Combination of cell culture and quantitative PCR (cc-qPCR) for assessment of efficacy of drugs and disinfectants against Cryptosporidium parvum

Cryptosporidium parvum is an obligatory intracellular parasitic protist that belongs to the phylum Apicomplexa. Cryptosporidiosis is an infection for which no satisfactory efficient curative treatment is known, especially in immunocompromised individuals. Furthermore, the parasite oocysts show considerable tenacity in the environment. Therefore, new potent drugs along with a simple and reliable experimental model for evaluation of anticryptosporidial measures are urgently needed. The present studies were undertaken to establish a combined cell culture and quantitative PCR assay (cc-qPCR) to assess efficacy of pharmacological compounds against C. parvum. Human ileocecal adenocarcinoma cells (HCT-8) were selected for culture of C. parvum. Oocysts were excysted directly on confluent monolayers for infection. After 3 h of incubation the non invasive parasite remains were removed by washing. At the end of the incubation period the cells were harvested and subjected to DNA extraction. Real time PCR was performed to quantify the target parasite DNA (fragments of 70 kDa heat shock protein gene) copy numbers. Each reaction was run in triplicate. A standard curve calculated on the basis of serial dilutions of plasmid DNA or infected control culture DNA was run in each experiment. A series of oocyst suspensions were applied to cell cultures to determine the sensitivity of the cc-qPCR assay and also to generate a calibration curve to calculate the infectivity of oocysts. A dilution series of heat inactivated oocysts (70°C for 1 h) were used to determine the size of the oocyst inoculum at which complete elimination of extracellular parasite material by washing is reliably achieved. The results obtained by the assays were reproducible and the method sensitive with a detection limit of infection with 10 oocysts 48 h post infection (p.i.) and with 100 oocysts 24 h p.i. Percent effects of drugs and disinfectants were enumerated by comparing DNA copies between treated and non treated samples. The suitability of cc-qPCR for screening of pharmacological compounds was validated by confirming the in vitro efficacy of monensin (98.15% ± 1.09 at 0.144 µM) and halofuginone (98.05% ± 0.59 at 25 µM) over the entire incubation period with a dose dependent reduction of parasite multiplication demonstrated 27 h p.i. The inhibition of parasite proliferation by 0.144 µM monensin in the period from 3 h p.i (time defined to represent the initial level of parasite development before drug application) to 27 h p.i. or 45 h p.i. was 97 and 99% respectively, and by 25 µM halofuginone 99% (27 h p.i.). Hexadecylphosphocholine (miltefosine), a new anti-leishmanial compound, was tested against cryptosporidia and provided a maximum of 98% reduction of parasite multiplication at 45 h p.i. The potential activity of curcumin (extract from the herb Curcuma longa) against C. parvum was also evaluated by cc-qPCR. Curcumin appeared to be sensitive to degradation after prolonged incubation and the observed inhibition of multiplication of C. parvum was significantly increased when medium was replaced by fresh medicated medium after 12 h of exposure. The effects on parasite multiplication (>95% inhibition with IC50 value of 13 µM) and on sporozoite invasion (assessed 3 h p.i.; 65% inhibition at 200 µM) suggest that further exploration of anticryptosporidial efficacy of curcumin may be rewarding. The cc-qPCR was further optimized to analyse inactivation measures directed against oocysts of C. parvum. The suitability of the assay for assessment of inactivation measures was confirmed by the reproducible demonstration of effectiveness of cresolic disinfectants at the recommended concentration of 4% and incubation period of 2 h (Neopredisan® 135-1, Menno Chemie, Norderstedt, Germany: 99.91% ± 0.08; Aldecoc® TGE, EWABO Chemikalien GmbH & Co. KG, Wietmarschen, Germany: 99.91± 0.05) and by using thermally inactivated oocysts (complete inactivation by 56°C and 70°C for 20 min). Based on the in vitro results and previously obtained data from the chicken infection model 99.5% inactivation is proposed as a suitable threshold value that needs to be consistently exceeded by a product to be considered efficient. Application of Neopredisan® 135- 1 and Aldecoc® TGE (4% for 2h) consistently inactivated more than 99.5% of oocysts while other disinfectants that are not certified as anticoccidial products like Aldecoc® XD (EWABO Chemikalien GmbH & Co. KG, Wietmarschen, Germany) and IGAVET® FF spezial (COS OHLSEN Chemie & Gerätevertrieb GmbH, Geltorf-Esprehm, Germany) and bleach (sodium hypochlorite) did not. It can be concluded that the cc-qPCR method is suited to easily and reliably assess anticryptosporidials in vitro. The method demonstrated that miltefosine and curcumin display anticryptosporidial efficacy under the applied conditions. The cc-qPCR is a highly standardized method supposedly appropriate to replace the chicken infection model for Eimeria tenella as currently practised for certification of anticoccidial disinfectants according to the guidelines of DVG (German Veterinary Society)