: AccuPower ZIKV(DENV, CHIKV) Multiplex Real-Time RT-PCR Kit. Copyright, Bioneer Corporation. All rights reserved.

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1 : AccuPower ZIKV(DENV, CHIKV) Multiplex Real-Time RT-PCR Kit Copyright, Bioneer Corporation. All rights reserved.

2 1. Offers superior diagnostic accuracy compared to immunoassay Allows precision diagnosis, opening up a possibility in early discussion on microcephaly in pregnancy Molecular testing (RT-PCR) gives improved accuracy over immunoassay due to serological cross reactivity among falviviruses 2. Allows simultaneous diagnosis of co-infection Molecular testing can differentiate individual flaviviruses sharing the common vector Early detection of co-infection is critical, as antibody to DENV enhances co-infection by ZIKV 3. Provides extreme sensitivity ZIKV RNA found in semen, potentiating non-vector mediated transmission, lasts in low copy numbers for up to 188 days from the onset of symptom RT-PCR based diagnostic kit is capable of sustained detection of ZIKV long after symptoms subside 2

3 There is clear association of newborn microcephaly with ZIKV infection during the first-trimester of pregnancy. Microcephaly Cases Relative to 1 st -Trimester Infection Rate Total number of microcephaly cases for each birth cohort 50% of total number (reflecting potential overreporting) The number of microcephaly case increases proportionally to estimated ZIKV infection rate during first-trimester and during second or third-trimester. It is thus advisable for a pregnant woman bitten by mosquito in her firsttrimester, to be tested for ZIKV infection. Ref. : Michael A. Johansson. Zika and the Risk of Microcephaly. N ENGL J MED, 375;1, July

4 ZIKV RNA can be detected up to 10 times longer In the serum of pregnant women than in that of non-pregnant women. ZIKV RNA was detected in the serum of a pregnant woman at 4 weeks and 10 weeks after the clinical onset of ZIKV infection. (ZIKV viremia lasts for less than a week after the on set of viremia in non-pregnancy.) However, we have detected ZIKV RNA in the serum of a pregnant woman at 4 weeks and 10 weeks after the clinical onset of ZIKV infection but not after delivery. We suspect that the persistent ZIKV viremia in the patient described here was a consequence of viral replication in the fetus or placenta, which had high viral loads. Therefore, In addition to current ZIKV diagnostics, the use of quantitative RT-PCR methods may be a potential diagnostic approach for ongoing placental or fetal infections in pregnant women. 4 Ref. : R.W. Driggers. Zika Virus Infection with Prolonged Maternal BIONEER s Viremia intellectual and Fetal Brain property. Abnormalities. Do not N distribute ENGL J MED, without 374;22, permission. June

5 CDC recommends RT-PCR confirmatory testing in conjunction with antibody testing. For persons with suspected Zika virus disease, a positive rrt-pcr result confirms Zika virus infection, and no antibody testing is indicated. However, because of the decline in the level of viremia over time and possible inaccuracy in reporting of dates of illness onset, a negative rrt-pcr result does not exclude Zika virus infection. Therefore, serum IgM antibody testing for Zika and dengue virus infections should be performed if rrt-pcr is negative. However, a negative IgM antibody test, in the absence of rrt-pcr testing, might reflect specimen collection before development of detectable antibodies and does not rule out infection with the viruses for which testing was performed.. Ref. : Ingrid B. Rabe et al., Interim Guidance for Interpretation of Zika Virus Antibody Test Results. MMWR, Vol.65, No.21, June

6 Antibodies to ZIKV and DENV cross-react, blunting the accuracy of immunoassays. Over half of the DENV reactive mabs bound with high affinity to ZIKV. DENV2 and ZIKV E proteins share a highly similar fold and 54% sequence identity. *E protein : envelope protein Ref. : Lalita Priyamvada. Human antibody responses after dengue virus infection are highly cross-reactive to Zika 6 virus. BIONEER s intellectual property. Do not distribute PNAS, vol. 113 without no. 28, permission

7 Due to high serological cross reactivity, immunoassays often fail to differentiate the diagnosis between ZIKV and DENV infections. This could, therefore, potentially lead to the erroneous conclusion that dengue was the cause of the outbreak. Serological cross-reactivity between ZIKV and DENV has been observed in earlier studies. This crossreactivity leads to the misdiagnosis of dengue because of the similarity of the clinical symptoms in addition to serological cross-reactivity. Ref. : C. Fernanda Estofolete. Clinical and laboratory profile of Zika virus infection in dengue suspected patients: A case series. Journal of Clinical Virology 81 (2016)

8 Molecular testing can diagnose simultaneously a case of co-infection with ZIKV, DENV and/or CHIKV We report for the first time in Colombia and Latin America a co-infection between DENV and CHIKV, and probably, among the first globally, with also ZIKV. The traveler was co-infected with ZIKV and DENV-3, and the local patient was co-infected with ZIKV and DENV-1. Ref. (Left): Villamil-Gómez WE, et al. Dengue, Chikungunya and Zika co-infection in a patient from Colombia. J Infect Public Health 8 (2015) Ref. (Right) : Myrielle Dupont-R, et al. Co-infection with Zika and Dengue Viruses in 2 Patients, BIONEER s New Caledonia, intellectual 2014 property. Emerging Infections Do not distribute Diseases, Vol. without 21, No. permission. 2, Feb 2015

9 Diagnosing infections with multiple pathogens require testing with multiplexing capability. Thus, clinicians should be aware of infections with multiple pathogens in the differential diagnosis of dengue-like illness, especially in patients who returned from tropical regions. This diagnostic procedure could be improved by using multiplex RT-PCR for travelers, given the frequent co-circulation of multiple arboviruses in tropical regions. Ref. : Myrielle Dupont-R, et al. Co-infection with Zika and Dengue Viruses in 2 Patients, New Caledonia, BIONEER s intellectual property. Emerging Infections Do not distribute Diseases, Vol. without 21, No. permission. 2, Feb 2015

10 Preexisting immunity to DENV causes ADE of ZIKV replication Here we took advantage of a panel of human mabs generated from DENV-infected subjects to demonstrate substantial crossreactivity between DENV and ZIKV. Most mabs to DENV also bound to ZIKV, but those that recognize the major linear fusion-loop epitope (FLE) did not neutralize ZIKV, whereas they showed neutralizing activity against DENV. DENV-immune plasma and mabs to DENV potently enhanced ZIKV infection, which suggests the possibility that preexisting immunity to DENV might increase ZIKV replication. Ref. : Wanwisa Dejnirattisai, et al. Dengue virus sero-cross-reactivity drives antibody dependent enhancement of infection with Zika 10 virus. BIONEER s intellectual property. Do not distribute Nature without immunology permission. (2016)

11 ZIKV RNA is detected up to 188 days after the onset of symptoms. A man in his early 30s reported in January 2016 a history of fever, asthenia and erythematous rash during a stay in Haiti. On his return to Italy, ZIKV RNA was detected in his urine and saliva 91 days after symptom onset, and in his semen on day 188, six months after symptom onset. Our findings support the possibility of sexual transmission of ZIKV and highlight the importance of continuing to investigate nonvector-borne ZIKV infection. Ref. : E Nicastri, et al. Persistent detection of Zika virus RNA in semen for six months after symptom onset in a traveler returning from Haiti to Italy, February Rapid Communications (2016) 11

12 Presence of low viral load during convalescent phase of ZIKV infection necessitates highly sensitive diagnostics The lack of isolation of ZIKV from the various biological samples of our patient, during the convalescent phase, is not unexpected. The high CT values found are consistent with a low Zika viral load during the convalescent phase of infection, making it difficult to obtain viral cultures and thus sequence data. Superior sensitivity is needed to diagnose ZIKV infection at a low viral load. Ref. : E Nicastri, et al. Persistent detection of Zika virus RNA in semen for six months after symptom onset in a traveler returning from Haiti to Italy, February Rapid Communications (2016) 12

13 Diagnosis of ZIKV, DENV and CHIKV Ability to distinguish individual viral RNA in a single reaction tube is the basis for simultaneous detection of DENV, CHIKV and ZIKV infections. DENV NTC IPC PC CHIKV IPC ZIKV DENV CHIKV ZIKV DENV positive DENV ZIKV positive ZIKV CHIKV positive CHIKV IPC IPC IPC *NTC: Non-template control *IPC: internal positive control *PC: positive control 13

14 Analytical Sensitivity ZIKV DENV CHIKV Matrix Target ZIKV(cp/ ml ) DENV(cp/ ml ) CHIKV(cp/ ml ) Serum cp/ ml (95% CI 9.77~25.11 cp/ ml ) cp/ ml (95% CI 10.7~22.9 cp/ ml ) cp/ ml (95% CI 12.3 ~ cp/ ml ) Plasma cp/ ml (95% CI 10.23~25.54 cp/ ml ) cp/ ml (95% CI 12.3~28.84 cp/ ml ) 19.5 cp/ ml (95% CI 13.49~28.18 cp/ ml ) Urine cp/ ml (95% CI 14.12~36.67 cp/ ml ) N/A* N/A* *Not applicable, There is no case of detecting DENV and CHIKV in urine sample. Therefore this experiment was not carried out. 14

15 Analytical Specificity Normal Serum (Negative sample) Human genomic DNA IPC Only IPC Only Sample Normal Serum IPC ct DENV,ZIKV,CHIKV ct UD UD UD Sample IPC ct DENV,ZIKV,CHIKV ct UD Human gdna UD UD HBV 1x10 5 IU/ml HIV 1x10 4 IU/ml HCV -1x 10 4 IU/ml IPC Only IPC Only IPC Only Sample IPC ct DENV,ZIKV,CHIKV ct UD HBV 35 UD UD * UD ; Undetermined Sample IPC ct DENV,ZIKV,CHIKV ct Sample IPC ct DENV,ZIKV,CHIKV ct UD UD HIV UD HCV UD UD UD 15

16 Analytical Specificity Zika virus Chikungunya virus Dengue virus IPC(Internal Positive Control) Dye 1( Purified Dengue RNA 5ul) Dye2 (Purified ZIKA RNA 5ul) Dye 3 (Purified Chikungunya RNA, 5ul) YK IPC Sample Multiplex kit Sample Multiplex kit Sample Multiplex kit Sample Multiplex kit DENG DENG-2 u.d DENG-2 u.d DENG DENG DENG-2 u.d DENG-2 u.d DENG DENG DENG-3 u.d DENG-3 u.d DENG DENG DENG-3 u.d DENG-3 u.d DENG CHK u.d CHK u.d CHK CHK CHK u.d CHK u.d CHK CHK CHK u.d CHK u.d CHK CHK CHK u.d CHK u.d CHK CHK ZIKV u.d ZIKV ZIKV u.d ZIKV ZIKV u.d ZIKV ZIKV u.d ZIKV ZIKV u.d ZIKV ZIKV u.d ZIKV ZIKV u.d ZIKV ZIKV u.d ZIKV

17 Summary AccuPower ZIKV(DENV, CHIKV) Multiplex Real-Time RT-PCR Kit the best in class ZIKV diagnostics! 17