The document summarizes the validation of the TOYO Anti-HCV Test for detecting antibodies to HCV. It assessed the test's sensitivity using 100 samples that were confirmed positive by other methods, finding the TOYO test identified all 100 correctly. It assessed specificity using 100 samples that were confirmed negative, finding the TOYO test identified all 100 correctly. One sample showed discrepant results between membrane-based and plastic-based technologies, demonstrating a difference between testing methods. The validation concluded the TOYO Anti-HCV Test showed 100% sensitivity and specificity.

1. The Validation of TOYO tests
diagnostic capacity for the detection
of antibodies to HCV
Tatiana Kuchuk
Republican Scientific Practical Center for Quality Control of Laboratory
Diagnostics (RCQCLD) of Infectious Diseases of the Ministry of Health of the
Kyrgyz Republic, Kyrgyzstan
Michael Favorov
DiaPrep System Inc.
July 25, 2018

2. Validation Conducted by Reference Laboratory of RCQCLD Infectious
Diseases of the Ministry of Health of the Kyrgyz Republic, Scientific
and Production Association "Preventive Medicine”
Lab description and credentials 1
1. Accredited under ISO / IEC 17025 General requirements for the
competence of testing and calibration laboratories (accreditation certificate
No. KG 417/КЦА.ИЛ.046) Scope of accreditation: testing of diagnostic medical
immunobiological medication for determination of antigens and antibodies of
infectious diseases:
– HIV‐ELISA, ICT (ET), IB;
– Viral hepatitis A, B, C, D, E‐IFA, ICT (ET); IB
– Syphilis ‐ ELISA, IHT (ET), RPGA, RPR, RMP;
– Chlamydia, toxoplasmosis, CMV, HSV, mycoplasmosis, ureoplasmosis‐ELISA, IHT (ET);
– Brucellosis ‐ ELISA, IHT (ET), RPGA, RA.
• Accreditation body ‐ Kyrgyz Center for Accreditation, the signatory of the ILAC
MRA Accreditation Agreement for Testing Laboratories in accordance with ISO /
IEC 17025

3. • Accredited under ISO / IEC 17043 “Conformity assessment ‐ general
requirements for professional testing” (accreditation certificate No. KG
417/КЦА.ППТ. 001)
• Scope of accreditation: programs for testing the quality of diagnosis of infectious
diseases using ELISA, ICT (ET), IB, PCR, RPGA. RPR, RMP, RA: HIV, HBV, HCV, HAV,
IOP, syphilis; brucellosis; HSV; CMV; chlamydia; toxoplasma; ureaplasma,
mycoplasma. Accreditation body – Kyrgyz Center for Accreditation
• USA CDC qualified preparedness for the International Accreditation
ISO/IEC 17043 under American Association for Laboratory
Accreditation (AALA) (pending for the visit 2018)
Validation Conducted by Reference Laboratory of RCQCLD of the
Ministry of Health of the Kyrgyz Republic, Scientific and Production
Association "Preventive Medicine
Lab description and credentials 2

4. EVALUATION OF ANTI‐HCV RAPID
DIAGNOSTIC TESTS PRODUCED BY
TURKLAB HTTPS://WWW.TURKLAB.COM.TR
DETERRING SENSITIVITY, SPECIFICITY,
ACCURACY, DIAGNOSTIC
PERFORMANCE, EASINESS OF USE IN
PRACTICE AND OTHER FOR HCV
INFECTIONS IDENTIFICATION
Goal of the project

5. The objectives:
1. Evaluation of Instructions for the use of TOYO test
cassettes;
2. Evaluation of completeness in accordance with the
operational documentation (instructions) of the
assessed test system;
3. Evaluation of compliance of internal control of the test
cassette with requirements of its operational
documentation (instructions);
4. Evaluation of sensitivity;
5. Evaluation of specificity.

6. Methods and interpretations
• Internal tests control – according the instruction the tests were
considered valid when any visible red line appeared in the control
window "C". The time for reaching the determined color is at least
15 minutes and a maximum of 20 minutes.
• A positive result is the appearance of two red lines of any
intensity: one in the "C" control window, the other in the "T"
result window.
• A negative result is the appearance of one line only in window
"C".
• Invalid result is the absence of a line in the control window "C",
even if there is a red line in the result window "T".

7. Evaluation of the “TOYO Anti‐HCV Test WD/S/P”.
Lot HC021802.
valid through March 2020
Sensitivity assessment based on positive samples:
a. Anti‐HCV Mixed Titer Performance Panel PHV207, SeraCare BBI Diagnostics, USA – 22
samples;
b. HCV AccuVert Seroconversion Panel Modified PHV 911(M) 0610‐0206, SeraCare BBI
Diagnostics, USA – 4 samples;
c. CQCLD standardized panel of native anti‐HCV positive blood sera collected from
individuals residence of in Kyrgyzstan ‐ 74 samples.
Specificity assessment based on the negative samples:
a. Anti‐HCV Mixed Titer Performance Panel PHV207, SeraCare BBI Diagnostics, USA – 3
samples;
b. CQCLD standardized panel of native negative blood sera collected from individuals
residence of in Kyrgyzstan ‐ 97 samples.

8. The validation of sensitivity of “TOYO” RDT based of
parallel testing with three groups of available
specimens in RCQCLD, Bishkek, Kyrgyzstan, 2018
Status of panel/sample Sample type: Serum / Plasma
Number of
examinations
Confirmation
methods used
Result
Anti‐HCV Mixed Titer
Performance Panel PHV207
22 EIA, RNA, WB/IB 100%
HCV Accu Vert Seroconversion
Panel Modified PHV 911(M)
0610‐0206
4 EIA, RNA 100%
(3 of 4)*
Kyrgyzstan seraplasma
National Panel
74 EIA, RT, RNA,
WB/IB
100%
*One discrepant test in one specimen “HCV AccuVert Seroconversion panel
PHV 911(M) 0610‐0206” will be described in next slide

9. Results of the sensitivity evaluation of the “TOYO Anti‐HCV Test
WD/S/P” by seroconversion panel
“HCV AccuVert SPanel Modified PHV 911(M) 0610‐0206”
HCV
AccuVert
Seroconversi
on Panel
Modified
PHV 911(M)
0610‐0206
Name of the test‐system
RT
«TOYO Anti‐
HCV
Test WD/S/P»
c ϵ
EIA
Dia Pro
Diagnostic
HCV Ab
c ϵ
RT
SD Bio Line
HCV (Multi)
PreQ ‐
WHO
RT
Human
Hexagon
HCV
c ϵ
RT
NADAL
HCV‐Test
(test
device)
non ‐ c ϵ
RNA
QIAGEN
Artus HCV
RT‐PCR
Kits
c ϵ
PHV911‐02 Neg 0.1 Neg Neg Neg Neg Pos
PHV911‐03 Pos 1.5 Pos Pos Neg Neg Pos
PHV911‐04 Pos 11,6 Pos Pos Pos Neg Pos
PHV911‐05 Pos 12,1 Pos Pos Pos Neg Pos

10. The validation of specificity of “TOYO” RDT based
of parallel testing with three groups of available
specimens in RCQCLD, Bishkek, Kyrgyzstan, 2018
Status of panel/sample Sample type: Serum / Plasma
Number of
examinations
Confirmation methods
used
Result
Anti‐HCV Mixed Titer
Performance Panel PHV207
3 EIA, RNA, WB 2 of 3* Neg
Kyrgyzstan seraplasma
National Panel
97 EIA, RT, RNA 97 Neg
Total negative
samples
100 100 neg
100%
(95% CI
96% to 100.00%)
*One inadequate test in one sample "HCC AccuVert Panel The seroconversion of PHV 911
(M) 0610‐0206" will be described in the next two slides

11. Speci
ment
ID
EIA WB/IB HCV
RNA
Abbott
HCV 2.0
Abbott
Architect
HCV
Abbott
AxSYM
HCV
Abbott
PRISM
HCV
Ortho
EIA HCV
3.0
Ortho
VITROS
HCV
Ortho
HCV
Enhan
sed
Siemens
ADVIA
HCV
Ortho RIBA
3.0
Roche
COBAS
PHV
207‐22
1,2 2,3 0,4 2,2 0,7 1,1 0,7 0,0 NS3+ BLD
Result pos pos neg pos neg pos neg neg IND* neg
Results of evaluation of the sample No. PHV207‐22 “Anti‐
HCV” from “Mixed Titer Performance Panel PHV207”
initial serum testing data
* ‐ indeterminate

12. Sample
No.
EIA WB/IB RT
Dia Pro
Diagnos
tic
HCV Ab
c ϵ
s/co
ДС‐
ИФА‐
HCV‐
АГ/АТ
s/co
INNO
LIA HCV
Score
c ϵ
Bio‐Rad
Deciscan
HCV PLUS
c ϵ
RT
SD Bio
Line
HCV
(Multi)
preQ‐
WHO
RT
Human
Hexagon
HCV
c ϵ
RT
NADAL
HCV‐Test
(test
device)
non c ϵ
RT
«TOYO
Anti‐HCV
Test
WD/S/P»
c ϵ
PHV207‐22 2,8
Pos
4,4
Pos
IND
One line
NS3+
Neg Neg Neg Neg Neg
Results of evaluation of the sample No. PHV207‐22 “Anti‐HCV”
from “Mixed Titer Performance Panel PHV207” in Republican
Scientific Practical Center for Quality Control of Laboratory
Diagnostics of Infectious Diseases of the Ministry of Health of
the Kyrgyz Republic

13. Discussion on sample #PHV207‐22 results
• Based on the results presented on last two slides, we the authors
believe:
‐ in the case of sample # PHV207‐22, a reaction to the single NS3 HCV
protein band takes place. That result is indeterminate (definitely not
positive) by test Ortho RIBA 3.0
• Thus, the difference between the ELISA and EDT testing methods was
demonstrated.
• The difference can possibly be due to the difference in membrane‐
based (nitrocellulose) and plastic‐based technology.
• Over all this particular specimen were positive (6 out of 10) by the
plastic‐based tests, the membrane tests have no positive result (0 of 5).
Even with a small sample size the difference is significant (P <0.05)
• Surely, suchlike blood can not be used for Blood Transfusion!
Nevertheless, there are no grounds to treat this patient follow the
current testing result from hepatitis C!

14. Conclusion:
1. Valid results were received in 100% cases :
– for “TOYO Anti‐HCV Test WD/S/P” 200 out of 200.
2. Sensitivity of the “TOYO Anti‐HCV Test WD/S/P”, Lot HC021802 test
cassettes is 100% for all positive samples, except for No. 02 from 4
samples of seroconversion panels. Specificity is 100%.
3. The comparative assessment of the sensitivity of the tests “anti‐HCV
Test WD/S/P" for "HCV AccuVert Seroconversion Panel" with RT of 3
manufacturers showed comparable results
4. One serum of seroconversion panel was positive by EECA producer
RDT that indicate exceeded the sensitivity of EECA RDT in comparison
with two Western manufacturers and exceeded CE certificate ELISA
used as assessment test for validation.
5. The RNA of HCV was determined by PCR in all 4 samples of the “HCV
AccuVert Seroconversion Panel Modified”.
6. Special sample #PHV207‐22 testing indicated relatively lower fuls
positivity rate by RDT used