Reduced susceptibility to praziquantel among naturally occurring Kenyan isolates of Schistosoma mansoni

Abstract

Background: The near exclusive use of praziquantel (PZQ) for treatment of human schistosomiasis has raised concerns about the possible emergence of drug-resistant schistosomes.

Methodology/principal findings: We measured susceptibility to PZQ of isolates of Schistosoma mansoni obtained from patients from Kisumu, Kenya continuously exposed to infection as a consequence of their occupations as car washers or sand harvesters. We used a) an in vitro assay with miracidia, b) an in vivo assay targeting adult worms in mice and c) an in vitro assay targeting adult schistosomes perfused from mice. In the miracidia assay, in which miracidia from human patients were exposed to PZQ in vitro, reduced susceptibility was associated with previous treatment of the patient with PZQ. One isolate ("KCW") that was less susceptible to PZQ and had been derived from a patient who had never fully cured despite multiple treatments was studied further. In an in vivo assay of adult worms, the KCW isolate was significantly less susceptible to PZQ than two other isolates from natural infections in Kenya and two lab-reared strains of S. mansoni. The in vitro adult assay, based on measuring length changes of adults following exposure to and recovery from PZQ, confirmed that the KCW isolate was less susceptible to PZQ than the other isolates tested. A sub-isolate of KCW maintained separately and tested after three years was susceptible to PZQ, indicative that the trait of reduced sensitivity could be lost if selection was not maintained.

Conclusions/significance: Isolates of S. mansoni from some patients in Kisumu have lower susceptibility to PZQ, including one from a patient who was never fully cured after repeated rounds of treatment administered over several years. As use of PZQ continues, continued selection for worms with diminished susceptibility is possible, and the probability of emergence of resistance will increase as large reservoirs of untreated worms diminish. The potential for rapid emergence of resistance should be an important consideration of treatment programs.

Figure 1. In vitro efficacy of 10⁻⁵ M PZQ in killing S. mansoni miracidia after 20 minutes of exposure in 96 well plates.

Miracidia were hatched from fecal samples collected from human patients from the Kisumu area in Kenya, and the assay run in triplicate. The percentage of miracidia dead at the end of the 20 min observation period is graphed. Error bars correspond to standard error of the mean. Numbers above bars correspond to number of PZQ treatments. Data for parasites obtained from sand harvesters are indicated by squares and circles for car washers.

Figure 2. Average number of S. mansoni adult worms perfused from mice, following treatment with 1000 mg/kg of PZQ in Cremaphor 2% EL.

Treatment was administered at 7.5 weeks p.i., and mice perfused 2 weeks after treatment. Efficacy of treatment was calculated as described in text. The p values for the comparison between mean # of worms recovered from control versus treated groups were as follows: KCW, p = 0.072; the sample size for KAS was too small to assess statistical significance; KNY, p<0.0001; PR1, p<0.0001; NMRI, p<0.0001. Error bars correspond to standard error of the mean.

Figure 3. Photographs of S. mansoni adult worms of KNY (plate A) and KCW isolates (plate B), before exposure to PZQ (left column), after 3 hrs of continuous exposure to PZQ (central column), and 24 hrs after removal from PZQ.

Concentration of PZQ is shown on the left side of the figure. The photographs show the effect of the drug on the length of the worms and the degree of recovery of the worm's length after removal from the sub-lethal concentrations of the drug. Note the difference in the response of the two isolates to the 8 µg/ml dose. The results for the PR1 strain which was also tested were similar to those shown for the KNY isolate.

Figure 4. In vivo susceptibility to PZQ of adult worms.

KCW (2008) refers to a subset of the KCW isolate kept in Kenya since 2005 that was tested in 2008, after 3 years of passage in laboratory mice and snails in Kenya. KCW (2006) refers to a subset of the KCW isolate brought to New Mexico in 2005 right after isolation from the patient, and that was tested in this assay in 2006. Also shown are results obtained with the KSH isolate and the PR1 lab strain. ^**Difference between average number of worms recovered from perfusion from control animals versus treated animal significant (α = 0.05). ET = Efficacy of treatment, was calculated as described in text.