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The current treatments have many serious drawbacks and new drugs are urgently required. In the UK, <jats:italic>T<\/jats:italic>. <jats:italic>cruzi<\/jats:italic> is classified by the Advisory Committee on Dangerous Pathogens (ACDP) as a Hazard Group 3 organism and strict safety practices must be adhered to when handling this pathogen in the laboratory. Validated inactivation techniques are required for safe <jats:italic>T<\/jats:italic>. <jats:italic>cruzi<\/jats:italic> waste disposal and removal from Containment Level 3 (CL3) facilities for storage, transportation and experimental analysis. Here we assess three <jats:italic>T<\/jats:italic>. <jats:italic>cruzi<\/jats:italic>. inactivation methods. These include three freeze-thaw cycles, chemical inactivation with Virkon disinfectant, and air drying on Whatman FTA cards (A, B, C, Elute) and on a Mitra microsampling device. After each treatment parasite growth was monitored for 4\u20136 weeks by microscopic examination. Three freeze-thaw cycles were sufficient to inactivate all <jats:italic>T<\/jats:italic>. <jats:italic>cruzi<\/jats:italic> CLBrener Luc life cycle stages and Silvio x10\/7 A1 large epimastigote cell pellets up to two grams wet weight. Virkon treatment for one hour inactivated <jats:italic>T<\/jats:italic>. <jats:italic>cruzi<\/jats:italic> Silvio x10\/7 subclone A1 and CLBrener Luc both in whole blood and cell culture medium when incubated at a final concentration of 2.5% Virkon, or at \u22651% Virkon when in tenfold excess of sample volume. Air drying also inactivated <jats:italic>T<\/jats:italic>. <jats:italic>cruzi<\/jats:italic> CLBrener Luc spiked blood when dried on FTA A, B or Elute cards for \u226530 minutes and on a Mitra Microsampler for two hours. However, <jats:italic>T<\/jats:italic>. <jats:italic>cruzi<\/jats:italic> CLBrener Luc were not inactivated on FTA C cards when dried for up to two hours. These experimentally confirmed conditions provide three validated <jats:italic>T<\/jats:italic>. <jats:italic>cruzi<\/jats:italic> inactivation methods which can be applied to other related ACDP Hazard Group 2\u20133 kinetoplastid parasites.<\/jats:p>","DOI":"10.1371\/journal.pone.0300021","type":"journal-article","created":{"date-parts":[[2024,4,18]],"date-time":"2024-04-18T18:17:57Z","timestamp":1713464277000},"page":"e0300021","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Validation of Trypanosoma cruzi inactivation techniques for laboratory use"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5691-5117","authenticated-orcid":true,"given":"Lorna M.","family":"MacLean","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-3521-6616","authenticated-orcid":true,"given":"Mark","family":"Ariyanayagam","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lalitha","family":"Sastry","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-4264-4860","authenticated-orcid":true,"given":"Christy","family":"Paterson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3171-3519","authenticated-orcid":true,"given":"Manu","family":"De Rycker","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5134-0329","authenticated-orcid":true,"given":"Alan H.","family":"Fairlamb","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2024,4,18]]},"reference":[{"key":"pone.0300021.ref001","unstructured":"WHO. 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