Demonstration of the efficacy of disinfectants against animal and human pathogens has become a requisite part of the documentation associated with licensed handling. In order to obtain a licence for working with trypanosomatid parasites, authorities request verification that the disinfectants and autoclaving conditions indicated in many standard operating procedures to inactivate the pathogens are indeed capable of efficient killing of the organisms. As such data has not been readily available either from manufacturers or as publication, in recent years each laboratory has been required to carry out inactivation experiments independently before further work can be undertaken. The purpose of this report is to confirm that disinfectants commonly used in laboratories and heat treatment result in killing of trypanosomatid parasites.
We tested the commercial disinfectant TriGene (MediChem International Ltd., U.K.), and bleach (sodium hypochlorite (NaOCl) solution; Fisher Scientific, U.K.) and ethanol as general laboratory disinfectants for their ability to kill bloodstream forms of Trypanosoma brucei (clone 427-221a [1]), epimastigotes of Trypanosoma rangeli (Choachi strain [2]) and promastigotes of Leishmania major (Friedlin strain [3]). In addition, we also investigated the effect of dilution in water, liquid hand soap (RBS HDS 10; Medline Scientific LTD., U.K.) and heat treatment on the parasites. The parasites were incubated at a cell density of 1 × 106/ml with various concentrations of the reagents in appropriate medium (T. brucei, Baltz medium plus 20% heat-inactivated foetal calf serum (iFCS) [4]; T. rangeli, Liver Infusion Tryptose medium plus 10% iFCS [2]; L. major, medium 199 plus 10% iFCS [5]) in a final volume of 1 ml at room temperature. The controls contained the corresponding amount of water (except for experiments testing the effect of dilution in water where the controls contained only medium). After 5 min incubation, live cells were counted using a Neubauer haemocytometer. The 50% lethal concentration (LC50), i.e. the reagent concentration necessary to kill 50% of the cells compared to the control, was determined by linear interpolation [6]. The 100% lethal concentration (LC100), i.e. the lowest concentration of a reagent at which all cells were killed, was determined microscopically. For heat treatment, parasites at a cell density of 1 × 106/ml in 1 ml appropriate medium were incubated at different temperature using a digital heater block (Grant Instruments, U.K.). Samples incubated at room temperature served as controls. After 5 min incubation, live cells were counted using a Neubauer haemocytometer. The 50% lethal temperature (LT50), i.e. the temperature necessary to kill 50% of the cells compared to the control, was determined by linear interpolation [6]. The 100% lethal temperature (LT100), i.e. the lowest temperature at which all cells were killed, was determined microscopically.
For TriGene, bleach and liquid hand soap, the same LC100 value was observed for all three parasites (Table 1). Based on LC50 values, T. brucei appears to be approximately 4-fold more resistant towards TriGene while T. rangeli are about 2-fold more sensitive towards liquid hand soap, compared with the other two parasites, respectively. Regarding ethanol and dilution in water, T. rangeli seems to be somewhat more resistant to these reagents than T. brucei and L. major (Table 1). The LC100 values for ethanol, TriGene and bleach are 4, 10 and 20 times higher than the recommended working concentrations of these disinfectants which are 70%, 2% and 1%, respectively. This shows that trypanosomatids are very sensitive to commonly used laboratory disinfectants. In the case of TriGene it has been shown that bloodstream forms of T. brucei are killed at a concentration of 0.1% within 20 s [7]. The finding that liquid hand soap efficiently destroys all three parasites suggests that soap solutions can be used as first aid measure to clean skin areas accidentally contaminated with the pathogens. The dilution experiment with water indicates that trypanosomatids cannot cope very well with hypoosmotic stress even though these parasites are capable of some kind of osmoregulation [8].
All three trypanosomatid parasites are equally sensitive to heat treatment (Table 2). No difference was observed for the LT100 and the LT50 varied only by 3°C. The finding that the parasites are already killed at 50°C indicates that trypanosomatids are very temperature sensitive and thus would certainly not survive normal autoclaving condition of 121°C and 1.4 bar for 15 min. Actually, it has been shown that cultures of bloodstream forms of T. brucei post autoclaving contained only cell debris and immotile, rounded-up cells [9].
In this study we have shown that bloodstream forms of T. brucei, epimastigotes of T. rangeli and promastigotes of L. major are quite fragile organisms which can be easily killed with disinfectants commonly used in laboratories and by heat treatment. All three parasite species exhibited very similar sensitivities for the reagents tested and temperature. As these three parasites are representatives of the Salivaria group (T. brucei brucei, T. brucei rhodesiense, T. brucei gambiense, T. congolense and T. vivax), the Stercoraria group (sibling species T. cruzi and T. rangeli) and the Leishmania genus (L. major, L. donovani, L. infantum, L. mexicana, L. braziliensis, L amazonensis etc.), our findings likely indicate that all other pathogenic trypanosomatids display similar susceptibilities for these disinfectant and temperature treatments. In conclusion, common laboratory disinfectant (at the indicated concentrations) and temperature treatment can be used for effective inactivation of waste liquid and general laboratory ware that has been contaminated with trypanosomatid parasites.
