Updated Clinical Validation Data

February 3rd, 2021

Overall Sensitivity and Specificity:

All Patients

API Covid-Rapid Antigen Test	BGI PCR Test	Total
	Positive	Negative
Positive	214	2	216
Negative	16	371	387
Total	230	373	603

Analysis of coincidence rate of API Covid-Rapid Antigen Test and PCR Test in nasal and nasopharyngeal swab samples:

Positive coincidence rate (Sensitivity): 214/ (214+16) × 100% = 93.0%

Negative coincidence rate (Specificity): 371/ (2+371) × 100% = 99.5%

Total coincidence rate (Accuracy): (214+371) / (214+16+2+371) × 100% = 97.0%

Asymptomatic Patients Overall

API Covid-Rapid Antigen Test	RT-PCR Test	Total
	Positive	Negative
Positive	13	2	15
Negative	0	124	124
Total	13	126	139

Analysis of coincidence rate of API Antigen Test and PCR in nasal swab samples of asymptomatic patients:

Positive coincidence rate (Sensitivity): 13/ (13+2) × 100% = 86.7%

Negative coincidence rate (Specificity): 124 / (124+0) × 100% = 100%

Total coincidence rate (Accuracy): (13+124) / (13+2+124+0) × 100% = 98.6%

I. Dallas, Texas Clinical Trial 

Using known positive clinical samples that had been frozen in viral transport media since procurement from August to October 2020, Kai Medical Labs in Dallas, Texas performed a validation on October 29-30, 2020 using antigen test lot number 20200918. 36 of 63 patients were asymptomatic and 12 were confirmed PCR positive. Our antigen test concurred on 11 of the 12 PCR positive, asymptomatic patients. (Ct=38 for the one “false” negative). All results are below which demonstrate high sensitivity and specificity. 

API Covid-Rapid Antigen Test	PCR Test	Total
	Positive	Negative
Positive	31	0	31
Negative	1	31	32
Total	32	31	63

Analysis of coincidence rate of API Antigen Test and PCR in nasal swab samples:

Positive coincidence rate (Sensitivity): 31/ (31+1) × 100% = 96.9%

Negative coincidence rate (Specificity): 31 / (31+0) × 100% = 100%

Total coincidence rate (Accuracy): (31+31) / (31+1+31+0) × 100% = 98.4%

II. China Clinical Trial 

435 total nasal and nasopharyngeal swabs were used. All assays during the period of the evaluation were double read by at least two technicians. All the specimens used in this evaluation were form The Red Flag Hospital of Mudanjiang at the Clinical Cooperation Unit of Norman, Hubei Hospital of Integrated Traditional Chinese and Western Medicine, and Wuhan Keyuan Hospital. More than 10 operators were used for the study. 

The evaluation was completed during 2 weeks in July and August 2020 for Red Flag Hospital. For the other sites, the antigen tests were performed in September and October 2020 from samples collected in April 2020 and stored frozen, with PCR results having been run in April. Specimens were collected from known PCR positive and known PCR negative patients’ samples. All samples were clinically derived, and none were contrived. Those samples that were old were stored frozen in viral transport medium. 

For those 100 positive samples that PCR cycle time was known, 7 of the 8 false negatives were cycle time >36 and the last was cycle time 33-36. All positive patients were symptomatic at some point. PCR comparator platform was BGI Genomics Co’s Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV.

API Covid-Rapid Antigen Test	BGI PCR Test	Total
	Positive	Negative
Positive	181	2	183
Negative	14	238	252
Total	195	240	435

Analysis of coincidence rate of API Covid-Rapid Antigen Test and BGI PCR Test in nasal and nasopharyngeal swab samples:

Positive coincidence rate (Sensitivity): 181/ (181+14) × 100% = 92.8%

Negative coincidence rate (Specificity): 238 / (2+238) × 100% = 99.16%

Total coincidence rate (Accuracy): (181+238) / (181+14+2+238) × 100% = 96.3%

Data for 180 PCR positives with known PCR cycle times. The sensitivity is 99.2% for those with PCR cycle time of 35 or less.

API Covid-Rapid Antigen Test	BGI PCR Test
	Ct <36	Ct>=36
True Positives	107	0
False Negatives	1	7
Total	108	7

III. Poland Clinical Trial 

Under the direction of Dr. Aleksandra Zimmer, Principal Investigator, we prospectively recruited 103 females over the age of 18 years except those who are part of a vulnerable population, such as prisoners or pregnant women. All patients over the age of 18 years will be recruited to participate in this study. Sampling was done by convenience samples from the population of patients that were enrolled at Wroclaw University Hospital, largely from the OB/GYN department. 

Patients were presented with the informational brochure to read and learned how the Antigen test works. They then performed their own nasal swab using the API Pharma Covid-Rapid Antigen test. They also performed all steps including adding reagent, adding to sample well, and interpreting results. They were asked their opinion of their results (pos, neg, invalid). A nurse monitored their progress to make sure they do it correctly and confirmed their results. For antigen tests, no viral transport media was used as tests were run immediately.

The healthcare provider then performed nasopharyngeal swab for PCR comparator. Swab was placed in viral transport media and sent to the nearest lab for processing. There was 100% concordance between the patient interpreted and nurse interpreted results. The healthcare provider then performed a nasopharyngeal swab for PCR comparator. Swab was placed in viral transport media and sent to the nearest lab for processing. The comparator PCR test was the VIASURE SARS-CoV-2 Real Time PCR Detection Kit with cycle time up to 38 cycles for positives.

API Covid-Rapid Antigen Test	PCR Test	Total
	Positive	Negative
Positive	2	0	2
Negative	1	102	103
Total	3	102	105

Analysis of coincidence rate of API Antigen Test and PCR VIASURE in nasal swab samples:

Positive coincidence rate (Sensitivity): 2/ (2+1) × 100% = 66%

Negative coincidence rate (Specificity): 102 / (102+0) × 100% = 100%

Total coincidence rate (Accuracy): (2+102) / (2+1+102+0) × 100% = 99.0%

IV. X-Prize Study:

89 blinded samples were sent to API Pharma labs for interpretation. Samples were in PBS, saliva, or nasal wash. For those samples that included whole virus with concentration of 200 copies per uL or more, Covid-Rapid detected 34 out of 40 samples, and it detected 33 out of 34 samples with concentration of 500 copies per uL or more. There were zero false positives and no cross reactivity with other viruses, including influenza and MERS coronavirus. The limit of detection was determined to be 500 copies per uL. The API Pharma Covid-Rapid Antigen Test was a semi-finalist in the X-prize competition, a top 200 test amongst nearly 700 international teams.

V. Point of care data:

Please see Poland Clinical Data results laid out above. The tests were performed by over a dozen nurses, seven doctors, and 103 individual patients. They were also interpreted by each of those individuals and all tests were interpreted twice—once by the patient and once by the provider. There was 100% concordance between providers and patients. While this was done in a hospital setting, it was not in the laboratory and the provider and patient interpretations clearly demonstrate that the test can be performed and interpreted in a point of care, CLIA-waived setting with accuracy and replicability.

For US based studies, we performed at Kai Medical Labs in Dallas, Texas:

POC Clinical Evaluation: A POC clinical evaluation should include 5 minimally trained operators at a non-laboratory (e.g., CLIA waived) sites in the United States. Each operator should test at least six positive and six negative specimens using only the test instructions and/or quick reference guide. As part of the EUA application, provide the detailed individual replicate result data and protocols for each of your studies, including: 

Detailed test procedure:

The POC studies were conducted with positive and negative controls. The negative control was a nuclease free water previously determined to be COVID-19 negative with PCR. The positive control included SARS-CoV-2 QC material from a PCR Amplification kit previously determined to be COVID-19 positive with PCR. All specimens were blinded and randomized and each operator tested six low positive and six negative specimens.  A low positive specimen has a Ct Score <36 but above 30.  Each sample was evaluated in 6 replicates for every experimental condition studied. All results, including invalid and incorrect results, were documented. 

All testing was conducted in a blinded fashion in which patients with an unknown SARS-CoV-2 status were presented to 5 minimally trained operators (two medical students and three nurses with no laboratory certifications). Patients were prospectively enrolled and tested sequentially and blindly.