compiled pursuant to current German law, European standards and

directives.

Curetis gives no guarantee or warranty, either with regard to this manual (in any form of presentation or distribution) or its

products. This also applies to marketability, applicability to a particular purpose, and the non-infringement of third-party

intellectual property rights. Curetis will provide immediate notification, if any conflicting rights become known.

CURETIS ACCEPTS NO LIABILITY FOR DAMAGE CAUSED AS A RESULT OF:

> improper installation by a person other than Curetis itself,

> failure to comply with the operating manual supplied with the instruments and cartridges,

> use of the devices or cartridges for applications other than the approved indication,

> untrained staff, or

> modifications to the products.

CURETIS

> is liable for any negligence or willful misconduct in the event that key contractual obligations are breached. Depending on the

amount, liability for negligence is limited to the amount of the order. Liability for consequential damage and financial losses

(e.g. loss of profit) is excluded in the case of negligence.

> is otherwise liable only for damage to materials or property caused by intent or gross negligence. Depending on the amount,

liability for negligence is limited to the amount of the order. Liability for consequential damage and financial losses (e.g. loss

of profit) is excluded in the case of negligence.

The aforementioned liability limitations and exclusions do not apply to claims brought under the Product Liability Act, claims

due to fraudulent behavior of a contractual partner, liability claims for guaranteed quality characteristics, or damages resulting

from injury to life, body or health.


1.5 SERVICE AND SUPPORT

If your region is under Curetis customer care, please contact Curetis support by phone or email. Have the serial number of

your devices at hand (on the rear of the Unyvero Cockpit and the Unyvero Lysator, and under the front cover of the Unyvero

Analyzer).

Phone: +49 (0) 7031 49195 55 (available Monday-Friday from 9 a.m. to 5 p.m. CET) or email: [email protected].

For all other regions, please ask your local contact for assistance.

1.6 E-LABELING

The Curetis online document service includes all documents for products that are subject to e-labeling. They are available in

electronic form at: ifu.curetis.com.

The KEY-CODE for this document is:

ifu.curetis.com

EU +800 135 79 135

GR 00 800 161 220 577 99

RO 0800 895 084

non-EU +31 20 794 7071

RU +800 135 79 135

Unyvero HPN Hospitalized Pneumonia

Application ManualCUR10046

SAFETY


2.1 INTENDED USE

This chapter provides information on how to use the Unyvero HPN Cartridge safely and describes the necessary safety pre-

cautions.

The Unyvero HPN Application is approved for use together with the Unyvero System. It is used to detect pneumonia-

associated pathogens and pathogen antibiotic resistance genes.

The Unyvero HPN Application is a qualitative DNA test based on the parallel execution of eight multiplex PCR reactions. It is

approved for the simultaneous detection of multiple pathogen-associated nucleic acids in sputum, respiratory aspirate and

bronchial lavage samples from patients with suspected pneumonia, with the aim to provide information on pathogen species

and antibiotic resistance genes. The identification of specific non-viral pathogen DNA in patients with signs and symptoms of

pneumonia helps to diagnose respiratory infections caused by bacteria and/or fungi, if these results are used in conjunction

with other clinical or laboratory methods. Additional cultures are required to cultivate microorganisms for further determination

of antibiotic resistance.

It is recommended that samples, which tested negative for microorganisms and antibiotic resistance genes with the Unyvero

HPN Application, are retested using microbiological cultures. In particular, a negative result for resistance genes does not rule

out antibiotic resistance, because other molecular mechanisms not tested using the Unyvero HPN Application can lead to

resistance. Positive findings do not rule out coinfections with viral or other bacterial microorganisms or fungi not analyzed in

the Unyvero HPN Panel. The detected microorganisms may not necessarily be the cause of the illness.

The Unyvero result should not be used alone for diagnosis and patient management.


2.2 RESPONSIBILITY OF THE USER

CAUTION

The Unyvero HPN Application may only be used for the approved indications. Curetis assumes no responsibility for the use of

the Unyvero HPN Application for any other purposes.

Do not use Unyvero consumables after the expiration date. Do not use consumables if fluid has leaked, or if the packaging is

damaged. Do not open seals or packaging until immediately before use.

The Unyvero HPN Cartridge, Unyvero Sample Tube, Unyvero Sample Tube Cap, Unyvero Master Mix Tube, and Unyvero

Sample Transfer Tool are intended for single use for one patient sample only. Components must be disposed of after use, and

cannot be reused.

Before using the Unyvero System, every user must be instructed how to operate the instruments. This training is only to be

provided by Curetis personnel, or persons trained by Curetis.

In addition, the user should have read the Unyvero System manual, and all relevant safety data sheets (Unyvero Cartridge,

Unyvero Sample Tube, and Unyvero Sample Tube Cap).


2.3 STORAGE AND USE

CAUTION

Store the Unyvero Cartridge, Unyvero Sample Tube, and Unyvero Sample Tube Cap at room temperature (15°C to 25°C).

The Unyvero Master Mix Tube is kept at -20°C for long-term (> 7 days) and at +4°C for short-term storage (< 7 days). Before

using the Unyvero Master Mix, let it thaw for 30 minutes at room temperature.

Do not open component packaging until shortly before an analysis, and use components within 30 minutes.

2.4 BIOLOGICAL SAFETY

WARNING

Biological samples can transmit infections. Treat all biological samples, including the sample containers, as potentially

infectious. Adhere to your local guidelines regarding the handling of biological material.

Wear disposable gloves, a lab coat, and safety glasses when handling samples and consumables of the Unyvero HPN

Application. Wash your hands thoroughly after handling samples and consumables of the Unyvero HPN Application. Discard

used consumables according to local safety guidelines, or instructions of your local regulatory authority.


2.5 CHEMICAL SAFETY

CAUTION

To minimize any danger when working with hazardous materials:

> avoid direct contact with chemicals

> always use personal protective equipment (safety glasses, gloves, protective clothing) when handling chemicals

> check regularly for any possible chemical leaks

In case any fluids leak, follow the cleaning instructions recommended in the safety data sheets (SDS). Adhere to local or

national guidelines when handling, storing, or disposing of chemicals. Discard used consumables according to local safety

guidelines, or instructions of your local regulatory authority.

NOTE

The SDS for the Unyvero Cartridge, Unyvero Sample Tube and Unyvero Sample Tube Cap are available from Curetis upon

request.


PACKAGING SYMBOLS

Product number Upper temperature limit

In-vitro diagnostic product Do not use if packaging is damaged.

CE marking Read the manual

Do not reuse Temperature limitations

Expiration date Content for <n> tests

Lot number Manufacturer

Harmful if swallowed.

Causes skin irritation.

Causes serious eye irritation.

Highly flammable liquid and vapor.

n

BASIC PRINCIPLE


The Unyvero HPN Application

includes the following components:

> Unyvero Lysator

> Unyvero Cockpit

> Unyvero Analyzer

> Unyvero OS and Unyvero HPN Plug-In

> Unyvero HPN Cartridge

> Unyvero T1 Sample Tube & T1 Sample Tube Cap

> Unyvero Master Mix Tube

> Unyvero Sample Transfer Tool

> Unyvero Sample Tube Holder

3.1 UNYVERO COMPONENTS

INSTRUMENTS AND SOFTWARE

The UNYVERO LYSATOR (1) lyses the sample. Based on newly developed

technologies, the Unyvero Lysator is able to process a wide range of clinical

sample types using a standardized protocol.

The UNYVERO ANALYZER (2) processes up to two Unyvero Cartridges in

random access mode and automatically performs DNA purification, specific

amplification, and detection.

The UNYVERO COCKPIT (3), is equipped with a touchscreen and barcode

reader, and connects the Unyvero Analyzer to the Unyvero Lysator. The Unyvero

OS and the application-specific Unyvero Plug-Ins make up the user interface,

which guides the user through the process step by step, and automatically

generates and displays test results.

1

23

UNYVERO A50 ANALYZERUNYVERO C8 COCKPITUNYVERO L4 LYSATOR

NOTE

The instruments are described in detail in the Unyvero System manual.

Figure 1 Unyvero System

The Unyvero HPN Cartridge contains reagents for DNA isolation

and purification, primers, hybridization buffers, wash buffers, and

oligonucleotides for detection.

The Unyvero T1 Sample Tube contains glass beads and buffer to lyse

bacteria and liquefy the sample.

The Unyvero T1 Sample Tube Cap seals the Unyvero Sample Tube. It

contains proteinase K, as well as a synthetic control gene for process

monitoring.

The Unyvero M1 Master Mix Tube contains reagents (polymerase,

nucleotides, buffer) for DNA amplification.

The Unyvero T1 Sample Transfer Tool is a tool to transfer samples, which

is connected to a 1 mL Luer-Lock syringe (not included). It helps transfer

the patient sample efficiently from the primary sample container to the

Unyvero Sample Tube.

The Unyvero Sample Tube Holder holds the Unyvero Sample Tube

securely while the patient material is transferred into the Unyvero Sample

Tube (accessory supplied with the Unyvero System).


CONSUMABLES AND ACCESSORIES

DNA Isolation

Multiplex endpoint PCR

Amplicon-detection by membrane array

hybridization


Figure 2 Unyvero HPN Cartridge

The Unyvero HPN Cartridge is equipped with integrated reagent containers, a DNA purification column, eight separate

PCR chambers, and a corresponding number of arrays. The cartridge contains buffers for DNA purification, reagents, and

fluorescence-labeled primers for PCR amplification, and probes for array hybridization.

The cartridge is assembled by inserting the Unyvero Sample Tube containing the lysed sample, and by inserting the Unyvero

Master Mix Tube. Once assembled, the cartridge is physically closed, minimizing the risk of contamination.

An internal control (a synthetic gene without any significant homology to known sequences) is included in the cartridge to

verify DNA purification, PCR, and array hybridization for each measurement. This gene is amplified in each of the eight PCR

chambers and hybridized on each array.

The Unyvero HPN Cartridge (Figure 2) integrates all relevant

analysis steps:

3.2 UNYVERO HPN HOSPITALIZED PNEUMONIA CARTRIDGE


3.3 WORKFLOW

The Unyvero HPN Application is easy to use and requires only five minutes of manual work. In the first step, as shown in

Figure 3, the sample is lysed in the Unyvero Lysator. The cartridge is then assembled by inserting the Unyvero Sample Tube

and Master Mix Tube for subsequent processing in the Unyvero Analyzer.

Figure 3 Workflow


3.4 UNYVERO SOFTWARE

The Unyvero Operating Software installed on the Unyvero

Cockpit generates the user interface that guides the user

through the process step by step, and displays the test

results.

Figure 4 shows the Active Tests overview screen of the

Unyvero OS. Every bar symbolizes one active measurement.

The color indicates the measurement status.

Bars are opened by tapping on them to display additional

test information. The type of information displayed depends

on the current status of the selected test.

Gray bars indicate lyses in progress, or those that were

completed. Purple bars represent analyses in progress.

Completed tests are shown in blue. Test results are

displayed when tapping on a blue bar. Unfinished tests that

were either terminated by the user, or not completed due to

a technical problem, are shown in orange.

1

2

3

4

Figure 4 Unyvero OS Active Tests overview screen

1 System status lights

2 Headline bar

3 Test bars

4 Function keys

Login / Logout To log the user in and out

Active Tests Displays all active tests

New Test Opens the input screen for a new test

Saved Tests Displays all saved tests

Advanced FunctionsIncludes a function to manage connected devices, configuration, and

system administration

Scroll buttons


The function keys are displayed at the bottom of the screen (Figure 5).

Figure 5 Function keys

FUNCTION KEYS

Symbol for the time period prior to lysator loading

Display during lysator processing of the sample tube

Display during the time of cartridge loading, scanning, and insertion into the analyzer

Symbol for the DNA purification process

Symbol for the multiplex PCR reaction process

Symbol for the array hybridization process


APPLICATION SOFTWARE SYMBOLS

When you tap on a bar of an active test, symbols are displayed that depict the respective process step. The logo for the

current step is green, steps that have been successfully completed have a check mark. Steps that were not successfully

completed are orange and marked by a cross. Tests that were not started yet are in black and white.


3.5 FACTS – PNEUMONIA

Pneumonia is an acute infection of the respiratory system with a high incidence rate, and in hospitalized patients is usually

caused by bacteria. 5% of hospitalized patients develop this rapidly progressive condition with mortality rates of up to 30%1,

and an average hospital stay of 11 to 14 days1.

Current standard diagnosis uses microbiological cultures to detect bacteria and antibiotic resistances. However, these results

are not available until one to five days later. Due to the lack of diagnostic results, most pneumonia cases are treated empirically.

This is accompanied by a high rate (~40%)2 of initially inadequate antibiotic treatments. Studies demonstrated that an early,

targeted antibiotic treatment can significantly reduce both the mortality rate and the length of hospital stay3.

The Unyvero HPN Application was developed to improve diagnosis of pathogens and resistances in acute bacterial pneumonia.

THE HPN APPLICATION

> allows the testing of samples from sputum, respiratory aspirates (tracheal and bronchial secretions), and bronchial lavages,

> combines all analytical steps in one closed test format, thus reducing the time and effort involved,

> requires just a few simple manual steps,

> detects various microorganisms and antibiotic resistance markers simultaneously in samples within approx. 4 - 5 hours.

1 Kollef et al., Chest, Vol 115 (1999)2 Kollef et al., Chest, Vol 128 (2005)3 Rello et al., Am J ResprCrit Care Med, Vol 176 (1997)


3.6 UNYVERO HPN PANEL

The detection panel for microorganisms and resistance markers was designed based on published incidence data, and the

following national and international guidelines:

> CDC: Prevention of Healthcare-Associated Pneumonia4

> American Thoracic Society: HAP, VAP, HCAP Guideline5

> CAP Guideline, Paul-Ehrlich-Gesellschaft (Germany)

> European Respiratory Society: Lower Respiratory Tract Infections

> British Society of Antimicrobial Chemotherapy: HAP Guideline

> Canadian Guideline Committee: VAP Diagnosis and Treatment

The Unyvero HPN Cartridge detects the following microorganisms:

Table 2

Microorganisms detected by the Unyvero HPN panel

GROUP PATHOGEN

Gram-positiveStaphylococcus aureus

Streptococcus pneumoniae

Enterobacteriaceae

Citrobacter freundii

Escherichia coli

Enterobacter cloacae complex 6

Proteus spp. 7

Klebsiella aerogenes(Enterobacter aerogenes)

Klebsiella pneumoniae 8

Klebsiella oxytoca

Klebsiella variicola 9

Serratia marcescens

Morganella morganii

GROUP PATHOGEN

Non-fermenting bacteria

Moraxella catarrhalis

Pseudomonas aeruginosa

Acinetobacter baumannii complex 10

Stenotrophomonas maltophilia

Legionella pneumophila

Other/fungi

Pneumocystis jirovecii

Haemophilus infl uenzae

Chlamydophila pneumoniae

Mycoplasma pneumoniae

4 Talan et al., MMWR Recomm Rep, Vol 53 (2004) 5 Siegel et al., Am J Infect Control, Vol 35 (2007)6 incl. E. cloacae, E. asburiae, E. hormaechei7 incl. P. vulgaris, P. mirabilis, P. penneri, P. hauseri8 inkl. Klebsiella pneumoniae Cluster kp I + II; Lit: Alves et al., J Clin Microbiol, 44(10), 20069 Klebsiella variicola (Cluster kpIII; formerly K. pneumoniae, 2004 classifi ed as a species of its own)10 incl. A. baumannii, A. oleivorans, A. calcoaceticus, A. pittii


Table 3

Resistence markers of the Unyvero HPN panel 11

The following genes associated with antibiotic resistance are detected simultaneously.

MARKER POSSIBLE RESISTANCE REFERENCE

ermB Macrolides / lincosamides Roberts et al., FEMS Microbiol Lett, 282(2), 2008

mecA Oxacillin Carvalho et al., Braz J Infect Dis, 14(1), 2010

mecC Oxacillin Katayama et al., Antimicrob Agents Chemother, 44(6), 2000

ctx-M 12 3rd gen. cephalosporins, class A Bonnet et al., Antimicrob Agents Chemother, 45(8), 2001

gyrA83 of E. coli and P. aeruginosa

FluoroquinolonesKugelberg et al., J Antimicrob Chemother, 55(1), 2005; Yu et al., J ClinMicrobiol, 42(9), 2004

gyrA87 of E. coli and P. aeruginosa

FluoroquinolonesKugelberg et al., J Antimicrob Chemother, 55(1), 2005; Yu et al., J ClinMicrobiol, 42(9), 2004

imp 13 Carbapenems, class B Kawa et al., J Clin Microbiol, 34(12), 1996

kpc 14 Carbapenems, class A Queenan et al., Clin Microbiol Rev, 20(3), 2007

ndm 15 Carbapenems, class B Cornaglia et al., Lancet Infect Dis, 11(5), 2011

oxa-23 Carbapenems, class D Walther-Rasmussen et al., J Antimicrob Chemother, 57(3), 2006

oxa-24 Carbapeneme, class D Walther-Rasmussen et al., J Antimicrob Chemother, 57(3), 2006

oxa-48 16 Carbapenems, class D Walther-Rasmussen et al., J Antimicrob Chemother, 57(3), 2006

oxa-58 Carbapenems, class D Walther-Rasmussen et al., J Antimicrob Chemother, 57(3), 2006

sul1 Sulfonamides Bennett et al., Br J Pharmacol, 153 Suppl 1, 2008

shv Penicillins, class A Gupta et al., Indian J Med Res, 126(5), 2007

tem Penicillins, class A Gupta et al., Indian J Med Res, 126(5), 2007

vim 17 Carbapenems, class B Cornaglia et al., Lancet Infect Dis, 11(5), 2011

11 The antibiotic resistances caused by ß-lactamases are classifi ed according to Ambler. BLAST searches were used to determine coverage. 12 including the clinically relevant variants ctx-M3, ctx-M10, ctx-M15, ctx-M55. Also, coverage of the following variants was determined in silico: ctx-M1, ctx-M23, ctx-M 28, ctx-M29, ctx-

M32, ctx-M36, ctx-M42, ctx-M44, ctx-M58, ctx-M61, ctx-M72, ctx-M79, ctx-M80, ctx-M89, ctx-M116, ctx-M158, ctx-M166, ctx-M175, ctx-M17913 Clinically relevant variants including imp1 to imp10, imp15, imp16, imp19, and imp2514 kpc1 to kpc2215 ndm-1 to ndm-1416 incl. oxa-162, oxa-181, oxa-232, oxa-24417 Coverage of the following variants was determined in silico: vim-1, vim-2, vim-3, vim-5, vim-6, vim-8, vim-9, vim-10, vim-11, vim-15, vim-16, vim-17, vim-18, vim-20, vim-23, vim-24,

vim-25, vim-30, vim-31, vim-36, vim-38, vim- 41, vim-44, vim-45, vim-46, vim-49, vim-50, vim-51

1

2

34 5

6

7

8


PCR CHAMBER DISTRIBUTION

The multiplex PCR reactions to detect microorganisms and

resistance markers are distributed across eight (independent)

PCR chambers (Figure 6). Table 4 shows the analyte distribution

across the individual chambers.

Figure 6 PCR chambers 1-8Table 4

Genetic markers per PCR chamber

CHAMBER 1 CHAMBER 2 CHAMBER 3 CHAMBER 4

gyrA (E. coli) Mycoplasma pneumoniae gyrA (E. coli) Proteus spp.

S. marcescens oxa-58 Chlamydophila pneumoniae gyrA (P. aeruginosa)

sul1 mecA imp vim

Acinetobacter baumannii complex

Klebsiella pneumoniae (kp I)Klebsiella aerogenes (Enterobacter aerogenes)

oxa-23

Klebsiella variicola ermB kpc

Escherichia coli

CHAMBER 5 CHAMBER 6 CHAMBER 7 CHAMBER 8

oxa-48 Pneumocystis jirovecii gyrA (P. aeruginosa) Stenotrophomonas maltophilia

Moraxella catarrhalis Staphylococcus aureus oxa-24 Citrobacter freundii

mecC Klebsiella oxytoca Haemophilus infl uenzae Legionella pneumophila

Morganella morganii Streptococcus pneumoniae Enterobacter cloacae complex ndm

Klebsiella pneumoniae (kp II) shv tem Pseudomonas aeruginosa

ctx-M


3.7 ANALYSIS PRINCIPLE

The Unyvero Application automates and integrates DNA purification, eight parallel multiplex endpoint PCR reactions, and the

qualitative detection of amplicons using hybridization on arrays in a single-use cartridge.

> The sample is transferred to the Unyvero Sample Tube, sealed with the Unyvero Sample Tube Cap, and lysed in the Unyvero

Lysator.

> The Unyvero Sample Tube and the Unyvero Master Mix Tube are then inserted into the cartridge.

> The Unyvero Cartridge is subsequently inserted into the Unyvero Analyzer, which automatically processes the cartridge.

The software guides the user through the entire process. A barcode reader allows the input of sample and consumable data.

The software also checks the expiration dates of the consumables, and stores them together with the lot number.

PERFORMING A TEST


4.1 PREPARATION

This chapter describes how to analyze a sample using the Unyvero HPN Application.

CAUTION

Never use components beyond their expiration date. Do not use consumables that appear damp, or have damaged packaging.

Do not open the component packaging until shortly before use.

The Unyvero HPN Cartridge, Unyvero T1 Sample Tube & Sample Tube Cap, Unyvero Sample Transfer Tool, and Unyvero M1

Master Mix Tube allow the testing of a single patient sample. Please do not reuse any of the components.

NOTE

Do not use the Unyvero HPN Application, if one of the components is past its expiration date.

UNYVERO MATERIALS

The following consumables are necessary to carry out the Unyvero HPN Application:

> 1× Unyvero HPN Cartridge

> 1× Unyvero T1 Sample Tube & T1 Sample Tube Cap

> 1× Unyvero M1 Master Mix Tube

> 1× Unyvero Sample Transfer Tool

NOTE

Safety data sheets (SDS) for the Unyvero Cartridge, Unyvero Sample Tube, and Unyvero Sample Tube Cap are available upon

request from Curetis, or your local contact.


ADDITIONAL MATERIALS

The following materials are needed to carry out an analysis:

> sample material

> 1 mL syringe with Luer-Lock connection (optional)

> personal protective equipment PPE (disposable gloves, lab coat, safety glasses), and equipment to handle infectious

materials pursuant to your local regulations

WARNING

Biological samples can transmit infections. Treat all biological samples, including their sample containers, as potentially

infectious. Please adhere to local guidelines when handling biological materials.

Wear disposable gloves, a lab coat, and safety glasses when handling samples and consumables of the Unyvero HPN

Application and wash your hands thoroughly afterwards. Discard used consumables according to local safety guidelines, or

instructions of your local regulatory authority.

UNYVERO SYSTEM

This section describes the basic steps for the initial operation of the instruments and software necessary for the Unyvero HPN

Application.

If the instruments have not yet been put into operation, switch them on in the following order:

1. Unyvero Cockpit, using the switch at the bottom right on the back of the device

2. Unyvero Analyzer, using the switch at the bottom right on the front of the device

3. Unyvero Lysator, using the switch at the bottom left on the back of the device

Further information on how to operate the devices can be found in the Unyvero System manual.

CAUTION

Before starting an analysis, ensure that the required application-specific plug-in (HPN) is activated.


Figure 7 Test input screen

NOTE

Refer to the Unyvero System manual to create a user.

NOTE

Before you start the lysing process, make sure that you took a Unyvero Master Mix Tube out of the freezer to thaw. See also

section 4.5: "Thawing the Unyvero Master Mix".

Tap on Login on the start screen.

In the next window, enter your user name and password

using the integrated on-screen keyboard, and then tap

on OK. You will then have access to the test input

screen (Figure 7).

LOGIN


Figure 8 Primary sample tubes

4.3 SAMPLE TRANSFER

The Unyvero HPN Application can process a variety of different samples. The sample transfer starts when the Unyvero Sample

Tube is placed in the Unyvero Sample Tube Holder. Samples can be transferred into the Unyvero Sample Tube with or without

the Unyvero Transfer Tool. Care should be taken to ensure that the sample is not contaminated with foreign microorganisms.

4.2 SAMPLE STORAGE

CAUTION

Store samples between 2°C and 8°C, even during transportation. Regardless of

the storage conditions, never use samples older than 12 hours for a test. Only

prepare as many samples as there are free slots in the Unyvero Analyzer.

NOTE

All samples that are diluted for microbiological testing should be used at the same dilution for analysis with the Unyvero

System. The maximum filling quantity of 180 μL must not be exceeded. Appropriate dilution media include TE buffer, 0.9%

NaCl, and Sputolysin.

CAUTION

Do not remove the Unyvero Sample Transfer Tool from its packaging prematurely.

1


Figure 9 Positioning the Unyvero Sample

Tube

2The Unyvero Sample Tube Holder has four slots for Unyvero Sample Tubes (1)

(Figure 9). Behind each slot, there are openings of various sizes into which primary

sample containers can be placed (2).

Place the sample into the top section of the Unyvero Sample Tube Holder.

CAUTION

Make sure to use the Unyvero T1 Sample Tube and T1 Sample Tube Cap.

CAUTION

Do not vortex or centrifuge the Unyvero Sample Tube with the Transport Cap.

Tap the top of the Unyvero Sample Tube to ensure that no fluid remains in the

Unyvero Transport Cap.

Place the Unyvero Sample Tube into an empty slot in the Unyvero Sample

Tube Holder (Figure 9).

Remove the Unyvero T1 Sample Tube from the packaging.

Remove and discard the Transport Cap. If necessary, remove any air bubbles from the Unyvero Sample Tube before filling

it with sample.


CAUTION

To prevent foreign genetic material from contaminating the sample, do not touch the Unyvero Sample Transfer Tool when

removing it from the packaging. Please observe the rules for sterile working conditions.

Open the packaging of the Unyvero Sample Transfer Tool, and leave as much of the packaging as possible on the front

part of the Unyvero Transfer Tool. Then attach the Unyvero Transfer Tool firmly to a 1 mL Luer-Lock syringe.

NOTE

If the Unyvero Transfer Tool is not attached tightly, it is not possible to draw up air or sample material.

Draw 50 μL of air into the syringe.

Guide the Unyvero Sample Transfer Tool into the sample and draw precisely 180 μL of the sample into the syringe

(Figure 10).

SAMPLE TRANSFER WITH TRANSFER TOOL

NOTE

For viscous samples, you can use the sharp end of the Unyvero Sample Transfer Tool to cut the sample on the inside wall of

the sample vial.

Figure 12 Unyvero Sample Tube – the line

indicates the maximum filling level

Figure 13 Unyvero T1 Sample Tube and

T1 Sample Tube Cap

2

1


Carefully insert the Unyvero Sample Transfer Tool as far as possible into the Unyvero Sample Tube.

Slowly transfer the sample into the Unyvero Sample Tube (Figure 11).

Figure 10 Drawing a patient sample

Figure 11 Transferring the patient sample

Dispose of the syringe and the Unyvero Sample Transfer Tool according to the

guidelines of your institution.

NOTE

There are two small openings at the bottom of the Unyvero Sample Tube Cap (1)

(Figure 13). At the top of the Unyvero Sample Tube are two small lock-in catches (2).

Pay attention to the maximum filling level (Figure 12).

Remove the packaging from the Unyvero Sample Tube Cap.

CAUTION

To prevent the Unyvero Sample Tube from overflowing, do not use more than

180 μL of the sample. The liquid should only reach up to the lower edge of the

Unyvero Sample Tube neck (Figure 12).


Press the Unyvero Sample Tube Cap onto the Unyvero Sample Tube until it

snaps into place (Figure 14).

Figure 14 Unyvero Sample Tube sealed with a Sample Tube Cap

WARNING

Any contact with patient samples carries the risk of infection during handling. Before continuing, make sure that the outside of

the Unyvero Sample Tube is clean. Please read the safety data sheet (SDS) for the Unyvero Sample Tube. After removing the

Transport Cap, chemicals may be exposed that irritate or damage the skin. If your skin or eyes have come into contact with

chemicals from the Unyvero Sample Tube, wash your skin/eyes thoroughly with water immediately.

CAUTION

Make sure that the Unyvero Sample Tube is securely sealed. If it is not properly sealed, contamination of the laboratory and

Unyvero Lysator is possible. It may also damage the lysator.

CAUTION

Do not attempt to reopen the Unyvero Sample Tube.

SAMPLE TYPE INPUT

Figure 16 Scanning the Unyvero Sample Tube

Select the application: HPN.

Select the sample type, e.g. Sputum.

Once all the information is entered, tap on Start.

Hold the bar code of the Unyvero Sample Tube in front of the bar code reader

and scan it (Figure 16).

NOTE

Once a button has been selected, it turns green and the other selection buttons become invisible. When the green button is

selected again, the invisible buttons will reappear.


4.4 STARTING THE TEST

Figure 15 Starting a new test

The input screen for a new test opens when tapping on New Test at the

botton of the screen (Figure 15). A new test screen opens.

The sample ID can be scanned or entered using the on-screen keyboard.

NOTE

Different sample types can be used. Please choose the appropriate sample material for the test.

NOTE

If the expiration date of the Unyvero Sample Tube and Cap has passed, a new Unyvero Sample Tube and Cap that has not

expired must be used for the test.


4.5 THAWING THE UNYVERO MASTER MIX

NOTE

The Unyvero Master Mix Tube must be stored at -20°C (> 7 days), but can be stored at +4°C for short-term storage (< 7 days).

Leave the Unyvero Master Mix Tube out to thaw for 30 minutes at room temperature. Do not shake the Unyvero Master

Mix Tube.


4.6 PERFORMING THE LYSIS

Figure 17 Prompt to load the Unyvero Lysator

Figure 18 Loading the Unyvero Lysator

Slide the safety latch backwards over the lysator slot.

Place the Unyvero Sample Tube into the opened

position (Figure 18). Then press the Unyvero Sample

Tube down firmly, as far as possible.

Allow the safety latch to slide forward again. Make sure

that it is back in the starting position and covers the

Unyvero Sample Tube.

Close the lid of the Unyvero Lysator.

NOTE

Before loading the Unyvero Lysator with the Unyvero Sample

Tube, make sure that the lysator slot is not contaminated

(see cleaning instructions at the end of this section).

Once the Unyvero Sample Tube was sucessfully scanned,

the Unyvero Lysator cover opens automatically, and a slot

is unlocked. An icon to insert the Unyvero Sample Tube into

the lysator appears on the screen (Figure 17).


NOTE

Only start a lysis, if free analyzer slots are available, or if an analyzer slot becomes available within 30 minutes (check the

remaining time on the Unyvero Analyzer screen).

NOTE

If a lysis in the Unyvero Lysator is already in progress, you must wait until the end of the lysis before another lysis can start in

this lysator.

NOTE

In the event of contamination, the slot must be cleaned with a cotton swab and 70% ethanol. Before continuing, make sure

that there is no ethanol residue left. Prior to using any cleaning or decontamination methods, except those recommended

by the manufacturer, users should check with the manufacturer that the proposed method will not damage the equipment.

NOTE

The lysis cannot start until the lysator lid is closed.

If another slot is free in both the Unyvero Lysator and Unyvero Analyzer, you will be asked whether you would like to perform

a new lysis.

If you intend to lyse another sample, tap on Yes and proceed as described above.

If you do not intend to lyse another sample, tap on No.

Once all Unyvero Sample Tubes are inserted, close the Unyvero Lysator lid. The lysis process starts automatically.


4.7 LYSIS STATUS

Figure 19 Lysator screen

4.8 REMOVING THE SAMPLE

Figure 20 Prompt to remove the Unyvero Sample Tube Figure 21 Lysator screen

The lysis process takes approximately 30 minutes and is

displayed on the overview screen by a gray bar for each

sample.

The remaining processing time and the loading status of the

lysator are also shown on the small screen on the Unyvero

Lysator (Figure 19).

When the lysis process is complete, the gray test bar in

the Unyvero Cockpit closes. If you tap on the bar, the

lid of the Unyvero Lysator opens automatically, and both

the Unyvero Cockpit (Figure 20) and the Unyvero Lysator

screen (Figure 21) prompt you to remove the sample tube.


In case several Unyvero Sample Tubes were lysed, start by assembling the cartridge with the first sample that you removed.

You can only proceed to remove the next Unyvero Sample Tube from the Unyvero Lysator after you inserted this cartridge into

the Unyvero Analyzer.

Proceed for each new sample in the same way as the previous one. After tapping on the lysis bar displayed on the screen,

the Unyvero Lysator opens again automatically and releases the next Unyvero Sample Tube.

CAUTION

Assemble the cartridge within 60 minutes at room temperature.

Tap on the gray test bar of the sample that is to be analyzed. The Unyvero Lysator will open.

Slide the safety latch backwards and remove the Unyvero Sample Tube. Allow the safety latch to slide back into position.

Then close the lid of the Unyvero Lysator.

43© Curetis GmbH, 2018 | Unyvero HPN Application Manual | Item no. 00255 Rev. 7.0 43

4.9 ASSEMBLING THE CARTRIDGE

WARNING

The Unyvero Cartridge contains chemicals that may irritate and damage the skin in case the cartridge is damaged. If your

skin or eyes come into contact with these chemicals, wash your eyes/skin immediately and thoroughly. Please do not use

damaged cartridges. Do not use a cartridge, if it appears damp or if its sealing film is broken. Do not open the cartridge.

CAUTION

Pay attention to the information in section 2.5. Check expiration dates on labels and packaging of the Unyvero HPN Cartridge,

Unyvero Sample Tube and Sample Tube Cap, as well as Unyvero Master Mix Tube. Analysis with the Unyvero HPN Application

cannot be conducted with expired consumables.

NOTE

Read also the safety data sheet for the Unyvero Cartridge.

Remove the packaging from the Unyvero HPN Cartridge.

Remove the packaging from the thawed Unyvero Master Mix Tube.

Tap the top of the Unyvero Master Mix Tube to ensure that the fluid collects in the lower part of the tube.


Figure 22 Prompt to assemble cartridge

CAUTION

Do not vortex or centrifuge the Unyvero Master Mix Tube.

NOTE

You can check the expiration dates of consumables using

the Check Consumable function (button at the bottom

of the opened bar for the current sample). Another option

to check consumables can be found under Advanced

Functions.

If, after assembly of the Unyvero HPN Cartridge, it appears

that the Unyvero HPN Cartridge or the Unyvero Master

Mix Tube are past their expiration dates, the test must be

restarted using a new Unyvero Sample Tube, Unyvero HPN

Cartridge, and Unyvero Master Mix Tube.

After removing the Unyvero Sample Tube from the Unyvero

Lysator and closing the lysator lid, the cartridge assembly

screen appears (Figure 22).

45© Curetis GmbH, 2018 | Unyvero HPN Application Manual | Item no. 00255 Rev. 7.0 45

Figure 23 Cartridge slots

Insert the Unyvero Sample Tube vertically into the Unyvero HPN Cartridge

(Figure 23).

Insert the Unyvero Master Mix Tube vertically into the cartridge (Figure 23).

As shown in Figure 24, hold the Unyvero HPN Cartridge facing the Unyvero

Cockpit barcode reader.

NOTE

When scanning the cartridge, place it on the narrow ledge of the Unyvero Cockpit to ensure problem-free scanning (Figure 24).

Figure 24 Scanning the cartridge

Sample TubeMaster Mix

All barcodes (Unyvero Sample Tube, Unyvero HPN Cartridge, Unyvero Master Mix

Tube) are scanned, checked, automatically linked by the Unyvero OS, and saved

under the sample ID.

NOTE

After scanning, press the Unyvero Sample Tube and Master Mix further into the

cartridge slots so that they are below the cartridge plastic rim.


4.10 MEASURING IN THE ANALYZER

Transfer the Unyvero HPN Cartridge to the slot selected by the Unyvero OS (Figure 25). The slot number is also displayed on

the screen of the Unyvero Analyzer and is indicated by a green arrow under the slot (Figure 26).

Figure 25 Prompt to load the Unyvero Analyzer Figure 26 Analyzer display


Slide the Unyvero HPN Cartridge as far as possible into the indicated slot

(Figure 27).

Figure 27 Inserting the cartridge into the Unyvero

Analyzer

4.11 ANALYSIS STATUS

Figure 28 Analyzer display

The processing of the Unyvero HPN Cartridge takes approximately five hours.

The remaining time of each analysis is displayed on the Unyvero Analyzer screen

(Figure 28) and in the Unyvero Cockpit test status bar. The processing progress of

the Unyvero HPN Cartridge in the Unyvero Analyzer is indicated by a purple status

bar on the overview screen.


Figure 29 Analyzer display

4.12 REMOVING THE CARTRIDGE

Remove the Unyvero HPN Cartridge.

Dispose of the Unyvero HPN Cartridge according to the guidelines of your institution.

CAUTION

The Unyvero HPN Cartridge is intended for single use and can only be used for one sample. The Unyvero HPN Cartridge

cannot be reused.

When the analysis is completed, you will be asked to remove the cartridge. The

Unyvero Analyzer slot appears blue on the small screen, and an arrow indicates

that the Unyvero HPN Cartridge can be removed (Figure 29).

As soon as the analysis is finished and the cartridge removed, the test bar on the Unyvero Cockpit screen appears blue.

NOTE

When the blue test bar of an analysis is opened and closed again, this analysis will be saved in the Saved Tests screen. Tap

on the Saved Tests button to open the display. If you do not close the blue bar and switch from the Active Tests screen to

another screen, the test is also moved to Saved Tests.

TEST RESULTS


Figure 30 Saved Tests screen

Figure 31 Results screen buttons

5.1 VIEWING RESULTS

To view a test result, tap on the corresponding blue test

bar (Figure 30).

Completed analyses are displayed by blue bars on the

overview screen (Active Tests or Saved Tests).

A screen appears with a new menu bar and the following

buttons (Figure 31):

> Overview

> Microorganisms

> Resistance Markers

> Test Information


5.2 READING THE RESULTS

Figure 32 Overview screen

Tap on the Overview button.

SUMMARY OF MEASUREMENT RESULTS

The result overview screen shows an overview of all

detected microorganisms and antibiotic resistances.

The microorganisms detected are shown in the top half

of the screen (Figure 32), while the bottom half features

a summary of the antibiotic classes and any detected

resistance genes.

© Curetis GmbH, 2018 | Unyvero HPN Application Manual | Item no. 00255 Rev. 7.052

Figure 33 Microorganisms result screen

Detected

Not detected

Invalid analyte

Information

DETAILED INFORMATION ON MICROORGANISMS

The microorganisms (Figure 33) are categorized as follows:

> Gram-positiv

> Non-fermenting bacteria

> Enterobacteriaceae

> Other/Fungi

Tap on the button Microorganisms to view a list of

all microorganisms analyzed by the Unyvero HPN

Application.

The presenence or absence of microorganisms is displayed

on the Microorganisms screen (Figure 33) as follows:

On the right-hand side, the measured signal intensities are shown by three small squares representing three levels. The more

squares are green, the higher is the signal intensity. To the right of these squares, a corresponding numerical signal intensity

is displayed. This number is also displayed, if the signal is below the threshold.

Tap on the button Resistance Markers to display

an overview of all resistance markers tested with the

Unyvero HPN Application.

Tap on the button Microorganisms to view a list of

all microorganisms analyzed by the Unyvero HPN

Application.


NOTE

Tap on the information icon to display detailed analyte information, such as a list of all species summarized under a particular

analyte.

Figure 34 Resistance Markers result screen

Detected

Not detected

Invalid analyte

Cannot be interpreted

DETAILS ON ANTIBIOTIC RESISTANCE MARKERS

Resistance markers (Figure 34) are categorized as follows:

1. resistance genes from transferable resistances

2. chromosomal mutations, non-transferable resistances

The presence or absence of resistance markers is shown

on the Resistance Markers screen (Figure 34):

Groups of microorganisms that may carry these resistance

genes are shown on the right-hand side of the screen. This

information is literature-based.

Tap on the button Resistance Markers to display

an overview of all resistance markers tested with the

Unyvero HPN Application.


TRANSFERABLE ANTIBIOTIC RESISTANCE (RESISTANCE GENE)

Most resistance markers that are detected using the Unyvero HPN Application are genes that are transferred by mobile

genetic elements (plasmids and integrons).18 The presence of such a gene correlates with the resistance against a particular

class of antibiotics.19 Table 5 lists the resistance markers detected by the Unyvero HPN Application.

Table 5

Resistance markers of the Unyvero HPN panel (without chromosomal resistance markers)

MARKER POSSIBLE RESISTANCE FREQUENT OCCURRENCE IN

ctx-M 3rd gen. cephalosporins Enterobacteriaceae, non-fermenting bacteria

ermB Macrolides / lincosamides Staphylococcus spp., Streptococcus spp.

imp Carbapenems Enterobacteriaceae, non-fermenting bacteria

kpc Carbapenems Enterobacteriaceae, non-fermenting bacteria

mecA Oxacillin Staphylococcus spp.

mecC Oxacillin Staphylococcus spp.

ndm Carbapenems Enterobacteriaceae, non-fermenting bacteria

oxa-23 Carbapenems Enterobacteriaceae, non-fermenting bacteria




shv Penicillins Enterobacteriaceae, non-fermenting bacteria

sul1 Sulfonamides Enterobacteriaceae, non-fermenting bacteria

tem Penicillins Enterobacteriaceae, non-fermenting bacteria, H. infl uenzae

vim Carbapenems Enterobacteriaceae, non-fermenting bacteria

NOTE

The analyte tem+shv is marked as "cannot be interpreted", if one of the two individual analytes (tem or shv) is positive and

the other is marked as invalid.

18 Roberts, Front Microbiol, Vol. 2 (2011)19 Maiden, MolBiotechnol, Vol. 18 (2001)


CHROMOSOMAL ANTIBIOTIC RESISTANCE (RESISTANCE GENES)

A common mechanism for resistance against gyrase inhibitors/fl uoroquinolones are mutations in the genes of bacterial gyrase.

These mutations have no effect on the function of these essential enzymes, but modify them so that they no longer bind

any gyrase inhibitors/fl uoroquinolones, and thus lose their effectiveness. This interaction between functional integrity and

resistance mechanism is caused by mutation of certain hotspots.

These mutations are point mutations, in which only one base of the DNA sequence is replaced, but in several variations. The

Unyvero HPN Application detects the wild type sequence coded for an enzyme sensitive to gyrase inhibitors, and thus detects

sensitivity to gyrase inhibitors/fl uoroquinolones rather than resistance to them.

Since these are mutations to chromosomal genes, the DNA sequences of these genes differ for the various microorganisms.

The Unyvero HPN Application limits the detection of resistance behavior to Escherichia coli and Pseudomonas aeruginosa.

> For Escherichia coli, subunits A of the gyrase are examined for codons 83 and 87 (gyrA83 and gyrA87).

> For Pseudomonas aeruginosa, subunits A of the gyrase are analyzed for codons 83 and 87 (gyrA83 and gyrA87).

Thus, in the case of Escherichia coli or Pseudomonas aeruginosa reacting sensitively to gyrase inhibitors/fl uoroquinolones, the

markers for the gyrases will produce a positive PCR signal. Even if only one of these resistance markers is not detectable, the

organism is considered to be resistant.

NOTE

When detecting any of these resistance markers, no reference to a microorganism can be made.


The presence or absence of the wild type sequence in chromosomal markers is shown as follows on the Resistance Marker

screen:

Wild type sequence of the marker was not detected (= resistance)

Wild type sequence of the marker was detected (= no resistance)

Invalid analyte

Cannot be interpreted

NOTE

If a resistance marker returned a positive measurement result, but the pathogen marker linked with it was invalid, then this

resistance marker is marked as "cannot be interpreted”. For example, a positive, specific gyr result is marked as "cannot

be interpreted", if E. coli or Pseudomonas aeruginosa are invalid. In the case of Pseudomonas aeruginosa, it must also be

noted that Pseudomonas aeruginosa has a lower detection threshold than the associated gyr resistance markers. If the result

for Pseudomonas aeruginosa below the detection threshold of the gyr resistance markers is positive, these markers are

displayed as "cannot be interpreted", if a resistance cannot be definitely excluded. At low pathogen concentrations, it cannot

be determined for certain whether a quinolone resistance is present.


5.3 INVALID RESULTS

The Unyvero System checks the quality of the analysis by different mechanisms, and marks individual analytes as invalid, if

certain criteria are not met, or if the cartridge could not be processed correctly.

> The functioning of the individual PCR chambers is checked by a specific control concept. Malfunctions in this control

concept can lead to invalid chambers.

> Proper detection cannot take place, if there are problems with the membrane array washing steps, or if holes, ruptures, or

dust particles appear unexpectedly on the membrane.

> A number of mechanisms monitor correct array detection by the optic module. If irregularities occur, these will result in

invalid analytes, because array detection could not be performed accurately.

PERFORMANCE EVALUATION STUDY


6.1 ANALYTICAL TESTING (ANALYTICAL PERFORMANCE DATA)

During the CE performance evaluation study, the Unyvero HPN Application was tested both analytically using bacterial

reference strains, isolated bacterial DNA or DNA fragments (amplicons), and clinically using patient samples with more than

900 cartridges. For the analysis of the clinical samples (sputum, tracheal aspirate and bronchial lavage samples), samples

were primarily used that had returned a positive result in clinical microbiology for the pathogens that are part of the HPN Panel

(a total of 319 pathogens).

ANALYTICAL SENSITIVITY

Detection thresholds for individual pathogens were tested with serial dilutions of living reference strains, or amplified DNA

fragments in buffer. The detection threshold for antibiotic resistance markers was performed with strains bearing resistance

markers. Table 6 lists the determined detection thresholds for all analytes.

Table 6

Data of analytical tests

ANALYTEDETECTION LIMIT

(PATHOGENS / mL)

Acinetobacter baumannii complex 104

Chlamydophila pneumoniae 104

Citrobacter freundii 105

Enterobacter cloacae complex 105

Escherichia coli 105

Haemophilus infl uenzae 104

Klebsiella aerogenes(Enterobacter aerogenes)

105

Klebsiella oxytoca 104

Klebsiella pneumoniae 105

Klebsiella variicola 105


(PATHOGENS / mL)

Legionella pneumophila 104

Moraxella catarrhalis 105

Morganella morganii 105

Mycoplasma pneumoniae 105

Pneumocystis jirovecii 105

Proteus spp. 104

Pseudomonas aeruginosa 104

Serratia marcescens 104

Staphylococcus aureus 105

Stenotrophomonas maltophilia 104

Streptococcus pneumoniae 105


Table 6

Data of analytical tests (cont.)

The indicated analytical detection limits for the resistance markers refer to the respectively tested concentration of a

corresponding pathogen. More detailed information can be requested from Curetis.


(PATHOGENS / mL)

ndm 104

oxa-23 107

oxa-24 104

oxa-48 107

oxa-58 107

sul1 104

shv 106

tem 104

vim 104


(PATHOGENS / mL)

ermB 106

mecA 105

mecC 107

ctx-M 104

gyrA83 of E. coli 104

gyrA83 of P. aeruginosa 105

gyrA87 of E. coli 105

gyrA87 of P. aeruginosa 105

imp 106

kpc 104

COVERAGE OF PATHOGENS AND RESISTANCES

> For the analyte Acinetobacter baumannii complex, A. lwoffi i and A. haemolyticus with a concentration of ≥104/mL is also

detected.

> For the analyte Haemophilus infl uenzae, the serotypes a, b and c are detected with a concentration of ≥104/mL.

> For the analyte Legionella pneumophila, the serotypes 1, 2, 3, 6, 8 and 10 are detected with a concentration of ≥104/mL.

> For the analyte Enterobacter cloacae complex, the strain Enterobacter asburiae is only detected with a reduced sensitivity

of ≥106/mL.

> For the analyte Klebsiella aerogenes (Enterobacter aerogenes), the strain ATCC®51697TM is only detected with a reduced

sensitivity of ≥107/mL.

> For the analyte ctx-M, the variant ctx-M2 is only detected with a reduced sensitivity of ≥107/mL.

> For the analyte Staphylococcus aureus, the strain Staphylococcus aureus ATCC®12600TM is only detected with a reduced

sensitivity of ≥107/mL.


EXCLUSION OF UNWANTED PATHOGENS AND MICROORGANISMS IN PATIENT BACTERIAL FLORA

Relevant microorganisms of the patient bacterial flora and closely related pathogens were tested up to a concentration of

107/ mL for possible cross reactions.

> For the microorganisms Actinomyces odontolyticus, Aggregatibacter actinomycetemcomitans, Aspergillus fumigatus,

Candida spp., Cardiobacterium hominis, Eikenella corrodens, Enterococcus spp., Fusobacterium nucleatum, Granulica-

tella adiacens, Kingella kingae, Lactobacillus acidophilus, Micrococcus luteus, Mycobacterium bovis, Neisseria lactamica,

Neisseria sicca, Pantoaea agglomerans, Peptostreptococcus stomatis, Porphyromonas gingivalis, Prevotella buccalis and

Raoultella planticola, no cross reactions were found with the analytes of the Unyvero HPN Panel.

> For the analyte Acinetobacter baumannii complex, no cross reaction was found with A. ursingii.

> For the analyte Citrobacter freundii, no cross reaction was found with C. koseri.

> For the analyte Haemophilus infl uenzae, no cross reactions were found with H. parainfl uenzae and H. parahaemolyticus.

> For the analyte Legionella pneumophila, no cross reactions were found with L. longbeachae, L. parisiensis and L. micdadei

(= Tatlockia micdadei).

> For the analyte Mycoplasma pneumoniae, no cross reaction was found with M. orale.

> For the analyte Pseudomonas aeruginosa, no cross reaction was found with P. putida.

> For the analyte Staphylococcus aureus, no cross reactions were found with various strains of coagulase-negative Staphylo-

cocci (S. epidermidis, S. haemolyticus, S. lugdunensis, S. capitis, S. saprophyticus).

> For the analyte Streptococcus pneumoniae, no cross reactions were found with other Streptococci (S. agalactiae, S. angi-

nosus, S. dysgalactiae, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. pseudopneumoniae,

S. pyogenes, S. salivarius, S. sanguinis, S. vestibularis).

POSSIBLE CROSS REACTIONS

At clinically relevant pathogen concentrations, the following possible cross reactions were observed with closely related

strains, or other analytes of the Unyvero HPN Panel:

> Possible cross reactions of the analyte Haemophilus influenzae with Aggregatibacter aprophilus at a concentration of

≥105/ mL and with Haemophilus haemolyticus at a concentration of ≥106/mL.

> For some Enterobacteriaceae (Serratia, Proteus, Citrobacter), Acinetobacter and Stenotrophomonas, very weak cross

reactions were observed to related strains in rare cases. Generally, such rare cross reactions appear as a signal below the

clinical threshold ("not detected").

Table 7

Possible interacting substances

RESPIRATORY MEDICATION AND ANTIBIOTICS

SAMPLE MEDIA AND LYSIS REAGENTS

Amikacin Lysis buffer (80%), incl. DTT (40 mM)

Ambroxol NaCl (5%)

Acetylcysteine Ringer‘s lactate solution (100%)

Ampicillin Ringer‘s solution

Beclomethasone (Vanceril) SAMPLE MATRIX COMPONENTS

Carbocysteine Blood (100%)

Cefuroxime DNA from human placenta (1 μg/μl)

Ciprofl oxacin Fish sperm DNA (4 μg/μl)

Clindamycin Mucin (25 mg/ml)

Dextromethorphan

Erythromycin

Guaifenesin (guajacol glyceryl ether)

Trimethoprim

Imipenem

Sulfamethoxazole

Salbutamol

Theophylline


6.2 INTERFERING SUBSTANCES

Possible interferences were verifi ed with respiratory medications, important antibiotics, sample storage media, the lysis buffer,

with blood, and human DNA. For this purpose, example analytes of the Unyvero HPN Panel were pooled and added to

potentially interfering matrices (undiluted or dissolved in PBS).

Table 7 lists the possible interfering substances that were tested. Maximum concentrations were used according to the CLSI

guideline „EP7-A2 Interference Testing in Clinical Chemistry”. No interferences were observed.


6.3 CLINICAL TRIAL (DIAGNOSTIC PERFORMANCE TEST)

In the clinical trial with 392 clinical samples (173 lavages, 140 aspirates, 75 sputa, 4 swabs) and with verifi ed positive

microbiological fi ndings, sensitivities between 88.9% and 100%, as well as specifi cities between 98.6% and 100% were found.

Results are shown in Table 8.

Table 8

Clinical trial data following investigation of discrepant findings

PATHOGENUNYVERO RESULT SENSITIVITY SPECIFICITY

TP FP FN TN (%) (%)

Acinetobacter baumannii complex 8 0 1 369 88.9 100.0

Chlamydophila pneumoniae 0 1 0 378 n.d. 99.7

Citrobacter freundii 2 2 0 367 100.0 99.5

Enterobacter cloacae complex 17 3 1 353 94.4 99.2

Escherichia coli 30 5 0 348 100.0 98.6

Haemophilus infl uenzae 17 0 1 348 94.4 100.0

Klebsiella aerogenes (Enterobacter aerogenes)

2 2 0 371 100.0 100.0

Klebsiella oxytoca 6 3 0 363 100.0 99.2

Klebsiella pneumoniae 8 2 2 366 80.0 99.5

Klebsiella variicola 5 0 0 381 100.0 100.0

Legionella pneumophila 1 1 0 369 100.0 99.7

Moraxella catarrhalis 12 2 0 337 100.0 99.4

Morganella morganii 2 2 0 357 100.0 99.4

Mycoplasma pneumoniae 9 1 0 374 100.0 99.7

Pneumocystis jirovecii 9 0 0 358 100.0 100.0

Proteus spp. 13 5 1 362 92.9 98.6

Pseudomonas aeruginosa 37 1 4 326 90.2 99.7

Serratia marcescens 18 3 2 356 90.0 99.2

Staphylococcus aureus 48 5 4 293 92.3 98.3

Stenotrophomonas maltophilia 12 1 1 346 92.3 99.7

Streptococcus pneumoniae 11 7 0 351 100.0 98.0

TP: true positive, FP: false positive, FN: false negative, TN: true negative, n.d. not determined


The Unyvero HPN Application detected in 127 cases additional pathogens that had not been seen in the microbiological

fi ndings. Further tests were performed for most of these discrepant fi ndings using manual PCRs and sequencing. These

tests showed that of the 127 analytes that had been assessed as false positives, at least 83 were actually verifi ed as positive,

which had been overlooked with conventional microbiological testing. Four analytes were found to have a specifi city of > 98%

(Escherichia coli, Staphylococcus aureus, Proteus spp. and Streptococcus pneumoniae). All other analytes had a specifi city

between 99% and 100%.

The data also suggested that Unyvero had returned 52 false negatives. For these discrepant fi ndings, additional investigations

were also conducted by manual PCR with at least two primer pairs against various genomic loci and sequencing. In the

process, 35 fi ndings were confi rmed as true negatives. Consequently, sensitivities between 80% and 100% were found.

However, only two analytes (Klebsiella pneumoniae and Acinetobacter baumannii complex) showed sensitivities between 80%

and 90%, while all other analytes were between 90% and 100%.

Clinical sensitivity could not be found for Chlamydophila pneumoniae, because positive clinical samples were not available.

Furthermore, Chlamydophila pneumoniae cannot be cultivated, meaning that it was not possible to spike negative sputa,

lavages or aspirates with the organism. As a result, only analytical performance evaluation with PCR amplicons as test material

was done for the Chlamydophila pneumoniae assay.

If resistance markers were detected together with their typical corresponding pathogens, it was verifi ed whether the expected

antibiotic resistances matched the microbiologically determined resistance data. Resistance marker detection together with

compatible pathogens were only found for tem, shv, ctx-M, kpc, oxa-24, mecA, sul1, and for quinolone resistance markers

in E. coli and Pseudomonas aeruginosa. The antibiogram matched the detection of the resistance marker for tem in 13 out of

16 cases (81.3%), for shv in 7 out of 10 cases (70.0%), for ctx-M in 1 out of 1 case, for kpc in 1 out of 2 cases (50.0%), for

oxa-24 in 2 out of 3 cases (66.7%), for mecA in 4 out of 11 cases (36.4%) for sul1 in 6 out of 9 cases (66.7%), for quinolone

resistance markers in E. coli in 9 out of 13 cases (69.2%), and for quinolone resistance markers in P. aeruginosa in 2 out of 3

cases (66,7%).

Detected resistance markers that did not match the antibiograms were confi rmed in almost all cases by additional tests.

In general, it should be noted that resistance markers may also originate from host bacterial fl ora and other pathogens

that are not included in the Unyvero HPN Panel. Thus, in the case of mecA, this resistance marker was connected with

Staphylococcus aureus. However, this resistance marker may also originate from strains of the host fl ora (e.g. in coagulase-

negative Staphylococci such as S. epidermidis). In such cases, the detection of mecA cannot be phenotypically determined as


resistance, if Staphylococcus aureus is detected in parallel.

In addition, the rate at which existing resistances are covered by the resistance markers included in the Unyvero HPN Panel

was determined. Quinolone resistances in E. coli were successfully detected in 9 out of 9 cases (100%) in investigations

with the Unyvero HPN Cartridge; quinolone resistances in P. aeruginosa were detected in 2 out of 3 cases (66.7%),

oxacillin resistances in S. aureus were detected in 4 out of 5 cases (80%), trimethoprim/sulfamethoxazole resistances (in

Enterobacteriaceae, Acinetobacter and Stenotrophomonas) were detected in 6 out of 13 cases (46.2%), carbapenem

resistances (in Enterobacteriaceae, Acinetobacter, Stenotrophomonas, Pseudomonas) were detected in 3 out of 12 cases

(25%), resistances to 3rd gen. cephalosporins (in Enterobacteriaceae, Acinetobacter, Stenotrophomonas, Pseudomonas)

were detected in 5 out of 26 cases (19.2%), and penicillin/ampicillin resistances (in Enterobacteriaceae, Acinetobacter,

Stenotrophomonas, Pseudomonas and in Haemophilus) were detected in 18 out of 63 cases (28.6%).



Curetis GmbH | Max-Eyth-Str. 42 | 71088 Holzgerlingen | Germany

phone: +49 (0)7031 49195 10 | Email: [email protected]

www.unyvero.com | www.curetis.com


Documents

UNYVERO HPN HOSPITALIZED PNEUMONIA APPLICATION …€¦ · 3.2 unyvero hpn hospitalized pneumonia cartridge 18 3.3 workflow 19 3.4 unyvero software 20 3.5 facts – pneumonia 23 3.6