Positive clinical experience with paromomycin sulfate in treatment of Balantioides coli (=Balantidium coli) natural infection in zoo-kept Mandrill monkeys (Mandrillus sphinx) and Western lowland gorillas (Gorilla gorilla gorilla).

Balantioides coli (=Balantidium coli), a large ciliated protozoan, is reported in multiple free-ranging and captive primate species, often in association with a clinical presentation that requires medical intervention. This report describes the clinical effectiveness of paromomycin sulfate against B.coli in zoo-kept mandrill monkeys (Mandrillus sphinx, at orally doses of 8-31 mg/kg, once daily (SID) for 7 days) and gorillas (Gorilla gorilla gorilla, at orally doses of 1.4-3.1 mg/kg, SID for 5 days).

Nanoemulsions containing amphotericin b and paromomycin for the treatment of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a vector-borne disease characterized by skin lesions that can evolve into high-magnitude ulcerated lesions. Thus, this study aimed to develop an innovative nanoemulsion (NE) with clove oil, Poloxamer® 407, and multiple drugs, such as amphotericin B (AmB) and paromomycin (PM), for use in the topical treatment of CL. METHODS: Droplet size, morphology, drug content, stability, in vitro release profile, in vitro cytotoxicity on RAW 264.7 macrophages, and antileishmanial activity using axenic amastigotes of Leishmania amazonensis were assessed for NEs. RESULTS: After optimizing the formulation parameters, such as the concentration of clove oil and drugs, using an experimental design, it was possible to obtain a NE with an average droplet size of 40 nm and a polydispersion index of 0.3, and these parameters were maintained throughout the 365 days. Furthermore, the NE showed stability of AmB and PM content for 180 days under refrigeration (4 °C), presented a pH compatible with the skin, and released modified AmB and PM. NE showed the same toxicity as free AmB and higher toxicity than free PM against RAW 264.7 macrophages. The same activity as free AmB, and higher activity than free PM against amastigotes L. amazonensis. CONCLUSION: It is possible to develop a NE for the treatment of CL; however, complementary studies regarding the antileishmanial activity of NE should be carried out.

Enhanced topical paromomycin delivery for cutaneous leishmaniasis treatment: Passive and iontophoretic approaches.

Conventional treatments for cutaneous leishmaniasis, a neglected vector-borne infectious disease, can frequently lead to serious adverse effects. Paromomycin (PAR), an aminoglycoside antibiotic, has been suggested for the topical treatment of disease-related lesions, but even when formulated in high drug-loading dosage forms, presents controversial efficacy. The presence of five ionizable amino groups hinder its passive cutaneous penetration but make PAR an excellent candidate for iontophoretic delivery. The objective of this study was to verify the feasibility of using iontophoresis for cutaneous PAR delivery and to propose a topical passive drug delivery system that could be applied between iontophoretic treatments. For this, in vitro iontophoretic experiments evaluated different application durations (10, 30, and 360 min), current densities (0.1, 0.25, and 0.5 mA/cm2), PAR concentrations (0.5 and 1.0 %), and skin models (intact and impaired porcine skin). In addition, 1 % PAR hydrogel had its penetration profile compared to 15 % PAR ointment in passive transport. Results showed iontophoresis could deliver suitable PAR amounts to dermal layers, even in short times and with impaired skin. Biodistribution assays showed both iontophoretic transport and the proposed hydrogel delivered higher PAR amounts to deeper skin layers than conventional ointment, even though applying 15 times less drug. To our knowledge, this is the first report of PAR drug delivery enhancement by iontophoresis. In summary, the association of iontophoresis with a topical application of PAR gel seems appropriate for improving cutaneous leishmaniasis treatment.

Safety and efficacy of paromomycin/miltefosine/liposomal amphotericin B combinations for the treatment of post-kala-azar dermal leishmaniasis in Sudan: A phase II, open label, randomized, parallel arm study.

BACKGROUND: Treatment for post-kala-azar dermal leishmaniasis (PKDL) in Sudan is currently recommended only for patients with persistent or severe disease, mainly because of the limitations of current therapies, namely toxicity and long hospitalization. We assessed the safety and efficacy of miltefosine combined with paromomycin and liposomal amphotericin B (LAmB) for the treatment of PKDL in Sudan. METHODOLOGY/PRINCIPAL FINDINGS: An open-label, phase II, randomized, parallel-arm, non-comparative trial was conducted in patients with persistent (stable or progressive disease for ≥ 6 months) or grade 3 PKDL, aged 6 to ≤ 60 years in Sudan. The median age was 9.0 years (IQR 7.0-10.0y) and 87% of patients were ≤12 years old. Patients were randomly assigned to either daily intra-muscular paromomycin (20mg/kg, 14 days) plus oral miltefosine (allometric dose, 42 days)-PM/MF-or LAmB (total dose of 20mg/kg, administered in four injections in week one) and oral miltefosine (allometric dose, 28 days)-LAmB/MF. The primary endpoint was a definitive cure at 12 months after treatment onset, defined as clinical cure (100% lesion resolution) and no additional PKDL treatment between end of therapy and 12-month follow-up assessment. 104/110 patients completed the trial. Definitive cure at 12 months was achieved in 54/55 (98.2%, 95% CI 90.3-100) and 44/55 (80.0%, 95% CI 70.2-91.9) of patients in the PM/MF and AmB/MF arms, respectively, in the mITT set (all randomized patients receiving at least one dose of treatment; in case of error of treatment allocation, the actual treatment received was used in the analysis). No SAEs or deaths were reported, and most AEs were mild or moderate. At least one adverse drug reaction (ADR) was reported in 13/55 (23.6%) patients in PM/MF arm and 28/55 (50.9%) in LAmB/MF arm, the most frequent being miltefosine-related vomiting and nausea, and LAmB-related hypokalaemia; no ocular or auditory ADRs were reported. CONCLUSIONS/SIGNIFICANCE: The PM/MF regimen requires shorter hospitalization than the currently recommended 60-90-day treatment, and is safe and highly efficacious, even for patients with moderate and severe PKDL. It can be administered at primary health care facilities, with LAmB/MF as a good alternative. For future VL elimination, we need new, safe oral therapies for all patients with PKDL. TRIAL REGISTRATION: ClinicalTrials.gov NCT03399955, https://clinicaltrials.gov/study/NCT03399955 ClinicalTrials.gov ClinicalTrials.gov.

Resistome in Streptomyces rimosus - A Reservoir of Aminoglycoside Antibiotics Resistance Genes.

Investigation of aminoglycoside acetyltransferases in actinobacteria of the genus Streptomyces is an integral part of the study of soil bacteria as the main reservoir and possible source of drug resistance genes. Previously, we have identified and biochemically characterized three aminoglycoside phosphotransferases, which cause resistance to kanamycin, neomycin, paromomycin, streptomycin, and hygromycin B in the strain Streptomyces rimosus ATCC 10970 (producing oxytetracycline), which is resistant to most natural aminoglycoside antibiotics. In the presented work, it was shown that the resistance of this strain to other AGs is associated with the presence of the enzyme aminoglycoside acetyltransferase, belonging to the AAC(2') subfamily. Induction of the expression of the gene, designated by us as aac(2')-If, in Escherichia coli cells determines resistance to a wide range of natural aminoglycoside antibiotics (neomycin, gentamicin, tobramycin, sisomycin, and paromomycin) and increases minimum inhibitory concentrations of these antibiotics.

Population pharmacokinetics of a combination of miltefosine and paromomycin in Eastern African children and adults with visceral leishmaniasis.

OBJECTIVES: To improve visceral leishmaniasis (VL) treatment in Eastern Africa, 14- and 28-day combination regimens of paromomycin plus allometrically dosed miltefosine were evaluated. As the majority of patients affected by VL are children, adequate paediatric exposure to miltefosine and paromomycin is key to ensuring good treatment response. METHODS: Pharmacokinetic data were collected in a multicentre randomized controlled trial in VL patients from Kenya, Sudan, Ethiopia and Uganda. Patients received paromomycin (20 mg/kg/day for 14 days) plus miltefosine (allometric dose for 14 or 28 days). Population pharmacokinetic models were developed. Adequacy of exposure and target attainment of paromomycin and miltefosine were evaluated in children and adults. RESULTS: Data from 265 patients (59% ≤12 years) were available for this pharmacokinetic analysis. Paromomycin exposure was lower in paediatric patients compared with adults [median (IQR) end-of-treatment AUC0-24h 187 (162-203) and 242 (217-328) µg·h/mL, respectively], but were both within the IQR of end-of-treatment exposure in Kenyan and Sudanese adult patients from a previous study. Cumulative miltefosine end-of-treatment exposure in paediatric patients and adults [AUCD0-28 517 (464-552) and 524 (456-567) µg·day/mL, respectively] and target attainment [time above the in vitro susceptibility value EC90 27 (25-28) and 30 (28-32) days, respectively] were comparable to previously observed values in adults. CONCLUSIONS: Paromomycin and miltefosine exposure in this new combination regimen corresponded to the desirable levels of exposure, supporting the implementation of the shortened 14 day combination regimen. Moreover, the lack of a clear exposure-response and exposure-toxicity relationship indicated adequate exposure within the therapeutic range in the studied population, including paediatric patients.

Self-application of aminoglycoside-based creams to treat cutaneous leishmaniasis in travelers.

BACKGROUND: In endemic foci, the use of an aquaphilic cream containing paromomycin with/without gentamicin to treat cutaneous leishmaniasis (CL) is safe, painless and cures 78-82% of patients with New and Old World CL. Self-application in travelers requires evaluation. METHODS: Travelers with 1-10 lesions of confirmed CL were prospectively treated with the paromomycin-gentamicin formulation (WR279396, 2012-2017, Group 1) and carefully follow up, or treated with a locally produced paromomycin-only cream (2018-2022, Group 2). The cream was applied once under supervision, then self-applied daily for 20-30 days. A cured lesion was defined as 100% re-epithelialization at day 42 without relapse at three months. RESULTS: Medical features were similar in Group 1 (17 patients), and Group 2 (23 patients). Patients were infected with either Leishmania major, L. infantum, L. killicki, L. guyanensis, L. braziliensis, or L. naiffi. Intention-to-treat and per-protocol cure rates were 82% (95% confidence interval (CI) [64.23;100.00]) and 87% (95% CI [71,29;100.00]) in Group 1, and 69% (95% CI [50.76; 88.37]) and 76% (95% CI [57.97; 94.41]) in Group 2. In the pooled Group 1&2, 75% (95% CI [61.58;88.42]) (30/40) and 81% (95% CI [68,46;93.6]) (30/37) of patients were cured in intention-to-treat and per-protocol, respectively. There were no significant differences observed in the success rates between Old World and New World CL (83.3% vs. 60%, p = 0.14). Prospective observations in Group 1 showed that adverse events were mainly pruritus (24%) and pain (18%) on lesions (all mild or moderate). No mucosal involvement was observed in either group. DISCUSSION: In this representative population of travelers who acquired CL either in the Old or New World, the 81% per-protocol cure rate of a self-applied aminoglycoside cream was similar to that observed in clinical trials.

Exploring Novel Drug Combinations: The Therapeutic Potential of Selanyl Derivatives for Leishmania Treatment.

This work describes the design, synthesis, and biological activities of new selenoester derivatives and its homologs thioesters. Thirty-two compounds were developed following an economical synthetic route, achieving small molecules, with structural characteristics similar to those present in antileishmanial drugs such as miltefosine (MIL) and paromomycin (PMN). These compounds were tested in vitro against strains of Leishmania major (L. major) and Leishmania infantum (L. infantum). The L. infantum strain (causative agent of visceral leishmaniasis) exhibited the highest sensitivity. Thus, four selanylacetic acid derivatives (A4, A5, A6 and A8) presented IC50 values below 40 µM in this strain. These derivatives also demonstrated low toxicity and high selectivity in PMA-differentiated THP-1 macrophages. The A4-A6 and A8 derivatives were evaluated in order to determine their pharmacological behavior, using drug combination studies with the reference drugs amphotericin B (AMB), MIL and PMN. Compounds A6 and A8 presented a potent synergistic interaction with MIL, which is the only oral drug available for the treatment of visceral leishmaniasis. Therefore, compounds A6 and A8 present significant potential as therapeutic candidates for the treatment of leishmaniasis based on their remarkable leishmanicidal characteristics and pharmacological synergism.

Evaluation of farnesol orally and topically against experimental cutaneous leishmaniasis: In -vivo analysis.

Leishmaniasis is a zoonotic disease transmitted by an obligate intra-macrophage protozoan of the genus Leishmania through the infective bite of a vector sandfly. This study investigated the therapeutic efficacy of farnesol, a sesquiterpene compound, for the treatment of cutaneous leishmaniasis (CL) using in vivo BALB/c mouse model. In this study, farnesol's efficacy was compared with the standard drug, paromomycin. It was observed that farnesol significantly reduced lesion sizes and footpad thickness compared to the control group (paromomycin). Lymph node size was also significantly reduced in farnesol-treated mice, indicating its ability to control infection spread. Combination therapy with farnesol and Paromomycin did not demonstrate synergistic effects. These results highlight the potential of farnesol as an alternative therapeutic agent for CL. Further investigations are required to elucidate its mechanism of action and assess potential off-target effects. Optimization of oral delivery methods should be explored to enhance bioavailability. Overall, our findings support farnesol's efficacy in CL treatment, offering promising prospects for improved disease management.

Cost-effective In Vivo and In Vitro Mouse Models for Evaluating Anticryptosporidial Drug Efficacy: Assessing Vorinostat, Docetaxel, and Baicalein.

BACKGROUND: Cryptosporidiosis is a significant diarrheal disease in humans and animals. Immunodeficient mice are the primary small animal models, but their high costs and specialized breeding/housing requirements limit in vivo drug testing. Numerous anticryptosporidial lead compounds identified in vitro remain untested in vivo. METHODS: Cryptosporidium tyzzeri, a natural mouse parasite closely related to Cryptosporidium parvum and Cryptosporidium hominis, was isolated to establish an infection model in immunocompetent mice. The model was validated using classic anticryptosporidial drugs (paromomycin and nitazoxanide) and then employed to assess the efficacy of 3 new leads (vorinostat, docetaxel, and baicalein). An in vitro culture of C. tyzzeri was also developed to complement the animal model. RESULTS: Chronic C. tyzzeri infection was established in chemically immunosuppressed wild-type mice. Paromomycin (1000 mg/kg/d) and nitazoxanide (100 mg/kg/d) demonstrated efficacy against C. tyzzeri. Vorinostat (30 mg/kg/d), docetaxel (25 mg/kg/d), and baicalein (50 mg/kg/d) were highly effective against C. tyzzeri infection. In vitro, nitazoxanide, vorinostat, docetaxel, and baicalein exhibited low to submicromolar efficacy against C. tyzzeri. CONCLUSIONS: Novel in vivo and in vitro models have been developed for cost-effective anticryptosporidial drug testing. Vorinostat, docetaxel, and baicalein show potential for repurposing and/or optimization for developing new anticryptosporidial drugs.

In Silico Selection and Validation of High-Affinity ssDNA Aptamers Targeting Paromomycin.

Glycans are promising for disease diagnosis since glycan biosynthesis is significantly affected by disease states, and glycosylation changes are probably more pronounced than protein expression during the transformation to the diseased condition. Glycan-specific aptamers can be developed for challenging applications such as cancer targeting; however, the high flexibility of glycosidic bonds and scarcity of studies on glycan-aptamer binding mechanisms increased the difficulty of screening. In this work, the model of interactions between glycans and ssDNA aptamers synthesized based on the sequence of rRNA genes was developed. Our simulation-based approach revealed that paromomycin as a representative example of glycans is preferred to bind base-restricted stem structures of aptamers because they are more critical in stabilizing the flexible structures of glycans. Combined experiments and simulations have identified two optimal mutant aptamers. Our work would provide a potential strategy that the glycan-binding rRNA genes could act as the initial aptamer pools to accelerate aptamer screening. In addition, this in silico workflow would be potentially applied in the more extensive in vitro development and application of RNA-templated ssDNA aptamers targeting glycans.

Using Entamoeba muris To Model Fecal-Oral Transmission of Entamoeba in Mice.

There are several Entamoeba species that colonize humans, but only Entamoeba histolytica causes severe disease. E. histolytica is transmitted through the fecal-oral route to colonize the intestinal tract of 50 million people worldwide. The current mouse model to study E. histolytica intestinal infection directly delivers the parasite into the surgically exposed cecum, which circumvents the natural route of infection. To develop a fecal-oral mouse model, we screened our vivarium for a natural murine Entamoeba colonizer via a pan-Entamoeba PCR targeting the 18S ribosomal gene. We determined that C57BL/6 mice were chronically colonized by Entamoeba muris. This amoeba is closely related to E. histolytica, as determined by 18S sequencing and cross-reactivity with an E. histolytica-specific antibody. In contrast, outbred Swiss Webster (SW) mice were not chronically colonized by E. muris. We orally challenged SW mice with 1 × 105 E. muris cysts and discovered they were susceptible to infection, with peak cyst shedding occurring between 5 and 7 days postinfection. Most infected SW mice did not lose weight significantly but trended toward decreased weight gain throughout the experiment compared to mock-infected controls. Infected mice treated with paromomycin, an antibiotic used against noninvasive intestinal disease, do not become colonized by E. muris. Within the intestinal tract, E. muris localizes exclusively to the cecum and colon. Purified E. muris cysts treated with bovine bile in vitro excyst into mobile, pretrophozoite stages. Overall, this work describes a novel fecal-oral mouse model for the important global pathogen E. histolytica. IMPORTANCE Infection with parasites from the Entamoeba genus are significantly underreported causes of diarrheal disease that disproportionally impact tropical regions. There are several species of Entamoeba that infect humans to cause a range of symptoms from asymptomatic colonization of the intestinal tract to invasive disease with dissemination. All Entamoeba species are spread via the fecal-oral route in contaminated food and water. Studying the life cycle of Entamoeba, from host colonization to infectious fecal cyst production, can provide targets for vaccine and drug development. Because there is not an oral challenge rodent model, we screened for a mouse Entamoeba species and identified Entamoeba muris as a natural colonizer. We determine the peak of infection after an oral challenge, the efficacy of paromomycin treatment, the intestinal tract localization, and the cues that trigger excystation. This oral infection mouse model will be valuable for the development of novel therapeutic options for Entamoeba infections.

A Systematic Review of Drug-Carrying Nanosystems Used in the Treatment of Leishmaniasis.

Leishmaniasis is an infectious disease responsible for a huge rate of morbidity and mortality in humans. Chemotherapy consists of the use of pentavalent antimonial, amphotericin B, pentamidine, miltefosine, and paromomycin. However, these drugs are associated with some drawbacks such as high toxicity, administration by parenteral route, and most seriously the resistance of some strains of the parasite to them. Several strategies have been used to increase the therapeutic index and reduce the toxic effects of these drugs. Among them, the use of nanosystems that have great potential as a site-specific drug delivery system stands out. This review aims to compile results from studies that were carried out using first- and second-line antileishmanial drug-carrying nanosystems. The articles referred to here were published between 2011 and 2021. This study shows the promise of effective applicability of drug-carrying nanosystems in the field of antileishmanial therapeutics, with the perspective of providing better patient adherence to treatment, increased therapeutic efficacy, reduced toxicity of conventional drugs, as well as the potential to efficiently improve the treatment of leishmaniasis.

Paromomycin and Miltefosine Combination as an Alternative to Treat Patients With Visceral Leishmaniasis in Eastern Africa: A Randomized, Controlled, Multicountry Trial.

BACKGROUND: This study aimed to determine whether paromomycin plus miltefosine (PM/MF) is noninferior to sodium stibogluconate plus paromomycin (SSG/PM) for treatment of primary visceral leishmaniasis in eastern Africa. METHODS: An open-label, phase 3, randomized, controlled trial was conducted in adult and pediatric patients at 7 sites in eastern Africa. Patients were randomly assigned to either 20 mg/kg paromomycin plus allometric dose of miltefosine (14 days), or 20 mg/kg sodium stibogluconate plus 15 mg/kg paromomycin (17 days). The primary endpoint was definitive cure after 6 months. RESULTS: Of 439 randomized patients, 424 completed the trial. Definitive cure at 6 months was 91.2% (155 of 170) and 91.8% (156 of 170) in the PM/MF and SSG/PM arms in primary efficacy modified intention-to-treat analysis (difference, 0.6%; 97.5% confidence interval [CI], -6.2 to 7.4), narrowly missing the noninferiority margin of 7%. In the per-protocol analysis, efficacy was 92% (149 of 162) and 91.7% (155 of 169) in the PM/MF and SSG/PM arms (difference, -0.3%; 97.5% CI, -7.0 to 6.5), demonstrating noninferiority. Treatments were well tolerated. Four of 18 serious adverse events were study drug-related, and 1 death was SSG-related. Allometric dosing ensured similar MF exposure in children (<12 years) and adults. CONCLUSIONS: PM/MF and SSG/PM efficacies were similar, and adverse drug reactions were as expected given the drugs safety profiles. With 1 less injection each day, reduced treatment duration, and no risk of SSG-associated life-threatening cardiotoxicity, PM/MF is a more patient-friendly alternative for children and adults with primary visceral leishmaniasis in eastern Africa. CLINICAL TRIALS REGISTRATION: NCT03129646.

RNA Chemical Labeling with Site-Specific, Relative Quantification by Mass Spectrometry for the Structural Study of a Neomycin-Sensing Riboswitch Aptamer Domain.

High-resolution mass spectrometry was used for the label-free, direct localization and relative quantification of CMC+ -modifications of a neomycin-sensing riboswitch aptamer domain in the absence and presence of the aminoglycoside ligands neomycin B, ribostamycin, and paromomycin. The chemical probing and MS data for the free riboswitch show high exposure to solvent of the uridine nucleobases U7, U8, U13, U14, U18 as part of the proposed internal and apical loops, but those of U10 and U21 as part of the proposed internal loop were found to be far less exposed than expected. Thus, our data are in better agreement with the proposed secondary structure of the riboswitch in complexes with aminoglycosides than with that of free RNA. For the riboswitch in complexes with neomycin B, ribostamycin, and paromomycin, we found highly similar CMC+ -modification patterns and excellent agreement with previous NMR studies. Differences between the chemical probing and MS data in the absence and presence of the aminoglycoside ligands were quantitative rather than qualitative (i. e., the same nucleobases were labeled, but to different extents) and can be rationalized by stabilization of both the proposed bulge and the apical loop by aminoglycoside binding. Our study shows that chemical probing and mass spectrometry can provide important structural information and complement other techniques such as NMR spectroscopy.

Triple-combination therapy for cutaneous leishmaniasis using detergent-free, hyaluronate-coated elastic nanovesicles.

Aim: To develop and evaluate detergent-free, triple-drug-loaded, hyaluronate-coated elastic nanovesicles (H-ENVs) for the topical treatment of cutaneous leishmaniasis. Materials & methods: H-ENVs were developed and evaluated for vesicle size, entrapment efficiency, skin permeation and antileishmanial potential. Results: A 15.7 and 28.6% decrease in the cytotoxicity of paromomycin and amphotericin B, respectively, was observed in detergent-free ENVs compared with conventional ENVs. H-ENVs improved the efficacy of paromomycin against promastigote and amastigote models of leishmaniasis by 4- and 7.5-fold, respectively. In vivo investigation of H-ENVs demonstrated efficient topical management of cutaneous leishmaniasis. Conclusion: The results indicate the potential of H-ENVs as a safe topical treatment choice for cutaneous leishmaniasis.

Application of topical gel is an attractive alternative to oral or intravenous administration of drugs and is likely to deliver a higher dose of the drug to the target site with only rare systemic adverse effects. Nanotechnology-based topical drug delivery is an attractive aspect of pharmaceutical sciences that expresses interest in the topical treatment of cutaneous leishmaniasis. The authors' research focuses on the development and evaluation of novel multidrug-loaded, detergent-free nanovesicles for the simple and effective topical treatment of cutaneous leishmaniasis.

Development and validation of an ultra-high performance liquid chromatography coupled to tandem mass spectrometry method for the quantification of the antileishmanial drug paromomycin in human skin tissue.

Bioanalytical assay development and validation procedures were performed to quantify antiprotozoal drug paromomycin in human skin tissue by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Paromomycin, an aminoglycoside drug, is administered intra-muscularly and used in the treatment of multiple clinical presentations of the neglected tropical disease leishmaniasis. It is currently studied in the treatment of post-kala-azar dermal leishmaniasis, a disease where the Leishmania parasites divide and reside in the skin. We present a target-site bioanalytical method to accurately quantify paromomycin in human skin tissue, with the clinical purpose of quantifying paromomycin in skin biopsies from post-kala-azar dermal leishmaniasis patients originating from Sudan. Enzymatic digestion using collagenase A incubated at 37 °C overnight was employed as homogenization method to produce skin tissue homogenates. Further sample preparation was performed by protein precipitation using trichloroacetic acid and a dilution step. Final extracts were injected onto a C18 analytical column and isocratic heptafluorobutyric acid ion-pair separation and elution were employed. The chromatography system was coupled to a triple quadrupole mass spectrometer for detection. The method was validated in digestion solution over a linear range from 5 to 1000 ng/mL (r2 ≥ 0.9967) with the assay performance of accuracy and precision within acceptable criteria values as stated by the EMA guidelines. Furthermore, matrix effects were observed in human skin tissue and were corrected by the multiple deuterated paromomycin internal standard. No substantial IS-normalized matrix effect was detected along with relatively high sample preparation recovery. Consequently, digestion solution matrix serving as the preparation of calibration standards can be used as surrogate matrix for human skin tissue, which is convenient given the limited availability of control matrix. Finally, paromomycin was accurately quantified in skin of post-kala-azar dermal leishmaniasis patients originating from clinical trials in Sudan.

Genome-wide analysis reveals allelic variation and chromosome copy number variation in paromomycin-resistant Leishmania donovani.

In the absence of adequate diagnosis and treatment, leishmaniasis remains a major public health concern on a global scale. Drug resistance remains a key obstacle in controlling and eliminating visceral leishmaniasis. The therapeutic gap due to lack of target-specific medicine and vaccine can be minimized by obtaining parasite's genomic information. This study compared whole-genome sequence of paromomycin-resistant parasite (K133PMM) developed through in vitro adaptation and selection with sensitive Leishmania clinical isolate (K133WT). We found a large number of upstream and intergenic gene variations in K133PMM. There were 259 single nucleotide polymorphisms (SNPs), 187 insertion-deletion (InDels), and 546 copy number variations (CNVs) identified. Most of the genomic variations were found in the gene's upstream and non-coding regions. Ploidy estimation revealed chromosome 5 in tetrasomy and 6, 9, and 12 in trisomy, uniquely in K133PMM. These contain the genes for protein degradation, parasite motility, autophagy, cell cycle maintenance, and drug efflux membrane transporters. Furthermore, we also observed reduction in ploidy of chromosomes 15, 20, and 23, in the resistant parasite containing mostly the genes for hypothetical proteins and membrane transporters. We chronicled correlated genomic conversion and aneuploidy in parasites and hypothesize that this led to rapid evolutionary changes in response to drug induced pressure, which causes them to become resistant.

Antibiotic resistance genes and bacterial diversity: A comparative molecular study of treated sewage from different origins and their impact on irrigated soils.

Present study aims to investigate how is soil affected following irrigation with treated effluents of different origins by analysing the bacterial diversity, metabolic diversity and antibiotic resistance genes (ARGs). Comparative analysis with previously reported ARGs in effluents was performed to understand the mobility of ARGs from treated wastewater to the irrigated soil with respect to the control soil regimen. Acinetobacter, Burkholderia and Pseudomonas were observed as the most abundant genera in all the samples. The metabolic gene abundance of all the samples suggests a prominent contribution to natural mineral recycling. Most abundant ARGs observed encode resistance for clindamycin, kanamycin A, macrolides, paromomycin, spectinomycin and tetracycline. Treated effluent reuse did not appear to enhance the ARG levels in soils in most cases except for institutional treatment site (M), where the ARGs for aminoglycosides, ß-lactams and sulfonamides were found to be abundantly present in both treated effluent and the irrigated soil. This study finds the importance of wastewater treatment from different origins and the impact of treated wastewater reuse in irrigation. This study also emphasises on the better understanding of ARGs mobility from water to soil.

Isolation, typing, and drug susceptibility of Leishmania (Leishmania) infantum isolates from dogs of the municipality of Embu das Artes, an endemic region for canine leishmaniasis in Brazil.

The parasitic protozoa Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis and canine leishmaniasis in South America, where Brazil is the most affected country. This zoonotic disease is transmitted by the bite of an infected phlebotomine sand fly and dogs constitute the main domestic reservoir of the parasite. In this study, we screened 2348 dogs of the municipality of Embu das Artes, Brazil, for antibodies against the parasite. Prevalence for canine leishmaniasis seropositivity was 2.81%, as assessed using a Dual-Path Platform rapid test for canine leishmaniasis. Twenty-five seropositive dogs were euthanized for parasite isolation and 14 isolates were successful obtained. Nucleotide sequencing of the internal transcribed spacer confirmed the isolates to be L. (L.) infantum, and very low sequence variability was observed among them. The in vitro susceptibility to miltefosine and paromomycin was assessed and moderate variation in paromomycin susceptibility was found among the isolates in the promastigote and intracellular amastigote stages. On the other hand, in vitro susceptibility to miltefosine of these isolates was homogenous, particularly in the amastigote stage (EC50 values from 0.69 to 2.07 µM). In addition, the miltefosine sensitivity locus was deleted in all the isolates, which does not corroborate the hypothesis that the absence of this locus is correlated with a low in vitro susceptibility. Our findings confirm that the municipality of Embu das Artes is endemic for canine leishmaniasis and that isolates from this region are susceptible to paromomycin and miltefosine, indicating the potential of these drugs to be clinically evaluated in the treatment of human visceral leishmaniasis in Brazil.