Influenza antiviral susceptibility: methods and challenges to detect resistant virus
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1 SARINet Pan-American Health Organization - PAHO Influenza antiviral susceptibility: methods and challenges to detect resistant virus Brazilian experience Paola Cristina Resende, PhD Researcher National Influenza Center for the Brazilian MoH and WHO Oswaldo Cruz Institute - FIOCRUZ - Rio de Janeiro paola@ioc.fiocruz.br May, 2017
2 Summary To give a brief background on antivirals and methods to measure the drug viral susceptibility To update on the compiled data from Brazil
3 Available antivirals against Influenza viruses NIs are the primary influenza anti-viral therapeutics in use Neuraminidase inhibitors (Nis) Oseltamivir, Zanamivir, Peramivir, Lanamivir Antiviral Resistance M2 inhibitors Amantadine Rhimantadine High levels of resistance (H1N1 and H3N2, changes in amino acid - L26F, V27A and or S31N) Not effective against Influenza B Polymerase inhibitors T705 - Favipiravir Promising drug in advanced clinical trial phases
4 Neuraminidase inhibitors NA functions by cleaving the HA from the sialic acid receptor. NA inhibitors are sialic acid analogs which bind the NA active site preventing viral budding. Source: Moscona, A. (2005). Neuraminidase Inhibitors for Influenza. N Engl J Med 353:
5 Neuraminidase inhibitors They differ in their chemical structure, bioavailability and mode of administratio n Zanamivir Oseltamivir Peramivir Lanamivir Imagem adaptada de McKimm-Breschkin J, 2012 Active site N2 H274Y Seasonal N1- H275Y Pandemic N1- H275Y H5N1 H275Y Adaptado de Nguyen et al, Conformational changes may occur due to mutations in this site, It leads to a lack of interaction with the antiviral drug
6 METHODS FOR LABORATORY SURVEILLANCE OF ANTIVIRAL RESISTANCE OR REDUCED SUSCEPTIBILITY GENOTIPIC Assays PHENOTIPIC Assays
7 GENOTIPIC Assays 1. Sanger sequencing NA partial or full length Widely used Data can be to address for a number of questions Quantification of mixtures is difficult, slower than screening assays (1-5 days) 2. SNP detection by Pyrosequencing (PSQ) Screening of SNPs, fast (5-6 hours) Estimate relative allele proportion (viral subpopulations/quasispecies) Problems in regions with homopolymers (Eg. AAAAA) 3. SNP detection by Real time RT-PCR Screening of SNPs, fast (3-4 hours) Need for control virus Difficulties with probes specificity or SNP in the primers site may affect sensibility Lower capacity to distinguish between genotypes that have the same Tm
8 GENOTIPIC Assays Advantages Relatively simple and rapid. Methods can be applied directly with clinical material. Methods can be implemented using existing molecular technologies in the laboratory. Disadvantages Interpretation of mutations is difficult without phenotypic information. Lack of known mutations associated with antiviral resistance is not a guarantee of susceptibility to a particular drug. Interpretation of the significance of novel mutations is difficult. Challenges Understanding the relationship between virus drug susceptibility phenotype and genotype. Determining what proportion, resistant vs sensitive, of a mixed virus population is required to confer resistance.
9 PHENOTIPC Assays Advantages Enable detection of all resistancecausing mutations (including unidentified mutations) Disadvantages The viral isolation is necessary Classification of viruses based on their fold differences compared to the median of viruses from the same type/subtype/lineage showing NI. Normal inhibition (NI) Reduced inhibition (RI) Highly reduced inhibition (HRI) Influenza isolates NA activity and NA inhibition IC50 drug capacity of virus inhibition in 50% Tested with the NIs antiviral drugs: OST, ZAN,PER, LAN
10 GENOTIPIC Assays Types of technology: Fluorescence (FL) In-house and commercial kits Substrate used: MUNANA (methyl umbelliferone N-acetyl neuraminic acid) Instrumentation: Fluorimeter Advantages: low cost Flexibility Disadvantages: Need to prepare reagents in house Requires viruses with higher titers No standard universal protocol Chemiluminescent (CL) Commercial kits NA-Star and NA-XTD Substrate used: 1,2-dioxetane derivative of neuraminic acid Instrumentation: Luminometer Advantages: Greater linearity of signal Higher sensitivity in measuring NA activity Standard protocols by fabricant Disadvantages: More expensive than FL assays
11 Brazilian experience with the Influenza antiviral resistance surveillance
12 Brazilian experience with Influenza antiviral resistance T. M. Souza, M. Mesquita, P. Resende, et al (2011) 315 H1N1pdm09 strain analyzed from 2009 to 2010 All strains H275 November, International training of antiviral resistance for Latin American countries organized by FIOCRUZ, Rio de Janeiro and HPE, London
13 Brazilian experience with Influenza antiviral resistance First detection of resistant viruses in Brazil Community transmission?
14 Brazilian experience with Influenza antiviral resistance A/RJ/105/2013 Abr, 05 A/PR/409/2012 Jun, 22 A/SC/229/2012 Mai, 20 A/RS/124/2013 Abr, 05 Potential transmission of resistant variants (H275Y)
15 Brazilian experience with Influenza antiviral resistance Permissive mutations V241I e N369K, better fitness for H1N1 resistant viruses (H275Y) (Hurt, 2006; Takashita et al, 2015) Communitarian Clusters (H275Y) 31 cases of resistant virus from May to August, 2011 in Newcastle, Australia (Hurt et al 2011a; Hurt et al, 2012) 104 cases of resistant virus from September, 2013 to July, 2014 in Sapporo/Hokkaido, Japan (Takashita et al, 2015a) Possibility of emergence of a variant totally resistant to antiviral classes currently available
16 Brazilian experience with Influenza antiviral resistance Brazilian samples analyzed for H275Y in A/H1pdm09 from 2009 to 2016 Year A/H1pdm09 cases Studied A/H1pdm09 cases (for H275Y)* A/H1pdm09 carrying H275Y (%) (0.3%) (0.0%) (0.6%) (1.5%) (0.9%) (0.0%) (3.0%) (0.2%) Total (0.6%) *PSQ or Sanger sequencing
17 Brazilian experience with Influenza antiviral resistance Emergence of resistant virus by OST treatment pressure Resistant case from Immunocompromised patient (Cancer) Male 27 years-old 1 dose of OST Onset 75mg/day symptoms S1 2 dose of OST 75mg/day S2 Patient status: Recovered S3 Days: RT-PCR: Negative RT-PCR: H1N1pdm09 PSQ: 100% H275 ina: Normal inhibition (NI) OST, ZAN, LAN and PER RT-PCR: H1N1pdm09 PSQ: 36% H275 e 64% Y275 ina: Normal inhibition (NI) - ZAN and LAN Highly reduced inhibition (HRI) - OST and PER Additional phenotypic analyzes with ZAN, LAN and PER were performed by CDC WHOCC, Atlanta
18 Brazilian experience with Influenza antiviral resistance Influenza Vírus Genetic resistant markers against Oseltamivir (A) Samples PSQ per year (N) Samples Sanger per year (N) N of samples containing genetic resistance markers A H1N1pdm09 H275Y* I223K, Q313K+I427T S247N Total = Total = (I223K) 1 (S247N) e 1 (H275Y) 3 (S247N) e 2 (H275Y) A H3N2 E119V*, R292K, N294S D151V Total = (parciais) Total = B R152K, R371K, E119A/G, D198Y/E/N, N294S I222V, H274Y, G402S (B) Total = (D198N) 0 (A) Reduced inhibition (RI), Highly reduced inhibition (HRI) (B) Numbering N2 * SNP detected by Pirosequencing (PSQ) Phenotypc assay NIs: 111 were tested in presented Highly reduced inhibition (HRI) to the OST. 110 presented Normal inhibition to the OST.
19 Conclusions Considering the risk of emergence and spread of resistant viruses to the class of antiviral currently available it is important to strengthen viral surveillance and also to encourage and to carry out studies for new antiviral drugs. New challenges for the study of antiviral resistance markers: New antiviral drugs that act on other viral targets and in host factors Strategies for monitoring emergence of resistant strains can change soon
20 Antiviral susceptibility surveillance Useful websites: WHO Antiviral susceptibility surveillance ISIRV Antiviral Group Annual Review: Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, Hurt AC, et al. Antiviral Res Aug;132: doi: /j.antiviral
21 Acknowledgments NIC, FIOCRUZ team Marilda Siqueira Aline Matos Milene Mirada Cristiana Garcia David Brown Paola Resende Fernando Motta Prisicila Born Braulia Caetano Maria de Lourdes Brazilian MoH Dr. Sérgio Nishioka Walquiria Almeida Influenza team Surveillance Network States Central Labs CDC Influenza Division - WHOCC Dr. Larissa Gubareva Australian - WHOCC Dr. Aeron Hurt PAHO Influenza team Dr. Rakhee Palekar Dr. Juliana Leite Thank you! Muchas Gracias! Obrigada! paola@ioc.fiocruz.br aline.matos@ioc.fiocruz.br mmsiq@ioc.fiocruz.br
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