Rezultatele investigatiilor de laborator pentru gripa realizate in sezonul particularitati
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1 IC Rezultatele investigatiilor de laborator pentru gripa realizate in sezonul particularitati Laboratorul Infectii Respiratorii Virale Institutul National de Cercetare Cantacuzino Intalnirea anuala gripa si SARI, Bucuresti, 26 noiembrie 2015
2 CNG Misiune Detectie rapida a variantelor antigenice sau aparitia unui virus gripal nou in populatia umana prin tipizare/subtipare, secventiere si caracterizare antigenica, precum si detectia virusurilor mutante rezistente la agenti antivirali. Scop De a contribui la prevenirea si controlul bolilor cu etiologie gripala si de a sprijini OMS pentru luarea deciziei in recomandarea anuala a compozitiei vaccinului gripal precum si de a adauga informatii stiintifice pentru o mai buna intelegere a patogenilor virali.
3 ILI si SARI Teste de laborator
4 1. Diagnostic Molecular Probele biologice (tampoane nasofaringiene, aspirate traheobronsice, fragmente de organe) sunt examinate pentru prezenta virusurilor gripale tip A si tip B folosind metoda real-time RT-PCR in house, avand ca tinta gena matrix, specifica de tip. Daca este detectat virusul gripal tip A, o a doua analiza rrt-pcr este efectuata tintind gena HA pentru determinarea subtipului H1N1 pdm09 sau H3N2. Daca este detectat virusul gripal tip B, viruses are detected, o a doua analiza rrt-pcr este efectuata (SNP genotyping) pentru determinarea liniei genetice (Yamagata sau Victoria).
5 Alte virusuri respiratorii Testate prin kit comercial Multiplex RT-PCR SEEPLEX RV 15 ONE-STEP Kit (Seegene.Inc., Seoul, Koreea), bazat pe RT-PCR classic conform cu protocolul de lucru recomandat de producator. Multiple Duplex one step RT-PCR folosind kit Qiagen One- Step RT-PCR si primeri specifici din literatura: RSV/hMPV, CoV 229E/OC43, Piv 1/3.
6 Algorithm Sample ILI/SARI RRT-PCR for influenza virus type A and type B Type A positive Type B positive Subtype: A/H1N1pdm 09 and A/H3 SNP Oseltamivir (RRT-PCR) Genetic lineage (RRT-PCR ) SARI negative for influenza virus >10% of positive influenza virus Isolation (MDCK, MDCK Siat) RSV + hmpv Other viruses Coronavirus: Para 1,2,3 - OC43/229E - MERS CoV (screening and confirmation) HA/NA genes sequencing - genetic characterization - relevant mutations for antiviral resistance - HI assay (antigenic characterization) - Phenotypic test for antiviral sensitivity
7 Total samples ILI 2014/15 N = 913 % 559 ILI from sentinel system primary care (61.2%) 354 ILI from nonsentinel system (38.8%) Total pos A&B (54.2%) 197 (55.6) type A (62.4) 101 (51.3) A/H1 pdm (38.1) 38 (37.6) A/H (61.9) 63 (62.4) type B (37.6) 96 (48.7) Neg (45.8) 157 (44.4) De observat detectia tuturor virusurilor gripale circulante in populatia umana, cu o usoara dominanta a tipului A in raport cu tipul B si o dominanta a subtipului H3N2 in raport cu H1N1 pdm09, atat in sistemul sentinela cat si la pacientii din sistemul non-sentinela (in general spitalizati). Au fost detectate doua infectii mixte: A (H3N2) si B.
8 Distributia pe saptamani a virusurilor gripale detectate in sezonul in sistemele de supraveghere ILI si SARI ILI GPs: Total = Neg: 256 (45.8%) - Pos: 303 (54.2%) - H3: 117 (38.6%) - H1: 72 (23.8%) - B: 114 (37.6%) ILI hospitalized: Total = Neg: 157 (44.4%) - Pos: 197 (55.6%) - H3: 63 (32%) - H1: 38 (19.3%) - B: 96 (48.7%) SARI: Total = Neg: 209 (51.4%) - Total Pos: 198 (48.6%) Influenza: H3: 47 (28.7%) - H1: 52 (31.7%) - B: 65 (39.6%) Non-influenza : * H3 H1pdm09 B Others
9 # detections Weekly distribution of SARI cases " 6' 7 8*# ^ 9** H1N1pdm09 A/H3N2 B RSV hmpv PIV 1 PIV3 PIV 2 PIV 4 Corona ADV Rhino Boca HEV Distribution of respiratory viruses in SARI samples 9% 8% 26% 33% 24% IV H1pdm09 IV H3 IV type B RSV Other
10 2. Caracterizarea antigenica, genetica si sensibilitatea la antivirale, sezon Pentru a determina: - distantele antigenice si genetice fata de virusurile incluse in vaccinul antigripal sezonier - aparitia mutatiilor care pot afecta patogenitatea si sensibilitatea la antivirale Probe pozitive selectate (aprox. 10%) sunt supuse studiilor moleculare.
11 Metode de selectie pentru izolare si secventiere Numar proportional de specimene pentru fiecare tip/subtip pe perioada intregului sezon. Severitatea bolii Statusul vaccinal. Tratamentul antiviral Valoarea Ct <30
12 Influenta stocarii specimnelor asupra rezultatelor de laborator Probele respiratorii pot fi stocate la 2-8 C timp de maxim 5 zile dupa recoltare, fara afectarea rezultatului prin detectie moleculara (rrt-pcr), desi valoarea medie C t a probelor pozitive a crescut dupa 4 zile de stocare. Desi nesemnificativ statistic, pozitivitatea a scazut pentru A(H1N1)pdm09, dar nu pentru H3. Efectul asupra ratei de izolare a virusului gripal pare de asemenea sa fie zero daca stocarea la 4 C nu este mai lunga de 5 zile.
13 Izolare Pe MDCK (ATCC/CCL 34) si MDCK SIAT1 pentru virus gripal A(H3N2) S-a efectuat pentru 16% din specimenele pozitive, de regula dupa 1-2 pasaje. Virusurile A (H3N2) au fost izolate si catacterizate cu dificultate comparativ cu tipul B si A(H1N1 pdm09). Treizeci si trei de izolate reprezentative au fost trimise la WHO Collaborating Center for Reference and Research on Influenza, The Francis Crick Institute, Mill Hill Laboratory, London
14 Secventierea si analiza filogenetica Genele care codifică HA au fost analizate prin metoda Sanger de secventiere. Amplificarea a trei sau patru fragmente genice cu secvente terminale suprapuse a fost efectuată cu kitul Superscript III Platinum One Step Quantitative RT-PCR System reagents (Invitrogen) cu ajutorul mai multor perechi de primeri specifici (PHE, Londra, Marea Britanie). Secventierea a fost realizată cu kitul BigDye Terminator v3.1 Ready Reaction Cycle (Applied Biosystems Foster City, California), pe un analizor ABI PRISM 3100-Avant (Applied Biosystems Foster City, California) cu patru capilare. Pentru comparare cu genoame de referinta, secventele au fost preluate din baza de date genetice GISAID. Arborii filogenetici au fost construiti folosind RAxML 8.0, cu modelul de substitutie GTRGAMMA.
15 Rezultate 20 Distributia saptamanala a izolatelor de virus gripal in in sistemul ILI/SARI sentinela si nonsentinela, Romania (N = 111) H3 B H1pdm09 week
16 Treizeci si cinci de tulpini izolate au fost testate pentru sensibilitate la antivirale, oseltamivir si zanamivir, prin metoda fenotipica toate au fost sensibile. Cca 23% din probele pozitive pentru virus gripal B au fost testate pentru stabilirea liniei genetice toate au apartinut liniei B/Yamagata.
17 Tip/Subtip Caracterizare antigenica (N =111) Caracterizare genetica (N =67) H1N1 pdm09-31 izolate, A/California/7/09-like (tulpina vaccinala) - 21 specimene, grup 6B - 3C.3b: 6 specimene A/Newcastle/22/2014; H3N2 B - 6 izolate A/Texas/50/2012 like (tulpina vaccinala) - 12 izolate A/Switzerland/ /2013 like - 9 izolate B/Massachusetts/02/2012-like (tulpina vaccinala) - 53 izolate B/Phuket/3073/2013 like - 3C.3a: 8 specimene A/Switzerland/ /20 13; - 3C.2a: 6 specimene A/HongKong/5738/ specimene clada 3, B/Phuket/3073/2013
18 Analiza filogenetică a genei HA a virusului gripal H1N1 pdm09 comparativ cu tulpina vaccinala si tulpinile sezonul trecut
19 Rezultate virus gripal tip A, subtip H3N2 Tip/Subtip Caracterizare antigenica Caracterizare genetica H1N1 H3N2 B Tulpinile A(H3N2) s-au multiplicat la un titru scãzut. - Numai 6 izolate (1/3) au fost mai bine recunoscute de serul de referintã antivirusul vaccinal A/Texas/50/2012; - 12 izolate (2/3) au fost bine recunoscute de serul de referintã antivirusul vaccinal pentru emisfera sudicã A/Switzerland/ / C.3b: 6 specimene A/Newcastle/22/2014; - 3C.3a: 8 specimene A/Switzerland/ /13; - 3C.2a: 6 specimene A/HongKong/5738/2014
20 Analiza filogenetică a genei HA a virusului gripal H3N2 comparativ cu tulpina vaccinala si tulpinile sezonul trecut
21 Rezultate virus gripal tip B Tip/Subtip Caracterizare antigenica Caracterizare genetica H1N1 H3N2 62 tulpini de virus gripal tip B au apartinut liniei genetice B/Yamagata/16/88: B numai 15% (N = 9) au fost similar antigenic cu B/Massachusetts/02/2012 (clada 2, tulpinã vaccinalã); 85% (N = 53) din izolate au arãtat o bunã reactivitate cu antiserul de referinta fatã de B/Phuket/3073/2013. Toate cele 16 virusuri tip B secventiate s-au încadrat în clada 3, B/Phuket/3073/2013
22 Analiza filogenetică a genei HA a virusului gripal tip B comparativ cu tulpina vaccinala si tulpinile sezonul trecut
23 WHO: vaccines for use in the 2015/16 influenza season (northern hemisphere) : an A/California/7/2009 (H1N1)pdm09-like virus; an A/Switzerland/ /2013 (H3N2)-like virus; a B/Phuket/3073/2013-like virus. It is recommended that quadrivalent vaccines containing two influenza B viruses contain the above three viruses and a B/Brisbane/60/2008-like virus. WHO: vaccines for use in the 2016 influenza season (southern hemisphere winter): an A/California/7/2009 (H1N1)pdm09-like virus; an A/HongKong/5738/2014 (H3N2)-like virus; a B/Brisbane/60/2008-like virus. It is recommended that quadrivalent vaccines containing two influenza B viruses contain the above three viruses and a B/Phuket/3073/2013-like virus.
24 Concluzii - Co-circulație pe toată durata sezonului 3 virusuri gripale A(H3N2), A(H1N1)pdm09 și B - Virusurile A(H3N2) dar si B au suferit drift antigenic. - CNR trebuie sa fie capabil de caracterizarea antigenica si genetica a unui numar important de virusuri pentru a-si putea aduce contributia la dezvoltarea de vaccinuri sezoniere. - Supravegherea epidemiologică și virologică trebuie continuată și îmbunătățită stă la baza recomandarilor de vaccinare, profilaxie si tratament cu antivirale diversitatea genetica mare a virusurilor circulante face dificila stabilirea compozitiei vaccinului gripal.
25 Referinte Virology 376 (2008) Virology 413 (2011) ECDC. Surveillance Report. Influenza virus characterization. Summary Europe, June 2015 Eurosurveillance, Volume 19, Issue 36, 11 September 2014 J Clin Microbiol. 1977;6(1):19-22.
26 Multumiri WHO CC, The Francis Crick Institute, Mill Hill Laboratory, London WHO: IMOVE project Pacienti, MF, medici spitale, DSP, CNSCBT
27 Staff and main resposability Alexandrescu Viorel MD, Researcher I PhD, head of NIC, retired Lupulescu Emilia MD, Researcher III, head of lab. Cristina Tecu MD, Researcher II PhD - SARI Mihai Maria Elena, biochemist, Researcher III - NAI Alina Elena Ivanciuc, biologist, Researcher, PhD student IV detection Carmen Cherciu, biochemist, Researcher IV isolation Mihaela Lazãr, biologist, Researcher, PhD student - sequencing Necula Gheorghe Researcher, PhD sequencing, part-time Daniela Pitigoi MD, PhD, epidemiologist, research, part-time Two technicians and two persons qualified for laboratory work
28 Total samples IMOVE 272* % Total pos A&B type A A/H1 pdm A/H type B Neg * one coinfection H3&B In IMOVE study, the percentage of vaccinated subjects was 7.5: 4.1% of cases (N = 8) 14.3% of controls (N = 11) In ILI (N=687), the percentage of vaccinated subjects was 3.6 In general population Influenza vaccination uptake was 2.5% IVE against any influenza : 74.3 % (95%CI: 26.1; 91.4)
Report on virological surveillance of influenza activity in the Romania 2014/15 season
National Institute for Research Cantacuzino National Influenza Centre, Laboratory for Respiratory Viruses Spl.Independentei 103, sector 5, 050096 Bucuresti Romania Report on virological surveillance of
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