{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T20:27:28Z","timestamp":1792787248369,"version":"4.5.0"},"reference-count":25,"publisher":"American Society for Microbiology","issue":"10","license":[{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T00:00:00Z","timestamp":1791244800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"Swedish Heart Lung Foundation","award":["20220148"],"award-info":[{"award-number":["20220148"]}],"id":[{"id":"https:\/\/ror.org\/052261q33","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Swedish Heart Lung Foundation","award":["20220238, 20251638"],"award-info":[{"award-number":["20220238, 20251638"]}],"id":[{"id":"https:\/\/ror.org\/052261q33","id-type":"ROR","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100004359","name":"Swedish Research Council","doi-asserted-by":"publisher","award":["2022-00865"],"award-info":[{"award-number":["2022-00865"]}],"id":[{"id":"10.13039\/501100004359","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010805","name":"Medical Research Council of Southeast Sweden","doi-asserted-by":"publisher","award":["968810, 101639"],"award-info":[{"award-number":["968810, 101639"]}],"id":[{"id":"10.13039\/100010805","id-type":"DOI","asserted-by":"publisher"}]},{"name":"ALF \u00d6sterg\u00f6tland","award":["ALF-987589"],"award-info":[{"award-number":["ALF-987589"]}]},{"name":"ALF \u00d6sterg\u00f6tland","award":["R\u00d6-1005642"],"award-info":[{"award-number":["R\u00d6-1005642"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,10,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Rapid quantification of\n                      <jats:italic toggle=\"yes\">Mycobacterium tuberculosis<\/jats:italic>\n                      (Mtb) is important for monitoring bacterial load during treatment and in clinical studies. The molecular bacterial load assay detecting Mtb 16S ribosomal RNA (TB-MBLA) can quantify mycobacterial load within hours. As the current TB-MBLA protocol includes highly toxic chemicals and manual RNA extraction, we aimed to optimize TB-MBLA for routine clinical laboratories. We compared several strategies to inactivate Mtb in sputum samples, which would facilitate analysis outside a BSL3 laboratory. Heating at 80\u00b0C for 20 min was the most efficient inactivation strategy, whereas GTC-DTT and 1 M NaOH failed to inactivate Mtb sufficiently. We used Mtb-inoculated sputum samples to compare manual phenol-chloroform RNA extraction, as recommended by the TB-MBLA protocol, to an automated magnetic bead-based extraction. Automated RNA extraction reduced total analysis time by 2 h and performed equally to phenol-chloroform extraction, with a detection limit of close to 100 CFU in 10-fold dilution series (mean Ct value: 36.0 \u00b1 1.4 automated versus 33.5 \u00b1 3.6 manual,\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 6). Freezing samples in lysis buffer up to 14 days did not affect TB-MBLA performance (mean Ct value: 22.6 \u00b1 0.3 fresh versus 22.6 \u00b1 0.6 frozen,\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 6). The final TB-MBLA protocol quantified bacterial load up to 11 days faster than time-to-positive culture (TTP) (mean TTP: 11 days, 2\u201324 days,\n                      <jats:italic toggle=\"yes\">n<\/jats:italic>\n                      = 9) in sputum from patients with pulmonary TB. We adapted the TB-MBLA method for faster throughput in a routine clinical laboratory by using inactivation at 80\u00b0C for 20 min, followed by automated RNA extraction, achieving equal sensitivity compared to the original TB-MBLA method, and avoiding manual extraction and toxic reagents.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>The current tuberculosis molecular bacterial load assay (TB-MBLA) protocol relies on toxic chemicals and manual RNA extraction, making the procedure both time\u2011consuming and labor\u2011intensive. We optimized and validated a faster TB-MBLA strategy, which can be adapted for routine clinical laboratories, including inactivation by heating, to facilitate implementation in settings without a BSL3 laboratory.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.01018-26","type":"journal-article","created":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T13:00:17Z","timestamp":1787058017000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Adaptation of the molecular bacterial load assay for real-time quantification of\n                    <i>Mycobacterium tuberculosis<\/i>\n                    in the clinical routine laboratory"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9562-0872","authenticated-orcid":true,"given":"B.","family":"Andersson","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05ynxx418","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biomedical and Clinical Sciences, Division of Inflammation and Infection, Link\u00f6ping University","place":["Link\u00f6ping, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"V.","family":"Kholod","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05h1aye87","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Clinical Microbiology, Link\u00f6ping University Hospital","place":["Link\u00f6ping, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.","family":"Jonsson Nordvall","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05ynxx418","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biomedical and Clinical Sciences, Division of Inflammation and Infection, Link\u00f6ping University","place":["Link\u00f6ping, Sweden"]},{"id":[{"id":"https:\/\/ror.org\/05h1aye87","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Clinical Microbiology, Link\u00f6ping University Hospital","place":["Link\u00f6ping, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1784-2864","authenticated-orcid":true,"given":"C.","family":"Braian","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05ynxx418","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biomedical and Clinical Sciences, Division of Inflammation and Infection, Link\u00f6ping University","place":["Link\u00f6ping, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R.","family":"Kumar","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05ynxx418","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biomedical and Clinical Sciences, Division of Inflammation and Infection, Link\u00f6ping University","place":["Link\u00f6ping, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"K.","family":"Niward","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05ynxx418","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biomedical and Clinical Sciences, Division of Inflammation and Infection, Link\u00f6ping University","place":["Link\u00f6ping, Sweden"]},{"id":[{"id":"https:\/\/ror.org\/05ynxx418","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Infectious Diseases, Link\u00f6ping University Hospital, Link\u00f6ping University","place":["Link\u00f6ping, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"T.","family":"Sch\u00f6n","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05ynxx418","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biomedical and Clinical Sciences, Division of Inflammation and Infection, Link\u00f6ping University","place":["Link\u00f6ping, Sweden"]},{"id":[{"id":"https:\/\/ror.org\/05ynxx418","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Infectious Diseases, Link\u00f6ping University Hospital, Link\u00f6ping University","place":["Link\u00f6ping, Sweden"]},{"id":[{"id":"https:\/\/ror.org\/05ynxx418","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Infectious Diseases, Kalmar County Hospital, Link\u00f6ping University","place":["Kalmar, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","volume-title":"Global tuberculosis report 2025","author":"WHO","year":"2025","unstructured":"WHO. 2025. 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