K Number

Device Name

ID-FISH Plasmodium Genus Test Kit, ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit

Manufacturer

ID-FISH TECHNOLOGY, INC

Date Cleared

2017-08-18

(417 days)

Product Code

PYN

Regulation Number

866.3367

Intended Use

ID-FISH Plasmodium Genus Test Kit (PlasG) and ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit (PlasFV) are intended for in vitro diagnostic use in the clinical laboratory for detection of Plasmodium species in human venous whole blood (EDTA) samples from patients suspected of Plasmodium infection. The test kits are intended to aid in the diagnosis of malaria and to aid in the differential diagnosis of P. falciparum and P. vivax infection. The test kits should be used only on samples from patients with a clinical history, signs and symptoms consistent with malaria, and are not intended as a screen for asymptomatic patients. The ID-FISH Plasmodium Genus Test Kit is a qualitative test for detection of malaria parasites in blood smears. Positive results should be supplemented with the Plasmodium species specific test kit, ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit for identification and differentiation of Plasmodium falciparum and Plasmodium vivax. The results of these test kits should be used in conjunction with other diagnostic test results. Clinical performance has not been established for P. ovale, P. malariae, or P. knowlesi.

Device Description

The ID-FISH Plasmodium Genus Test Kit (PlasG) and ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit (PlasFV) are fluorescence in situ hybridization (FISH) assays to detect Plasmodium spp. or P. falciparum or P. vivax parasites in thin film blood smears prepared from EDTA venous whole blood samples.

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Device Name

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Third Party Reviewed

Expedited Review

Predicate Devices

Not Found

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The document describes a fluorescence in situ hybridization (FISH) assay and does not mention any AI or ML components.

Therapeutic

No.
This device is an in vitro diagnostic test intended to aid in the diagnosis of malaria, not to treat it.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the test kits "are intended to aid in the diagnosis of malaria and to aid in the differential diagnosis of P. falciparum and P. vivax infection."

SaMD (Software as a Medical Device)

The device is described as a "fluorescence in situ hybridization (FISH) assay" and a "test kit," which are physical components used in a laboratory setting to perform a diagnostic test on blood samples. This indicates it is a hardware-based device, not software-only.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the test kits "are intended for in vitro diagnostic use in the clinical laboratory for detection of Plasmodium species in human venous whole blood (EDTA) samples from patients suspected of Plasmodium infection."
Device Description: The description details a laboratory-based assay (fluorescence in situ hybridization) performed on biological samples (blood smears).
Intended User / Care Setting: The device is intended for use in a "Clinical laboratory / For professional use only," which is typical for IVDs.
Performance Studies: The document describes clinical performance studies comparing the device to established diagnostic methods (Giemsa microscopy and PCR/sequencing) using patient samples. This is a standard requirement for demonstrating the performance of an IVD.
Key Metrics: The performance studies report metrics like Sensitivity, Specificity, PPA, and NPA, which are commonly used to evaluate the performance of diagnostic tests.

All these elements align with the definition and characteristics of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The ID-FISH Plasmodium Genus Test Kit is a qualitative test for detection of malaria parasites in blood smears. Positive results should be supplemented with the Plasmodium species specific test kit, ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit for identification and differentiation of Plasmodium falciparum and Plasmodium vivax. The results of these test kits should be used in conjunction with other diagnostic test results. Clinical performance has not been established for P. ovale, P. malariae, or P. knowlesi.

Product codes (comma separated list FDA assigned to the subject device)

PYN

Device Description

Materials provided:
ID-FISH Plasmodium Genus Test Kit

Plasmodium Genus Hyb A
Plasmodium Hyb B
5x Plasmodium Wash Buffer
10x Plasmodium Rinse Buffer
Plasmodium Counterstain

ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit

Plasmodium falciparum / P. vivax Hyb A
Plasmodium Hyb B
5x Plasmodium Wash Buffer
10x Plasmodium Rinse Buffer
Plasmodium Counterstain

Materials not provided, but available for separate purchase:

SPR Smear Preparation Reagent.
Dual Band Filter Set specific to ID-FISH hybridization probes

Materials required but not provided:

Fluorescence microscope with 100x oil objective.
37±1° C Incubator
Negative Control Smears (made with non-malarial EDTA blood less than four days old and SPR)
Positive Control Smears (made with EDTA blood infected with P.f. or P.v. parasites less than four days old with SPR)

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Fluorescence microscopy

Anatomical Site

Human venous whole blood (EDTA)

Indicated Patient Age Range

The clinical study included patients from 29 days old to 92 years old.

Intended User / Care Setting

Clinical laboratory; For professional use only

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

A total of 1067 leftover venous whole blood (EDTA) samples were collected from patients with malaria-like symptoms at different times between 2013 and 2015. Endemic patient ages ranged from 29 days old to 92 years old, and non-endemic patient ages ranged from two years old to 92 years old. Of the 917 patient samples enrolled from malaria endemic regions, 300 sequential samples were collected from a site in Mangalore India, 395 sequential samples were collected from three Kenyan sites, and 222 samples were collected from Iquitos, Peru. The 222 samples from Peru were selectively enrolled (non-sequential) based on Giemsa results as follows: 22 Giemsa positive samples, and an additional 100 Giemsa positive samples with 100 matched Giemsa negative samples. A total of 150 non-endemic symptomatic patient samples were collected from the United States. An additional 21 samples that were collected from Kenya were excluded from analysis because the patients had received malaria treatment within the six weeks prior to sample collection.
Giemsa thick and thin smear malaria microscopy testing was conducted at each collection site using a uniform protocol and pre-defined, expert adjudication by up to three microscopists. All samples were blinded and coded prior to testing, and all FISH testing and comparator method testing procedures were conducted by independent technicians. PCR testing followed by bi-directional sequencing was used for discrepant analysis.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Analytical performance:

Reproducibility: A study was conducted to evaluate the reproducibility of the ID-FISH Plasmodium Genus Test Kit (PlasG) and the ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit (PlasFV). A panel of five samples was contrived in human EDTA whole blood at the following parasite concentrations: Moderate positive P. falciparum 480 parasites/uL, Moderate positive P. vivax 440 parasites/uL, Low positive P. falciparum 156 parasites/uL, Low positive P. vivax 160 parasites/uL, Negative 0 parasites/uL. One hundred and eighty (180) replicate slides containing two smears per slide were prepared from each sample at two separate sites (900 slides total). The randomized and blinded panel was distributed to three sites (300 slides per site) and tested over five non-consecutive days, with two operators per site performing two runs per day. For each run, each blinded panel member was tested in triplicate with both the PlasG and PlasFV test kits using two smears per slide. For the PlasG test kit, one negative smear gave a false positive result in one run for a negative agreement of 99.4% (178/179). There was 100% agreement with the expected result with the PlasG test kit for all other panel members across runs, days, and sites. For the PlasFV test kit, one false positive result was observed for one negative smear (99.4% agreement). One low positive P. falciparum sample produced a false negative result in one run for an agreement of 99.4% (177/178). There was 100% agreement with expected results for other panel members.
Detection Limit: The analytical limit of detection (LoD) was estimated for the PlasG and PlasFV test kits using well defined clinical samples serially diluted in EDTA whole blood. The P. falciparum LoD for both PlasG FISH and PlasFV FISH was 143 parasites/uL. The P. vivax LoD for both PlasG FISH and PlasFV FISH was 126 parasites/uL.
Analytical Reactivity (Inclusivity): Evaluated with 11 additional Plasmodium-positive blood samples (4 P. falciparum, 2 P. vivax, 2 P. ovale, 2 P. malariae, 1 P. knowlesi). All Plasmodium positive samples were detected by PlasG. All P. falciparum and P. vivax samples were correctly identified by PlasFV probes.
Analytical Specificity (Cross Reactivity): Evaluated with a panel of 29 pathogenic bacteria, parasites, and viruses, and with 76 blood samples from patients with non-malarial medical conditions. No off-target organism produced a positive result with PlasG or PlasFV test kits. No false positive results were observed with blood samples from patients with non-malarial medical conditions.
Interfering Substances: Evaluated in the presence of various endogenous and exogenous substances. Each Plasmodium positive sample was detected by the expected PlasG and PlasFV test kit probes in the presence of the spiked substances. No false positive results were observed in the uninfected control blood samples.
Specimen Stability: Blood smear slides prepared from EDTA venous whole blood stored at 15-30°C for 5000 parasites/uL.
- PlasFV test kit for P. falciparum: 93.3% sensitivity at 101-500 parasites/uL, and 100% at >500 parasites/uL.
- PlasFV test kit for P. vivax: 92.6-97.5% sensitivity at 1001-10000 parasites/uL, and 100% at higher concentrations.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

PlasG test kit vs. Giemsa microscopy (Combined Indian and Kenyan Sample Results):
- Sensitivity: 95.7%
- Specificity: 92.6%
PlasFV test kit vs. Giemsa Microscopy - P. falciparum (Combined Indian and Kenyan Sample Results):
- Sensitivity: 97.4%
- Specificity: 96.1%
PlasFV test kit vs. Giemsa Microscopy - P. vivax (Combined Indian and Kenyan Sample Results):
- Sensitivity: 91.2%
- Specificity: 98.3%
PlasG test kit vs. Giemsa microscopy (Peruvian Sample Results):
- PPA: 95.9%
- NPA: 100%
PlasFV test kit vs. Giemsa Microscopy - P. falciparum (Peruvian Sample Results):
- PPA: 100%
- NPA: 100%
PlasFV test kit vs. Giemsa Microscopy - P. vivax (Peruvian Sample Results):
- PPA: 90.6%
- NPA: 100%

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

Not Found

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 866.3367 Device to detect and identify microbial nucleic acids by FISH in clinical specimens.

(a)
Identification. A device to detect and identify microbial nucleic acids by fluorescence in situ hybridization (FISH) in clinical specimens is an in vitro diagnostic device intended for the detection and identification of microbial pathogens in specimens collected from patients with signs and symptoms of infection. The device is intended to aid in the diagnosis of human disease in conjunction with clinical signs and symptoms and other laboratory findings.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Design verification and validation must include the following:
(i) Detailed device description documentation, including the device components, instrument requirements, ancillary reagents required but not provided, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens, probe sequences, and rationale for probe sequence selection;
(ii) Detailed description of the fluorophores, signal source, detection mechanism, and method of result interpretation;
(iii) Detailed documentation from the following analytical studies: analytical sensitivity (Limit of Detection), inclusivity, reproducibility, interference, cross reactivity, and specimen stability; and
(iv) Detailed documentation from a clinical study that includes prospective (sequential) samples. The study, performed on a study population consistent with the intended use population, must compare the device performance to results obtained from appropriate and well-accepted comparator methods.
(2) The labeling required under § 809.10(b) of this chapter must include:
(i) A statement that the device is intended to be used in conjunction with clinical history, signs, symptoms, and the results of other diagnostic testing;
(ii) A detailed explanation of the interpretation of results and acceptance criteria for any quality control testing; and
(iii) A limitation that negative results do not preclude the possibility of infection.

Summary

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR ID-FISH Plasmodium Genus Test Kit ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit DECISION SUMMARY

A. DEN Number:

DEN160025

B. Purpose for Submission:

De novo request for evaluation of automatic class III designation for the ID-FISH Plasmodium Genus Test Kit and the ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit

C. Measurands:

Ribosomal RNA sequences of Plasmodium falciparum, Plasmodium vivax, and other Plasmodium species

D. Type of Test:

Fluorescence In Situ Hybridization (FISH) assay using fluorescently labeled DNA probes

E. Applicant:

ID-FISH Technology, Inc.

F. Proprietary and Established Names:

ID-FISH Plasmodium Genus Test Kit (PlasG)

ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit (PlasFV)

G. Regulatory Information:

1. Regulation section:
  21 CFR 866.3367
1. Classification:
  Class II (Special Controls)

3. Product code(s):

PYN

1. Panel:
  83- Microbiology

H. Indications for Use:

1. Indications for use:
  ID-FISH Plasmodium Genus Test Kit (PlasG) and ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit (PlasFV) are intended for in vitro diagnostic use in the clinical laboratory for detection of Plasmodium species in human venous whole blood (EDTA) samples from patients suspected of Plasmodium infection. The test kits are intended to aid in the diagnosis of malaria and to aid in the differential diagnosis of P. falciparum and P. vivax infection. The test kits should be used only on samples from patients with a clinical history, signs and symptoms consistent with malaria, and are not intended as a screen for asymptomatic patients.

1. Special conditions for use statement(s):
  For in vitro diagnostic use only For prescription use only For professional use only The product has not been validated with specimens other than venous whole blood (EDTA)

3. Special instrument requirements:

Fluorescence microscope equipped with the following:

Mercury arc lamp or LED light powered illumination
Dual band filter set compatible with ID-FISH hybridization probes ■
100x oil objective

I. Device Description:

The ID-FISH Plasmodium Genus Test Kit (PlasG) and ID-FISH Plasmodium falciparum and

P. vivax Combo Test Kit (PlasFV) are fluorescence in situ hybridization (FISH) assays to detect Plasmodium spp. or P. falciparum or P. vivax parasites in thin film blood smears prepared from EDTA venous whole blood samples.

Materials provided:

ID-FISH Plasmodium Genus Test Kit

Plasmodium Genus Hyb A
Plasmodium Hyb B ●
5x Plasmodium Wash Buffer
10x Plasmodium Rinse Buffer ●
Plasmodium Counterstain ●

ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit

Plasmodium falciparum / P. vivax Hyb A ●
Plasmodium Hyb B
5x Plasmodium Wash Buffer ●
10x Plasmodium Rinse Buffer
Plasmodium Counterstain ●

Materials not provided, but available for separate purchase:

SPR Smear Preparation Reagent .
. Dual Band Filter Set specific to ID-FISH hybridization probes

Materials required but not provided:

Fluorescence microscope with 100x oil objective .
37±1° C Incubator ●
Negative Control Smears (made with non-malarial EDTA blood less than four days old and SPR)
Positive Control Smears (made with EDTA blood infected with P.f. or P.v. parasites less ● than four days old with SPR)

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

Not applicable

K. Test Principle:

Fluorescent in situ hybridization (FISH) is a microscopic technique in which a DNA probe is labeled with a fluorescent dye and then hybridized on a slide with a target region of nucleic acid of the pathogen in a clinical sample. Venous whole blood from a patient (EDTA preserved) suspected of malaria is mixed with a fixative (smear preparation reagent), smeared onto a glass slide in a thin film, air-dried, and preserved with methanol. The smear

is then hybridized with a fluorescently-labeled DNA probe complementary to the rRNA of Plasmodium genus, P. falciparum, or P. vivax, depending on the probe used. Plasmodium parasites are visible in the sample when viewed with a fluorescence microscope equipped with the appropriate filters, and non-Plasmodium organisms do not produce a signal under the filters. Plasmodium species, if present, will appear green with the Plasmodium genus probe in the PlasG test kit. P. falciparum will appear green with the P. falciparum probe, and P. vivax will appear orange with the P. vivax probe in the PlasFV test kit. Ribosomal RNA is present in the cytoplasm of the parasite and is visible within the host red blood cell so that the entire parasite will give a fluorescent signal. Both sexual forms of Plasmodium spp., P. falciparum and P. vivax parasites are labelled by the respective PlasG and PlasFV test kit probes. Samples from patients who are receiving anti-malarial drug treatment may have negative FISH results, while Giemsa microscopy and PCR may remain positive for longer periods of time after treatment.

L. Performance Characteristics:

1. Analytical performance:

a. Reproducibility
A study was conducted to evaluate the reproducibility of the ID-FISH Plasmodium Genus Test Kit (PlasG) and the ID-FISH Plasmodium falciparum and P. vivax Combo Test Kit (PlasFV). A panel of five samples was contrived in human EDTA whole blood at the following parasite concentrations:
Moderate positive P. falciparum 480 parasites/uL ●
Moderate positive P. vivax 440 parasites/uL ●
Low positive P. falciparum 156 parasites/uL ●
Low positive P. vivax 160 parasites/uL ●
Negative 0 parasites/uL .

One hundred and eighty (180) replicate slides containing two smears per slide were prepared from each sample at two separate sites (900 slides total). The randomized and blinded panel was distributed to three sites (300 slides per site) and tested over five non-consecutive days, with two operators per site performing two runs per day. For each run, each blinded panel member was tested in triplicate with both the PlasG and PlasFV test kits using two smears per slide (3 replicates x 2 operators x 2 runs x 5 days x 3 sites = 180 observations per kit).

Of the 900 slides, three smears (one negative smear and two low positive P. falciparum smears) fell off the slides at one site and so could not be read (3/900 = 0.3%). These smears were excluded from the analysis.

For the PlasG test kit, one negative smear gave a false positive result in one run for a negative agreement of 99.4% (178/179). There was 100% agreement with the expected result with the PlasG test kit for all other panel members across runs, days, and sites. See Table 1.

For the PlasFV test kit, one false positive result was observed for one negative smear (note: the same negative smear that also gave a false positive result with the PlasG test kit) for a negative agreement of 99.4% (178/179). One low positive P. falciparum

sample produced a false negative result in one run for an agreement of 99.4% (177/178). There was 100% agreement with the expected P. falciparum, P. vivax, or negative results with the PlasFV test kit for all other panel members across operators, runs, days, and sites. See Table 2.

| Sample | Agreement with expected result (%) | | | | | | Overall | 95%
Confidence
Interval |
|-----------|------------------------------------|----------------|----------------|----------------|----------------|-------------------|---------------------|-------------------------------|
| | Site 1 | | Site 2 | | Site 3 | | | |
| | Op 1 | Op 2 | Op 1 | Op 2 | Op 1 | Op 2 | | |
| Negative | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 28a/29b
(96.6) | 178a/179b
(99.4) | 96.9 - 99.9% |
| Low P.f. | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 28/28b
(100) | 178/178b
(100) | 97.9 – 100% |
| Mod. P.f. | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 180/180
(100) | 97.9-100% |
| Low P.v. | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 180/180
(100) | 97.9-100% |
| Mod. P.v. | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 30/30
(100) | 180/180
(100) | 97.9-100% |

Table 1. PlasG test kit reproducibility

a One negative smear gave a positive result for Operator 2 at Site 3.

b Smear(s) fell off.

Table 2. PlasFV test kit reproducibility

	Agreement with expected result (%)
	Site 1		Site 2		Site 3			95%
Sample	Op 1	Op 2	Op 1	Op 2	Op 1	Op 2	Overall	Confidence
Interval
Negative	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	28a/29b
(96.6)	178a/179b
(99.4)	96.9 - 99.9%
Low P.f.	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	27c/28b
(96.4)	177c/178b
(99.4)	96.9 - 99.9%
Mod. P.f.	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	180/180
(100)	97.9 - 100%
Low P.v.	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	180/180
(100)	97.9 - 100%
Mod. P.v	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	30/30
(100)	180/180
(100)	97.9 - 100%

a One negative smear gave a false positive result for Operator 2 at Site 3.

b Smear(s) fell off.

C One low positive P.f. smear gave a false negative result for Operator 2 at Site 3.

b. Linearity/assay Reportable Range:

Not Applicable

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

Recommended External Controls:

Control material should be tested in accordance with the guidelines or requirements of local, state, and/or federal regulations or accrediting organizations. To monitor the

assay, reagent performance and day-to-day variation, positive controls for P. falciparum or P. vivax along with a negative control (non-malarial blood) must be tested with each run. Negative controls can be prepared from EDTA whole blood collected from normal subjects. Positive slides can be prepared from previously positive patient blood samples. Control slides should be prepared in the same manner as patient samples and they are stable for five years if stored in a dry place at room temperature. During the clinical testing conducted at three sites, external positive and negative FISH controls were tested for each run as per kit instructions, and no control failures were observed for the 61 days of non-consecutive clinical testing across all sites.

d. Detection Limit:
The analytical limit of detection (LoD) was estimated for the PlasG and PlasFV test kits using well defined clinical samples serially diluted in EDTA whole blood. A P. falciparum positive sample with a neat parasitemia of 18,375 parasites per microliter and a P. vivax positive sample with a neat parasitemia of 5,040 parasites per microliter were tested. The P. falciparum sample was serially diluted as follows: 1:16, 1:32, 1:64, 1:128, and 1:256. The P. vivax sample was serially diluted as follows: 1:5, 1:10, 1:20, 1:40, 1:80, 1:160, and 1:320. Triplicate smears were prepared from each dilution and tested with both kits, and the highest dilution (i.e. lowest parasite concentration) which produced a positive result for all three replicates was considered the preliminary LoD. The LoD was confirmed by testing an additional 20 smears prepared from blood containing parasites at this concentration to demonstrate a detection rate of greater than or equal to 95%. The LoD for each kit is summarized in Table 3.

Table 3. Limit of detection (parasites/uL)

| | P.
falciparum | P. vivax |
|-------------|------------------|----------|
| PlasG FISH | 143 | 126 |
| PlasFV FISH | 143 | 126 |

e. Analytical Reactivity (Inclusivity):
The inclusivity of the PlasG and PlasFV test kits was evaluated with an additional 11 blood samples positive for Plasmodium species. Ten human clinical blood samples from different geographic regions were tested including four P. falciparum, two P. vivax, two P. ovale, and two P. malariae positive samples. One monkey blood P. knowlesi positive sample was also tested. All Plasmodium positive samples were detected by the PlasG test kit. All P. falciparum and P. vivax positive samples were correctly identified by the respective probes in the PlasFV test kit.
Analytical Specificity: f.

Cross reactivity

The cross reactivity of the PlasG and PlasFV test kits was evaluated with a panel of 29 pathogenic bacteria, parasites, and viruses that may be found in human blood

samples. Microorganisms were spiked into whole blood samples and then blood smear slides were prepared according to the kit instructions. Except as noted, all parasites and bacteria were tested at concentrations greater than 10° organisms per milliliter and viruses were tested at greater than 10° PFU or copies per milliliter. For the PlasFV test kit. off-target Plasmodium species were also evaluated (e.g., non-P. vivax species to evaluate the P. vivax probe cross reactivity). See Table 4. No offtarget organism produced a positive result with PlasG or PlasFV test kits.

Table 4. Cross reactivity panel

Microorganisms
Anaplasma phagocytophilum
Babesia duncani
Babesia microti
Bartonella henselae
Borrelia burgdorferi
Bwamba Fever Virus
Chikungunya virus
Cytomegalovirus (CMV)
Dengue virus
Ehrlichia chaffeensis
Epstein-Barr virus (EBV)
Hepatitis B
HIV-1a
Influenza A
Influenza B
Leishmania major (amastigotes)
Leishmania major (promastigotes)
Leptospira interrogans
Rickettsia rickettsii
Rickettsia typhi
Trypanosoma cruzi
West Nile virus
Yellow Fever virus
Plasmodium berghei a,b
Plasmodium falciparum a,b
Plasmodium malariae a,b
Plasmodium knowlesi a,b
Plasmodium ovale a,b
Plasmodium vivax a,b

a Tested at > 104 organisms/mL

6 All on-target Plasmodium species were detected with the expected PlasG and PlasFV test kit probes.

Additionally, cross reactivity was evaluated with a panel of 76 blood samples from patients with non-malarial medical conditions that were processed as blood smears according to the kit instructions. These included 42 blood samples from patients with positive antibody test results for one or more of the following tick-borne diseases: B. burgdorferi, Babesia spp, B. henselae, A. phagocytophilum, E. chaffeensis, and Rickettsig spp .; 20 samples from patients with positive antimuclear antibody test results; four samples from patients with hepatitis B infection; and ten blood samples spiked with plasma from ten patients with HIV 1 infection. No false positive results were observed with the PlasG or PlasFV test kits.

Interfering Substances

The performances of PlasG and PlasFV test kits were evaluated in the presence of potentially interfering exogenous and endogenous substances that may occur in blood. Human whole blood samples spiked with P. falciparum, P. vivax, or unspiked controls were prepared as smears according to the kit instructions and tested in the presence of potential interferents at the concentrations shown in Tables 5 and 6. Each Plasmodium positive sample was detected by the expected PlasG and PlasFV test kit probes in the presence of the spiked substances. No false positive results were observed in the uninfected control blood samples in the presence of the spiked substances.

Substance	Concentration
Triglycerides	37 mmol/L
Hemoglobin	2 g/L
Bilirubin	342 µmol/L
Serum albumin	60 g/L

Table 5. Endogenous potential interfering substances

	Table 6. Exogenous potential interfering substances
--	-----------------------------------------------------	--	--	--

Substance	Concentration
Acetaminophen	1 mg/mL
Aspirin	1 mg/mL
Ibuprofen	1 mg/mL
Artesunate	1 mg/mL
Chloroquine	1 mg/mL
Doxycycline	1 mg/mL
Primaquine	1 mg/mL
Quinine	1 mg/mL
Sulfadoxine	1 mg/mL
Amoxicillin	100 µg/mL
Cephalexin	100 µg/mL
Ciprofloxacin	100 µg/mL
Erythromycin	100 ug/mL

g. Assay Cut-off:
n/a

h. Specimen Stability

The specimen collection and handling instructions recommend that blood smear slides for the PlasG and PlasFV test kits should be prepared and fixed as soon as possible after venous whole blood sample collection into EDTA vacutainer tubes.

In the clinical study, blood smears were prepared from leftover EDTA venous whole blood samples that were stored at 15 – 30 °C for less than 24 hours or stored at 4 °C for less than four days. The clinical study results supported the storage of whole blood under these conditions prior to preparing blood smear slides.

To evaluate the stability of blood smear slides that have been prepared and fixed according to package insert instructions, a real-time sample stability study was conducted. Multiple slide replicates were prepared from each of four P. falciparum and 10 P. vivax positive blood samples that had different concentrations of parasites as determined by microscopic examination (low, medium, or high parasite concentrations. The slides were prepared and stored with desiccant in the dark at 15 -30 °C. The PlasG and PlasFV test kits were used to stain selected slide replicates every three months for the first year, and once per year thereafter up to five years. All samples produced the expected test kit result at each time point without a noticeable reduction in staining intensity. The results supported the storage of fixed slides for specimens or quality controls for up to five years prior to staining.

Fresh versus Frozen Study i.
n/a
1. Comparison Studies:
- a. Clinical Comparison:

See section L3

b. Matrix Equivalence Study
n/a
1. Clinical Studies:
  The clinical performances of the PlasG and PlasFV test kits were evaluated in a multicenter study conducted in malaria endemic and non-endemic regions. The test kit results were compared to Giemsa thick and thin smear malaria microscopy results that followed a uniform protocol with pre-defined Giemsa adjudication among multiple readers. PCR testing followed by bi-directional sequencing was used for discrepant analysis.

Sample collection

A total of 1067 leftover venous whole blood (EDTA) samples were collected from patients with malaria-like symptoms at different times between 2013 and 2015 (See Table 9). Endemic patient ages ranged from 29 days old to 92 years old, and non-endemic patient ages ranged from two vears old to 92 vears old (See Tables 7 - 8). Of the 917 patient samples enrolled from malaria endemic regions. 300 sequential samples (i.e., all comers meeting inclusion criteria) were collected from a site in Mangalore India, 395 sequential samples were collected from three Kenyan sites that receive samples from western and central Kenya (one site in Kisumu, Kenya and two sites in Nairobi, Kenya), and 222 samples were collected from Iquitos, Peru. The 222 samples from Peru were selectively enrolled (non-sequential) based on Giemsa results as follows: 22 Giemsa positive samples, and an additional 100 Giemsa positive samples with 100 matched Giemsa negative samples. A total of 150 non-endemic symptomatic patient samples were collected from the United States. An additional 21 samples that were collected from Kenya were excluded from analysis as per the study protocol because the patients had received malaria treatment within the six weeks prior to sample collection.

| Patient Age group | Kenya | India | Peru | Total
Samples
Tested | Percent |
|-----------------------|-------|-------|------|----------------------------|---------|
| 29 days - P. f. Pos | P.f. Neg | Grand Total |
| PlasFV FISH | P.f. Pos | 152 | 21a | 173 |
| | P.f. Neg | 4 | 518 | 522 |
| | Grand Total | 156 | 539 | 695 |

Sens	97.4%	(152/156)	95% CI	93.6%	99.0%
Spec	96.1%	(518/539)	95% CI	94.1%	97.4%

a Of the 21 samples negative for P. falciparum by Giemsa and positive for P. falciparum by the PlasFV test kit, 17 samples were positive for P. falciparum DNA by PCR/sequencing.

	Table 12. PlasFV test kit vs. Giemsa Microscopy - P. vivax: Combined Indian and
Kenyan Sample Results
	Carrely Company Component Company Comments of Children

		Giemsa Microscopy
		P.v. Pos	P.v. Neg	Grand Total
PlasFV FISH	P.v. Pos	156	9a	165
	P.v. Neg	15	515	530
	Grand Total	171	524	695

Sens	91.2%	(156/171)	95% CI	86.0%	94.6%
Spec	98.3%	(515/524)	95% CI	96.8%	99.1%

a Of the 9 samples negative for P. vivax by Giemsa negative and positive for P. vivax by the PlasFV test kit, 6 samples were positive for P. vivax DNA by PCR/sequencing. One sample that was positive for P. falciparum by Giemsa and positive for P. falciparum DNA by PCR/sequencing is included in this count of samples negative for P. vivax by Giemsa.

Sample Results - India

Table 13. PlasG test kit vs. Giemsa microscopy - All Plasmodium sp .: Indian Sample
Results

	Giemsa Microscopy
	Pos	Neg	Grand Total
PlasG FISH	Pos	179	9a	188
	Neg	11	101	112
	Grand Total	190	110	300

Sens	94.2%	(179/190)	95% CI	89.9% 96.7%
Spec	91.8%	(101/110)	95% CI	85.2% 95.6%

a Of the 9 samples with Giemsa negative and FISH positive results, 7 samples were positive for Plasmodium species DNA by PCR/sequencing: 6 samples positive for P. vivax DNA and 1 sample positive for P. falciparum DNA.

Table 14. PlasFV test kit vs. Giemsa Microscopy - P. falciparum: Indian Sample Results

		Giemsa Microscopy
		P. f. Pos	P.f. Neg	Grand Total
PlasFV FISH	P.f. Pos	19	5a	24
	P.f. Neg	0	276	276
	Grand Total	19	281	300

Sens	100%	(19/19)	95% CI	83.2%	100.%
Spec	98.2%	(276/281)	95% CI	95.9%	99.2%

a All 5 samples with Giemsa negative and FISH positive results were positive for P. falciparum DNA by PCR/sequencing.

		Giemsa Microscopy
		P.v. Pos	P.v. Neg	Grand Total
PlasFV FISH	P.v. Pos	156	9a	165
	P.v. Neg	15b	120	135
	Grand Total	171	129	300

Table 15. PlasFV test kit vs. Giemsa Microscopy - P. vivax: Indian Sample Results

Sens	91.2%	(156/171)	95% CI	86.0%	94.6%
Spec	93.0%	(120/129)	95% CI	87.3%	96.3%

a Of the 9 samples Giemsa negative and FISH positive for P. vivax, 6 samples were positive for P. vivax DNA by PCR/sequencing. One sample that was positive for P. falciparum DNA by PCR/sequencing and Giemsa positive for P. falciparum is included in the count of samples negative for P. vivax by Giemsa.

b Of the 15 samples that were positive for P. vivax by Giemsa and negative for P. vivax by the PlasFV test kit, 4 samples were positive for Plasmodium sp. by the PlasG test kit. All 4 samples were positive for P. falciparum DNA by PCR/sequencing; however, dual infection with P. vivax could not be ruled out by this method. 3 of the 4 sample were positive for P. falciparum by the PlasFV test kit

Sample Results - Kenya

		Table 16. PlasG Kit vs. Giemsa microscopy - All Plasmodium sp .: Combined Kenyan
Sample Results

		Giemsa Microscopy
		Pos	Neg	Grand Total
PlasG FISH	Pos	135b	19a	154
	Neg	3	238	241
	Grand Total	138	257	395

Sens	97.8%	(135/138)	95% CI	93.8%	99.3%
Spec	92.6%	(238/257)	95% CI	88.7%	95.2%

a Of the 19 samples with Giemsa negative and positive PlasG test kit results, 13 samples were positive for Plasmodium species DNA by PCR/sequencing: 12 samples positive for P. falciparum DNA and 1 sample positive for P. malariae DNA.

b Of the 135 Giemsa positive and FISH positive results, 2 samples were positive for P. ovale DNA and 1 sample was positive for P. malariae DNA by PCR/sequencing.

Table 17. PlasFV test kit vs. Giemsa Microscopy - P. falciparum: Combined Kenyan Sample Results

		Giemsa Microscopy
		P. f. Pos	P.f. Neg	Grand Total
PlasFV FISH	P.f. Pos	133	16a	149
	P.f. Neg	4b	242	246
	Grand Total	137	258	395

Sens	97.1%	(133/137) 95% CI	92.7%	98.9%
Spec	93.8%	(242/258) 95% CI	90.2%	96.2%

a Of the 16 samples with Giemsa negative and FISH positive results, 12 samples were positive for P. falciparum DNA by PCR/sequencing.

b Of the 4 samples positive for P. falciparum by Giemsa and negative for P. falciparum by the PlasFV test kit, 1 sample was positive for Plasmodium sp. by the PlasG test kit. This sample was positive for P. ovale DNA by PCR/sequencing.

Table 18. PlasFV test kit vs. Giemsa Microscopy - P. vivax: Combined Kenyan Sample Results

		Giemsa Microscopy
		P.v. Pos	P.v. Neg	Grand Total
PlasFV FISH	P.v. Pos	0	0	0
	P.v. Neg	0	395	395
	Grand Total	0	395	395

Sens	n/a
Spec	100%	(395/395)	95% CI	99.0%	100%

Malaria endemic region sample results (selective enrollment Peru)

The Peru sample results are presented separately as positive percent agreement and negative percent agreement (PPA, NPA) with adjudicated Giemsa microscopy because this sample set was selectively enrolled based on Giemsa results (Peru n=222, Tables 19 -21).

PlasG FISH test kit

The PlasG test kit had a PPA of 95.9% (117/122) 95% CI 90.8 - 98.2% and a NPA of 100% (100/100) 95% CI 96.3 - 100% in comparison to Giemsa results for detecting any Plasmodium sp. with the samples collected from Peru (Table 19).

PlasFV FISH test kit – P. falciparum

The PlasFV test kit had a PPA of 100% (5/5) 95% CI 56.6 - 100% and a NPA of 100% (217/217) 95% CI 98.3 - 100% for P. falciparum in comparison to Giemsa results with the samples collected from Peru (Table 20).

PlasFV FISH test kit – P. vivax

The PlasFV test kit had a PPA of 90.6% (106/117) 95% CI 84.0 - 94.7% and a NPA of 100% (105/105) 95% CI 96.5 -100% for P. vivax in comparison to Giemsa results with the samples collected from Peru. Of the 11 samples reported as Giemsa positive and PlasFV test kit negative for P. vivax, seven samples were positive for Plasmodium sp. by the PlasG test kit (Table 21)

Sample Results - Peru

Table 19. PlasG test kit vs. Giemsa microscopy - All Plasmodium sp .: Peruvian Sample Results

	Giemsa Microscopy
	Pos	Neg	Grand Total
PlasG FISH	Pos	117	0	117
	Neg	5	100	105
	Grand Total	122	100	222

	PPA	95.9%	(117/122)	95% CI	90.8%	98.2%
	NPA	100%	(100/100)	95% CI	96.3%	100%

Table 20. PlasFV test kit vs. Giemsa Microscopy - P. falciparum: Peruvian Sample Results

		Giemsa Microscopy
		P. f. Pos	P.f. Neg	Grand Total
PlasFV FISH	P.f. Pos	5	0	5
	P.f. Neg	0	217	217
	Grand Total	5	217	222

PPA	100%	(5/5)	95% CI	56.6%	100%
NPA	100%	(217/217)	95% CI	98.3%	100%

Table 21. PlasFV test kit vs. Giemsa Microscopy - P. vivax: Peruvian Sample Results

	Giemsa Microscopy
PlasFV FISH		P.v. Pos	P.v. Neg	Grand Total
	P.v. Pos	106	0	106
	P.v. Neg	11a	105	116
	Grand Total	117	105	222

PPA	90.6%	(106/117)	95% CI	84.0%	94.7%
NPA	100%	(105/105)	95% CI	96.5%	100%

a Of the 11 samples reported as Giemsa positive and FISH negative for P. vivax by the PlasFV test kit, 7 samples were positive for Plasmodium sp. by the PlasG test kit.

Positive results stratified by parasitemia

Kit performance for all positive samples combined is also presented stratified by the Giemsa microscopy quantified parasite level (PlasG FISH Tables 22 - 24, and PlasFV FISH Tables 25 - 26).

The sensitivity point estimate for the PlasG test kit for all species combined was 94.7% at parasite concentrations of 101 - 500 parasites per microliter, and 100% at concentrations above 5000 parasites per microliter (Table 22).

The sensitivity point estimate of the PlasFV test kit for P. falciparum was 93.3% at parasite concentrations of 101 - 500 parasites per microliter, and 100% at concentrations above 500 parasites per microliter (Table 25).

The sensitivity point estimate of the PlasFV test kit for P. vivax was 92.6 -97.5% at parasite concentrations between 1001 - 10,000 parasites per microliter, and 100% at higher concentrations (Table 26).

Combined Results Stratified by Parasitemia - PlasG test kit

| Parasites/µl
blood | Sensitivity | Giemsa sp. Pos) | 95% CI | |
|-----------------------|------ | >10000 | 100% | 5001-10000 | 100% | 1001-5000 | 95.8% | 501-1000 | 98.4% | 101-500 | 94.7% | 10000 | 100% | 5001-10000 | 100% | 1001-5000 | 100% | 501-1000 | 100% | 101-500 | 96.7% | 10000 | 100% | 5001-10000 | 100% | 1001-5000 | 93.8% | 501-1000 | 97.3% | 101-500 | 93.3% | 10000 | 100% | 5001-10000 | 100% | 1001-5000 | 100% | 501-1000 | 100% | 101-500 | 93.3% | 10000 | 100% | 5001-10000 | 97.5% | 1001-5000 | 92.6% | 501-1000 | 83.8% | 101-500 | 88.9% | (PlasG FISH Pos/
-------|-------------------------------------|--------|-------|
| (63/63) | 94.3% | 100% |
| (99/99) | 96.3% | 100% |
| (113/118) | 90.5% | 98.2% |
| (62/63) | 91.5% | 99.7% |
| (71/75) | 87.1% | 97.9% |
| (38/38) | 90.82% | 100% |
| (17/17) | 81.6% | 100% |
| (37/37) | 90.6% | 100% |
| (26/26) | 87.1% | 100% |
| (29/30) | 83.3% | 99.4% |
| (25/25) | 86.7% | 100% |
| (81/81) | 95.5% | 100% |
| (76/81) | 86.4% | 97.3% |
| (36/37) | 86.2% | 99.5% |
| (42/45) | 82.1% | 97.7% |
| (38/38) | 90.8% | 100% |
| (17/17) | 81.6% | 100% |
| (37/37) | 90.6% | 100% |
| (26/26) | 87.1% | 100% |
| (28/30) | 78.7% | 98.2% |
| (25/25) | 86.7% | 100% |
| (79/81) | 91.4% | 99.3% |
| (75/81) | 84.8% | 96.6% |
| (31/37)b | 68.9% | 92.4% |
| (40/45) | 76.5% | 95.2% |