Drug resistance in the treatment of leishmaniasis in animals and humans could have an increasingly significant impact on clinical management, just as antibiotic resistance has done for bacterial infections. Whilst we still have numerous active substances across various classes available for bacterial infections, we currently have very few effective active substances for the treatment of leishmaniasis.

From a One Health perspective, it will become increasingly important to optimise the use of antimicrobial drugs and implement targeted therapeutic strategies only where truly necessary, reducing treatment in seropositive patients who are not ill or the excessive duration of treatment in infected individuals.

Indeed, in recent years, the indiscriminate treatment of patients, even if only SEROPOSITIVE, has led to the progressive development of drug resistance.

It has recently been demonstrated that certain genetic variants of Leishmania can develop resistance to drugs used in veterinary medicine (antimonials, miltefosine and allopurinol). Recent studies on clinical samples have shown that this resistance is more common than previously thought.

A new commercial test is now available exclusively from Mylav for the detection and identification of resistant genetic variants.

Figure 1: Some resistance mechanisms triggered by protozoa of the genus Leishmania when subjected to environmental, immune and toxic stress

Objectives of the LeishGenR™ Test

The purpose of the test is to identify mutations in the DNA of protozoa of the genus Leishmania (variations in the number of copies of certain genes) that are associated with resistance to antimonials, miltefosine and allopurinol.

Procedure for performing the LeishGenR™ Test

The test is performed on DNA extracted from a biological sample containing protozoa of the genus Leishmania.

As a minimal quantity of protozoa is required to analyse the DNA of these pathogens, the test must necessarily be performed on tissue samples characterised by a high protozoal load, such as, for example, lymph node cytological samples or bone marrow blood. Performing the test on biological fluids such as blood and serum is not always possible, as the quantity of Leishmania may be insufficient for adequate analysis.

For these reasons, the LeishGenR™ test must always be preceded by quantitative real-time PCR to detect Leishmania. If the amount of protozoan DNA extracted from the sample is insufficient, it is not possible to proceed with the LeishGenR™ test.

Figure 2: An illustrative diagram of the LeishGenR™ test procedure. Leishmania DNA is extracted from a sample rich in protozoa and subjected to standard PCR. If the result is positive and there is a high concentration of parasite DNA, further PCR reactions can be performed to detect specific resistance genes and quantify them.

Clinical interpretation and optimisation of treatment

The presence of mutant variants of protozoa may promote resistance to one or more of the three anti-Leishmania drugs: it is therefore possible that the patient may not respond adequately to the recommended treatment protocols or may experience earlier relapses. The presence of mutations is not in itself automatically associated with a reduced therapeutic response or an unfavourable prognosis; however, their identification plays a crucial role. Mutations indicate that the protozoa present in the patient are genetically predisposed to developing drug resistance. Although such resistance may not always manifest in vivo, its detection allows for a more accurate assessment of the risk of treatment failure and supports more targeted and timely therapeutic decisions, making the test particularly relevant in the patient’s diagnostic and clinical pathway.

Furthermore, failure to respond to a correctly administered recommended therapy may depend on at least three main factors: the presence of resistance mutations, the presence of concomitant systemic diseases, and an inadequate immunological response in the patient.

Example of use of the LeishGenR™ Test

Patient with a first diagnosis of leishmaniasis: the test is indicated to check for the presence of variants that have already mutated and are therefore potentially more difficult to treat. Although the presence of resistance mutations does not preclude treatment with that drug, the test result can be used to select the most suitable active ingredient, to monitor the patient over time and, where appropriate, to obtain potentially useful prognostic data.

Patient with recurrence/reinfection or an inadequate therapeutic response: the LeishGenR™ test is strongly recommended to help decide whether a change to the initial treatment protocol and active ingredient is advisable. Based also on the result of the LeishGenR™ test at initial diagnosis, it is also possible to determine whether the inadequate response may be attributable to the development of resistance or whether other causes should instead be investigated (for example, a concomitant systemic disease).

FREQUENTLY ASKED QUESTIONS

Q: At present, which leishmanicidal drugs are most frequently associated with resistance?

A: Resistance is most common with allopurinol and antimonials, and less common with miltefosine

Q: The result provides a numerical value which, if higher than a ‘threshold value’, indicates the presence of drug resistance. Does a high numerical value correlate with greater resistance?

A: No, the value is not currently correlated with greater resistance; it merely indicates whether resistance is present or not.

Q: Can there be dogs that are clinically resistant to treatment without there being a genetic mutation?

A: Yes, it is possible for some patients to be resistant to drug therapy or to respond poorly, without there being a mutation. This may potentially – as already mentioned – be due to the presence of other concomitant diseases, or to an ineffective immune response against leishmaniasis, or to other types of non-genetic treatment failure.

Q: Are there protozoa that are potentially genetically resistant to all three drugs?

A: Yes, there are potentially patients who exhibit genetic resistance to two or three drugs (allopurinol, antimonials, miltefosine)

Q: Can the test also be requested in cases of leishmaniasis treatment for feline patients?

A: Yes, in any animal infected with Leishmania infantum, because the test is performed using the parasite’s DNA.

Q: Can the vaccine affect the results of these genetic tests?

A: No, receiving the vaccine in the preceding months or years does not affect the results of the genetic test.

Q: I have a patient who is clinically resistant to the treatment. If I still wish to or need to use the drug to which they are resistant, should I adjust the dosage and timing of administration?

A: No, the dosage and timing of administration should not be altered even if a patient shows genetic resistance. However, the first recommendation would be to switch to a different drug, if possible.