(200 days)
The GenMark ePlex® Blood Culture Identification Gram-Negative (BCID-GN) Panel is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on GenMark's ePlex Instrument for simultaneous qualitative detection and identification of multiple potentially pathogenic gram-negative bacterial organisms and select determinants associated with antimicrobial resistance in positive blood culture. In addition, the ePlex BCID-GN Panel is capable of detecting several gram-positive bacteria (Pan Gram-Positive assay) and several Candida species (Pan Candida assay). The ePlex BCID-GN Panel is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system and which contain gram-negative organism.
The following bacterial organisms and genes associated with antibiotic resistance are identified using the ePlex BCID-GN Panel: Acinetobacter baumannii, Bacteroides fragilis, Citrobacter, Cronobacter sakazakii. Enterobacter cloacae complex, Enterobacter (non-cloacae complex), Escherichia coli, Fusobacterium necrophorum, Fusobacterium nucleatum, Haemophilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae group, Morganella morganii, Neisseria meningitidis, Proteus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella, Serratia, Serratia marcescens, Stenotrophomas maltophilia, СТХ-М (blactх-м), IMP (blамм) , КРС (blakec) , NDM (bland), OXA (blaoxa) (OXA-23 and OXA-48 groups only), and VIM (blaviм).
The ePlex BCID-GN Panel contains assays for the detection of genetic determinants associated with resistance to antimicrobial agents including CTX-M(blactx.M), which is associated with resistance to extended spectrum beta-lactamase (ESBL)-mediated resistance to penicillins, cephalosporins and monobactams, as well as OXA (blaoxA) (OXA-23 and OXA-48 groups only), KPC (blakpc), and metallo-beta-lactamases IMP (blavM), VIM (blavM), and NDM (blaNDM), which is associated with carbapenemase-mediated resistance. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance assays do not indicate susceptibility, as there are multiple mechanisms of resistance in gram-negative bacteria.
The ePlex BCID-GN Panel also contains targets designed to detect a broad range of organisms with a potentially misleading Gram stain result or organisms that may be missed by Gram staining altogether, for example in the case of co-infections. These include a broad Pan Gram-Positive assay (which is designed to detect Bacillus cereus group, Bacillus subtilis group, Enterococcus, Staphylococcus, and Streptococcus), as well as a Pan Candida assay, which is designed to detect four Candida species: Candida albicans, Candida glabrata, Candida krusei, and Candida parapsilosis.
The detection and identification of specific bacterial and fungal nucleic acids from individuals exhibiting signs and/or symptoms of bloodstream infection aids in the diagnosis of bloodstream infection when used in conjunction with other clinical information. The results from the ePlex BCID-GN Panel are intended to be interpreted in conjunction with Gram stain results and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
Negative results in the setting of a suspected bloodstream infection may be due to infection with pathogens that are not detected by this test. Positive results do not rule out co-infection with other organisms; the organism(s) detected by the ePlex BCID-GN Panel may not be the definite cause of disease. Additional laboratory testing (e.g. sub-culturing of positive blood cultures for identification of organisms not detected by ePlex BCID-GN Panel and for susceptibility testing, differentiation of mixed growth, and association of antimicrobial resistance marker genes to a specific organism) and clinical presentation must be taken into consideration in the final diagnosis of bloodstream infection.
The ePlex Blood Culture Identification Gram-Negative (BCID-GN) Panel is based on the principles of competitive nucleic acid hybridization using a sandwich assay format, wherein a single-stranded target binds concurrently to a sequence-specific solution-phase signal probe and a solid-phase electrode-bound capture probe. The test employs nucleic acid extraction, target amplification via polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) and hybridization of target DNA. In the process, the double-stranded PCR amplicons are digested with exonuclease to generate single-stranded DNA suitable for hybridization.
Nucleic acid extraction from biological samples occurs within the cartridge via cell lysis, nucleic acid capture onto magnetic beads, and release for amplification. The nucleic acid extraction is processed through microfluidic liquid handling. Once the nucleic acid targets are captured and inhibitors are washed away, the magnetic particles are delivered to the electrowetting environment on the printed circuit board (PCB) and the targets are eluted from the particles and amplified.
During hybridization, the single-stranded target DNA binds to a complementary, single-stranded capture probe immobilized on the working gold electrode surface. Single-stranded signal probes (labeled with electrochemically active ferrocenes) bind to specific target sequence / region adjacent to the capture probe. Simultaneous hybridization of target to signal probes and capture probe is detected by alternating current voltammetry (ACV). Each working electrode on the array contains specific capture probes, and sequential analysis of each electrode allows detection of multiple analyte targets.
Here's a summary of the acceptance criteria and study information for the ePlex Blood Culture Identification Gram Negative (BCID-GN) Panel, extracted from the provided text:
Acceptance Criteria and Device Performance for ePlex Blood Culture Identification Gram Negative (BCID-GN) Panel
The ePlex BCID-GN Panel is a qualitative nucleic acid multiplex in vitro diagnostic test for the simultaneous qualitative detection and identification of multiple potentially pathogenic gram-negative bacterial organisms and select determinants associated with antimicrobial resistance in positive blood cultures. Performance characteristics were evaluated through clinical and analytical studies.
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria are generally implied by the achieved performance metrics, which show high Positive Percent Agreement (PPA) for sensitivity and Negative Percent Agreement (NPA) for specificity. While explicit numerical acceptance thresholds are not directly stated as "acceptance criteria" in a dedicated section, the provided tables demonstrate the device's performance against its intended use. For brevity, the "Overall" performance (combining prospective, retrospective, and contrived samples where applicable) is used here to represent the device's reported performance against implied high accuracy criteria.
| Target (Organism/Resistance Gene) | Implied Acceptance Criteria (High Accuracy) | Reported Device Performance (Overall PPA % (95% CI)) | Reported Device Performance (Overall NPA % (95% CI)) |
|---|---|---|---|
| Acinetobacter baumannii | High Sensitivity & Specificity | 100 (95.1-100) | 99.9 (99.7-100) |
| Bacteroides fragilis | High Sensitivity & Specificity | 95.6 (87.8-98.5) | 99.9 (99.6-100) |
| Citrobacter | High Sensitivity & Specificity | 98.6 (92.2-99.7) | 99.8 (99.4-99.9) |
| Cronobacter sakazakii | High Sensitivity & Specificity | 100 (92.3-100) | 100 (99.8-100) |
| Enterobacter cloacae complex | High Sensitivity & Specificity | 95.3 (89.4-98.0) | 99.6 (99.2-99.8) |
| Enterobacter (non-cloacae complex) | High Sensitivity & Specificity | 96.6 (88.3-99.0) | 99.9 (99.7-100) |
| Escherichia coli | High Sensitivity & Specificity | 96.9 (94.4-98.3) | 99.8 (99.4-99.9) |
| Fusobacterium necrophorum | High Sensitivity & Specificity | 98.0 (89.3-99.6) | 100 (99.8-100) |
| Fusobacterium nucleatum | High Sensitivity & Specificity | 100 (93.1-100) | 99.9 (99.7-100) |
| Haemophilus influenzae | High Sensitivity & Specificity | 100 (93.5-100) | 100 (99.8-100) |
| Klebsiella oxytoca | High Sensitivity & Specificity | 89.6 (80.0-94.8) | 99.8 (99.5-99.9) |
| Klebsiella pneumoniae group | High Sensitivity & Specificity | 97.9 (95.2-99.1) | 99.7 (99.3-99.9) |
| Morganella morganii | High Sensitivity & Specificity | 100 (94.2-100) | 99.9 (99.7-100) |
| Neisseria meningitidis | High Sensitivity & Specificity | 100 (92.0-100) | 99.9 (99.7-100) |
| Proteus | High Sensitivity & Specificity | 97.7 (92.0-99.4) | 100 (99.8-100) |
| Proteus mirabilis | High Sensitivity & Specificity | 97.6 (91.6-99.3) | 100 (99.8-100) |
| Pseudomonas aeruginosa | High Sensitivity & Specificity | 95.8 (90.6-98.2) | 99.7 (99.4-99.9) |
| Salmonella | High Sensitivity & Specificity | 96.4 (87.9-99.0) | 100 (99.8-100) |
| Serratia | High Sensitivity & Specificity | 100 (95.4-100) | 99.9 (99.7-100) |
| Serratia marcescens | High Sensitivity & Specificity | 100 (94.2-100) | 99.9 (99.7-100) |
| Stenotrophomonas maltophilia | High Sensitivity & Specificity | 94.0 (83.8-97.9) | 99.9 (99.7-100) |
| Pan Candida | High Sensitivity & Specificity | 62.5 (30.6-86.3) | 99.7 (98.4-99.9) (Prospective All) |
| Pan Gram-Positive | High Sensitivity & Specificity | 78.2 (67.8-85.9) | 97.9 (95.6-99.0) (Prospective All) |
| CTX-M (Overall) | High Sensitivity & Specificity | 93.1 (88.1-96.1) | 100 (99.7-100) |
| IMP (Overall) | High Sensitivity & Specificity | 100 (91.2-100) | 100 (99.7-100) |
| KPC (Overall) | High Sensitivity & Specificity | 98.1 (89.9-99.7) | 99.9 (99.6-100) |
| NDM (Overall) | High Sensitivity & Specificity | 100 (93.4-100) | 100 (99.7-100) |
| OXA (Overall) | High Sensitivity & Specificity | 94.0 (83.8-97.9) | 99.8 (99.4-100) |
| VIM (Overall) | High Sensitivity & Specificity | 100 (91.6-100) | 100 (99.7-100) |
2. Sample Sizes Used for the Test Set and Data Provenance
The test set comprised a combination of clinical and contrived samples:
- Prospective Clinical Samples: 349 evaluable samples (167 fresh, 182 frozen). Patients were of all ages and genders. Collected at 7 clinical sites.
- Retrospective Clinical Samples: 577 evaluable samples. Collected at 10 clinical sites.
- Contrived Samples: 777 samples.
Data Provenance: The clinical samples (prospective and retrospective) were collected at multiple clinical sites, suggesting data from different countries/regions, but specific countries of origin are not detailed. Both prospective and retrospective collection methods were used.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts
The document does not explicitly state the number of experts or their specific qualifications (e.g., radiologist with 10 years of experience) used to establish the ground truth. It refers to "standard laboratory procedures for identification of blood culture isolates, including traditional and automated identification methods, MALDI-TOF IVD, and microbiological and biochemical techniques" as the comparator method. For Acinetobacter baumannii and Candida parapsilosis, as well as antibiotic resistance genes, PCR assays followed by bi-directional sequencing were used for confirmation. This implies a reliance on established laboratory protocols and potentially expert interpretation within those processes, but specific expert count or detailed qualifications are not provided.
4. Adjudication Method for the Test Set
The document does not describe an explicit adjudication method (e.g., 2+1, 3+1). The "Comparator Method" served as the reference standard and was used to determine true positive (TP), false negative (FN), true negative (TN), and false positive (FP) results. Discrepancies were investigated using PCR/sequencing (as noted in footnotes for various tables), suggesting a re-evaluation process for conflicting results but not a formal multi-reader adjudication scheme for the initial ground truth establishment.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done
No MRMC comparative effectiveness study involving human readers and AI assistance was performed or described in the provided text. The study focuses on the standalone performance of the ePlex BCID-GN Panel against laboratory reference methods.
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done
Yes, the study described is a standalone performance study of the ePlex BCID-GN Panel. The performance metrics (PPA and NPA) are calculated by comparing the device's output directly against the "Comparator Method" without human interpretation influencing the device's result.
7. The Type of Ground Truth Used
The ground truth was established using expert consensus methods based on standard laboratory procedures for organism identification and analytically validated molecular assays (qPCR followed by bi-directional sequencing) for confirming specific organisms and resistance genes. This includes:
- Traditional and automated identification methods
- MALDI-TOF IVD
- Microbiological and biochemical techniques
- PCR/sequencing for specific organisms and resistance markers.
8. The Sample Size for the Training Set
The document does not mention a distinct "training set" in the context of the clinical performance evaluation. The clinical studies (prospective, retrospective, and contrived samples) are described as evaluation/test sets. However, the "Analytical Reactivity (Inclusivity)" and "Limit of Detection (LoD)" studies (Tables 56 and 57) describe testing of various strains and isolates to establish the device's analytical performance and broad reactivity. These analytical studies are crucial for the development and "training" (in a broader sense of assay design and validation) of such molecular diagnostic panels.
Specifically:
- Limit of Detection (LoD): At least 20 replicates per target were tested for each condition using quantified reference strains (Table 56 lists 38 organisms/targets with specific strains).
- Analytical Reactivity (Inclusivity): A panel of 336 strains/isolates was evaluated (Table 57 provides a partial list of these validated strains).
- Predicted (in silico) Reactivity: Bioinformatic analysis covered many additional variants for genus/group assays and resistance markers (Tables 58-76).
9. How the Ground Truth for the Training Set Was Established
For the analytical "training" aspects:
- LoD: The ground truth for LoD was established by using quantified reference strains with known concentrations in simulated blood culture sample matrix. The lowest concentration detected in ≥95% of tested replicates was defined as the LoD.
- Analytical Reactivity (Inclusivity): The ground truth was established by testing well-characterized strains/isolates with known identities at specified concentrations, confirming their detection by the panel.
- Predicted (in silico) Reactivity: This involved bioinformatic analysis of existing sequence data for various organisms and resistance gene variants to predict the panel's ability to detect them. This is a computational method for establishing theoretical ground truth based on genetic sequences.
§ 866.3365 Multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures.
(a)
Identification. A multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures is a qualitative in vitro device intended to simultaneously detect and identify microorganism nucleic acids from blood cultures that test positive by Gram stain or other microbiological stains. The device detects specific nucleic acid sequences for microorganism identification as well as for antimicrobial resistance. This device aids in the diagnosis of bloodstream infections when used in conjunction with other clinical and laboratory findings. However, the device does not replace traditional methods for culture and susceptibility testing.(b)
Classification. Class II (special controls). The special control for this device is FDA's guideline document entitled “Class II Special Controls Guideline: Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures.” For availability of the guideline document, see § 866.1(e).