unyvero uti urinary tract infection ......12 x 10066), unyvero m1 master mix (item no. 10011,...

© Curetis GmbH, 2018 | Unyvero UTI Application Manual | Item no. 00536 Rev. 1.0 – EN – G1

UNYVERO

UTI URINARY TRACT INFECTION

APPLICATION MANUAL

2 © Curetis GmbH, 2018 | Unyvero UTI Application Manual | Item no. 00536 Rev. 1.0

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 as "Curetis")

Last updated: 04 / 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 UTI Application and should only be used by qualified health

professionals.

© Curetis GmbH, 2018 | Unyvero UTI Application Manual | Item no. 00536 Rev. 1.0 3

TABLE OF CONTENTS

INTRODUCTION

1.1 PATENTS 5

1.2 USED SYMBOLS 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 UTI URINARY TRACT INFECTION CART-

RIDGE 18

3.3 WORKFLOW 19

3.4 UNYVERO SOFTWARE 20

3.5 FACTS – URINARY TRACT INFECTIONS 23

3.6 UNYVERO UTI PANEL 24

3.7 ANALYSIS PRINCIPLE 28

PERFORMING A TEST

4.1 PREPARATION 30

4.2 SAMPLE STORAGE 33

4.3 SAMPLE TRANSFER 33

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 SAMPLES 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 56

PERFORMANCE EVALUATION STUDY

6.1 ANALYTICAL TESTING (ANALYTICAL PERFOR-

MANCE DATA) 58

6.2 INTERFERING SUBSTANCES 61

6.3 CLINICAL TRIAL (DIAGNOSTIC PERFORMANCE

TEST) 61

INTRODUCTION

5© Curetis GmbH, 2018 | Unyvero UTI Application Manual | Item no. 00536 Rev. 1.0

1.1 PATENTS

The Unyvero System is protected by the following patents: 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, 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 2014/0242678 A1, CA 2,839,951, ZL 201180072624,9, JP

6130831, 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, EU002210401-0002, EU002210401-0003, EU002210401-0004, DE 30 2008 010

146.6/44, CH 603 237, US 77/930,558, EU 007 160 955, CN 16871246, CN 16871247, CN 16871248, CN 16871249,

DE 30 2010 032 715.4/01, CH 613 203, US 85/068,577, EU 009 532 029, CN 16871250, CN 16871251, CN 16871252.

1.2 USED SYMBOLS

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 UTI Application uses the following components: The Unyvero UTI Cartridge Set (item no. 10079, contains

12 x Unyvero UTI Cartridges item no. 10078), Unyvero T1 Sample Tubes & T1 Sample Tube Caps (item no. 10069, contains

12 x 10066), Unyvero M1 Master Mix (item no. 10011, contains 12 x 10002), Unyvero Sample Pre-Treatment Tool (item no.

10043, contains 12 x 10042), as well as the Unyvero UTI Plug-In.

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 UTI Urinary Tract Infection Cartridge Unyvero UTI Cartridge

Unyvero UTI Plug-In Unyvero UTI 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 Pre-Treatment Tool Sample Pre-Treatment 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 the cartridge

> 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:

Unyvero UTI Urinary Tract Infection Application Manual

CUR10078

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

SAFETY


2.1 INTENDED USE

This chapter provides information on how to use the Unyvero UTI Cartridge safely, and describes the necessary safety

precautions.

The Unyvero UTI Application is approved for use together with the Unyvero System. The Unyvero UTI Application is handled

to detect pathogens and antibiotic resistance genes in clinical sample materials taken from patients with complicated urinary

tract infections.

The Unyvero UTI 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 serious urinary tract infection-

specific solid and liquid samples, with the objective to provide information on pathogen species and antibiotic resistance genes.

The identification of specific non-viral pathogen DNA in samples from patients with signs and symptoms of a serious urinary

tract infection caused by bacteria and/or fungi helps to diagnose these infections, if these results are used in conjunction with

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

of antibiotic resistances.

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

UTI Application, are retested using microbiological cultures, or appropriate detection tests. In particular, a negative result

for resistance genes does not rule out antibiotic resistance, because other molecular mechanisms not tested with the

Unyvero UTI Application can lead to resistances. Positive findings do not preclude co-infections with viral or other bacterial

microorganisms, or fungi, that are not analyzed as part of the Unyvero UTI Panel. The detected microorganism may not

necessarily be the cause of illness.

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


2.2 RESPONSIBILITY OF THE USER

CAUTION

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

the Unyvero UTI 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 UTI Cartridge, Unyvero Sample Tube, Unyvero Sample Tube Cap, Unyvero Master Mix Tube, and Unyvero

Sample Pre-Treatment 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, Unyvero Sample Tube Cap, and Unyvero Pre-Treatment Tool).


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). Do

not store any components in direct sunlight.

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 UTI

Application. Wash your hands thoroughly after handling samples and consumables of the Unyvero UTI 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, Unyvero Sample Tube Cap, and Unyvero Pre-Treatment Tool are

available from Curetis upon request.


Pay attention to warnings

In-vitro diagnostic product

CE marking

Do not reuse

Expiration date

Lot number

Product number

Flammable

Do not use if packaging is damaged

Read manual

Temperature limitations

Content for <n> tests

Manufacturer

PACKAGING SYMBOLS

BASIC PRINCIPLE


The Unyvero UTI Application includes

the following components:

> Unyvero Lysator

> Unyvero Cockpit

> Unyvero Analyzer

> Unyvero OS and Unyvero UTI Plug-In

> Unyvero UTI Cartridge

> Unyvero T1 Sample Tube & T1 Sample Tube Cap

> Unyvero Sample Pre-Treatment Tool

> Unyvero Master Mix Tube

> 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 Unyvero UTI Plug-In form 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

Figure 1 Unyvero System


NOTE

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

CONSUMABLES AND ACCESSORIES

The Unyvero UTI 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 is filled with reagents for DNA amplification.

The Unyvero T1 Sample Pre-Treatment Tool is an aid to prepare and process sample materials.

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).

The consumables listed below are necessary to use the Unyvero System. They are available separately:

DNA isolation

Multiplex-endpoint-PCR

Amplicon-detection by membrane array

hybridization


Figure 2 Unyvero UTI Cartridge

The Unyvero UTI 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 UTI Cartridge (Figure 2) integrates all relevant

analysis steps:

3.2 UNYVERO UTI URINARY TRACT INFECTION CARTRIDGE


3.3 WORKFLOW

The Unyvero UTI 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.

Figure 4 Unyvero Unyvero OS Active Tests overview screen

1 System status lights

2 Headline bar

3 Test bars

4 Function keys

1

2

3

4


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

Login / Logout

Active Tests

New Test

Saved Tests

Figure 5 Function keys

FUNCTION KEYS

To log the user in and out

Displays all active tests

Opens the input screen for a new test

Displays all saved tests

Includes a function to manage connected devices,

configuration, and system administration

Scroll buttons

Advanced Functions


APPLICATION SOFTWARE SYMBOLS

When you tap on a bar of an active test, symbols are displayed that depict the respective process step (Table 2). 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.

Symbolizes the time period prior to lysator loading

Displayed during lysator processing of the sample tube

Displayed during the time of cartridge loading, scanning,

and insertion into the analyzer

Symbolizes the DNA purification process

Symbolizes the multiplex PCR reaction process

Symbolizes the array hybridization process

Table 2

Process Step Symbols


3.5 FACTS – URINARY TRACT INFECTIONS

Complicated courses of urinary tract infections (UTI) that include serious and acute complicated cases, are infections of the

urine-forming organs and urinary tract system, which in serious cases result in urosepsis. In many cases infections are caused

by bacteria, seldom by fungi, viruses, or parasites.

Current standard diagnostic methods use microbiological cultures to detect microorganisms and antibiotic resistances.

However, results are generally not available for several days. Because of this, most cases of complicated urinary tract infections

are treated empirically.

The Unyvero UTI Application was developed to improve the diagnosis of pathogens and resistances. This application is

intended as an aid to diagnose complicated urinary tract infections, acute pyelonephritis, acute bacterial prostatitis, and

urosepsis.

THE UTI APPLICATION

> allows the testing of different samples, such as midstream, suprapubic and fresh catheter urines, as well as tissues

> 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. 5 hours


3.6 UNYVERO UTI PANEL

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

following national and international guidelines:

> ECDC: Annual epidemiological report, Antimicrobial resistance and healthcare-associated infections, 2014

> ECDC: Annual epidemiological report, Healthcare-associated infections, acquired in intensive care units, 2015

> MiQ: Urinary Tract Infections: Qualitätsstandards in der mikrobiologisch-infektiologischen Diagnostik, 2 ed., 2005

> EAU Guidelines 2015: EAU Guidelines on Urological Infections, Grabe et al.

> EAU Guidelines 2016: EAU Guidelines on Urological Infections, Pickard et al.

> Horan, T.C. et al. CDC/NHSN surveillance definition of healthcare-associated infection and criteria for specific types of

infections in the acute care setting. Am J Infect Control, 2008. 36: 309

The Unyvero UTI Cartridge detects the microorganisms listed in Table 3.

Table 3

Microorganisms Detected by the Unyvero UTI Panel

GROUP PATHOGEN

Universal Bacteria 1

Gram-positive

Coagulase negative staphylococci 2

Staphylococcus aureus

Staphylococcus saprophyticus

Streptococcus agalactiae

Enterococcus spp.3

Enterococcus faecalis

Enterococcus faecium

Corynebacterium urealyticum

Anaerobes Bacteroides spp. / Prevotella spp.4

Non-fermenting bacteriaAcinetobacter baumannii complex 5

Pseudomonas aeruginosa

1 More information on this analyte can be found in the text below. 2 incl. S. saprophyticus, S. hominis, S. epidermidis, S. warneri, S.haemolyticus, S. capitis, S. lugdunensis3 incl. E. faecalis, E. faecium, E. gallinarum, E. casselifl avus, E. avium, E. hirae, E. durans, E. raffi nosus, E. cecorum4 incl. B. fragilis, B. dorei, B. faecis, B. heparinolyticus, B. ovatus, B. pyogenes, B. thetaiotaomicron, B. uniformis, B. vulgatus, B. intestinalis, Parabacteroides distasonis, P. bivia, P. buccae,

P. denticola, P. disiens, P. intermedia, P. jejuni, P. melaninogenica, P. nanceiensis, P. nigrescens, P. oris, P. timonensis, P. amnii, Prevotella sp. Marseille P3667, Alloprevotella tannerae, P. buccalis

5 incl. A. baumannii, A. oleivorans, A. calcoaceticus, A. pittii, A. bereziniae


Candida albicans, Candida tropicalis, Candida orthopsilosis, Candida parapsilosis, Candida dubliniensis, Candida viswanathii,

Candida metapsilosis, Candida labiduridarum, Candida theae. Coverage of the closely related species Lodderomyces

elongisporus, Schwanniomyces etchellsii and Millerozyma farinosa (Pichia farinosa) was also determined in silico with this

analyte. These organisms were described to cause bloodstream infections15, and cholecystitis16. According to the database

Schwanniomyces occidentalis, Yamadazyma triangularis (Candida polymorpha) and Phaeosphaeria nodorum were also

detected. There is no data on human pathogenicity of these organisms. These species are combined under the Candida spp.

analyte.

The Unyvero UTI Cartridge includes a universal primer pair

to detect bacteria (Universal Bacteria analyte).

The PCR product of this primer pair is detected by multiple

universal bacteria probes. The coverage of prokaryotic

target sequences in the SILVA database was determined

to be 75.2% for these probes (SILVA data base13 search,

accessed: 25 November, 2015).

The Unyvero UTI Cartridge also contains a universal primer

pair to detect fungi. The PCR product of this primer pair is

not used for universal detection. Specific members of the

Candida genus and close relatives are detected instead.

Coverage of the following species of Candida spp. was

determined by searching the SILVA database13 (accessed:

27 November 2015), as well as BLAST14 analysis:

GROUP PATHOGEN

Enterobacteriaceae

Enterobacteriaceae 6

Citrobacter freundii / koseri

Enterobacter aerogenes

Enterobacter cloacae complex 7

Escherichia coli

Klebsiella oxytoca

Klebsiella pneumoniae 8

Klebsiella variicola 9

Proteus spp.10

Providencia spp.11

Fungi

Candida spp.12

Candida albicans

Candida auris

Candida glabrata

6 incl. E. coli, Enterobacter cloacae, Klebsiella pneumoniae, Serratia marcescens, Morganella morganii, Citrobacter freundii, Citrobacter koseri, Providenica regretteri, Proteus vulgaris, Providenica alcalifaciens, Providenica stuartii, Providenica rustigianii, Klebsiella oxytoca, Klebsiella variicola, Proteus mirabilis

7 incl. E. cloacae, E. asburiae, E. hormaechei, E. kobei8 incl. Klebsiella pneumoniae Cluster kp I + II, Alves et al., J Clin Microbiol, 44(10), 2006; incl. Klebsiella pneumoniae subsp. ozaenae9 incl. Klebsiella variicola (Cluster kp III; formerly K. pneumoniae, 2004 classifi ed as separate species)10 incl. P. vulgaris, P. mirabilis, P. penneri, P. hauseri11 incl. P. rettgeri, P. alcalifaciens, P. rustigiani, P. stuartii12 More information on this analyte can be found in the text.13 Quast et al. (2013) Nucleic Acids Res. 41(Database issue):D590-6. doi: 10.1093/nar/gks1219.14 Altschul et al. (1990) J Mol Biol. 215(3):403-410.15 Lockhart et al. (2008) J Clin Microbiol. 46(1):374-376; Adler et al. (2007) J Clin Microbiol. 45(10):3456-3458. 16 Relich et al. (2015) Diagn Microbiol Infect Dis. doi: 10.1016/j.diagmicrobio.2015.11.010.

Table 3

Microorganisms Detected by the Unyvero UTI Panel (cont.)


Table 4

Unyvero UTI Panel Resistance Markers17

At the same time, the following genes associated with antibiotic resistances are detected.

MARKER POSSIBLE RESISTANCE REFERENCE

mecA Oxacillin18 Fernandes et al.; J of Antimik. Chemoth; 506-510, 2005

vanA Glycopeptides Cetinkaya et al., Clin Microbiol Rev. 13(4):686-707, 2000

vanB Glycopeptides Cetinkaya et al., Clin Microbiol Rev. 13(4):686-707, 2000

ctx-M 19 3rd generation cephalosporins Wie-Hua Zhao and Zhi-Qing Hu, Crit rev Microbiol, 39(1): 79-101, 2013

imp 20 Carbapenems, class B Kawa et al., J Clin Micobiol., 34(12), 1996

kpc 21 Carbapenems, class A Queenan et al., Clin Microbiol Rev, 20(3), 2007

mcr-1 Polypeptides / polymyxins Liu et al., Lancet Infect Dis. 16(2):161-8., 2016

ndm 22 Carbapenems, class B Yong et al. Antimikr. Agents Chemoth., Noo 12 5046-5054, 2009

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

oxa-24/40 Carbapenems, class D Walther-Rasmussen et al., J Antimicrob Chemother, 57(3), 2006

oxa-48 23 Carbapenems, class D Walther-Rasmussen et al., J Antimicrob Chemother, 57(3), 2006

qnrB Fluoroquinolones Hooper & Jacoby, Ann N Y Acad Sci. 1354:12-31, 2015

qnrS Fluoroquinolones Hooper & Jacoby, Ann N Y Acad Sci. 1354:12-31, 2015

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

vim 24 Carbapenems, class B Cornaglia et al., Lancet Infect Dis, 11 (5), 2011

17 The antibiotic resistances caused by ß-lactamases are classifi ed according to Ambler. BLAST searches were used to determine coverage.18 Also possible is a resistance against the indicator substance cefoxitin.19 incl. the clinically relevant variants ctx-M3, ctx-M10, ctx-M14, ctx-M15, ctx-M55. Additionally, in silico coverage of the following variants was found: ctx-M1, ctx-M23, ctx-M28, 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-M179.20 Clinically relevant variants incl. imp-1 to imp-10, imp-15, imp-16, imp-19 and imp-2521 kpc-1 to kpc-2222 ndm-1 to ndm-1423 incl. oxa-162, oxa-181, oxa-232, oxa-24424 In silico coverage of the following variants was found: 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


PCR-CHAMBER DISTRIBUTION

The multiplex PCR reactions to detect microorganisms and

resistance markers are distributed across eight (independent)

PCR chambers (Figure 6). Table 5 shows the analyte distribution

across the individual chambers.

Figure 6 PCR chambers 1-8Table 5

Genetic Markers per PCR Chamber

CHAMBER 1 CHAMBER 2 CHAMBER 3 CHAMBER 4

Citrobacter freundii / koseri Universal Bacteria Corynebacterium urealyticum Coagulase negative staphylococci (CNS)

Enterobacter cloacae complexBacteroides spp. / Prevotella spp.

Enterococcus faecium Enterococcus faecalis

Escherichia coli Candida auris mecA Providencia spp.

kpc Candida glabrata Staphylococcus aureus sul1

vim Candida spp. vanB

Staphylococcus saprophyticus

CHAMBER 5 CHAMBER 6 CHAMBER 7 CHAMBER 8

Acinetobacter baumannii complex

Candida albicans ctx-M Enterobacteriaceae

ctx-M Enterococcus spp. Enterobacter aerogenes mcr-1

Klebsiella pneumoniae kpI imp Klebsiella oxytoca ndm

Klebsiella variicola Klebsiella pneumoniae kpII Proteus spp. qnrB

oxa-23 oxa-48 Pseudomonas aeruginosa qnrS

oxa-24/40 Streptococcus agalactiae

vanA


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 UTI 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 UTI Cartridge, Unyvero T1 Sample Tube & Sample Tube Cap, Unyvero Pre-Treatment 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 UTI Application, if one of the components is past its expiration date.

UNYVERO MATERIALS

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

> 1× Unyvero UTI Cartridge

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

> 1× Unyvero M1 Master Mix Tube

> 1× Unyvero Pre-Treatment Tool (according to sample)

NOTE

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

Pre-Treatment Tool are available upon request from Curetis, or your local contact.


ADDITIONAL MATERIALS

To carry out an analysis, the following materials are needed:

> sample material

> pipette, scalpel, tweezers, etc. (depending on sample)

> 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 UTI

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 UTI

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 (UTI) is activated.


LOGIN

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).

2


Figure 9 Positioning the Unyvero T1 Sample

Tube

Figure 8 Urin samples4.3 SAMPLE TRANSFER

The Unyvero UTI Application can process a variety of different samples. Please

pay attention to the different sample types listed in the sections below. Sample

transfer starts when the Unyvero Sample Tube is placed in the Unyvero Sample

Tube Holder.

The Unyvero Sample Tube Holder has four Unyvero Sample Tube slots (1)

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

primary sample containers can be placed (2).

25 Interdisziplinäre S3 Leitlinie Epidemiologie, Diagnostik, Therapie, Prävention und Management unkomplizierter, bakterieller, ambulant erworbener Harnwegsinfektionen bei erwachsenen Patienten Aktualisierung 2017 (Interdisciplinary S3 guideline, diagnostics, therapy, prevention and management of uncomplicated, bacterial, community acquired urinary tract infections in adult patients. Up-date 2017).

4.2 SAMPLE STORAGE

CAUTION

Urine samples should be processed immediately after sampling. If this is not

possible, it is recommended to store them refrigerated at max. 8°C immediately

after sampling.25 Regardless of 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.

1


The general procedure for sample transfer is described independently of the sample type. Information on transferring particular

sample types can be found in the relevant subchapters.

CAUTION

Make sure to use the T1 Sample Tube.

CAUTION

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

Figure 10 Unyvero Sample Tube – the line

indicates the maximum filling level

Figure 11 Unyvero T1 Sample Tube and

T1 Sample Tube Cap

Place the sample in the respective sample holder in the top section of the Unyvero Sample Tube Holder.

Remove the Unyvero T1 Sample Tube from the packaging. Tap on the top of the Unyvero Sample Tube to ensure that no

fluid remains in the Unyvero Transport Cap.

Remove and discard the Transport Cap. If necessary, remove any air bubbles

from the Unyvero Sample Tube before filling it with sample.

Transfer the sample into the Unyvero Sample Tube, as described in the

following sections for the different sample types.

Pay attention to the different ways of sample handling, volumes, as well as

maximum filling levels in each case (Figure 10).

Remove the packaging from the Unyvero Sample Tube.

Place the Unyvero T1 Sample Tube into an empty slot in the Unyvero Sample Tube Holder (Figure 9).

1

2


Figure 12 Closed T1 Unyvero Sample Tube

with T1 Sample Tube Cap

NOTE

There are two small openings at the bottom of the Unyvero Sample Tube Cap (1) (Figure 11). At the top of the Unyvero Sample

Tube are two small lock-in catches (2). Make sure that they fully snap into place.

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 that irritate or damage the skin may be exposed. 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.

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

snaps into place (Figure 12).


LIQUID SAMPLES

NATIVE SOLID SAMPLES

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 10).

CAUTION

The size of the solid sample must not exceed 3 mm x 3 mm x 3 mm (27 mm3,

max. 30 mg).

Figure 13 Transferring a liquid sample into

the Unyvero T1 Sample Tube

Figure 14 Transferring a solid sample

Liquid samples such as midstream urine, catheter urine, and suprapubic

aspirations can be transferred directly into the Unyvero T1 Sample Tube.

Transfer 180 μL of sample material into the Unyvero T1 Sample Tube

(Figure 13).

Solid samples, such as tissue, can be transferred directly into the Unyvero T1

Sample Tube. Cut the material to fit into the tube.

37© Curetis GmbH, 2018 | Unyvero UTI Application Manual | Item no. 00536 Rev. 1.0 37

Transfer the sample into the Unyvero Sample Tube (Figure 15) using sterile

tweezers (Figure 14), or other suitable tools. Add up to 150 μL buffer from the

Unyvero Sample Pre-Treatment Tool. Make sure that you do not overfill the

Unyvero Sample Tube. The liquid should only reach up to the lower edge of

the Sample Tube neck (Figure 10).

Figure 15 Transferring a solid sample

4.4 STARTING THE TEST

Figure 16 Starting a new test

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

botton of the screen (Figure 16). A new test screen opens.

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

NOTE

Sample types are processed individually in each cartridge. Make sure to select the appropriate sample material for the test.


SAMPLE TYPE INPUT

Figure 17 Scanning the Unyvero Sample

Tube

Select the application (UTI).

Select the indication, e.g. Urine.

Select the sample type, e.g. Midstream.

Once all of the information has been entered, tap on Start.

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

and scan it (Figure 17).

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.

4.5 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 18 Prompt to load Unyvero Lysator

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 18).

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).

Figure 19 Loading the Unyvero Lysator

Slide the safety latch backwards over the lysator slot.

Place the Unyvero Sample Tube into the opened

position (Figure 19). 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

You can start a lysis only, 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 20 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 20).

4.8 REMOVING THE SAMPLES

Figure 21 Prompt to remove the Unyvero Sample Tube

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 21) and the Unyvero Lysator screen

(Figure 22) prompt you to remove the sample tube.

Figure 22 Lysator screen


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.


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 packagings of the Unyvero UTI Cartridge,

Unyvero Sample Tube, and Unyvero Master Mix Tube. Analysis with the Unyvero UTI Application cannot be conducted with

expired consumables.

NOTE

Also read the safety data sheet for the Unyvero Cartridge.

Remove the packaging from the Unyvero UTI 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.


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 UTI Cartridge, it appears

that the Unyvero UTI Cartridge or the Unyvero Master

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

restarted using a new Unyvero Sample Tube, Unyvero UTI

Cartridge, and Unyvero Master Mix Tube.

Figure 23 Prompt to assemble cartridge

After removing the Unyvero Sample Tube from the Unyvero

Lysator and closing the lysator lid, the cartridge assembly

screen appears (Figure 23).


12

Figure 24 Cartridge slots

1 Sample Tube slot

2 Master Mix Tube slot

Insert the Unyvero Sample Tube vertically into position 1 of the Unyvero UTI

Cartridge (Figure 24).

Insert the Unyvero Master Mix Tube into position 2 of the cartridge (Figure 24).

As shown in Figure 25, hold the Unyvero UTI 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 25).

Figure 25 Scanning the cartridge

All barcodes (Unyvero Sample Tube, Unyvero UTI 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 UTI Cartridge to the slot selected by the Unyvero OS (Figure 26). The slot number is also displayed on

the screen of the Unyvero Analyzer and is indicated by a green arrow under the slot (Figure 27).

Figure 26 Prompt to load the Unyvero Analyzer Figure 27 Analyzer display


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

(Figure 28).

Figure 28 Inserting the cartridge into the Unyvero

Analyzer

4.11 ANALYSIS STATUS

Figure 29 Analyzer display

The processing of the Unyvero UTI Cartridge takes approximately 4-5 hours. The

remaining analysis time is displayed on the Unyvero Analyzer screen (Figure 29)

and in the cockpit test status bar. The processing progress of the Unyvero UTI

Cartridge in the analyzer is indicated by a purple status bar on the overview screen.


Figure 30 Analyzer display

4.12 REMOVING THE CARTRIDGE

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 UTI Cartridge can be removed (Figure 30).

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

Remove the Unyvero UTI Cartridge.

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

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.

CAUTION

The Unyvero UTI Cartridge is intended for single use and can only be used for one sample. The Unyvero UTI Cartridge cannot

be reused.

TEST RESULTS


Figure 31 Saved Tests screen

Figure 32 Results screen buttons

5.1 VIEWING RESULTS

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 32):

> Overview

> Microorganisms

> Resistance Markers

> Test Information

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

bar (Figure 31).


5.2 READING THE RESULTS

Figure 33 Overview screen

SUMMARY OF MEASUREMENT RESULTS

Tap on the Overview button.

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 33), while the bottom half features

a summary of the antibiotic classes and any detected

resistance genes.

© Curetis GmbH, 2018 | Unyvero UTI Application Manual | Item no. 00536 Rev. 1.052

Figure 34 Microorganism results screen

DETAILED INFORMATION ON MICROORGANISMS

The microorganisms (Figure 34) are categorized as follows:

> Universal Bacteria

> Gram-positive

> Anaerobes

> Non-fermenting bacteria

> Enterobacteriaceae

> Fungi

Tap on the button Microorganisms to view a list

of all microorganisms analyzed by the Unyvero UTI

Application.

The presenence or absence of microorganisms is displayed

as follows on the Microorganisms screen (Figure 34):

Detected

Not detected

Invalid analyte

Information

Positive, specific detection of bacterial pathogens - see result report


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. For the analyte Universal Bacteria, the result

is shown only as being positive or negative.

NOTE

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

analyte.

CAUTION

The universal test for bacterial DNA only displays a positive or negative result, if all of the panel's specific bacterial assays are

negative and/or invalid. If a specific bacterial assay is positive, this is indicated in the result field for this specific result (a circular

symbol is displayed for the Universal Bacteria and Enterobacteriaceae analytes). This does not rule out the possibility that, in

addition to the specifically detected pathogens, DNA from other bacteria is also present.


Figure 35 Resistance Marker results screen

DETAILS ON ANTIBIOTIC RESISTANCE MARKERS

The presence or absence of resistance markers is shown

on the Resistance Markers screen (Figure 35):

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 UTI Application.

Detected

Not detected

Invalid analyte


TRANSFERABLE ANTIBIOTIC RESISTANCE (RESISTANCE GENE)

The resistance markers that are detected using the Unyvero UTI Application are genes transferred by mobile genetic elements

(plasmids and integrons).26 The presence of such a gene correlates with resistance against a particular antibiotic class.27

Table 6 lists the resistance markers detected by the Unyvero UTI Application.

Table 6

Unyvero UTI Panel Resistance Markers

MARKER POSSIBLE RESISTANCE FREQUENT OCCURRENCE IN

mecA Oxacillin Staphylococcus spp.

vanA Glycopeptides Staphylococcus spp., Streptococcus spp., Enterococcus spp.

vanB Glycopeptides Staphylococcus spp., Streptococcus spp., Enterococcus spp.

ctx-M 3rd generation cephalosporins Enterobacteriaceae, non-fermenting bacteria

imp Carbapenems, class B Enterobacteriaceae, non-fermenting bacteria

kpc Carbapenems, class A Enterobacteriaceae, non-fermenting bacteria

mcr-1 Polypeptides / polymyxins Enterobacteriaceae

ndm Carbapenems, class B Enterobacteriaceae, non-fermenting bacteria

oxa-23 Carbapenems, class D Enterobacteriaceae, non-fermenting bacteria

oxa-24/40 Carbapenems, class D Enterobacteriaceae, non-fermenting bacteria

oxa-48 Carbapenems, class D Enterobacteriaceae, non-fermenting bacteria

qnrB Fluoroquinolones Enterobacteriaceae

qnrS Fluoroquinolones Enterobacteriaceae

sul1 Sulfonamide Enterobacteriaceae, non-fermenting bacteria

vim Carbapenems, class B Enterobacteriaceae, non-fermenting bacteria

NOTE

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

26 Roberts, Front Microbiol, Vol. 2 (2011)27 Maiden, MolBiotechnol, Vol. 18 (2001)


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

ANALYTEDETECTION LIMIT (PATHOGENS / ML)

POSITIVITY RATE FOR 1/10 DETECTION LIMIT [%]

Universal Bacteria 103 n.d.

Coagulase negative staphyococci (CNS)28 105 80.0

Staphylococcus aureus 104 85.0

Staphylococcus saprophyticus 104 70.0

Staphylococcus agalactiae 104 90.0

Enterococcus spp.29 104 65.0

Enterococcus faecalis 104 75.0

Enterococcus faecium 104 50.0

Corynebacterium urealyticum 105 20.0

Bacteroides spp. / Prevotella spp.30 104 52.5

Acinetobacter baumannii complex 104 47.5

Pseudomonas aeruginosa 103 n.d.

Enterobacteriaceae 4 x 103 n.d.

Citrobacter freundii/koseri 31 104 72.5

Enterobacter aerogenes 103 n.d.


6.1 ANALYTICAL TESTING (ANALYTICAL PERFORMANCE DATA)

During the CE performance evaluation study, the Unyvero UTI Application was tested both analytically using microbial reference

strains or DNA fragments (amplicons) and clinically using samples on more than 1180 cartridges. For the analysis of clinical

samples (midstream urine, catheter urine), primarily those samples were used that had a positive clinical microbiology result

of the pathogens present on the UTI Panel.

ANALYTICAL SENSITIVITY

Detection limits of individual pathogens were tested with serial dilutions of reference strains in buffer. The following limits of

detection were reproducibly verified with a positivity rate of at least 95% using the Unyvero UTI Cartridge (Table 7):

Table 7

Data of Analytical Tests

n.d. not determined28 Detection limit determined with Staphylococcus lugdunensis and Staphylococcus saprophyticus29 Detection limit determined with Enterococcus faecalis and Enterococcus faecium30 Detection limit determined with Bacteroides fragilis and Prevotella melaninogenica31 Detection limit determined with Citrobacter freundii and Citrobacter koseri


Table 7

Data of Analytical Tests (cont.)

n.d. not determined

ANALYTEDETECTION LIMIT (PATHOGENS / ML)

POSITIVITY RATE FOR 1/10 DETECTION LIMIT [%]

Enterobacter cloacae complex 32 104 80.0

Escherichia coli 104 93.3

Klebsiella oxytoca 103 n.d.

Klebsiella pneumoniae 104 52.5

Klebsiella variicola 104 65.0

Proteus spp.33 104 45.0

Providencia spp.34 103 n.d.

Candida spp.35 103 5.0

Candida albicans 103 0.0

Candida auris 103 70.0

Candida glabrata 104 90.0

mecA 104 80.0

vanA 105 80.0

vanB 104 30.0

ctx-M 104 62.5

imp 105 80.0

kpc 104 85.0

ndm 104 70.0

oxa-23 105 90.0

oxa-24/40 103 n.d.

oxa-48 103 n.d.

vim 104 85.0

qnrB 103 n.b.

qnrS 105 35,0

mcr-1 104 65.0

sul1 104 75.0

32 Detection limit determined with Enterobacter cloacae33 Detection limit determined with Proteus vulgaris34 Detection limit determined with Providencia rettgeri35 Detection limit determined with Candida albicans


COVERAGE OF PATHOGENS AND RESISTANCES

> The Acinetobacter baumannii complex analyte is also used to detect Acinetobacter ursingii, Acinetobacter bereziniae and

Acinetobacter haemolyticus.

> Staphylococcus pettenkoferi is not detected with the coagulase-negative Staphylococci (CNS) analyte.

> The analyte Bacteroides spp. / Prevotella spp. is detected with specific probes for these genera, which hybridize with the

Universal Bacteria PCR fragment. The following species tested positive for the analyte Bacteroides spp. / Prevotella spp: B.

fragilis, B. dorei, B. ovatus, B. heparinolyticus, B. intestinales, B. pyogenes, B. thetaiotaomicron, B. uniformis, B. vulgatus,

Parabacteroides distasonis, Alloprevotella tanneri, P. amni, P. bivia, P. buccae, P. buccalis, P. disiens, P. intermedia, P. jejuni,

P. melaninogenica, P. nanceiensis, P. nigrescens, P. oris, P. timonensis, Prevotella sp. Marseille P3667.

> With the analyte Enterobacteriaceae the following Enterobacteriaceae strains were detected/covered experimentally:

Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, Morganella morganii, Citrobacter

freundii, Citrobacter koseri, Providencia rettgeri, Proteus vulgaris, Providencia alcalifaciens, Providencia stuartii, Providencia

rustigianii, Klebsiella oxytoca, Klebsiella variicola, Proteus mirabilis.

> With the analyte Providencia spp. the following Providencia strains were detected/covered: Providencia rettgeri, Providencia

alcalifaciens, Providencia stuartii, Providencia rustigianii.

> With the analyte Enterobacter cloacae complex also Enterobacter kobei is detected.

> With the analyte Enterococcus spp. also Enterococcus cecorum is detected.

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 UTI Panel:

> Possible cross-reactions of Staphylococcus saccharolyticus (coagulase-positive) with the analyte coagulase-negative

staphylococci (CNS)

> Possible cross-reactions of Porphyromonas endodontalis, Porphyromonas somerae, Porphyromonas uenonis and

Porphyromonas asaccharolytica with the analyte Bacteroides spp./ Prevotella spp..


6.2 INTERFERING SUBSTANCES

Possible interferences with sample matrix components and a typical additive in urine collection containers were examined.

Sample matrix components were tested in twice the physiologically normal concentration. Table 8 lists the tested substances.

No interferences were observed.

Table 8

Substances with possible interactions

SAMPLE MATRIX COMPONENTSSAMPLE COLLECTION CONTAINTER

ADDITIVE

Urea (35 g/L) Boric acid (15 mg/mL; 30 mg/mL)

Calcium (7.5 mMol)

It was additionally tested, if a concentration of the sample infl uenced the analysis. For this purpose, urine samples were

concentrated 5-fold. No interferences were observed

6.3 CLINICAL TRIAL (DIAGNOSTIC PERFORMANCE TEST)

For the clinical trial, 443 samples were analyzed (clinical samples with verifi ed, microbiologically positive fi ndings). Of these 443

samples, 300 samples were measured retrospectively and 143 samples prospectively. The results of the clinical trial are listed

in Table 9.


Table 9

Clinical Trial Data Following Investigation of Discrepant Findings

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

PATHOGENUNYVERO RESULT SENSITIVITY SPECIFICITY

TP FP FN TN (%) (%)

Universal Bacteria 386 0 27 30 93.5 100.0

Coagulase negative staphyococci (CNS) 88 5 2 348 97.8 98.6

Staphylococcus aureus 33 1 1 407 97.1 99.8

Staphylococcus saprophyticus 11 0 0 432 100.0 100.0

Streptococcus agalactiae 16 0 0 422 100.0 100.0

Enterococcus spp. 145 8 2 286 98.6 97.3

Enterococcus faecalis 134 6 0 303 100.0 98.1

Enterococcus faecium 33 2 1 406 97.1 99.5

Corynebacterium urealyticum 10 10 0 422 100.0 97.7

Bacteroides spp. / Prevotella spp. 62 28 0 353 100.0 92.7

Acinetobacter baumannii complex 4 0 0 439 100.0 100,0

Pseudomonas aeruginosa 34 3 0 404 100.0 99.3

Enterobacteriaceae 235 3 33 167 87.7 98.2

Citrobacter freundii/koseri 27 0 0 416 100.0 100.0

Enterobacter aerogenes 8 0 2 431 80.0 100.0

Enterobacter cloacae complex 27 1 0 415 100.0 99.8

Escherichia coli 178 0 3 262 98.3 100.0

Klebsiella oxytoca 30 1 0 410 100.0 99.8

Klebsiella pneumoniae 41 0 1 399 97.6 100.0

Klebsiella variicola 6 0 0 420 100,0 100,0

Proteus spp. 49 0 2 390 96.1 100.0

Providencia spp. 11 0 0 432 100.0 100.0

Candida spp. 18 2 1 422 94.7 99.5

Candida albicans 21 0 0 420 100.0 100.0

Candida auris 0 0 0 443 n.d. 100.0

Candida glabrata 4 0 0 439 100.0 100.0


SELECTED RESISTANCE MARKER DATA

In 660 cases the Unyvero UTI Application detected additional pathogens that had not been present in the microbiological

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

showed that 590 of the analytes assessed as false positive were actually verifi ed as positive, which had been missed in the

microbiological tests.

The data further suggested that Unyvero had returned 76 false negatives. For these discrepant fi ndings, also further tests using

manual PCRs and sequencing were conducted. Of these results 75 were confi rmed as true negative. Most of the false negative

results were found for the Enterobacteriaceae analyte. The detection threshold of this group analyte is higher than that of some

of the covered individual species, so that false negative results are possible. Also, several false negative results were found for

the Universal Bacteria analyte. For this analyte false negative results are to be anticipated in some cases, since their coverage

determined in silico is around 75.2%, due to the broad spectrum of prokaryotic target sequences.

Overall, 355 midstream urine and 88 catheter urine samples were measured for the clinical validation. In addition, further tests

demonstrated that the Unyvero UTI Application can be used with other sample materials, such as tissue and suprapubic

aspirations.

The following resistance markers of the Unyvero UTI Panel were detected in the clinical trial: mecA, ctx-M, oxa-23, oxa-48,

qnrB, qnrS, sul1. Additionally, it was examined if phenotypic resistances of a specifi c pathogen correspond to the genotypic

analysis of the resistance marker and pathogen. Antibiograms were not available for all pathogen fi ndings. The positive Unyvero

results for resistance markers, as well as the number of positive results for detected resistance markers, found together with a

pathogen with antibiogram are listed in Table 10.


Table 11

PPV and NPV Values for Some Resistance Markers

TP: true positive, FP: false positive, FN: false negative, TN: true negative, PPV: positive predictive value, NPV: negative predictive value

RESISTANCE TO THE FOLLOWING CLASSES OF ANTIBIOTICS/ RESISTANCE MARKERS OF THE UNYVERO UTI PANEL

NUMBER OF POSITIVE UNYVERO-RESULTS

NUMBER OF POSITIVE UNYVERO-RESULTS DETECTED WITH

PATHOGENS WITH MATCHING ANTIOGRAM

3rd generation cephalosporins / ctx-M 38 14

Carbapenems / imp, kpc, ndm, vim, oxa-23, oxa-24/40, oxa-48

3 0

Oxacilline / mecA 94 9

Polymyxin / polypeptides / mcr-1 0 0

Vancomycin / vanA, vanB 0 0

Fluorquinolone / qnrB, qnrS 27 9

Sulfonamide / sul1 77 38

Table 10

Positive Unyvero Results for Resistance Markers

Due to the low prevalence, a suffi cient number of sample measurements with antibiogram could only be achieved for some

resistance markers in order to determine positive and negative predictive values (PPV, NPV). The corresponding results are

presented in Table 11.

RESISTANCE MARKERTYPICAL CORRESPONDING

PATHOGENSTP TN FP FN PPV NPV

mecA Staphylococcus spp. 4 14 5 0 44.4 100.0

ctx-M Enterobacteriaceae 11 128 3 7 78.8 94.8

sul1 Enterobacteriaceae 27 87 11 18 71.1 82.9

qnrB Enterobacteriaceae 5 109 2 30 71.4 78.4

qnrS Enterobacteriaceae 1 110 2 29 33.3 79.1

unyvero uti urinary tract infection ......12 x 10066), unyvero m1 master mix (item no. 10011,...

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