SMART LEISH, MODEL LGM1-050

{0} 1 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY AND INSTRUMENT COMBINATION TEMPLATE A. 510(k) Number: K081868 B. Purpose for Submission: To obtain a 510(k) clearance for a new device. C. Measurand: Leishmania species DNA and L. major DNA D. Type of Test: Real-time polymerase chain reaction assay E. Applicant: Department of the Army U.S. Army Medical Research and Matériel Command 1430 Veterans Drive Fort Detrick, MD 21702-5012 F. Proprietary and Established Names: SMART Leish G. Regulatory Information: 1. Regulation section: 866.3870 Trypanosoma spp. serological reagents 2. Classification: Class I 3. Product code: OUZ 4. Panel: Microbiology 83 {1} H. Intended Use: 1. Intended use: The SMART Leish is a qualitative diagnostic real-time PCR test for the rapid detection of *Leishmania* species and the identification of *L. major* in skin lesion scrapings and punch biopsies from individuals suspected of having cutaneous leishmaniasis. The test utilizes real-time polymerase chain reaction assay on the Cepheid SmartCycler® II Dx to detect *Leishmania* species and *L. major*. The SMART Leish is intended for use only by trained laboratory personnel in Department of Defense laboratories. Clinical performance has not been established with strains other than *L. major*. 2. Indications for use: The SMART Leish is a qualitative diagnostic real-time PCR test for the rapid detection of *Leishmania* species and the identification of *L. major* in skin lesion scrapings and punch biopsies from individuals suspected of having cutaneous leishmaniasis. The test utilizes real-time polymerase chain reaction assay on the Cepheid SmartCycler® II Dx to detect *Leishmania* species and *L. major*. The SMART Leish is intended for use only by trained laboratory personnel in Department of Defense laboratories. Clinical performance has not been established with strains other than *L. major*. 3. Special conditions for use statement(s): The device is for prescription use only. 4. Special instrument requirements: Cepheid SmartCycler® II Dx I. Device Description: The SMART Leish consists of an assay reagent kit and assay definition files for the polymerase chain reaction (PCR) instrument platform. The kit contains sufficient reagents, in lyophilized bead form, to qualitatively assay 50 clinical samples for both *Leishmania* genus and *L. major* targets. Additional required accessories that are specified include the PCR instrument platform, a deoxyribonucleic acid (DNA) purification kit, and a positive extraction control. J. Substantial Equivalence Information: 1. Predicate device name(s): Amizyme-Leishmania spp. Test Kit {2} 2. Predicate 510(k) number(s): K842526 3. Comparison with predicate: Similarities | Device Aspect | SMART Leish | Reference Device | | --- | --- | --- | | Intended Use | Detection of Leishmania spp. | Detection of Leishmania spp. | | Assay Type | Qualitative | Qualitative | | Agent measured | Leishmania genus and L. major specific DNA | Leishmania spp. | | Technology | Qiagen QIAamp DNA Mini Kit | None | | Controls | External – positive and negative controls for both the extraction process and detection assay runs Internal – positive control for PCR inhibitors in sample | External positive and negative controls | | Differences | | | | Instrument Platform | Cepheid SmartCycler | None | | Automation | Automated DNA amplification cycling, probe detection, and interpretation of results | Manual method | | Sample Collection | Skin punch biopsy or scrapings, stored in ethanol | Serum | | Endpoint detection method | PCR amplification of DNA sequences unique to the organisms of interest with detection by hybridization probes incorporating reporter dyes | Indirect Immunofluorescence (IFA) Fluorescent conjugate secondary antibodies on a prepared slide | # K. Standard/Guidance Document Referenced (if applicable): N/A # L. Test Principle: For the Leishmania assays, a tissue specimen (skin scraping or punch biopsy) from an individual suspected of being infected with Leishmania species or $L$ . major is collected in $70\%$ or $100\%$ ethanol, and the DNA is extracted from the specimen using the Qiagen QIAamp DNA Mini Kit. An aliquot of this DNA is tested using the Leishmania genus assay, which will amplify a portion of DNA encoding the Leishmania species 16S ribosomal ribonucleic acid (rRNA) gene if present. Amplified targets are detected using a TaqMan hybridization probe with 6-carboxy-fluorescein (FAM) reporter dye and a Black Hole Quencher (BHQ). This assay also contains a positive internal control consisting of a nonsensical, non-naturally {3} occurring DNA sequence, with Texas Red reporter dye and BHQ, used to detect evidence of PCR inhibition and confirm the integrity of assay reagents in negative specimens. If the sample tests positive for Leishmania species, another aliquot of DNA may be tested using the $L$ . major assay, which will amplify a portion of DNA encoding the GPI gene that is specific to $L$ . major. Amplified targets are detected using a TaqMan hybridization probe with FAM reporter dye and a BHQ. The DNA amplification, detection of fluorescence, and interpretation of signals are done automatically by the SmartCycler instrument for each assay. The thermocycling protocols take approximately 45-55 minutes. # M. Performance Characteristics (if/when applicable): # 1. Analytical performance: # a. Precision/Reproducibility: Studies were conducted to evaluate the reproducibility of the SMART Leish assay. The precision of the Leishmania genus and L. major assays between laboratories was evaluated using purified L. major DNA and cultured L. major parasites. Additionally, between-laboratory precision of the assays was evaluated using mock human samples (punch biopsies from porcine tissue injected with L. major parasites). Reproducibility Results for the Leishmania Genus Assay | Sample Type | Sample ID | Concentration | Number of Replicates (over 5 days) | (WRAIR) | (BAMC) | (MAMC) | Total Agreement | % Total Agreement | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | genome equivalents/μL | | | | | | | | Purified DNA | Low | 2.0 | 20 | 20/20 | 20/20 | 20/20 | 60/60 | 100% | | | Medium | 20 | 20 | 20/20 | 20/20 | 20/20 | 60/60 | 100% | | | High | 200 | 20 | 20/20 | 20/20 | 20/20 | 60/60 | 100% | | Positive Agreement for Purified DNA | | | | | | | 180/180 | 100% | | | | (parasites/extraction) | | | | | | | | Cultured Parasites | Low | 1,000 | 20 | 20/20 | 20/20 | 20/20 | 60/60 | 100% | | | Medium | 10,000 | 20 | 20/20 | 20/20 | 20/20 | 60/60 | 100% | | | High | 25,000 | 20 | 20/20 | 20/20 | 20/20 | 60/60 | 100% | | Positive Agreement for Cultured Parasites | | | | | | | 180/180 | 100% | | | | (parasites/extraction) | | | | | | | {4} 5 | Mock Human Samples | Negative | 0 | 10 | 10/10 | 10/10 | 10/10 | 30/30 | 100% | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | Positive | 1.1x10^6 | 14 | 14/14 | 13/14 | 14/14 | 41/42 | 97.6% | b. Linearity/assay reportable range: The assay linearity of SMART Leish for Leishmania genus and Leishmania major was determined using eight different concentrations of purified L. major DNA tested in a 10-fold dilution series from 14.26 ng to 0.001426 pg and 21.39ng to 0.002139 pg, respectively, DNA per reaction. c. Traceability, Stability, Expected values (controls, calibrators, or methods): Not applicable d. Detection limit: In addition to the LOD determination, six different concentrations of purified L. major parasites were tested in a 10-fold dilution of 5 X 10² parasites/mL (equivalent to 10⁷ to 10² parasites/extraction) to determine the minimum number of L. major parasites that can be taken through the extraction and testing procedure and still give a positive result for the SMART Leish assays (extraction LOD). The following tables summarize the results of the assay LOD for the L. genus and L. major assays, and summarizes the results of the extraction LOD (eLOD) study. Summary of the Measuring Range and LOD for the Leishmania genus and L. major Assays (Values are per assay reaction) | Assay | Linear Range¹ | Assay LOD¹,² | LOD Equates to | | --- | --- | --- | --- | | Leishmania Genus Assay | 0.014–14,000 pg | 0.14 pg | 4 genome copies with ~200 copies/genome of target gene | | L. major Assay | 0.2139–21,390 pg | 2.1 pg | 62 genome copies | ¹ Amount of L. major DNA in the PCR reaction. LOD refers to the minimum amount of L. major DNA that can be put into a SMART Leish PCR reaction and still result in a positive SMART Leish test greater than or equal to 95% of the time. ² 95% accuracy across different operators and instruments and from multiple L. major strains. {5} Summary of Assay eLOD for the Leishmania genus and L. major Assays Using Parasite Samples. Values are per extraction. | Assay | eLOD^{1} | LOD Equates to^{2} | | --- | --- | --- | | Leishmania Genus Assay | 250 parasites | ~5 genome copies at assay reaction stage | | L. major Assay | 1,000 parasites | ~30 genome copies at assay reaction stage | $^{1}$ 95% accuracy across different operators and days and from multiple L. major strains. Extraction LOD refers to the minimum number of L. major parasites that can be taken through the Qiagen extraction procedure and still consistently result in a positive SMART Leish test greater than or equal to 95% of the time. Extraction LOD was determined across different operators, different days, and using multiple L. major strains. e. Analytical specificity: A total of 121 DNA samples were evaluated in the specificity study. The DNA from 11 Leishmania major strains tested in this study gave robust positive results with the Leishmania major PCR assay. Twenty-five non-Leishmania major samples tested negative; hence the analytical specificity was 100%. The percentage of false positives was 0%, and the percentage of false negatives is 0%. In the Leishmania genus study, 85 of the 121 samples were non-target DNA samples. Eighty-five were negative, 2 false positives and 36 were positive. The analytical specificity of the Leishmania genus PCR assay was 98.3%, false positive rate was 2.4% and the false negative rate was 0%. See the following table for a list of organisms used in the study. Nucleic acids from non-target organisms were tested for both the Leishmania genus and Leishmania major assays. DNA samples with concentrations used in this study included purified DNA from bacteria (52; conc = 50 - 46,250 pg/uL), fungi (11; conc = 50,000 - 97,000 pg/uL), viruses (7; conc = 5,950 - 30,990 pg/uL), mammals (3; conc = 20 pg/uL (human, bovine, and murine)), human melanoma cell lines (3; conc = 10,200 - 25,200 pg/uL), Leishmania major (11; conc = 21,540 - 83,000 pg/uL), 25 additional Leishmania species (not-L. major) (conc = 16,680 - 592,00 pg/uL), and 9 trypanosomes representing 6 species (conc = 12,760 - 95,000 pg/uL). {6} 7 # List of Organisms Used in Specificity Study | Bacteria | Staphylococcus aureus (4) | Trichophyton mentagrophytes | | --- | --- | --- | | Acinetobacter baumannii | Staphylococcus hominis | Trichophyton soudane | | Bacillus anthracis (3) | Stenotrophomonas maltophilia | Trichophyton sonda | | Bacillus cereus (2) | Streptococcus pyogenes (2) | Arthroderma benhamiae | | Bacillus subtilis var.niger | Streptococcus sp. (B) | Sporothrix schenckii | | Bacillus thuringiensis | Streptococcus (F2) | Microsporum canis | | Brucella abortus | Yesinia pestis | | | Brucella melitiensis | Yersinia | Mammalian Bovine | | Brucella susi | | Human | | Clostridium botulinum type A | Mycobacteria | Murine | | Clostridium botulinum type B | Mycobacteria abscessus | Human melanoma cell line (3) | | Clostridium perfringens (2) | Mycobacteria fortuitum | | | Clostridium sordellii | Mycobacteria marinum | | | Entrbacter aerogenes | Mycobacteria spp | | | Enterococcus durans | Mycobacteriia tuberculosis | Trypanosoma | | Enterococcus faecalis | Mycobacteria ulcerans | | | Escherichia coli | Viruses | Trypanosoma cruzi (2) | | Francisella tularensis (2) | Human Papilloma Virus | Trypanosoma rhodesiense | | Haemophilus influenzae | Herpes Simplex Virus Type I | Trypanosoma rangeli (2) | | Klebsiella oxytoca | Herpes Simplex Virus Type II | Trypanosomal lewisi | | Klebsiella pneumoniae | (2) | lincicome | | Moraxella cattaharalis | Varicella Zoster Virus (2) | Trypanosoma brucei gambiense | | Neisseria lactamica | Fungi | Crithidia fasciculata (2) | | Pasteurella multocida | Microsporium gypseum | | | Proteus mirabilis | Cladophialophora carrionii | | | Proteus vulgaris | Fonsecaea pedrosoi | | | Providencia stuartii | Rhinocladiella compacta | | | Pseudomonas aeruginosa | Phialophora verrucosa | | | | Trichophyton tonsurans | | Two strains of *C. fasciculata* were determined to be low-level cross-reactors based on false-positive results observed. The degree of cross-reactivity of the *Leishmania* genus assay for *C. fasciculata* can be considered low level or weak. No cross-reactors were determined for the *L. major* assay through the study. Supplementary in silico analysis was done comparing the Smart Leish primer and probe sequences to sequences from the following organisms that could potentially result in a clinical presentation similar to cutaneous leishmaniasis: Corynebacterium diphtheria (veldt sores), Mycobacteria (Tropical Ulcer), Mycobacterium tuberculosis (Lupus vulgaris), Treponema pallidum (Tertiary Syphilis), Treponema pertenue (Yaws), and Blastomyces (Blastomycosis). Each Smart Leish primer and probe contained at least four mismatches to those {7} sequences. Based on these alignments, no cross-reactivity was predicted with the Smart Leish primers and probes. f. Assay cut-off: Not applicable # 2. Comparison studies: a. Method comparison with predicate device: Clinical performance of the SMART Leish was compared to culture and microscopy. b. Matrix comparison: Not applicable # 3. Clinical studies: a. Clinical Sensitivity: The clinical performance of the SMART Leish assay was established in a multi-center, retrospective clinical study conducted at two military U.S. hospital sites. A total of 303 prospective specimens were collected from adults 18 years or older and evaluated in the SMART Leish assay and compared to culture and/or microscopy. Evaluated specimens were skin lesion scrapings and punch biopsies from individuals suspected of having cutaneous leishmaniasis. The patient population consisted entirely of military personnel who were at the time, or had previously been, deployed to Southwest Asia—primarily Afghanistan and Iraq. SMART Leish performance versus culture and microscopy, including $95\%$ confidence intervals, is detailed below. For the BAMC study, DNA was extracted from specimens and Leishmania genus and $L$ . major assays were run. For the WRAIR study, Leishmania genus and $L$ . major assays were run on archived DNA samples that were previously extracted from clinical specimens. For both studies, the results were compared to "clinical truth" that had been established for each specimen or sample. Summary of Smart Leish WRAIR Studies for the Leishmania genus Assay | | Clinical Truth | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | Smart Leish – Leishmania Genus Assay | Positive | 160 | 29 | 189 | | | Negative | 1 | 56 | 57 | {8} 9 | | Total | 161 | 85 | 246 | | --- | --- | --- | --- | --- | | Sensitivity = 99.4% (96.1% - 99.9%) Specificity = 65.9% (54.7% - 75.6%) | | | | | ## Summary of Smart Leish WRAIR Studies for the Leishmania major Assay | | Clinical Truth | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | Smart Leish – L. major | Positive | 70 | 8 | 78 | | | Negative | 4 | 77 | 81 | | | Total | 74 | 85 | 159 | | Sensitivity = 94.6% (86.0% - 98.3%) Specificity = 90.6% (81.8% - 95.6%) | | | | | ## Summary of Smart Leish BAMC Studies for the Leishmania genus Assay | | Clinical Truth | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | Smart Leish – Leishmania Genus | Positive | 63 | 0 | 63 | | | Negative | 1 | 2 | 3 | | | Total | 64 | 2 | 66 | | Sensitivity = 98.4% (90.5% - 99.9%) Specificity = 100% (19.8% - 100%) | | | | | ## Summary of Smart Leish BAMC Studies for the Leishmania major Assay | | Clinical Truth | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | Smart Leish – L. major | Positive | 22 | 0 | 22 | | | Negative | 0 | 6 | 6 | | | Total | 22 | 6 | 28 | | Sensitivity = 100.0% (81.5% - 100.0%) Specificity = 100.0% (51.7% - 100.0%) | | | | | {9} 10 Summary of SMART Leish Combined Studies for the Leishmania genus Assay | | Clinical Truth | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | SMART Leish - Leishmania Genus Assay | Positive | 223 | 29 | 252 | | | Negative | 2 | 58 | 60 | | | Total | 225 | 87 | 312 | | * Sensitivity = (99.1% (96.5 - 99.8)) | | | | | | Specificity = (67% (55% - 76%) | | | | | Diagnosis of leishmaniasis by culture and slide techniques results in lower clinical sensitivity than PCR assays (46%-84% and 33%-74% respectively). In this clinical data set there were 29 false positives generated for the Leishmania Genus assay. Twenty seven of these Genus false positives were confirmed to be Genus positive using sequencing methods. Clinical and specificity values were recalculated counting the 27 Genus false positives that were confirmed by sequencing to be Leishmania as true positives. These "adjusted" calculation results are as follows: Sensitivity Genus: 99.2% (96.9 - 99.9); Specificity Genus: 96.7% (87.5 - 99.9). *Diagnosis of leishmaniasis by culture and slide techniques results in lower clinical sensitivity than PCR assays (46%-84% and 33%-74% respectively) Summary of SMART Leish Combined Studies for the Leishmania major Assay | | Clinical Truth | | | | | --- | --- | --- | --- | --- | | | | Positive | Negative | Total | | SMART Leish - L. major Assay | Positive | 92 | 8 | 100 | | | Negative | 4 | 83 | 87 | | | Total | 96 | 91 | 187 | | * Sensitivity = = 95.8% (89.1 - 98.7) | | | | | | * Specificity = = 91.2% (82.9 - 95.9) | | | | | In this clinical data set there were eight false positives generated for the $L.$ major assay, of which three of were confirmed to be $L.$ major using sequencing methods. Clinical and specificity values were recalculated counting the 27 three $L.$ major false positives that were confirmed by sequencing to be $L.$ major as true positives. These "adjusted" calculation results are as follows: Sensitivity $L.$ major: $96.0\%$ (89.4 - 98.7); Specificity $L.$ major: $94.3\%$ (86.6 - 97.9). b. Clinical specificity: Not applicable {10} c. Other clinical supportive data (when a. and b. are not applicable): None 4. Clinical cut-off: Not applicable 5. Expected values/Reference range: In the clinical studies conducted, for this study, on U.S. military personnel that served in the Middle East and Afghanistan, 57% of 2,783 samples tested using an in-house developed PCR assay in an accredited clinical laboratory were positive for Leishmania major. In the investigational study of the Smart Leish assay, 72% of 312 skin scrapings and punch biopsy specimens from U.S. service members that served in the Middle East and Afghanistan, and which had a skin lesion consistent with cutaneous leishmaniasis, tested positive compared to the gold standard of culture and/or microscopy. N. Instrument Name: SmartCycler Dx System O. System Descriptions: The SMART Leish consists of an assay reagent kit and assay definition files for the polymerase chain reaction (PCR) instrument platform. The kit contains sufficient reagents, in lyophilized bead form, to qualitatively assay 50 clinical samples for both Leishmania genus and L. major targets. Additional required accessories that are specified include the PCR instrument platform, a deoxyribonucleic acid (DNA) purification kit, and a positive extraction control. The device components and required accessories are listed as follows. The SMART Leish Assay contains the following reagents packaged in a test kit for detection of Leishmania DNA. - SmartMix™ HM Lyophilized PCR Master Mix - Leishmania Genus Primer and Probe Set - Leishmania major Primer and Probe Set - Leishmania Positive and Negative Control DNA Beads Modes of Operation: The assay is performed on the automated Cepheid SmartCycler Dx System. The SmartCycler Dx System is a real time thermal cycler used for identifying DNA or RNA from prepared samples. Each instrument contains 16 independently programmable I-CORE modules, each with one reaction site. Thermally optimized proprietary reaction tubes combined with the unique design of the I-CORE modules allow very rapid cycling and faster amplification and detection. Up to six SmartCycler II processing blocks can be daisy-chained together, 11 {11} allowing simultaneous analysis of up to 96 discrete samples. The SmartCycler Dx System is suited to *in vitro* diagnostic assays that require automatic, repeated, rapid heating and cooling cycles for test samples, such as PCR and RT-PCR. Specific sequences can be detected using hybridization probes. The system has the capacity to store any number of runs, limited only by available disk space. All assay data and results are stored in a database. ## Software: FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types: Yes ___ x ___ or No ___ The software controls the operation of the I-CORE module, and collects, analyzes and interprets the acquired optical data. This software has been determined to be a moderate level of concern. ## 3. Specimen Identification: - User enters the sample identifiers under the “Sample ID Column” before starting the run. - User inserts each reaction tube into the assigned site of the SmartCycler. ## 4. Specimen Sampling and Handling: - Collected specimens should be stored in 70% - 100% ethanol. During shipment, the samples can be kept at ambient temperature, 20°C – 29°C. - Once received, samples should be stored at 2°C–8°C until tested. - Extracted DNA samples must be stored in the AE buffer provided in the Qiagen kit and may be stored at 2°C–8°C for up to 3 days. - DNA extracts in AE buffer may be stored for more than 3 days in a –20°C freezer. Avoid multiple freeze-thaw cycles of the samples. ## 5. Calibration: The thermistors used to monitor the reaction chamber temperature are calibrated to ±0.50°C using National Institute of Standards and Technology (NIST)-traceable standards. The optical system is calibrated using standard concentrations of the individual unquenched dye-oligo standard to determine the spectral characteristics. The instrument performs optics self-test before each run start to verify that the optical system is functioning properly. 12 {12} 6. Quality Control: Positive DNA controls are provided in the assay kit. The positive extraction control (Leishmania mexicana parasites) and negative controls (molecular biology grade water) are materials required but not supplied. One positive DNA Control and one negative DNA Control are processed for each assay run on the SmartCycler Dx System. The software automatically assigns the positive DNA Control to the second to last position, and the negative DNA control to the last position. The positive DNA control monitors for reagent failure and operator error. The negative DNA control monitors reagent or environmental contamination (or carry-over). The positive extraction control monitors for extraction failure and operator error. The negative extraction control monitors for reagent contamination and environmental contamination in the extraction process. The internal control (IC) verifies functional PCR reagents and the absence of inhibition that would prevent PCR amplification. DNA for the IC is included in one of the reagent beads within the Smart Leish Assay. The response of the IC in the presence of inhibitors correlates with the response of target DNA in the presence of inhibitors. Before start of PCR reaction, the SmartCycler Dx System is programmed to perform an optical measurement or probe check on the optical channels associated with Leishmania target and Internal Control detection. The Probe Check control verifies reagent bead rehydration, appropriate tube filling, probe integrity, and dye stability. Probe Check is considered to PASS if optical measurements meet the validated acceptance criteria. If the Probe Check fails in either optical channel, the test will not continue. The System Control Check for Temperature Control ensures that the SmartCycler Dx Instrument is operating within specification. P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above: None Q. Proposed Labeling: The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10. 13 {13} R. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. 14