CL DETECT RAPID TEST

K141341 · InBios International, Inc. · PIT · Nov 14, 2014 · Microbiology

Device Facts

• Record ID: K141341
• Device Name: CL DETECT RAPID TEST
• Applicant: InBios International, Inc.
• Product Code: PIT · Microbiology
• Decision Date: Nov 14, 2014
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3870
• Device Class: Class 1

Indications for Use

The CL Detect™ Rapid Test is a qualitative, in vitro immunochromatographic assay for the rapid detection of Leishmania species antigen in ulcerative skin lesions. The test is intended for use with dental broach samples from less than four month old ulcerative skin lesions that are obtained from patients with suspected cutaneous leishmaniasis (CL). The test targets the peroxidoxin antigen of Leishmania species that may cause CL. The CL Detect™ Rapid Test is intended to aid in the diagnosis of CL, and must be interpreted within the context of all relevant clinical and laboratory findings.

Device Story

Device is a qualitative, membrane-based immunochromatographic assay for detecting Leishmania amastigote peroxidoxin antigen in ulcerative skin lesions. Input: dental broach sample from lesion, processed in lysis buffer. Operation: sample applied to test strip; capillary action moves sample across membrane; antigen binds to dye-conjugated monoclonal antibody and affinity-purified polyclonal antibody on test line. Output: visual red line at test region (positive) or control region (validity check). Used in clinical settings by trained professionals. Output aids diagnosis of CL; must be interpreted alongside clinical/laboratory findings. Benefits: rapid, point-of-care detection of Leishmania species.

Clinical Evidence

Prospective clinical study in endemic (Tunisia, n=168) and non-endemic (US, n=150) populations. Endemic study: sensitivity 100% (95% CI: 97.1-100%), specificity 84.2% (95% CI: 62.4-94.5%) compared to Giemsa microscopy. Non-endemic study: specificity 96.0% (95% CI: 91.6-98.2%). Analytical performance included reproducibility, limit of detection across various Leishmania species, and cross-reactivity/interference testing.

Technological Characteristics

Immunochromatographic assay; lateral flow format; detects peroxidoxin antigen of Leishmania species. In vitro diagnostic device.

Indications for Use

Indicated for patients with suspected cutaneous leishmaniasis (CL) presenting with ulcerative skin lesions less than four months old. Used as an aid in diagnosis.

Regulatory Classification

Identification

Trypanosoma spp. serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies to Trypanosoma spp. in serum. The identification aids in the diagnosis of trypanosomiasis, a disease caused by parasitic protozoans belonging to the genus Trypanosoma. Trypanosomiasis in adults is a chronic disease characterized by fever, chills, headache, and vomiting. Central nervous system involvement produces typical sleeping sickness syndrome: physical exhaustion, inability to eat, tissue wasting, and eventual death. Chagas disease, an acute form of trypanosomiasis in children, most seriously affects the central nervous system and heart muscle.

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM ASSAY ONLY TEMPLATE

A. 510(k) Number:
K141341

B. Purpose for Submission:
To obtain a substantial equivalence determination for a new device

C. Measurand:
Antigen from species of *Leishmania* that cause cutaneous leishmaniasis

D. Type of Test:
Qualitative immunochromatographic assay

E. Applicant:
InBios International, Inc.

F. Proprietary and Established Names:
CL Detect™ Rapid Test for Cutaneous Leishmaniasis

G. Regulatory Information:
1. Regulation section:
21 CFR 866.3870 *Trypanosoma spp.* serological reagents
2. Classification:
Class I
3. Product code:
PIT– Reagent, *Leishmania spp.* antigen detection
4. Panel:
83 Microbiology

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H. Intended Use:

1. Intended use(s):

The CL Detect™ Rapid Test is a qualitative, in vitro immunochromatographic assay for the rapid detection of *Leishmania* species antigen in ulcerative skin lesions. The test is intended for use with dental broach samples from less than four month old ulcerative skin lesions that are obtained from patients with suspected cutaneous leishmaniasis (CL). The test targets the peroxidoxin antigen of *Leishmania* species that may cause CL. The CL Detect™ Rapid Test is intended to aid in the diagnosis of CL, and must be interpreted within the context of all relevant clinical and laboratory findings.

2. Indication(s) for use:

The CL Detect™ Rapid Test is a qualitative, in vitro immunochromatographic assay for the rapid detection of *Leishmania* species antigen in ulcerative skin lesions. The test is intended for use with dental broach samples from less than four month old ulcerative skin lesions that are obtained from patients with suspected cutaneous leishmaniasis (CL). The test targets the peroxidoxin antigen of *Leishmania* species that may cause CL. The CL Detect™ Rapid Test is intended to aid in the diagnosis of CL, and must be interpreted within the context of all relevant clinical and laboratory findings.

3. Special conditions for use statement(s):

For prescription use only

4. Special instrument requirements:

Not applicable

I. Device Description:

The CL Detect™ Rapid Test is a qualitative membrane-based immunoassay for the detection of antigens expressed by *Leishmania* amastigotes present in skin lesions of infected individuals. The test strip membrane is pre-coated with an affinity-purified polyclonal antibody to a *Leishmania* antigen (peroxidoxin) on the test line region and goat anti-mouse IgG on the control line region. The test strip also contains a control line in an area pre-coated with a dye-conjugated mouse monoclonal antibody to the amastigote peroxidoxin antigen. The control line serves as verification of sufficient sample volume, proper sample flow, and also as a control for the reagents.

Kit Components:

1. Twenty-five (25) individually pouched test strips or twenty-five (25) test strips in a vial.

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2. One (1) vial of Lysis Buffer, $6\mathrm{mL}$ .
3. One (1) vial of Chase Buffer solution, $6\mathrm{mL}$ .
4. One (1) vial of Positive Control solution, $6\mathrm{mL}$ .
5. One (1) vial of Negative Control solution, $6\mathrm{mL}$ .
6. Twenty-five (25) sterile dental broaches for sample collection.

# J. Substantial Equivalence Information:

1. Predicate device name(s):

Kalazar Detect™ Rapid Test for Visceral Leishmaniasis

2. Predicate 510(k) number(s):

K023483

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | CL Detect™ Rapid Test for Cutaneous Leishmaniasis | Kalazar Detect™ Rapid Test for Visceral Leishmaniasis |
| Intended Use | Aid in the diagnosis of Leishmania spp. infection | Same |
| Technology | Immunochromatographic assay | Same |
| Dye conjugate | Colloidal gold | Same |
| Interpretation | Qualitative | Same |
| Reading method | Visual, manual | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | CL Detect™ Rapid Test for Cutaneous Leishmaniasis | Kalazar Detect™ Rapid Test for Visceral Leishmaniasis |
| Indication | Aid in the diagnosis of cutaneous leishmaniasis | Aid in the presumptive diagnosis of visceral leishmaniasis |
| Specimen type | Dental broach samples from ulcerative skin lesions | Serum |
| Analyte | Antigen from Leishmania spp. | Antibodies to members of the L. donovani complex |
| Test line capture reagent | Anti-Leishmania peroxidoxin polyclonal antibody | Recombinant rK39 antigen |
| Conjugate reagent | Anti-Leishmania peroxidoxin monoclonal antibody conjugate | Protein A conjugate |

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K. Standard/Guidance Document Referenced (if applicable):

Not applicable

L. Test Principle:

Sample collection:

Only ulcerative skin lesions less than four months old may be tested with this kit. A sample should be obtained from a skin lesion by a trained medical professional. After cleaning, debriding, and anesthetizing the lesion and surrounding skin if necessary, the sample is collected by twisting a sterile dental broach at the border of the lesion. The broach is then placed into a sample cup containing three drops of lysis buffer. Material should remain in lysis buffer for at least five to ten minutes but no longer than 30 minutes prior to testing.

Test procedure:

During testing, 20 microliters of the sample collected in lysis buffer is pipetted onto the sample pad of the test strip. The strip is then placed into a cup containing two to three drops of chase buffer to facilitate capillary action. The mixture reacts with the dye-conjugate monoclonal antibody on the test strip and migrates upward on the membrane chromatographically. When Leishmania amastigote antigens are present, the mixture reacts with the affinity-purified polyclonal antibody to generate a red line at the test line region of the membrane. Regardless of the presence of amastigote antigens, the mixture continues to migrate across the membrane to the immobilized goat anti-mouse IgG region where a red line at the control line region is expected to appear. The presence of the control line serves as verification of sufficient sample volume, proper sample flow, and also as a control for the reagents. The presence of the control line in the presence of the test line indicates a positive result. The presence of the control line in the absence of the test line indicates a negative result. The absence of the control line indicates an invalid result.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

A reproducibility study of the CL Detect™ Rapid Test kit was performed at three sites by two operators at each site for five days. Each site was provided sufficient material to run the assay in triplicate, for a total of 90 tests per panel sample. Each site was given a blinded coded panel of samples containing varying amounts of microscopy-quantified L. major promastigotes spiked into lysis buffer (six negative, six low positive, and six medium positive panel samples). Reproducibility was 98.9% for two of the negative panel samples and 100% for each of the other positive and negative panel samples. An additional study was performed in-house with six operators over five days using a blinded coded panel of lysed L. major promastigotes spiked at levels close to the estimated assay detection limit for that strain (negative, 300 parasite equivalents/test strip, and 450 parasite equivalents/test strip). A total of

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90 tests were run per panel sample. Reproducibility was 97.8% for the negative sample, 80% for the 300 parasite equivalents/test strip sample, and 92.2% for 450 parasite equivalents/test strip sample.

b. Linearity/assay reportable range:

Not applicable

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

Controls:

One vial each of positive and negative control is provided with each test kit. Positive and negative controls should be used to test each new kit of 25 test strips to ensure kit integrity. If either the positive or negative control fails, retest with new test strips. If kit controls fail a second time and tips of bottles have not been contaminated, then open a new kit box for testing.

Sample stability:

The test should be performed as soon as possible after sample collection. Do not leave samples at room temperature for prolonged periods. Material should remain in lysis buffer for at least five to ten minutes but no longer than 30 minutes prior to testing.

High-dose hook effect:

Purified Leishmania peroxidoxin antigen produced reduced color intensity test line but otherwise positive results with the CL Detect™ when tested at 100 µg/mL, a concentration projected to exceed the concentrations expected in clinical samples. No false negative results were observed and all positive test lines remained positive up to purified antigen concentrations of 100 µg/mL.

d. Detection limit:

The limit of detection was estimated by an expert operator using promastigotes from culture isolates of various Leishmania species that may cause cutaneous leishmaniasis. Quantified parasites were serially diluted in lysis buffer and simulated sample matrix, and three replicates of each dilution were tested with the CL Detect. The estimated limit of detection for each culture isolate (detected at least 95% of the time after testing an additional 20 replicates by an expert operator) is reported in Table 1.

Table 1. Estimated limit of detection
| Species | Parasite equivalents / test |
| --- | --- |
| L. tropica (WR-2995) | 187 |
| L. major (9/22/09 YS) | 200 |
| L. donovani (WR-378) | 380 |

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| Species | Parasite equivalents / test |
| --- | --- |
| L. panamensis (WR-2307) | 1080 |
| L. mexicana (WR-2798) | 1440 |
| L. braziliensis (WR-2353) | 1440 |

# e. Analytical specificity:

# Cross reactivity:

A cross-reactivity study was performed to determine the effects of potentially cross-reactive species with the CL Detect™ Rapid Test kit. The potentially cross-reactive species are listed in Table 2 below and included organisms that cause the majority of secondary infections in CL. Bacteria were diluted to $10^{4}$ , $10^{5}$ and $10^{6}$ CFU equivalents per mL. Viruses were diluted to $10^{3}$ , $10^{4}$ and $10^{5}$ pfu per mL. Mammalian cells were diluted to $10^{4}$ , $10^{5}$ and $10^{6}$ cells per mL. Fungi were diluted to $10^{4}$ , $10^{5}$ and $10^{6}$ spores/mL and parasites to $10^{4}$ , $10^{5}$ and $10^{6}$ parasites/mL. Species were tested in duplicate. Four mammalian cell lines [HeLa (human cervical cancer), MCF-7 (human breast cancer), WI-38 (human fibroblast) and WM-115 (human melanoma)] resulted in slight cross-reactivity with CL Detect™ at the highest concentration tested ( $10^{6}$ cells/mL). With all four cell lines, testing was negative once cells were diluted to $10^{5}$ cells/mL. None of the bacterial, viral and fungal species commonly known to cause secondary infections in CL patients, and none of the closely related parasite species cross-reacted with the CL Detect™ Rapid Test kit at any concentration tested.

Table 2. Cross reactivity

| Bacteria | Mammalian | Mycobacteria |
| --- | --- | --- |
| Acinetobacter baumannii | WI-38 (human fibroblast) | Mycobacteria abscessus |
| Bacillus cereus | MCF7 (human breast cancer) | Mycobacteria fortuitum |
| Bacillus subtilis | HeLa (human cervical cancer) | Mycobacteria marinum |
| Bacillus thuringiensis | 293T/17 (HEK293 human embryonic kidney) | Mycobacteria tuberculosis (attenuated) H37Ra-1 |
| Clostridium perfringens | WM-115 (human melanoma) | Mycobacteria ulcerans |
| Clostridium sordellii | U-937 (human lymphoma) | |
| Enterobacter aerogenes | | Viruses |
| Enterococcus durans | Fungi | HSV-1 |
| Enterococcus faecalis | Cladophialophora carrionii | HSV-2 |
| Escherichia coli (Clinical Isolate) | Fonsecaea pedrosoi | VZV (Ellen) |
| Haemophilus influenzae | Microsporum canis | VZV (Isolate D) |
| Klebsiella pneumoniae | Phialophora verrucosa | |
| Moraxella catarrhalis | Rhinocladiella compacta | Trypanosoma |
| Neisseria lactamica | Sporothrix schenckii | Crithidia fasciculate |
| Pasteurella multocida | Arthroderma benhamiae | Trypanosoma cruzi |
| Proteus mirabilis | Trichophyton soudanense | Trypanosoma lewisi |
| Proteus vulgaris | Trichophyton tonsurans | Trypanosoma rhodesiense |
| Proteus rufus | Trichophyton rubrum | Trypanosoma rufus |
| Proteus vulgaris | Trichophyton rubrum | Trypanosoma rufus |
| Proteus vulgaris | Trichophyton rubrum | Trichophyton rubrum |
| Proteus vulgaris | Trichophyton rubrum | Trichophyton rubrum |

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| Providencia stuartii | Microsporum gypseum | Trypanosoma rangeli |
| --- | --- | --- |
| Pseudomonas aeruginosa | | Trypanosoma gambiense |
| Staphylococcus aureus | | |
| Staphylococcus hominis | | |
| Stenotrophomonas maltophilia | | |
| Streptococcus pyogenes | | |
| Streptococcus agalactiae | | |
| Streptococcus sp. ATCC 12392 | | |
| Klebsiella oxytoca | | |

Interference study:

Blood components and topical treatments were evaluated for potential interference with the CL Detect™ Rapid Test kit. Potentially interfering substances were tested at concentrations expected to exceed those encountered in clinical samples as a "worst case scenario." Lysed L. major promastigotes were diluted to generate a sample panel that included a negative sample and three positive samples (500, 1000, and 10,000 parasite equivalents per test strip). Final concentration of interfering substances in each sample was 5% in lysis buffer. Results are summarized in Table 3 below. Betadine and mercurochrome should not be used on lesions, as even minute traces can detrimentally impact performance of CL Detect™. This information is included in the limitations section of the package insert.

Table 3. Interfering substances

| Substance | Results |
| --- | --- |
| Whole Blood | Interference observed^{1} |
| Buffy Coat | No interference |
| Plasma | No interference |
| Betadine (10% povidone-iodine) | Interference observed^{2} |
| Hydrogen peroxide (3%) | No interference |
| 70% isopropyl alcohol | No interference |
| Saline | No interference |
| Mercurochrome (2%) | Interference observed^{3} |
| EMLA cream (2.5% lidocaine, 2.5% prilocaine) | No interference |
| Xylocaine (lidocaine) | No interference |
| Antibacterial hand sanitizer (70% ethanol) | No interference |
| Neosporin (0.8% bacitracin, 0.05% polymyxin B, 0.5% neomycin) | No interference |
| Hydrocortisone (2.5%) | No interference |
| Boil-Ease (20% benzocaine) | No interference |

1. Whole blood at ≥5% final concentration showed some interference with the reading of low positive samples. No interference was observed with whole blood tested at 2.5% final concentration.

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2 False positives observed with betadine at $>0.0008\%$ final concentration.
3 False negatives observed with mercurochrome at $>0.025\%$ final concentration.

# Strain reactivity:

The analytical reactivity of the CL Detect™ Rapid Test was evaluated with promastigotes from various Leishmania culture isolates. Reactivity was reported for the lowest concentration of lysed parasite equivalents per test strip that was detected by the CL Detect™ in three out of three replicates. Results are summarized in Table 4 below.

Table 4. Analytical reactivity

| Species | Parasite Equivalents / test strip |
| --- | --- |
| L. tropica (WR-3064) | 312 |
| L. tropica (WR-1091) | 312 |
| L. amazoniensis (WR-404) | 156 |
| L. amazoniensis (WR-2837) | 626 |
| L. amazoniensis (WR-2767) | 1500 |
| L. donovani (WR-2712) | 626 |
| L. donovani (WR-2801) | 626 |
| L. infantum (WR-2719) | 626 |
| L. infantum (WR-2455) | 1250 |
| L. infantum (WR-2317) | 1500 |
| L. mexicana (WR-2800) | 312 |
| L. mexicana (WR-3006) | 2500 |
| L. guyanensis (WR-3017) | 750 |
| L. guyanensis (WR-2334) | 1250 |
| L. guyanensis (WR-2949) | 2500 |
| L. major (WR-3087) | 376 |
| L. major (WR-779) | 1250 |
| L. major (WR-1088) | 2500 |
| L. braziliensis (WR-2356) | 1250 |
| L. braziliensis (WR-3088) | 5000 |
| L. panamensis (WR-2306) | 2500 |
| L. panamensis (WR-3098) | 5000 |
| L. peruviana (WR-2334) | 1500 |
| L. peruviana (WR-2771) | 2500 |
| L. peruviana (WR-2851) | 5000 |

# f. Assay cut-off:

Not applicable

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# 2. Comparison studies:

a. Method comparison with predicate device:

Not applicable

b. Matrix comparison:

Not applicable

# 3. Clinical studies:

a. Clinical Sensitivity:

# Clinical performance - Endemic population

A prospective clinical study was conducted in two ex-US sites endemic for cutaneous leishmaniasis (Sidi Bouzid and Gafsa, Tunisia). These regions are known to be endemic for $L$ major. One hundred sixty eight (168) patients with suspected CL lesions were tested with the CL Detect™ Rapid Test and with the reference method, Giemsa microscopy. For each patient, a sample was collected with a dental broach and tested with the CL Detect™ Rapid Test. An additional sample from the same site was collected by scraping for microscopic identification of amastigotes. All 149 microscopy-positive samples also tested positive with CL Detect™. The sensitivity of the CL Detect™ was $100\%$ , $95\%$ C.I. [97.6%, 100%]. Out of 19 microscopy-negative samples, three samples tested positive with CL Detect™, so that the specificity in this endemic population was $84.2\%$ , $95\%$ C.I. [62.4%, 94.5%]. One microscopy-negative and CL Detect™-positive sample was positive by subsequent culture analysis. Results are summarized in Tables 5, 6, and 7 below.

It is likely that only patients infected with $L$ major were enrolled in the clinical study. Clinical performance has not been established for other Leishmania species that cause cutaneous leishmaniasis.

Table 5. Clinical performance endemic site 1:

| | Microscopy | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| CL Detect™ Rapid Test | Positive | 92 | 1 | 93 |
| | Negative | 0 | 12 | 12 |
| | Total | 92 | 13 | 105 |
| | | | | |
| Sensitivity: | | 100% (92/92, 95% C.I.: 96.0% - 100%) | | |
| Specificity: | | 92.3% (12/13, 95% C.I.: 66.7% - 98.6%) | | |

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Table 6. Clinical performance endemic site 2:

| | Microscopy | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| CL DetectTM Rapid Test | Positive | 57 | 2 | 59 |
| | Negative | 0 | 4 | 4 |
| | Total | 57 | 6 | 63 |
| | | | | |
| Sensitivity: | | 100% (57/57, 95% C.I.: 93.7% - 100%) | | |
| Specificity: | | 66.7% (4/6, 95% C.I.: 30% - 90.3%) | | |

Table 7. Combined endemic population performance

| | Microscopy | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| CL DetectTM Rapid Test | Positive | 149 | 3* | 152 |
| | Negative | 0 | 16** | 16 |
| | Total | 149 | 19 | 168 |
| | | | | |
| Sensitivity: | | 100% (149/149, 95% C.I.: 97.1% - 100%) | | |
| Specificity: | | 84.2% (16/19, 95% C.I.: 62.4%-94.5%) | | |

* One sample was positive with subsequent culture analysis.
** Two samples were positive with subsequent culture analysis.

# Clinical performance - Non-endemic population

A prospective clinical study was conducted in one non-endemic site in the US. Although this site observes occasional patients with microscopically confirmed CL, the probability of CL in this non-endemic population is very low. One hundred fifty (150) samples from patients with skin lesions that had a clinical presentation similar to cutaneous leishmaniasis were tested with the CL DetectTM Rapid Test and by Giemsa microscopy. All 150 samples were negative for CL by microscopy. One hundred forty-four (144) samples tested negative with the CL DetectTM, and six samples were false positive. Therefore, the specificity of the CL DetectTM in this non-endemic population was $96.0\%$ , $95\%$ C.I. $[91.6\%, 98.2\%]$ . Results are summarized in Table 8 below.

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Table 8. Clinical performance non-endemic population

| | Microscopy | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| CL Detect™ Rapid Test | Positive | 0 | 6 | 6 |
| | Negative | 0 | 144 | 144 |
| | Total | 0 | 150 | 150 |
| | | | | |
| Sensitivity: | | n/a* | | |
| Specificity: | | 96.0% (144/150, 95% C.I.: 91.6% - 98.2%) | | |

* No microscopy-positive CL lesions were observed in this population.

b. Clinical specificity:

See section M3a.

c. Other clinical supportive data (when a. and b. are not applicable):

Not applicable

4. Clinical cut-off:

Not applicable

5. Expected values/Reference range:

In an endemic population in Tunisia, CL Detect™ Rapid Test demonstrated positive results in 90.5% (152/168) of human ulcerative skin lesion samples. The endemic study population was 55.4% female and 44.6% male with an age range of 18 to 79 years old.

In a non-endemic population in the United States, CL Detect™ Rapid Test demonstrated positive results in 4.0% (6/150) of human ulcerative skin lesion samples. The non-endemic study population was 57.3% female and 42.7% male with an age range of 18 to 92 years old.

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.