Bundle TestedSamples Tested

Samples with Detections

Detection Rate

Aspergillus flavus and Clavispora lusitaniae 3 0 0%Caulobacter segnis, Aspergillus niger, and Pedobacter heparinus 3 0 0%Shewanella oneidensi, Streptomyces griseus, and Candida kefyr 3 0 0%Influenza, Parainfluenza, Adenovirus, and Adenovirus 31 3 0 0%Parvovirus B19, Coronavirus, and HHV-1 3 0 0%

Candida spp.

Moraxella catarrhalis/nonliquefaciens

Corynebacterium striatum

Neisseria flavescens/subflava

Eikenella corrodensPrevotella bivia/denticola

Enterococcus durans/hirae/mundtii

Propionibacterium acnes

Fusobacterium nucleatum

Rothia dentocariosa/mucilaginosa

Gemella haemolysans/sanguinis

Staphylococcus epidermidis

Granulicatella adiacensSalivarius Group Streptococci

Haemophilus parahaemolyticus

Viridans Streptococci Lactobacillus spp. Veillonella dispar/parvula

Variable (Identity) # of Detections/ # of Tests ReproducibilityOperator 1 73 / 75 97.3%Operator 2 72 / 75 96.0%Operator 3 73 / 75 97.3%Lot/Instrument 1 75 / 76 98.7%Lot/Instrument 2 72 / 75 96.0%Lot/Instrument 3 71 / 74 95.9%Overall 218 / 225 96.9%

Substance Concentration Substance ConcentrationAcetaminophen 1324 µmol/l Cyclosporine 6690 ng/mlAcetylsalicylic acid 3.62 mmol/l Digoxin 7.8 nmol/lAcyclovir 4.83 µg/ml Fluconazole 245 µmol/lAdalimumab 14.1 µg/ml Fluticasone 558 pg/mlAlbuterol 1.67 µmol/l Ipratropium 246 pg/mlAmpicillin 152 µmol/l Lopinavir and Ritonavir 46.5 µg/mlAtenolol 37.6 µmol/l Oseltamivir 195.6 ng/mlAzathioprine 10.8 µmol/l Prednisone 0.84 µmol/lAzithromycin 15.3 µmol/l Sulfamethoxazole/Trimethoprim 1580/138 µmol/lCeftazidime 117 µg/ml Vancomycin 69 µmol/lCiprofloxacin 30.2 µmol/l Warfarin 32.5 µmol/lSirolimus 289.5 ng/ml Zidovudine (AZT)Cisplatin 25.2 µmol/l Whole Blood 25% v/v

Substances were spiked at the listed (CLSI-recommended) test concentration for the entire 5 mL sample. As the volume of actual patient specimen used in the BAC LRT Assay is only 100 µl (BAL) or 50 µl (ETA) and the remainder is sample diluent (Negative Control), the functional test concentrations tested were 50X (for BAL) and 100X (for ETA) the listed concentrations.

6 µg/ml

*Not available in the USACopyright © 2015 Ibis Biosciences, an Abbott Company

Performance characterization of the IRIDICA™ BAC LRT Assay* for detection and identification of bacteria associated with lower respiratory tract infection

Objectives: Identifying organisms in nonsterile respiratory specimens through culture-based methods is time-consuming and challenging. These methods are often rendered ineffective by antibiotic pre-treatment and biased growth rates which can result in overgrowth of initially less common and less relevant organisms. In order to address these issues, a broad-spectrum molecular assay based on a robust universal lysis method, broad-spectrum mismatch- tolerant PCR, and electrospray ionization mass spectrometry (PCR/ESI-MS) was developed. In the work described here, we explore the analytical sensitivity, specificity, robustness, reproducibility, and breadth of coverage of the method, and compare its performance to that of traditional culture-based methods in a collection of clinical waste bronchoalveolar lavage (BAL) and endotracheal aspirate (ETA) specimens.

Conclusion: The IRIDICA™ BAC LRT Assay (Not available in the USA) is capable of detecting and identifying diverse, potentially pathogenic organisms in bronchoalveolar lavage and endotracheal aspirate specimens, can detect slow growing and unculturable organisms, and is unaffected by potential growth inhibitors such as antibiotics. The clinical utility of molecular techniques such as this should be investigated further.

Results: The BAC LRT Assay* was able to detect and identify all tested organisms at concentrations of 5 to 1000 CFU/sample (Figure 1). The assay was able to detect organisms in the presence of potentially interfering substances (Table 1). No carryover or cross-reactivity to untargeted organisms, viruses, or human DNA was observed (Table 2), and the reproducibility rate at moderate positive test concentrations was 97%. The assay yielded an overall calculated positive agreement of 100% with culture-based identifications of ≥ 10,000 CFU/ml in all cases where the presence of co-infecting organisms or normal flora did not prevent comparison (Figure 2, Table 3). Calculated negative agreement was 97% when all possible organism detections were considered. The BAC LRT Assay* detected difficult-to- culture organisms in clinical specimens, including Bordetella, Legionella, Mycobacterium, and Tropheryma species.

Methods: The IRIDICA™ BAC LRT Assay (08N40-10, CE-IVD)* was designed to accommodate 100µl of unconcentrated BAL fluid or 50µl of ETA digested with 50µl of dithiothreitol. Such clinical samples were then diluted with 4.9ml of sterile sample diluent to bring the total volume to 5ml. The limit of detection (LOD) of the BAC LRT Assay was tested for 23 bacteria and Candida species in a sterile sample diluent and/or the saline fluid used to collect BAL specimens. A further 47 species were tested at single concentrations to confirm the ability to detect and identify bacteria and Candida throughout the designed breadth of coverage of the assay. The robustness of the assay was challenged through studies of interfering substances, carryover, potentially cross-reacting organisms, and a reproducibility analysis using multiple instruments, reagent lots, and users. Results from the developed assay were compared to those from traditional culture-based methods using 136 de-identified (waste) clinical BAL and ETA specimens.

Megan Rounds,1 Mark W. Frinder,1 Gregory S. Richmond,1 David Metzgar,1 Heather E. Carolan,1 Donna M. Toleno,1 Karen C. Carroll,2 Richard E. Rothman,3 Britany N. Zeglin,3 Stephen Peterson,3 Franco D’Alessio,3 David J. Ecker,1 Rangarajan Sampath,1 Lawrence B. Blyn1

1Ibis Biosciences, an Abbott Company, Carlsbad, CA, USA; 2The Johns Hopkins Hospital Clinical Microbiology Laboratory, Baltimore, MD, USA; 3Department of Emergency Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

Figure 1: LODs determined for the IRIDICA BAC LRT Assay. Core LODs (bolded black text) and analytical LODs (green text) were determined using quantified microbial stocks. Each organism was tested in 20 replicates at multiple concentrations to confirm LOD titers. LODs were defined as the lowest concentration at which ≥95% of replicates tested positive. The results of Inclusivity study were used to verify the assay’s ability to detect and identify an additional 47 organisms (blue and purple text), tested at single concentrations. These organisms also include bioinformatic challenge organisms representing poor matches between assay primers and the genomic sequences of the targets (purple text). The BAC LRT Assay was able to detect and identify all tested organisms at concentrations of 50 to 10,000 CFU/sample.

Bacillales

Lactobacillales

Clostridiales

Actinobacteria

Bacteroidetes

Beta-proteobacteria

Epsilon-proteobacteria

Fusobacteria

Alpha-proteobacteria

Gamma-proteobacteria

Firmicutes

Proteo-bacteria

Color Key

Fungi

CM Mar 30 2015

Mycoplasma bovis

Corynebacterium urealyticum

Helicobacter pylori

Fusobacterium nucleatum

Campylobacter jejuni

Challenge organisms (5000 CFU/ml)

Listeria monocytogenes

Legionella pneumophila

Enterobacter aerogenes

Lactobacillus acidophilus

Staphylococcus lugdunensis Staphylococcus saprophyticus

Staphylococcus haemolyticus

Stenotrophomonas maltophilia

Salmonella entericaShigella sonnei

Streptococcus agalactiae

Veillonella dispar

Vibrio parahaemolyticus

Yersinia pseudotuberculosis

Neisseria meningitidis

Pasteurella multocida

Proteus vulgaris

Bordetella pertussisEikenella corrodens

Morganella morganiiProvidencia stuartii

Moraxella catarrhalis

Burkholderia cepacia

Campylobacter coli

Clostridium perfringens

Bacteroides fragilis *

Citrobacter freundii

Acinetobacter lwoffii

Bartonella henselaeBartonella quintana

Propionibacterium acnesNocardia nova

Arcanobacterium haemolyticum

Mycobacterium chelonaeMicrococcus luteusMicrobacterium sp.

Rothia dentocariosa

Streptococcus mutans

Streptococcus gordonii

Brucella neotomae

Candida tropicalisCandida parapsilosis

Breadth of coverage (5000 CFU/ml)

* Organism detected at 200 CFU/mL

Staphylococcus epidermidis (6400)

Enterococcus faecalis (1600)

Proteus mirabilis (400)

Escherichia coli (1600)Enterobacter cloacae (6400)

Serratia marcescens (1600)

Klebsiella oxytoca (1600)

Acinetobacter baumannii (3200)Pseudomonas aeruginosa (400)

Candida glabrata (200)

Streptococcus pyogenes (100)Streptococcus pneumoniae (12.5)

Bacillus cereus (1600)Clostridium difficile (1600)

Haemophilus influenzae (400)

Aeromonas hydrophila (400)

Aerococcus viridans (1600)

Analytical LOD organisms (CFU/ml)

Corynebacterium diphteriae (6400)Corynebacterium jeikeium (1600)

Staphylococcus aureus (mecA +)(800)

Candida albicans (800)

Enterococcus faecium (vanA +, vanB +)(1600)

Klebsiella pneumoniae (KPC +)(800)

Core LOD organisms (BAL LOD [CFU/ml])

Figure 2: Clinical sample performance. The overall positive and negative percent agreement (by detection) of the IRIDICA BAC LRT Assay was evaluated by testing 136 de-identified (waste) clinical bronchoalveolar lavage (BAL) or digested endotracheal aspirate (ETA) specimens which were collected from patients suspected of pulmonary infection. A total of 18 true negative samples (samples which are negative for all tests) was observed during this study. Both the IRIDICA LRT Assay and culture utilize single reagents to detect many or all targets. Therefore, the presence of detected organisms can mask the detection of other organisms through competitive interference. For the analysis presented here, results were considered negative only if negative for all analytes, while positive samples were considered positive for all detected analytes and excluded from analyses of other analytes. In order to account for detections that had no valid comparator, any positive result by either technology in a sample that was negative for that analyte but positive for another by the other technology was considered to be an “additional detection” in the final comparison.

Reproducibility: Assay reproducibility was evaluated at 1 testing site. Panels containing organisms at moderate concentrations were run by 3 operators on 3 sets of IRIDICA instruments using 3 lots of Assay Strip on each of 5 days. Expected results were retrieved in 218/225 tests (96.9%).

Carryover: Potential sample carryover within the IRIDICA BAC LRT Assay was evaluated by testing high concentration samples (1 x 108 CFU/sample) interspersed within adjacent negative samples. The assay did not exhibit detectable carryover from high positive samples to negative samples. The observed carryover rate was 0% (0/104).

Table 3: Normal respiratory flora. Eight samples had Normal Respiratory Flora (NRF) detections on the BAC LRT Assay that were matched (>10,000 CFU) with standard of care results. Species found in unmatched NRF detections (96 samples) were not reported in Figure 2 but are listed below.

Table 1: Potential interfering substances. The susceptibility of the IRIDICA BAC LRT Assay to interference by elevated levels of exogenous and endogenous substances was evaluated by testing 3X LOD samples of the 4 core organisms. No interference was observed at the concentrations shown below.

Table 2: Potentially cross-reacting organisms. The viruses and microorganisms shown below were evaluated for potential cross-reactivity with the IRIDICA BAC LRT Assay. The organisms were tested at targeted concentrations of 1 x 105 CFU/ml, copies/ml, or TCID50/ml for bacteria, viruses, and fungi. No cross-reactivity was observed in the performance of the assay in the presence of the potential cross-reactants for all positive and negative samples tested.

CM Jan 29 2015

Color Key

Additional detections

Acinetobacter baumanniiAcinetobacter sp.Escherichia coliEnterobacter cloacaeHaemophilus influenzaeKlebsiella pneumoniaePseudomonas aeruginosaPseudomonas mendocinaShigella dysenteriaeStaphylococcus aureusStreptococcus pneumoniaeGroup B streptococcus

110001011210

101110100202

Organism IRIDICA Culture

Corynebacterium pseudodiphteriticum (1)Corynebacterium striatum (1)

Escherichia coli (1)Enterobacter cloacae complex (2)

Klebsiella oxytoca (1)

Acinetobacter baumannii (1)

Streptococcus oralis/pneuminiae (1)Streptococcus pneumoniae (22)

Finegoldia magna (1)

Haemophilus parainfluenzae (1)

Pseudomonas alcaliphila (1)

Streptococcus pseudopneumoniae (3)

E.coli/Shigella flexneri (1)Haemophilus influenzae (5)

Pseudomonas aeruginosa (7)

Legionella pneumophila (1)

Sneathia sp. (1)

Stenotrophomonas maltophilia (2)

Shigella boydii (1)

Group G Streptococcus (1)

Pasteurella multocida (1)

Bordetella bronchiseptica/pertussis (1)

Kluyvera intermedia (1)Citrobacter koseri (1)

Nocardia sp. (1)Mycobacterium sp. (1)Gardnerella vaginalis (3)Kitococcus schroeteri (1)

Tropheryma whipplei (1)

Streptococcus mitis (1)

IRIDICA detections, unmatched

Aggregatibacter segnis (3)

Enterococcus faecium (2)

Klebsiella pneumoniae (1)

Staphylococcus aureus (6)

Corynebacterium amycolatum (1)

Corynebacterium striatum (1)

Enterobacter aerogenes (1)

Stenotrophomonas maltophilia (1)

Proteus mirabilis (1)Serratia marcescens (1)

Haemophilus influenzae (2)

IRIDICA detections, matched

Pseudomonas aeruginosa (3)

Staphylococcus aureus (2)

Klebsiella pneumoniae (2)