Dengue & Chikungunya In Asia Strategies for Testing
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1 Dengue & Chikungunya In Asia Strategies for Testing Sally Lam Blood Services Group Health Sciences Authority Singapore 1
2 The Viruses - Arboviruses Flavivirus DENGUE Enveloped virus, ~50 nm in diameter with single-stranded RNA, ~10.7 kb in length 4 serotypes based on immunological properties (DEN-1, DEN-2, DEN-3 & DEN-4) Mainly maintained in urban cycle : CHIKUNGUNYA Alphavirus Enveloped virus, spherical particle, nm in diameter, single-stranded RNA, ~11.8 kb length 3 lineages from phylogenetic analysis : West Africa; Asia; East, Central & South African (ECSA) Mainly maintained in sylvatic cycle : 2
3 The Vector DENGUE Primary vector : Aedes aegypti mosquito Highly domesticated, prefers to rest indoors Predominantly daytime feeder, peak biting activity at dawn and dusk Prefers to breed in artificial containers Multiple feeding within a single blood meal, with preference for human host, infected mosquitoes may take longer to complete blood meal CHIKUNGUNYA Vectors : Aedes aegypti and Aedes albopictus Prefers outdoor environment - suburban, forests Opportunistic feeding on wide range of hosts Breeds in both natural and artificial containers Dispersal even more widespread than Ae aegypti 3
4 The Disease Onset DENGUE Homologous immunity to single serotype complete and lifelong, cross-protection between serotypes lasts less than 12 weeks Primarily diseases of children High fever, serve headache, pain behind the eyes, muscle & joint pains and rash. Clinical Illness : undifferentiated fever; dengue fever (DF); dengue hemorrhagic fever (DHF) & dengue shock syndrome (DSS) DSS is a medical emergency associated with high mortality rate and DIC, requires intensive blood transfusion support. Supportive treatment only CHIKUNGUNYA Single serotype - life long immunity High fever rapidly emerges associated with headache, back pain, myalgia & intense joint pain Fatalities rare, 1:1000 cases classified as severe, - elderly, pregnant women, immunosuppressed patients at risk majority do not require blood transfusion Treatment symptomatic 4
5 Average Annual Number of DF/DHF Cases Reported to WHO & Average Number of Countries Reporting Dengue
6 Since 1970s leading cause of hospitalization and death in children in SEA Of those at risk, >70% in the Region Even temperate countries are affected e.g. Bhutan, Nepal
7 Hyperendemic for Dengue : Singapore DENV-1 DENV-2 Lab-based dengue surveillance of dengue patient samples from TTSH from *10% of all dengue cases. Source : Emerging Infectious Diseases, Vol 16, No. 5, May 2010
8 1958 Bangkok 1963 India 1998 Malaysia 2001 Indonesia ~ mainly Asian strains with small outbreaks A226V variant of ECSA for 08/ 09 outbreak found in Thailand, Malaysia, Singapore, Bangladesh, Taiwan Asian genotype 8
9 Aggressive vector control & active case detection/isolation of patients Ae. Albopictus Suburban residential areas A226V variant Ae. Aegypyti Urban areas ECSA type 9
10 Factors Contributing to Expansion Convenient global travel Population growth Urbanization Low or no herd immunity Changes in weather patterns Inadequate water management Lack of effective mosquito control Increasing rate of viral genetic change and emergence of strains/genotypes with greater epidemic potential
11 Transmission of DEN/CHIK by Blood Transfusion 2 documented cases (Hong Kong & Singapore) for Dengue by blood transfusion, viral load not determined. No reports of transfusion transmission from CHIKV. Reunion Island epidemic peak : 2 out of 568 blood donors RT-PCR positive. One donor was in presymptomatic phase of infection, the other never developed symptoms Singapore 2008 outbreak : Active CHIK surveillance using RT-PCR, 2 viremic cases founded 1 day before onset of illness. Viral load level were 40 pfu/ml and 750 pfu/ml respectively. Estimated risk of viremic blood donation was high in endemic countries, but low compared to the risk of mosquito-borne DEN/CHIKV transmission. 11
12 Strategies for Testing 12
13 Dengue Diagnosis: Primary infection Incubation Disease Recovery Intrinsic Viremia FEVER IgM IgA IgG Days Tests for Viraemic/Fever phase Tests for Seroconversion - Virus isolation - NAT Detect NS1 Ag IgM ELISA / duo cassette IgG ELISA 13
14 Tests for Viremic/Fever Phase : Dengue TMA (Gen-Probe) Transfusion 2008; 48: , Linnen et al Analytical sensitivity : 95% detection limit of 14.9 copies/ml 50% detection limit of 3.5 copies/ml. Detects all 4 serotypes at 0.11 PFU/ml. Single donation testing required. Multiplex RT-PCR J. Clinical Microbiol : , Lai et al. use of SYBR Green for detection of all four DENV serotypes and fluorogenic probebased (FRET) for DENV serotyping Detects 0.1 PFU/ml for DEN 1-3; 0.01 PFU/ml for DEN-4 88% positive correlation between virus isolation and RT-PCR with regard to dengue virus detection Single donation testing required. 14
15 Tests for Viremic/Fever phase : Dengue NS1 antigen assays NS1 membrane/secreted NS1, secreted NS1 could be detected at day 1-9 after fever onset; surrogate marker of dengue viremia. 2 platforms by Biorad : ELISA & lateral flow rapid test (strip) test sensitivity : 97.3% for Primary Infection; 70% for Secondary Infection (Kumarasamy et al, 2007) NS1 more sensitive than EHI multiplex PCR (72.5%) A negative test does not exclude recent infection Platelia Dengue NS1 Ag assay -FDA allows IND for blood screening (May 2010) Ms Anti-NS1 Mab -HRP Serum Biotin Bound Antiden NS1 Ig Gold particles coated with streptavidin Capillary action Ms Anti-den NS1 Mab Diluted serum Unbound gold labeled antiden NS1 Ig Biorad NS1 Ag detection platform 15
16 Chikungunya Diagnosis Incubation Disease Recovery Intrinsic Viremia FEVER IgM IgA IgG Days Tests for Viraemic/Fever phase - Virus isolation - NAT Tests for Seroconversion Euroimmun IFT IgM/IgG In-house IgG Dot Blot Virus Neutralisation assay 16
17 Tests for Viremic/Fever phase : Chikungunya RT-PCR Good & Definitive test Most conclusive useful for initial viremic phase (day 0 day 7) Used in active surveillance during the Singapore outbreak No Antigen tests available. Adapted protocols with EHI in-house evaluation 1. Hasebe F et al. Combined Detection and Genotyping of CHIKV by a Specific Reverse Transcription- Polymerase Chain Reaction. Journal of Medical Virology 67: Detection limit of 100 pfu/ml. Though is less sensitive, 2 cases were detected as PCR positive 1 day before onset of fever 2. Pastorino B et al., Development of a TaqMan RT-PCR assay without RNA extraction step for the detection and quantification of African CHIKV. Journal of Virological Methods Taqman probe, very sensitive method, detection limit of 0.1 pfu/ml (Protocol kindly provided by Dr Tim Barkham, TTSH) 17
18 Stratification of Countries Category A - major public health problem, leading cause of hospitalization and death among children, cyclical epidemics in urban areas, spreading to rural areas, multiple circulating serotype Category B - cyclical epidemics becoming more frequent, multiple circulating serotypes, expanding geographically within country Category C - endemicity uncertain Category D - no evidence of endemicity
19 Strategies No Specific Measures Endemic Countries Risk of DEN/CHIK TTI increased, dependant on prevalence in donor population, and proportion of donors with asymptomatic infection. No direct cost to blood service indirect cost from patient morbidity from TTIs loss of confidence in blood supply safety. Non-Endemic Countries Risk of DEN/CHIK TTI low, depends on donor population exposure through travel. No direct cost to blood service indirect cost from loss of confidence in blood supply safety in event of a TTI occurring. 19
20 Strategies - Donor Qualification Deferral Endemic Countries Deferral based on exposure not feasible when disease is endemic. Unable to exclude early and asymptomatic infection. Non-specific, leads to high donor loss. Low cost effectiveness. Non-Endemic Countries Deferral based on exposure feasible, able to reduce risk of accepting donations from early and asymptomatic infected donors. Low donor loss, dependant on proportion of donor population likely to travel to endemic countries. Cost effective. 20
21 Strategies Testing by NAT / Ag Endemic Countries Non-Endemic Countries Able to detect asymptomatic infection. Donor loss dependant on specificity of test system. Universal testing. NAT - Expensive. Antigen testing affordable. Cost effectiveness depends on prevalence of asymptomatic infected donors. Able to detect asymptomatic infection. Donor loss dependant on specificity of test system. Selective testing. NAT - Expensive. Antigen testing affordable. Low cost effectiveness. Singapore BSG conducted a donor prevalence study, 5,204 donations ( ) screened by DEN Fourplex RT-PCR (Lanciotti et al, 2005), no cases detected. No licensed NAT / Ag assays for blood donation screening diagnostic tests need to be validated for blood screening prior to use. 21
22 Strategies - Pathogen Reduction Endemic Countries Able to reduce transmission risks. Expensive and only available for platelets and plasma currently. May result in reduced product yields. Increased cost effectiveness depends on ability to reduce risks of other TTDs as well. Non-Endemic Countries Able to reduce transmission risks. Expensive and only available for platelets and plasma currently. May result in reduced product yields. Increased cost effectiveness depends on ability to reduce risks of other TTDs as well. 22
23 Evaluation of the INTERCEPT and Mirasol PR Systems on the Inactivation of DENV-2 and CHIKV in Single-Donor Platelet Concentrates Blood Services Group, Health Sciences Authority; Environmental Health Institute, National Environment Agency APLT suspended in 100% plasma 4 sets of double APLT used for each virus study (3 test and 1 control) screened for DENV/CHIKV virus and antibodies 23
24 INTERCEPT SDP sample Mirasol SDP Sample DENV-2 Inactivation - Plaque Assay Pre-treatment Virus Titre (log pfu/ml) Pre-treatment Virus Titre (log pfu/ml) Post-treatment Virus Titre (log pfu/ml) Day 1 Day 3 Day 5 Day 7 Post-treatment Virus Titre (log pfu/ml) Day 1 Day 3 Day 5 Day 7 Log-reduction 294 (I) 3.71 ND ND ND ND > (I) 1.7 ND ND ND ND > (I) 2.44 ND ND ND ND > (I) ctrl ND ND ND 0 Log-reduction 294 (M) ND ND ND (M) 1.88 ND ND ND ND > (M) ND ND ND (M) ctrl ND ND ND 0.41 ND: not detected. >, inactivation to below the limit of detection. No virus was detected in the volume assayed.
25 INTERCEPT SDP sample Pre-treatment Virus Titre (log pfu/ml) Post-treatment Virus Titre (log pfu/ml) Day 1 Day 3 Day 5 Day 7 Log-reduction ND ND ND ND > ND ND ND ND > ND ND ND ND > (ctrl) 4.63 not collected Mirasol SDP sample CHIKV Inactivation - Plaque Assay Pre-treatment Virus Titre (log pfu/ml) Post-treatment Virus Titre (log pfu/ml) Day 3: 0.49 Day 5: 1.27 Day 7: 1.43 Log-reduction Day 1 Day 3 Day 5 Day ND ND ND ND > ND ND ND ND > ND ND ND ND > (ctrl) Day 1: 0.48 Day 3: 1.06 Day 5: 1.16 Day 7: 1.34 ND: not detected. >, inactivation to below the limit of detection. No virus was detected in the volume assayed.
26 Determining Appropriate Measures for Blood Supply Prevalence of infection in population Prevalence of infection in donor population Effectiveness of existing strategies Effectiveness/availability of testing/ pathogen reduction Impact on blood supply availability Clinical importance in transfusion recipients Cost effectiveness in reducing disease morbidity in relation to overall health situation in the country 26
27 Acknowledgment Dr Diana Teo Group Director, Blood Services Group, Health Sciences Authority Dr Ng Lee Ching Head, Environmental Health Institute, National Environmental Agency Dr Tan Li Kiang Research Scientist, Environmental Health Institute, National Environmental Agency 27
28 Thank You 28
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