UNYVERO UTI URINARY TRACT INFECTION APPLICATION MANUAL

Transcription

1 UNYVERO UTI URINARY TRACT INFECTION APPLICATION MANUAL Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0 EN G1

2 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 Holzgerlingen Germany Phone +49 (0) Fax +49 (0) (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. 2 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

3 TABLE OF CONTENTS INTRODUCTION 1.1 PATENTS USED SYMBOLS CONVENTIONS USED LIABILITY SERVICE AND SUPPORT E-LABELING 8 SAFETY 2.1 INTENDED USE RESPONSIBILITY OF THE USER STORAGE AND USE BIOLOGICAL SAFETY CHEMICAL SAFETY 13 BASIC PRINCIPLE 3.1 UNYVERO COMPONENTS UNYVERO UTI URINARY TRACT INFECTION CART- RIDGE WORKFLOW UNYVERO SOFTWARE FACTS URINARY TRACT INFECTIONS UNYVERO UTI PANEL ANALYSIS PRINCIPLE 28 PERFORMING A TEST 4.1 PREPARATION SAMPLE STORAGE SAMPLE TRANSFER STARTING THE TEST THAWING THE UNYVERO MASTER MIX PERFORMING THE LYSIS LYSIS STATUS REMOVING THE SAMPLES ASSEMBLING THE CARTRIDGE MEASURING IN THE ANALYZER ANALYSIS STATUS REMOVING THE CARTRIDGE 48 TEST RESULTS 5.1 VIEWING RESULTS READING THE RESULTS INVALID RESULTS 56 PERFORMANCE EVALUATION STUDY 6.1 ANALYTICAL TESTING (ANALYTICAL PERFOR- MANCE DATA) INTERFERING SUBSTANCES CLINICAL TRIAL (DIAGNOSTIC PERFORMANCE TEST) 61 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

4 INTRODUCTION

5 1.1 PATENTS The Unyvero System is protected by the following patents: PCT/EP/2011/02303, WO 2011/144304, EP , EP A1, US 9,598,721, SG , JP , (Div) JP , HK , AU , ES , PCT/EP/2011/002507, WO 2011/144345, EP , EP , IN: 9813/DELNP/2012, US 9,592,511, ZL , HK Nr A, AU , SG , JP , CA 2,799,676, EP , WO2013/013687A1, AU , US 2014/ A1, CA 2,839,951, ZL ,9, JP , JP , EU , US D683,044, CH , EU , US 29/433,060, D723,180, CH , EU , EU , EU , EU , DE /44, CH , US 77/930,558, EU , CN , CN , CN , CN , DE /01, CH , US 85/068,577, EU , CN , CN , CN 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. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

6 1.3 CONVENTIONS USED SHORT NAMES Table 1 Unyvero Components FULL NAMES Unyvero A50 Analyzer Unyvero L4 Lysator Unyvero C8 Cockpit Unyvero M1 Master Mix Tube Unyvero Operating Software Unyvero UTI Urinary Tract Infection Cartridge Unyvero UTI Plug-In Unyvero Sample Tube Holder Unyvero T1 Sample Tube Unyvero T1 Sample Tube Cap Unyvero T1 Transport Cap Unyvero Sample Pre-Treatment Tool SHORT NAMES Unyvero Analyzer Unyvero Lysator Unyvero Cockpit Unyvero Master Mix Unyvero OS Unyvero UTI Cartridge Unyvero UTI Plug-In Sample Tube Holder T1 Sample Tube T1 Sample Tube Cap T1 Transport Cap Sample Pre-Treatment Tool The Unyvero System consists of the Unyvero Analyzer (item no ), the Unyvero Lysator (item no ), the Unyvero Cockpit (item no ) incl. Unyvero OS and the Unyvero Sample Tube Holder (item no ). The Unyvero UTI Application uses the following components: The Unyvero UTI Cartridge Set (item no , contains 12 x Unyvero UTI Cartridges item no ), Unyvero T1 Sample Tubes & T1 Sample Tube Caps (item no , contains 12 x 10066), Unyvero M1 Master Mix (item no , contains 12 x 10002), Unyvero Sample Pre-Treatment Tool (item no , contains 12 x 10042), as well as the Unyvero UTI Plug-In. 6 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

7 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. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

8 1.5 SERVICE AND SUPPORT If your region is under Curetis customer care, please contact Curetis support by phone or . 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) (available Monday-Friday from 9 a.m. to 5 p.m. CET) or support@curetis.com. 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 GR RO non-eu RU Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

9 SAFETY

10 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 infectionspecific 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. 10 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

11 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). Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

12 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. 12 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

13 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. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

14 PACKAGING SYMBOLS Pay attention to warnings Flammable In-vitro diagnostic product Do not use if packaging is damaged CE marking Read manual Do not reuse Temperature limitations Expiration date Content for <n> tests Lot number Manufacturer Product number 14 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

15 BASIC PRINCIPLE

16 3.1 UNYVERO COMPONENTS The Unyvero UTI Application includes the following components: > Unyvero Lysator 3 2 > Unyvero Cockpit > Unyvero Analyzer 1 > Unyvero OS and Unyvero UTI Plug-In > Unyvero UTI Cartridge > Unyvero T1 Sample Tube & T1 Sample Tube Cap > Unyvero Sample Pre-Treatment Tool UNYVERO L4 LYSATOR Figure 1 Unyvero System UNYVERO C8 COCKPIT UNYVERO A50 ANALYZER > Unyvero Master Mix Tube > Unyvero Sample Tube Holder 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. 16 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

17 The consumables listed below are necessary to use the Unyvero System. They are available separately: 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). NOTE The instruments are described in detail in the Unyvero System manual. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

18 3.2 UNYVERO UTI URINARY TRACT INFECTION CARTRIDGE The Unyvero UTI Cartridge (Figure 2) integrates all relevant analysis steps: 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. 18 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

19 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 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

20 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. 4 Figure 4 Unyvero Unyvero OS Active Tests overview screen 1 System status lights 2 Headline bar 3 Test bars 4 Function keys 20 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

21 The function keys are displayed at the bottom of the screen (Figure 5). Figure 5 Function keys 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 Functions Includes a function to manage connected devices, configuration, and system administration Scroll buttons Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

22 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. Table 2 Process Step Symbols 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 22 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

23 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 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

24 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, : 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 Coagulase negative staphylococci 2 Staphylococcus aureus Staphylococcus saprophyticus Streptococcus agalactiae Gram-positive Enterococcus spp. 3 Enterococcus faecalis Enterococcus faecium Corynebacterium urealyticum Anaerobes Bacteroides spp. / Prevotella spp. 4 Acinetobacter baumannii complex 5 Non-fermenting bacteria 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. lugdunensis 3 incl. E. faecalis, E. faecium, E. gallinarum, E. casselifl avus, E. avium, E. hirae, E. durans, E. raffi nosus, E. cecorum 4 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 24 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

25 Table 3 Microorganisms Detected by the Unyvero UTI Panel (cont.) GROUP PATHOGEN The Unyvero UTI Cartridge includes a universal primer pair to detect bacteria (Universal Bacteria analyte). Enterobacteriaceae Fungi 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 Candida spp. 12 Candida albicans Candida auris Candida glabrata 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 base 13 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 database 13 (accessed: 27 November 2015), as well as BLAST 14 analysis: 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 infections 15, and cholecystitis 16. 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. 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. kobei 8 incl. Klebsiella pneumoniae Cluster kp I + II, Alves et al., J Clin Microbiol, 44(10), 2006; incl. Klebsiella pneumoniae subsp. ozaenae 9 incl. Klebsiella variicola (Cluster kp III; formerly K. pneumoniae, 2004 classifi ed as separate species) 10 incl. P. vulgaris, P. mirabilis, P. penneri, P. hauseri 11 incl. P. rettgeri, P. alcalifaciens, P. rustigiani, P. stuartii 12 More information on this analyte can be found in the text. 13 Quast et al. (2013) Nucleic Acids Res. 41(Database issue):d doi: /nar/gks Altschul et al. (1990) J Mol Biol. 215(3): Lockhart et al. (2008) J Clin Microbiol. 46(1): ; Adler et al. (2007) J Clin Microbiol. 45(10): Relich et al. (2015) Diagn Microbiol Infect Dis. doi: /j.diagmicrobio Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

26 At the same time, the following genes associated with antibiotic resistances are detected. Table 4 Unyvero UTI Panel Resistance Markers 17 MARKER POSSIBLE RESISTANCE REFERENCE meca Oxacillin 18 Fernandes et al.; J of Antimik. Chemoth; , 2005 vana Glycopeptides Cetinkaya et al., Clin Microbiol Rev. 13(4): , 2000 vanb Glycopeptides Cetinkaya et al., Clin Microbiol Rev. 13(4): , 2000 ctx-m 19 3 rd generation cephalosporins Wie-Hua Zhao and Zhi-Qing Hu, Crit rev Microbiol, 39(1): , 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 , 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 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), 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-m Clinically relevant variants incl. imp-1 to imp-10, imp-15, imp-16, imp-19 and imp kpc-1 to kpc ndm-1 to ndm incl. oxa-162, oxa-181, oxa-232, oxa 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 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

27 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. Table 5 Genetic Markers per PCR Chamber Figure 6 PCR chambers 1-8 CHAMBER 1 CHAMBER 2 CHAMBER 3 CHAMBER 4 Citrobacter freundii / koseri Universal Bacteria Corynebacterium urealyticum Coagulase negative staphylococci (CNS) Enterobacter cloacae complex Bacteroides 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 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

28 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. 28 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

29 PERFORMING A TEST

30 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. 30 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

31 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. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

32 LOGIN 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). 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". 32 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

33 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. 4.3 SAMPLE TRANSFER Figure 8 Urin samples 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. 2 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). 1 Figure 9 Positioning the Unyvero T1 Sample Tube 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). Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

34 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. 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. Place the Unyvero T1 Sample Tube into an empty slot in the Unyvero Sample Tube Holder (Figure 9). CAUTION Make sure to use the T1 Sample Tube. CAUTION Do not vortex or centrifuge the Unyvero Sample Tube with the Transport Cap. Remove and discard the Transport Cap. If necessary, remove any air bubbles from the Unyvero Sample Tube before filling it with sample. Figure 10 Unyvero Sample Tube the line indicates the maximum filling level 2 Figure 11 Unyvero T1 Sample Tube and T1 Sample Tube Cap 1 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. 34 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

35 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. Press the Unyvero Sample Tube Cap onto the Unyvero Sample Tube until it snaps into place (Figure 12). Figure 12 Closed T1 Unyvero Sample Tube with T1 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 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. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

36 LIQUID SAMPLES 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). Figure 13 Transferring a liquid sample into the Unyvero T1 Sample Tube 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). NATIVE SOLID SAMPLES Solid samples, such as tissue, can be transferred directly into the Unyvero T1 Sample Tube. Cut the material to fit into the tube. CAUTION The size of the solid sample must not exceed 3 mm x 3 mm x 3 mm (27 mm 3, max. 30 mg). Figure 14 Transferring a solid sample 36 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no 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 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. Figure 16 Starting a new test NOTE Sample types are processed individually in each cartridge. Make sure to select the appropriate sample material for the test. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

38 SAMPLE TYPE INPUT 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. Figure 17 Scanning the Unyvero Sample Tube 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. 38 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

39 4.6 PERFORMING THE LYSIS 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 18 Prompt to load 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. Figure 19 Loading the Unyvero Lysator Close the lid of the Unyvero Lysator. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

40 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. NOTE The lysis cannot start until the lysator lid is closed. 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. 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. 40 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

41 4.7 LYSIS STATUS The lysis process takes approximately 30 minutes and is displayed on the overview screen by a gray bar for each sample. Figure 20 Lysator screen 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 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 21 Prompt to remove the Unyvero Sample Tube Figure 22 Lysator screen Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

42 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. 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. 42 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

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 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. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

44 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. After removing the Unyvero Sample Tube from the Unyvero Lysator and closing the lysator lid, the cartridge assembly screen appears (Figure 23). Figure 23 Prompt to assemble cartridge 44 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

45 1 2 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). Figure 24 Cartridge slots 1 Sample Tube slot 2 Master Mix Tube slot 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). 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. Figure 25 Scanning the cartridge Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

46 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 46 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

47 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 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 29 Analyzer display Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

48 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). Remove the Unyvero UTI Cartridge. Figure 30 Analyzer display 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. Dispose of the Unyvero UTI Cartridge according to the guidelines of your institution. 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. 48 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

49 TEST RESULTS

50 5.1 VIEWING RESULTS Completed analyses are displayed by blue bars on the overview screen (Active Tests or Saved Tests). To view a test result, tap on the corresponding blue test bar (Figure 31). A screen appears with a new menu bar and the following buttons (Figure 32): > Overview > Microorganisms > Resistance Markers > Test Information Figure 31 Saved Tests screen Figure 32 Results screen buttons 50 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

51 5.2 READING THE RESULTS 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. Figure 33 Overview screen Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

52 DETAILED INFORMATION ON MICROORGANISMS Tap on the button Microorganisms to view a list of all microorganisms analyzed by the Unyvero UTI Application. The microorganisms (Figure 34) are categorized as follows: > Universal Bacteria > Gram-positive > Anaerobes > Non-fermenting bacteria > Enterobacteriaceae > Fungi The presenence or absence of microorganisms is displayed as follows on the Microorganisms screen (Figure 34): Detected Not detected Figure 34 Microorganism results screen Invalid analyte Information Positive, specific detection of bacterial pathogens - see result report 52 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

53 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. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

54 DETAILS ON ANTIBIOTIC RESISTANCE MARKERS Tap on the button Resistance Markers to display an overview of all resistance markers tested with the Unyvero UTI Application. 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. Detected Not detected Invalid analyte Figure 35 Resistance Marker results screen 54 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

55 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) Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

56 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. 56 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

57 PERFORMANCE EVALUATION STUDY

58 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 ANALYTE DETECTION LIMIT (PATHOGENS / ML) POSITIVITY RATE FOR 1/10 DETECTION LIMIT [%] Universal Bacteria 10 3 n.d. Coagulase negative staphyococci (CNS) Staphylococcus aureus Staphylococcus saprophyticus Staphylococcus agalactiae Enterococcus spp Enterococcus faecalis Enterococcus faecium Corynebacterium urealyticum Bacteroides spp. / Prevotella spp Acinetobacter baumannii complex Pseudomonas aeruginosa 10 3 n.d. Enterobacteriaceae 4 x 10 3 n.d. Citrobacter freundii/koseri Enterobacter aerogenes 10 3 n.d. 28 Detection limit determined with Staphylococcus lugdunensis and Staphylococcus saprophyticus 29 Detection limit determined with Enterococcus faecalis and Enterococcus faecium 30 Detection limit determined with Bacteroides fragilis and Prevotella melaninogenica 31 Detection limit determined with Citrobacter freundii and Citrobacter koseri n.d. not determined 58 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

59 Table 7 Data of Analytical Tests (cont.) ANALYTE DETECTION LIMIT (PATHOGENS / ML) POSITIVITY RATE FOR 1/10 DETECTION LIMIT [%] Enterobacter cloacae complex Escherichia coli Klebsiella oxytoca 10 3 n.d. Klebsiella pneumoniae Klebsiella variicola Proteus spp Providencia spp n.d. Candida spp Candida albicans Candida auris Candida glabrata meca vana vanb ctx-m imp kpc ndm oxa oxa-24/ n.d. oxa n.d. vim qnrb 10 3 n.b. qnrs ,0 mcr sul Detection limit determined with Enterobacter cloacae n.d. not determined 33 Detection limit determined with Proteus vulgaris 34 Detection limit determined with Providencia rettgeri 35 Detection limit determined with Candida albicans Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

60 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.. 60 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

61 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 Urea (35 g/l) SAMPLE MATRIX COMPONENTS SAMPLE COLLECTION CONTAINTER ADDITIVE 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. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

62 Table 9 Clinical Trial Data Following Investigation of Discrepant Findings PATHOGEN UNYVERO RESULT SENSITIVITY SPECIFICITY TP FP FN TN (%) (%) Universal Bacteria Coagulase negative staphyococci (CNS) Staphylococcus aureus Staphylococcus saprophyticus Streptococcus agalactiae Enterococcus spp Enterococcus faecalis Enterococcus faecium Corynebacterium urealyticum Bacteroides spp. / Prevotella spp Acinetobacter baumannii complex ,0 Pseudomonas aeruginosa Enterobacteriaceae Citrobacter freundii/koseri Enterobacter aerogenes Enterobacter cloacae complex Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae Klebsiella variicola ,0 100,0 Proteus spp Providencia spp Candida spp Candida albicans Candida auris n.d Candida glabrata TP: true positive, FP: false positive, FN: false negative, TN: true negative, n.d. not determined 62 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

63 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. SELECTED RESISTANCE MARKER DATA 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. Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

64 Table 10 Positive Unyvero Results for Resistance Markers 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 Carbapenems / imp, kpc, ndm, vim, oxa-23, oxa-24/40, oxa Oxacilline / meca 94 9 Polymyxin / polypeptides / mcr Vancomycin / vana, vanb 0 0 Fluorquinolone / qnrb, qnrs 27 9 Sulfonamide / sul 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. Table 11 PPV and NPV Values for Some Resistance Markers RESISTANCE MARKER TYPICAL CORRESPONDING PATHOGENS TP TN FP FN PPV NPV meca Staphylococcus spp ctx-m Enterobacteriaceae sul1 Enterobacteriaceae qnrb Enterobacteriaceae qnrs Enterobacteriaceae TP: true positive, FP: false positive, FN: false negative, TN: true negative, PPV: positive predictive value, NPV: negative predictive value 64 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0

65 Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev

66 Curetis GmbH Max-Eyth-Str Holzgerlingen Germany Tel.: +49 (0) Curetis GmbH, 2018 Unyvero UTI Application Manual Item no Rev. 1.0