The effects of formulation on the pharmacokinetics of itraconazole and amiodarone in dogs after oral administration of a combination product, commercial products, and compounded products.

This study evaluated four different formulations of itraconazole and amiodarone. Formulation 1 was Vida's combination tablet containing both active pharmaceutical ingredients (APIs). Formulation 2 was separate, commercially available human generic capsules and tablets of itraconazole and amiodarone, respectively. Formulation 3 was separate, compounded suspensions of itraconazole and amiodarone. Formulation 4 was a compounded chewable tablet of itraconazole. Eight female dogs were dosed with 5 mg/kg of itraconazole and 15 mg/kg amiodarone (except for formulation 4, which only received 5 mg/kg itraconazole) once weekly for 4 weeks using a modified Latin Square design, ensuring that all dogs received all formulations with a 7-day washout between treatments. Animals were fasted overnight prior to each dose administration, with food returned to all animals 4 h post-dose. Blood samples (3 mL) were collected pre-treatment (0) and at appropriate time points over 72 h after each dose for a total of 14 samples per dog per treatment. There was high variability in the serum concentration data within treatment groups for itraconazole. The compounded suspensions were difficult to dose due to the nature of the formulations. The volumes dosed were accurate and consistent, but the suspension was thin and settled immediately when shaking was stopped for both itraconazole and amiodarone. All serum samples following itraconazole chewable tablet administration were not detectable or just above itraconazole's LOQ and thus did not allow for pharmacokinetic determination.

Validation of Oxford nanopore sequencing for improved New World Leishmania species identification via analysis of 70-kDA heat shock protein.

BACKGROUND: Leishmaniasis is a parasitic disease caused by obligate intracellular protozoa of the genus Leishmania. This infection is characterized by a wide range of clinical manifestations, with symptoms greatly dependent on the causal parasitic species. Here we present the design and application of a new 70-kDa heat shock protein gene (hsp70)-based marker of 771 bp (HSP70-Long). We evaluated its sensitivity, specificity and diagnostic performance employing an amplicon-based MinION™ DNA sequencing assay to identify different Leishmania species in clinical samples from humans and reservoirs with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). We also conducted a comparative analysis between our novel marker and a previously published HSP70 marker known as HSP70-Short, which spans 330 bp. METHODS: A dataset of 27 samples from Colombia, Venezuela and the USA was assembled, of which 26 samples were collected from humans, dogs and cats affected by CL and one sample was collected from a dog with VL in the USA (but originally from Greece). DNA was extracted from each sample and underwent conventional PCR amplification utilizing two distinct HSP70 markers: HSP70-Short and HSP70-Long. The subsequent products were then sequenced using the MinION™ sequencing platform. RESULTS: The results highlight the distinct characteristics of the newly devised HSP70-Long primer, showcasing the notable specificity of this primer, although its sensitivity is lower than that of the HSP70-Short marker. Notably, both markers demonstrated strong discriminatory capabilities, not only in distinguishing between different species within the Leishmania genus but also in identifying instances of coinfection. CONCLUSIONS: This study underscores the outstanding specificity and effectiveness of HSP70-based MinION™ sequencing, in successfully discriminating between diverse Leishmania species and identifying coinfection events within samples sourced from leishmaniasis cases.

A Combination of Itraconazole and Amiodarone Is Highly Effective against Trypanosoma cruzi Infection of Human Stem Cell-Derived Cardiomyocytes.

Trypanosoma cruzi is the etiologic agent of Chagas disease (CD), which can result in severe cardiomyopathy. Trypanosoma cruzi is endemic to the Americas, and of particular importance in Latin America. In the United States and other non-endemic countries, rising case numbers have also been observed. The currently used drugs are benznidazole (BNZ) and nifurtimox, which have limited efficacy during chronic infection. We repurposed itraconazole (ICZ), originally an antifungal, in combination with amiodarone (AMD), an antiarrhythmic, with the goal of interfering with T. cruzi infection. Human pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes (hiPSC-CMs). Vero cells or hiPSC-CMs were infected with T. cruzi trypomastigotes of the II or I strain in the presence of ICZ and/or AMD. After 48 hours, cells were Giemsa stained, and infection and multiplication were evaluated microscopically. Trypanosoma cruzi infection and multiplication were evalutated also by electron microscopy. BNZ was used as a reference compound. Cell metabolism in the presence of test substances was assessed. Itraconazole and AMD showed strain- and dose-dependent interference with T. cruzi infection and multiplication in Vero cells or hiPSC-CMs. Combinations of ICZ and AMD were more effective against T. cruzi than the single substances, or BNZ, without affecting host cell metabolism, and better preserving host cell integrity during infection. Our in vitro data in hiPSC-CMs suggest that a combination of ICZ and AMD might serve as a treatment option for CD in patients, but that different responses due to T. cruzi strain differences have to be taken into account.

PCR monitoring of parasitemia during drug treatment for canine Chagas disease.

To date, there is no clear standard to monitor drug treatment for canine Chagas disease. We used 2 real-time PCR (rtPCR) assays targeting Trypanosoma cruzi kinetoplast DNA (kDNA) and nuclear satellite DNA (nDNA) to detect T. cruzi in canine whole blood. Samples were collected randomly from 131 untreated dogs with unknown T. cruzi infection status in Texas. The kDNA-based rtPCR was slightly more sensitive (diagnostic sensitivity of kDNA = 49% vs. nDNA = 44%; p = 0.5732) but slightly less specific (diagnostic specificity of kDNA = 96% vs. nDNA = 97%; p > 0.9999) than the nDNA-based rtPCR. However, the differences in sensitivity and specificity between the nDNA- and kDNA-based rtPCR assays were not statistically significant. Using the nDNA- and kDNA-based qualitative rtPCR assays to monitor parasitemia from 137 itraconazole- and amiodarone-treated cases with nDNA- and kDNA-based PCR-positive baselines showed that the PCR positive rate decreased to 0% in 30 d. Using kDNA-based quantitative rtPCR to monitor normalized T. cruzi DNA copies in 4 representative dogs demonstrated that drug treatment could reduce parasite loads within 7-30 d. The kDNA-based qualitative rtPCR may be used for routine parasitemia screening of drug-treated Chagas-positive dogs, whereas nDNA-based qualitative rtPCR may be used for confirmation.

Investigation of a combination of amiodarone and itraconazole for treatment of American trypanosomiasis (Chagas disease) in dogs.

OBJECTIVE: To evaluate clinical, serologic, parasitological, and histologic outcomes of dogs with naturally occurring Trypanosoma cruzi infection treated for 12 months with amiodarone and itraconazole. ANIMALS: 121 dogs from southern Texas and southern Louisiana. PROCEDURES: Treatment group dogs (n = 105) received a combination of amiodarone hydrochloride (approx 7.5 mg/kg [3.4 mg/lb], PO, q 24 h, with or without a loading dosage protocol) and itraconazole (approx 10 mg/kg [4.5 mg/lb], PO, q 24 h, adjusted to maintain a plasma concentration of 1 to 2 µg/mL) for 12 months. Control group dogs (n = 16) received no antitrypanosomal medications. Serologic assays for anti-T cruzi antibodies, PCR assays for T cruzi DNA in blood, and physical evaluations were performed 1, 6, 9, 12, and 24 months after study initiation. Adverse events were recorded. Outcomes of interest were recorded and compared between groups. RESULTS: 86 of 105 treatment group dogs and 8 of 16 control group dogs survived and completed the study (5/19 and 6/7 deaths of treatment and control group dogs, respectively, were attributed to T cruzi infection). Mean survival time until death attributed to T cruzi was longer (23.19 vs 15.64 months) for the treatment group. Results of PCR assays were negative for all (n = 92) tested treatment group dogs (except for 1 dog at 1 time point) from 6 to 24 months after study initiation. Clinical improvement in ≥ 1 clinical sign was observed in 53 of 54 and 0 of 10 treatment and control group dogs, respectively; adverse drug events were minor and reversible. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested efficacy of this trypanocidal drug combination for the treatment of T cruzi infection in dogs.