Validation of Trypanosoma cruzi inactivation techniques for laboratory use

Lorna M. MacLean , Mark Ariyanayagam, Lalitha Sastry, Christy Paterson, Manu De Rycker, Alan H. Fairlamb

PLOS ONE 19(4): e0300021.


Trypanosoma cruzi (Tcruzi) is the causative agent of Chagas’ disease, a parasitic infection responsible for significant morbidity and mortality in Latin America. The current treatments have many serious drawbacks and new drugs are urgently required. In the UK, Tcruzi 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 Tcruzi waste disposal and removal from Containment Level 3 (CL3) facilities for storage, transportation and experimental analysis. Here we assess three Tcruzi. 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 4–6 weeks by microscopic examination. Three freeze-thaw cycles were sufficient to inactivate all Tcruzi 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 Tcruzi 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 ≥1% Virkon when in tenfold excess of sample volume. Air drying also inactivated Tcruzi CLBrener Luc spiked blood when dried on FTA A, B or Elute cards for ≥30 minutes and on a Mitra Microsampler for two hours. However, Tcruzi CLBrener Luc were not inactivated on FTA C cards when dried for up to two hours. These experimentally confirmed conditions provide three validated Tcruzi inactivation methods which can be applied to other related ACDP Hazard Group 2–3 kinetoplastid parasites.