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

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

Additional file 3: Figure S2. of Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis

Correlation in the eggs per gram quantified in the HRM qPCRs for the ITS1 and 18S of S. lupi in fecal samples from dogs with spirocercosis. Logarithmic scales are used in both x- and y-axes. (PNG 16 kb

Additional file 2: Figure S1. of Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis

Limit of detection of the cox1 semi-nested PCR for S. lupi. DNA-standards with known epg concentration were run by triplicates, and the detection limit was estimated as the last concentration of which more than 67% of the samples were still positive. (PNG 867 kb

A-C. Individual relapse pattern.

<p>The absolute spirochete numbers per ml blood of three selected mice that had received 1 x 10⁶ <i>B</i>. <i>persica</i>/mouse (Y-axis) are plotted against the blood sampling days (X-axis). Mouse two (Fig 2A) and mouse 12 (Fig 2B) were part of subgroup one; mouse 18 (Fig 2C) was part of subgroup two. During the first 14 days, subgroups were bled according to an alternating sample collection schedule.</p

Organ dissemination of <i>B</i>. <i>persica</i> following inoculation with different doses of spirochetes.

<p>Organ dissemination of <i>B</i>. <i>persica</i> following inoculation with different doses of spirochetes.</p

A-C. Antibody response against <i>B</i>. <i>persica</i>.

<p>Fig 3A. Antibody levels of pooled plasma samples (infection dose 1 x 10⁶ <i>B</i>. persica/mouse). The mean (calculated out of the values of the subgroup-specific pooled plasma samples) and the standard deviation (Y-axis) are plotted against the blood sampling days (X-axis). The logistic fitting is shown as a continuous line. Fig 3B. Antibody levels of all experimental infection dose groups are shown as box plots; serum samples from final days and pooled plasma samples from days 35 and 28. Fig 3C. Western blot of final sera from two randomly selected mice out of each infection dose group. Mw = molecular weight standard; 1, 2 = 1 x 10⁶ <i>B</i>. <i>persica</i>/mouse; 3, 4 = 1 x 10⁴ <i>B</i>. <i>persica</i>/mouse; 5, 6 = 1 x 10² <i>B</i>. <i>persica</i>/mouse; 7, 8 = 4 x 10⁰ <i>B</i>. <i>persica</i>/mouse; 9, 10 = negative mice.</p

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

Additional file 1: of Renal dialysis and long-term treatment of a dog with kidney disease associated with canine leishmaniosis

Table S1. Follow up of clinical findings and treatment of the dog during 15 weeks after its discharge from the Hebrew University Veterinary Teaching Hospital. Earlier findings are described in the text. (DOCX 14 kb

A-C. Spirochete burden in the blood.

<p>Results of real-time PCR detecting the <i>flaB</i> gene of <i>B</i>. <i>persica</i> in blood from C3H/HeOuJ mice that had received 1 x 10⁶ (Fig 1A), 1 x 10⁴ (Fig 1B) or 1 x 10² (Fig 1C) <i>B</i>. <i>persica</i>/mouse. The absolute spirochete numbers in each blood sample (Y-axis, log₁₀-scale) are plotted as box plots against the blood sampling days (X-axis).</p