Promastigote growth curves of strains from the non-treated (NT) and treated relapsed (TR) groups.

<p>No significant differences were found between promastigote counts for any of the isolates on respective days (Repeated measures ANOVA with Greenhouse-Geisser tests, <i>P =</i> 0.784).</p

Allopurinol Resistance in <i>Leishmania infantum</i> from Dogs with Disease Relapse

<div><p>Background</p><p>Visceral leishmaniasis caused by the protozoan <i>Leishmania infantum</i> is a zoonotic, life threatening parasitic disease. Domestic dogs are the main peridomestic reservoir, and allopurinol is the most frequently used drug for the control of infection, alone or in combination with other drugs. Resistance of <i>Leishmania</i> strains from dogs to allopurinol has not been described before in clinical studies.</p><p>Methodology/Principal Findings</p><p>Following our observation of clinical disease relapse in dogs under allopurinol treatment, we tested susceptibility to allopurinol of <i>L</i>. <i>infantum</i> isolated from groups of dogs pre-treatment, treated in remission, and with disease relapse during treatment. Promastigote isolates obtained from four treated relapsed dogs (TR group) showed an average half maximal inhibitory concentration (IC50) of 996 μg/mL. A significantly lower IC50 (<i>P</i> = 0.01) was found for isolates from ten dogs before treatment (NT group, 200 μg/mL), as well as for five isolates obtained from treated dogs in remission (TA group, 268 μg/mL). Axenic amastigotes produced from isolates of the TR group also showed significantly higher (<i>P</i> = 0.002) IC50 compared to the NT group (1678 and 671 μg/mL, respectively). The lower sensitivity of intracellular amastigotes from the TR group relative to those from the NT group (<i>P</i> = 0.002) was confirmed using an infected macrophage model (6.3% and 20% growth inhibition, respectively at 300 μg/mL allopurinol).</p><p>Conclusions</p><p>This is the first study to demonstrate allopurinol resistance in <i>L</i>. <i>infantum</i> and to associate it with disease relapse in the canine host. These findings are of concern as allopurinol is the main drug used for long term control of the disease in dogs, and resistant <i>L</i>. <i>infantum</i> strains may enhance uncontrolled transmission to humans and to other dogs.</p></div

Induction of allopurinol resistance in <i>Leishmania infantum</i> isolated from dogs

<div><p>Resistance to allopurinol in zoonotic canine leishmaniasis has been recently shown to be associated with disease relapse in naturally-infected dogs. However, information regarding the formation of resistance and its dynamics is lacking. This study describes the successful <i>in-vitro</i> induction of allopurinol resistance in <i>Leishmania infantum</i> cultured under increasing drug pressure. Allopurinol susceptibility and growth rate of induced parasites were monitored over 23 weeks and parasite clones were tested at selected time points and compared to their parental lines, both as promastigotes and as amastigotes. Allopurinol resistance was formed in strains from two parasite stocks producing a 20-fold rise in IC₅₀ along three distinct growth phases. In addition, characteristic differential clustering of single nucleotide polymorphisms (SNP) was found in drug sensitive and resistant parasite clones. Results confirm that genetic polymorphism, as well as clonal heterogeneity, contribute to <i>in-vitro</i> resistance to allopurinol, which is likely to occur in natural infection.</p></div

Allopurinol susceptibility of <i>L</i>. <i>infantum</i> parental strains and clones at different time points during induction of resistance.

<p>(A, B) Results for promastigotes, numbers indicate IC₅₀ values of the post-thaw parent cultures at the respective time points. See also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005910#pntd.0005910.s001" target="_blank">S1 Table</a>. (C) Allopurinol percent inhibition results for intracellular amastigotes. An induced resistant culture, 5 of its respective clones and its drug-free control culture were tested for each parental line (NT4.L and NT5.L) at one time point (day 104 and 86, respectively). Values indicated by distinct letters differ significantly (Tukey HSD test, p<0.05).</p

Maximum likelihood analysis of six induced-resistance clonal strains for all SNP detected.

<p>SNPs were found by comparing mapping results of the strains to the <i>L</i>. <i>infantum</i> JPCM5 reference genome. Bootstrapping (n = 100) was used.</p

Changes in allopurinol susceptibility and growth rates in <i>Leishmania infantum</i> cultures NT4.L and NT5.L under drug pressure.

<p>(A, B) IC₅₀ of promastigotes cultured under drug pressure (NT4.L, NT5.L) as compared to controls (NT4, NT5) cultured in medium alone over time. Only IC₅₀ values with R square >0.9 are presented. The allopurinol concentrations in medium are also shown. Distinct letters refer to significant differences in IC₅₀ values between time points within each induced culture, NT4.L (A) or NT5.L (B). Asterisk (*) refers to differences in IC₅₀ values between the induced and respective control culture (for e.g. NT4.L and NT4) for the same time point. Large squares indicate points of cloning. (C, D) Average step growth rate (ASGR) values for cultures under drug pressure, showing two distinct phases. Values expressed in 10⁶ promastigotes/mL/day. Dotted lines and labels represent average ASGR values for respective phase. § Difference in ASGR averages between phases.</p