Trypanosoma cruzi survival following cold storage: possible implications for tissue banking

Abstract

While Trypanosoma cruzi, the etiologic agent of Chagas disease, is typically vector-borne, infection can also occur through solid organ transplantation or transfusion of contaminated blood products. The ability of infected human cells, tissues, and cellular and tissue-based products (HCT/Ps) to transmit T. cruzi is dependent upon T. cruzi surviving the processing and storage conditions to which HCT/Ps are subjected. In the studies reported here, T. cruzi trypomastigotes remained infective 24 hours after being spiked into blood and stored at room temperature (N = 20); in 2 of 13 parasite-infected cultures stored 28 days at 4°C; and in samples stored 365 days at -80°C without cryoprotectant (N = 28), despite decreased viability compared to cryopreserved parasites. Detection of viable parasites after multiple freeze/thaws depended upon the duration of frozen storage. The ability of T. cruzi to survive long periods of storage at +4 and -80°C suggests that T. cruzi-infected tissues stored under these conditions are potentially infectious.

Figure 1. Vero cell culture infected with Brazil strain T. cruzi showing asynchronous amastigote nests inside cell.

(1) uninfected cell; (2) very early infection, (3) more advanced and (4) a cell in which amastigotes have completely transformed to trypomastigotes. Note that other infected cells are present in this view as well. Photo was taken at 400× magnification.

Figure 2. Infectivity of parasites stored 24 hours at RT in decaying blood product.

One ml of heparinized (n = 5) or non-heparinized (n = 7) blood was spiked with 2×10⁶ Y or Tc23 T. cruzi trypomastigotes. Blood was cultured over FS9 or HMEC-1 cells and examined for amastigote nests after 4–7 days. Pictured is a representative amastigote nest of Y strain parasites in FS9 cells. Photos were taken at 400× magnification.

Figure 3. T. cruzi-infected culture cells were stored for various lengths of time at 4°C.

A. Vero cells infected with Brazil T. cruzi after 24 h at 4°C, then re-cultured at 37°C. Figure is representative of N = 7. B. HMEC-1 cells infected with Tc23 T. cruzi were stored 5 d at 4°C, then the supernatants were re-cultured over fresh HMEC-1 cells. Figure is representative of N = 10 stored cultures and positive re-cultures.

Figure 4. Viable parasites recovered after one year storage at −80°C without cryoprotection.

A. T. cruzi (Tc23 stain)-infected HMEC-1 cells were stored in RPMI-10 at −80°C for greater than one year, then re-cultured in HMEC-1 cells. Amastigote nests were photographed at 400× magnification. Data are representative of N = 19 cultures. B. T. cruzi trypomastigotes (Tc23 strain) were stored with cryoprotectant (freezing media, FM) or FBS at −80°C for the indicated time period. Samples were thawed, washed in RPMI-10, and counted on a hemocytometer. Data represent at least N = 4 per group. C. T. cruzi trypomastigotes (Tc23 strain) were stored in RPMI-10 at -80°C for greater than one year, and then re-cultured in HMEC-1 cells. Infected cells were photographed at 400×. Data are representative of N = 5 cultures. D. T. cruzi trypomastigotes (Tc23 strain) were stored in FM, FBS, or RPMI-10 at −80°C for greater than one year. Samples were thawed, washed in RPMI-10, and counted on a hemocytometer. Data represent N = 4 per group.