Detection and identification of travel-acquired Zika virus infections

Transcription

1 Detection and identification of travel-acquired Zika virus infections Sarah K. White, MPH Department of Environmental and Global Health College of Public Health and Health Professions November 4, 2016

2 Background Case introductions Results Conclusions

3 Common symptoms Fever 1-3 days Maculopapular rash 3-7 days Malaise/fatigue 5-10 days Body aches or joint pain/swelling 3-10 days* Maculopapular rash, Cherabuddi et al., 2016, under review

4 Samples January March 2016 Recent travel history Haiti (n=2) Colombia (n=1) Whole blood/serum 1 Saliva swab 2 Urine 3

5 Testing Sample preparation Whole Blood (Serum 1 case) Saliva Urine RNA Extract RT-PCR 5 Culture MRC-5, LLC-MK2, Vero E6, and C6/36* cell lines 1. Culture methods 2. Molecular methods RNA Extract RT-PCR and real-time RT-PCR 6.

6 Zika virus detection Cell culture Observe changes in cells induced by virus infection Cytopathic effects (CPE) Molecular detection Not necessarily detecting infectious virus +ssrna

7 Culture methods Why culture? Low virus concentration Cell lines used: MRC-5 LLC-MK2 Vero E6 C6/36 only for Albert s samples C C6/36 at 28 C Grown up to 28 days

8 Cell culture Vero E6 C6/36 LLC-MK2 200X MRC-5 200X 200X 100X 200X

9 Molecular detection Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Exponential amplification of a highly specific/conserved sequence Primers and reagents Binary results is the sequence present or not

10 Molecular detection Real-time RT-PCR Detection of amplification after each cycle via fluorescent probe Qualitative Lower Ct value = more +ssrna present in sample Threshold Cycle (Ct) value

11 Cases Case 1: Alberta missioned in Haiti Case 2: Albert visited Colombia Case 3: Dave traveled to Haiti

12 Case 1: Alberta Worked in Haiti for 2 weeks in January Symptom onset 2 days after returning to UF Common symptoms Rash ankles, spread upward Provided blood sample, saliva swab, and urine sample when rash appeared

13 Alberta s test results RT-PCR: Blood, saliva and urine Zika positive Cultured in cells Recovered infective zika virus from saliva and urine 9 days post-infection (dpi) to 16dpi LLC-MK2 and MRC-5 cell lines Real-time RT-PCR: Saliva and urine MRC-5, 16dpi Ct= 4 and 13 Urine LLC-MK2, 16dpi Ct= 8

14 Alberta s samples in culture Mock infected, LLC-MK2 9 dpi Saliva, LLC-MK2 9dpi Urine, LLC-MK2 9dpi

15 Alberta s follow-up Saliva swab and urine sample 5 days after initial sample Urine still positive (RT-PCR) Saliva and urine grown in culture RT-PCR positive Saliva swab and urine sample 14 days after initial sample RT-PCR negative Grown in culture, urine RT-PCR positive, 20dpi

16 Case 2: Albert Colombia for 5 days, March Symptom onset 7 days after returning to the swamp Symptoms Fever Rash torso Severe, persistent arthralgia Provided blood, saliva swab, and urine sample when rash appeared

17 Albert s results RT-PCR: Blood, saliva, and urine Zika positive Cultured in cells Recovered infective zika virus from blood, saliva and urine 10 dpi Blood and saliva, C6/36 and LLC-MK2 Urine, LLC-MK2 and Vero E6 Real-time RT-PCR Blood and saliva, C6/36, 10dpi Ct= 15 and 23 Bood, saliva, and urine, LLC-MK2, 10dpi Ct= 20, 13, and 24 Urine, Vero E6, 10dpi Ct= 18

18 Albert s sample in culture Cherabuddi et al., 2016; under review

19 Albert s co-infection Plaque assay Urine LLC-MK2, 10dpi Real-time RT-PCR Zika Ct= 18 Chikungunya Ct= 33 White SK, 2016; unpublished

20 Case 3: Dave Haiti for 11 days, January Symptoms started the evening he returned to UF Common symptoms Rash torso, spread Conjunctivitis Dave got Dengue Provided serum, saliva swab, and urine sample when rash appeared Serum and urine sent to State Diagnostic Lab Don t be like Dave

21 Dave s results 7 RT-PCR State Lab - Blood was Dengue 2 positive, Zika negative Urine Dengue 2 and Zika negative Lednicky Lab - Blood was Dengue 2 positive, Zika negative Saliva and urine Dengue 2 negative, Zika positive Culture Recovered infective zika virus from saliva and urine Both in MRC-5 Urine only in LLC-MK dpi

22 Dave s results continued Zika virus Real-time RT-PCR Saliva MRC-5 19dpi Ct= 18 Urine MRC-5 19dpi Ct= 20 Urine LLC-MK2 16dpi Ct= 12

23 Dave s sample in culture

24 Dave s follow-up 7 Saliva swab and urine sample 36 days after symptom onset RT-PCR negative Culture negative Real-time RT-PCR negative

25 Conclusions Imported Zika virus cases Active transmission in Haiti and Colombia Zika virus presence in samples Persistence still being investigated Whole blood vs. plasma Urine testing less invasive No commercial diagnostic State/CDC testing overwhelmed

26 Co-infections conclusions questions Zika co-infections Stop at identification of one pathogen Differential diagnosis challenging Antibody-dependent enhancement Chronology of infections, sequential or simultaneous One or multiple mosquitoes

27 References 1. Murray KO, Gorchakov R, Carlson AR, Berry R, Lai L, Natrajan M, et al. Prolonged detection of Zika virus in vaginal secretions and whole blood. Emerg Infect Dis Jan [23 Oct 2016] Barzon L, Pacenti M, Berto A, Sinigaglia A, Franchin E, Lavezzo E, et al. Isolation of infectious zika virus from saliva and prolonged viral RNA shedding in treveller returning from the Dominican Republic to Italy, January Euro Serveill. 2016:21(10). Doi: / ES Bingham AM, Cone M, Mock V, Heberlein-Larson L, Stanek D, Blackmore C, et al. Comparison of test results for zika virus RNA in urine, serum, and saliva specimens from persons with travel-associated zika virus disease Florida, MMWR Morb Mortal Wkly Rep May 13;65(18): doi: /mmwr.mm6518e2. 4. Gaskell KM, Houlihan C, Nastouli E, Checkley AM. Persistent Zika virus detection in semen in a traveler returning to the United Kingdom from Brazil, Emerg Infect Dis Jan [23 Oct 2016] Balm MN, Lee CK, Lee HK, Chiu L, Koay ES, Tang JW. A diagnostic polymerase chain reaction assay for Zika virus. J Med Virol 2012; 84: doi: /jmv Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, Stanfield SM, Duffy MR Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia. Emerg Infect Dis 2007; 14: Iovine N, Lednicky J, Cherabuddi K, Crooke H, White SK, Loeb JC, et al Co-infection with zika and dengue-2 viruses in a traveler returning from Haiti, 2016: Clinical presentation and genetic analysis. Clin Infect Dis. [Epub ahead of print]