Date Published: January 6, 2016
Publisher: Public Library of Science
Author(s): Daniel Yasur-Landau, Charles L. Jaffe, Lior David, Gad Baneth, Eric Dumonteil. http://doi.org/10.1371/journal.pntd.0004341
Abstract: BackgroundVisceral leishmaniasis caused by the protozoan Leishmania infantum 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 Leishmania strains from dogs to allopurinol has not been described before in clinical studies.Methodology/Principal FindingsFollowing our observation of clinical disease relapse in dogs under allopurinol treatment, we tested susceptibility to allopurinol of L. infantum 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 (P = 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 (P = 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 (P = 0.002) was confirmed using an infected macrophage model (6.3% and 20% growth inhibition, respectively at 300 μg/mL allopurinol).ConclusionsThis is the first study to demonstrate allopurinol resistance in L. infantum 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 L. infantum strains may enhance uncontrolled transmission to humans and to other dogs.
Partial Text: Visceral leishmaniasis caused by L. infantum is a potentially fatal disease, which is a serious public health concern in Europe, Asia, North Africa and Latin America. The dog is the main reservoir for this zoonotic infection and it has been estimated that in Southwestern Europe alone there are about 2.5 million infected dogs [1,2].
This is the first detailed report of resistance to allopurinol in L. infantum parasites isolated from dogs and associated with clinical relapse. All dogs included in this study were seropositive at diagnosis, had typical clinical manifestations of CanL at the time of their initial diagnosis or when they relapsed, and were positive by lymph node culture. The clinical score was able to reliably reflect the clinical status of each group, while serology, complete blood count results, as well as the biochemical indices tested, did not show significant differences between groups that could be beneficial for their clinical characterization. A previous study has demonstrated that an increase in parasite DNA loads in lymph nodes, but not blood, was correlated with the reappearance of clinical signs in relapsing dogs . Our results shows that parasitemia seems to be greatly reduced or absent following treatment, which may be attributed to the effect of allopurinol, causing a total reduction in parasite load, as reported previously . Thus, these results do not indicate that any ancillary test employed in the study is superior to the clinical score in the diagnosis of relapse in leishmaniotic dogs. Although it is widely accepted that dogs treated for visceral leishmaniasis using the currently available drugs frequently remain infected and often relapse [8,13], only a small number of relapse cases have been described so far [14,15,16,17,18], and no characterization of parasite strains from relapsed dogs was made. As opposed to the large volume of evidence on drug resistance in human leishmaniasis, little is known regarding the development of drug resistance in CanL. One study demonstrated that L. infantum isolates obtained from naturally infected dogs after a therapeutic course of meglumine antimoniate were less susceptible to the drug compared to isolates from the same dogs before treatment . Similar results were also demonstrated in a canine experimental L. infantum infection model followed by meglumine antimoniate treatment . Differences in susceptibility to meglumine antimoniate among L. infantum isolates belonging to two different zymodemes from an area lacking drug pressure have also been shown and were hypothesized to represent an inherent resistance to this drug . As part of a larger study on drug susceptibility in humans and dogs, allopurinol susceptibility was evaluated in two L. infantum isolates from dogs . The IC50 value of an isolate from a non-treated dog was found to be lower compared to an isolate from a dog undergoing treatment. However, the clinical status of the latter dog was not discussed, and no association was made between clinical relapse and decreased drug susceptibility of parasites. We have found a significant 3 to 4 fold increase in allopurinol IC50 for promastigote strains isolated from relapsed dogs (TR) undergoing treatment with this drug compared to non-treated dogs. This increase was evident when testing axenic amastigotes and intracellular amastigotes. Although we did not find a significant correlation between treatment duration and drug IC50, the fact that increase in drug resistance was found in parasites isolated from treated dogs suggests that resistance may at least in some cases be caused by selection under drug pressure. Resistance development over time under the pressure of drug treatment has been described for L. donovani and antimonials . In addition, inherent increased resistance to drugs must also be considered, in view of the considerable variation in IC50 and the % inhibition values demonstrated within each group in all parasite life stages tested. The dissemination of drug resistant parasites may provide another possible explanation for this variation. Cases such as the TR4 isolate, which presented increased drug resistance while being under drug pressure for only short time, may be attributed to either of the above options. Although relapse was associated in this study with parasite drug resistance, a future study with a larger number of relapsed dogs may identify animals with lower IC50’s that relapsed due to other potential causes such as concurrent immune-suppressing conditions and neoplasia. By widening the database of dogs and parasites we may better understand the origin of these resistant parasites and take measures to minimize their prevalence. The association found in this study between clinical relapse and resistant parasites may affect current drug selection and treatment regimens. Since no significant difference was found in serology values between the groups and no parasite DNA was detected in the blood of most relapsed dogs, these routine tests are ineffective in discriminating between infection with sensitive and resistant parasites. Relapse in leishmaniotic dogs presents a clinical challenge, since possible effects of deteriorating leishmanial infection such as kidney disease, as well as nonrelated conditions such as other diseases, should be assessed before parasite drug resistance is defined as the cause of relapse. Despite that, our results suggest that allopurinol resistance should be considered, and that an affordable methodology to test the presence of allopurinol resistant L. infantum in dogs should be available, once clinical relapse is suspected.