{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T03:20:45Z","timestamp":1789960845013,"version":"4.0.1"},"reference-count":29,"publisher":"American Society for Microbiology","issue":"1","license":[{"start":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T00:00:00Z","timestamp":1767657600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T00:00:00Z","timestamp":1767657600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["1DP1HD115427"],"award-info":[{"award-number":["1DP1HD115427"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["1K01TW011482"],"award-info":[{"award-number":["1K01TW011482"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01AI137527"],"award-info":[{"award-number":["R01AI137527"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100005627","name":"Thrasher Research Fund","doi-asserted-by":"publisher","award":["01098"],"award-info":[{"award-number":["01098"]}],"id":[{"id":"10.13039\/100005627","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100005627","name":"Thrasher Research Fund","doi-asserted-by":"publisher","award":["14323"],"award-info":[{"award-number":["14323"]}],"id":[{"id":"10.13039\/100005627","id-type":"DOI","asserted-by":"publisher"}]},{"name":"NIAID CEIRR CRIPT Data Management","award":["75N93021C00014"],"award-info":[{"award-number":["75N93021C00014"]}]},{"name":"St. Judes\/Pediatric Infectious Disease Society"},{"name":"NIGMS IDeA Alaska INBRE","award":["P20GM103395"],"award-info":[{"award-number":["P20GM103395"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,1,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Alaska has the highest incidence of tuberculosis (TB) in the United States, with 8% mortality while undergoing TB treatment. With a quarter of TB cases lacking sputum culture to enable drug resistance testing, FreezeTB aimed to develop tools tailored to meet the challenges in Alaska while being translatable to other settings. We designed a rapid and cost-effective laboratory workflow and software to identify drug-resistant mutations in\n                      <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                      using targeted next-generation sequencing (tNGS). FreezeTB, a Fast, Reliable, Economical Evaluation tool to Zap Endemic Tuberculosis, amplifies 22 gene loci associated with resistance to 16 anti-tuberculosis drugs.\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      isolates from Alaska (2011\u20132024) were blinded and underwent analysis with FreezeTB, then compared with phenotypic drug susceptibility testing (pDST) and whole genome sequencing (WGS). Compared with WGS (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 79), FreezeTB provided the same mutations in 96% (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 76\/79) of samples with 100% lineage agreement (\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 79\/79). Compared with pDST using Cohen\u2019s kappa, FreezeTB had almost perfect agreement for rifampin (RIF, 0.90;\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 97\/98) and ethambutol (EMB, 1.00;\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 98\/98), strong agreement for isoniazid (INH, 0.80;\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 88\/98), moderate agreement for ethionamide (ETO, 0.78;\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 95\/98), weak agreement for streptomycin (STR, 0.56;\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 95\/98), and no agreement for pyrazinamide (PZA, 0.20;\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 91\/98). Using a portable nanopore sequencer, each sample cost under $30 for sequencing, which included a flow cell, a barcoding kit, a flow cell wash kit, a polymerase, and primers. FreezeTB provides a portable, 1-day laboratory and bioinformatic workflow for sequencing\n                      <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                      . Freeze TB can accurately determine mycobacterial lineage and resistance for RIF, INH, ETH, and ETO at a low cost.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Globally, tuberculosis is the leading infectious cause of mortality with 10.8 million new cases and 1.25 million deaths occurring in 2024. Targeted next-generation sequencing (tNGS) is a rapid, cost-effective method for identifying mutations in the\n                        <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                        genome associated with drug resistance. FreezeTB was created to provide low-cost, portable sequencing and tNGS analysis for drug resistance. FreezeTB selectively amplifies and sequences 24 targets of the\n                        <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                        genome that cover regions the WHO has designated as containing mutations conferring drug resistance, as well as provides species confirmation and rapid lineage determination. Freeze TB is a laboratory workflow and free, open-source (OS) low dependency bioinformatic software that can be downloaded onto a computer with no further need for internet access. Using\n                        <jats:italic toggle=\"yes\">M. tuberculosis<\/jats:italic>\n                        isolates from Alaska, FreezeTB provided the same mutations in 96% (\n                        <jats:italic toggle=\"yes\">n<\/jats:italic>\n                        = 76\/79) of samples with 100% lineage agreement (\n                        <jats:italic toggle=\"yes\">n<\/jats:italic>\n                        = 79\/79) compared with whole genome sequencing.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.02335-25","type":"journal-article","created":{"date-parts":[[2025,11,17]],"date-time":"2025-11-17T13:00:16Z","timestamp":1763384416000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Application of FreezeTB, a targeted nanopore sequencing assay, for identification of drug resistance and lineages among pulmonary tuberculosis cases in Alaska"],"prefix":"10.1128","volume":"14","author":[{"given":"Bryce","family":"Inman","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03k3c2t50","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Alaska","place":["Anchorage, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeremy","family":"Butler","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03k3c2t50","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Alaska","place":["Anchorage, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Soren","family":"George-Nichol","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03k3c2t50","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Alaska","place":["Anchorage, USA"]},{"id":[{"id":"https:\/\/ror.org\/03k3c2t50","id-type":"ROR","asserted-by":"publisher"}],"name":"College of Fisheries and Ocean Sciences, University of Alaska","place":["Fairbanks, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ganna","family":"Kovalenko","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03k3c2t50","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Alaska","place":["Anchorage, USA"]},{"id":[{"id":"https:\/\/ror.org\/04gyf1771","id-type":"ROR","asserted-by":"publisher"}],"name":"Wen School of Public Health, University of California","place":["Irvine, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Theresa","family":"Savidge","sequence":"additional","affiliation":[{"name":"Alaska TB Program, Division of Public Health, Alaska Department of Health","place":["Anchorage, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yvette","family":"Vergnetti","sequence":"additional","affiliation":[{"name":"Alaska TB Program, Division of Public Health, Alaska Department of Health","place":["Anchorage, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Catherine","family":"Pongratz","sequence":"additional","affiliation":[{"name":"Alaska TB Program, Division of Public Health, Alaska Department of Health","place":["Anchorage, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth","family":"Bee","sequence":"additional","affiliation":[{"name":"Alaska TB Program, Division of Public Health, Alaska Department of Health","place":["Anchorage, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew R.","family":"DiNardo","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02pttbw34","id-type":"ROR","asserted-by":"publisher"}],"name":"Global Tuberculosis Program, Baylor College of Medicine","place":["Houston, USA"]},{"name":"Radboud University","place":["Nijmegen, the Netherlands"]},{"name":"Baylor Children's Foundation","place":["Mbabane, Swaziland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Kay","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02pttbw34","id-type":"ROR","asserted-by":"publisher"}],"name":"Global Tuberculosis Program, Baylor College of Medicine","place":["Houston, USA"]},{"name":"Baylor Children's Foundation","place":["Mbabane, Swaziland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Mandalakas","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02pttbw34","id-type":"ROR","asserted-by":"publisher"}],"name":"Global Tuberculosis Program, Baylor College of Medicine","place":["Houston, USA"]},{"name":"Baylor Children's Foundation","place":["Mbabane, Swaziland"]},{"id":[{"id":"https:\/\/ror.org\/036ragn25","id-type":"ROR","asserted-by":"publisher"}],"name":"Division of Clinical Infectious Diseases, Research Center Borstel, Leibniz Lung Center","place":["Borstel, Germany"]},{"id":[{"id":"https:\/\/ror.org\/028s4q594","id-type":"ROR","asserted-by":"publisher"}],"name":"German Center for Infection Research (DZIF), Partner Site Hamburg-L\u00fcbeck-Borstel-Riems","place":["Borstel, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1376-0012","authenticated-orcid":false,"given":"Eric","family":"Bortz","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03k3c2t50","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biological Sciences, University of Alaska","place":["Anchorage, USA"]},{"name":"Department of Microbiology, University of Washington School of Medicine","place":["Seattle, 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