U N I V E R S I T Y O F B E R G E N Nye diagnostiske metoder for TB Tehmina Mustafa Professor, Senter for internasjonal helse, UiB Overlege, Lungeavdelingen, HUS
Agenda Diagnostiske metoder og ny utvikling Ny diagnostiske metode- EPTB basert på påvisning av mykobakteriell antigen
Laboratorieundersøkelser Direkt: Mykobakteria eller antigener Mikroskopi Bakteriologisk dyrkning NAAT (PCR) Antigen påvisning Indirekt: Basert på immunrespons TST/Mantoux Interferon-gamma release assays (IGRAs) Histology/cytology Hematologiske og biokjemiske prøver Serologi- ikke anbefalt
Mikroscopi- AFB Sentralt i global TB kontroll Billig - 0.50 USD Rask Behandlingsresponse Fanger opp smitteførende 1 smitteførende 15-20 nye smitte 1 ny smitteførende Begrensninger: Lav sensitivitet - krever 10,000 bacilli per ml Lav spesifisitet: Atypiske mykobakteria er AFB Utvikling: Automatisering Analysert-datamakin
Dyrkning Gull standard Fast eller flytende medium Flytende-MGIT-Raskere -WHO anbefaler SN 80-98% (10-100 bakterier/ml), SP 90-100% Fenotypiske resistens, behandlingsresponsen Begrensening: Tar tid: 4-8 uker Rask identifisering - culture isolate Strip speciation test TB-specific antigen (MPT 64)-15 min
Nucleic-Acid Amplification Tests (NAAT)- PCR basert metoder Molekylær diagnose i TB- veien fremover Høy SN 77-100%, Høy SP 83-100% Nyttig- mikroskopi-negativ; Barn, HIV-infiserte, EPTB Rask Mulighet for samtidig påvisning av genotypiske resistens -spesifikke mutasjoner (INH, Rif) In-house eller kommersiell 3 metoder Kassett-basert (Xpert MTB/RIF) Line probe assay (LPA) Loop-mediated amplification (LAMP)
Xpert MTB/RIF assay Stor fremskritt Kassett-basert lukket sytem- ikke kontaminering Rask (2 t) Samtidig påvisning av MTB & RIF resistens (MDR-TB) Mikroskopi-negativ ekspektorat Sensitivitet: 57% -83% WHO anbefaler
Center for International Health, Department of Global Public HEalth & Primary Care De nyeste versjonene av Xpert «Xpert-Ultra»-kassetter- høyere SN «Expert Omni»- bærbar- point-of-care «Xpert Xpand»- XDR-TB- mutasjoner resistanse mot isoniazid, fluorokinoloner og 2.linje sprøytemidler Flere i «pipe-line»
Center for International Health, Department of Global Public HEalth & Primary Care Utfordringer-implementering i høy TB-endemiske land
NAAT- Line probe assay (LPA) Linje probe assays: samtidig påvisning av M.TB & genotypiske resistens -spesifikke mutasjoner (INH, Rif) Anvendes -mikroskopi-positive ekspektorat 5 timer Begrensning: Ikke egnet for mikroskopi-negativ prøver
Loop-mediated isothermal amplification (LAMP) simple visual colorimetric read-out No repeated heating and cooling cycles low power requirement WHO- not yet recommended
TB i Norge-organfordeling Kilde: FHI
Ekstrapulmonal tuberkulose Diagnostiske utfordringer Paucibacillary Mikrobiologiske metoder- lav sensitivitet direkte mikroskopi (0-10%) dyrkning(0-22%) NAAT baserte metoder: Bedre men varierende sensitivitet, Kontaminering, kostbar Histologi: ikke spesifikk Immunesvekkede- atypisk histologi
Detection of MPT64-antigen from the biopsies, aspirates, cell smears by immunohistochemistry Robust, rapid, sensitivity/specificity comparable to sensitive PCR-based tests
Center for International Health, Department of Global Public HEalth & Primary Care MPT64 in HIV-TB coinfected pleural TB lesions
MPT64 antigen in HIV-TB coinfected pleural TB lesions- atypical histology H&E stain Acid-fast stain IHC-MPT64 IHC- MPT64
Immunocytochemical staining of mycobacterial antigen MPT64 Lymfeknute Aspirat Pleuravæske
MPT64 antigen detection test- previous studies Country Site of disease Reference standard India South Africa HIV-coinfected Lymph node, Pleural fluids, Abdominal TB (peritoneum, intestinal wall, lymph nodes, ascitic fluid), CNS (CSF) Pleural biopsies ZN sensitivity < 10%, Culture sensitivity < 28% SN SP N-PCR 88-93% 81-98 Response treatment 80% 100% Tanzania LN biopsies N-PCR 90% 83% Norway LN biopsies N-PCR 95% 62% Ethiopia Pleural fluid aspirates & Lymph node aspirates PCR 88 % 90% 5 Publications: Diagnostic Pathology 2007, 2:36, Appl Immunohistochem Mol Morphol.2008.16:554, Diagn Cytopathol. 2012.40:782, BMC Infectious Diseases 2014, 14:585, Appl Immunohistochem Mol Morphol 2017;25:282 288
U N I V E R S I T Y O F B E R G E N Center for International Health, Department of Global Public Health & Primary Care Project Title: Improving diagnosis of extrapulmonary tuberculosis by implementation of a sensitive and specific antigen detection test in routine diagnostic settings Support: Research Council of Norway through the Global Health and Vaccination Programme (GLOBVAC), project number 234457. Study is part of the EDCTP2 programme supported by the European Union 2015-2018 Helse Vests forskningsmidler 2014-2017
Primary objective: To improve the diagnosis of extrapulmonary TB by introducing a sensitive and specific test in the selected tertiary care hospitals in high TB and high-low HIV burden countries using a routine TB control programme.
Center for International Health, Department of Global Public HEalth & Primary Care Partners: Pakistan, India, Tanzania, Zanzibar, Norway Zanzibar
Study design Center for International Health, Department of Global Public HEalth & Primary Care Biobank
Center for International Health, Department of Global Public HEalth & Primary Care Implementaion of test in zanzibar Melissa Davidsen Jørstad-PhDcandidate Mariam Abdallah doctor at MMH
Performance of test in zanzibar Center for International Health, Department of Global Public HEalth & Primary Care Number specimens Number of specimens (%) positive by ZN LJ culture GenXpert MPT64 test TB, all specimens 69 12 13 16 65 FNAC TB-LN 34 18 17 27 76 Non-TB, all specimens 76 0 0 0 4 FNAC non-tb LN 32 0 0 0 0 All case: Sensitivity (SN) 69%, Specificity (SP) 95% Lymphadenitis: SN 79%, SP 97% Pediatric Lymphadenitis: SN 100%, SP 96%
Acknowledgement Center for International Health, Department of Global Public HEalth & Primary Care Professor Lisbet Sviland, Department of Pathology, Haukeland University Hospital Professor Harald Wiker, Department of Clinical Science, UiB Professor Anne Ma Dyrhol-Riise, Department of Infectious Diseases, UiO/ OUS Melissa D Jørstad, UiB, PhD scholar Masafiri Marijani, Pathologist, Mnazi Mmoja Hospital, Zanzibar