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Quantitative monitoring of experimental and human leishmaniasis employing amastigote-specific genes.Parasitology Jul 2022The gold standard for diagnosis of leishmaniasis is the microscopic detection of amastigotes/Leishman Donovan (LD) bodies, but its moderate sensitivity necessitates the...
The gold standard for diagnosis of leishmaniasis is the microscopic detection of amastigotes/Leishman Donovan (LD) bodies, but its moderate sensitivity necessitates the development of molecular approaches. This study aimed to quantify in experimental animal models and human leishmaniasis the expression of amastigote-specific virulence genes, and by droplet digital polymerase chain reaction (ddPCR). Total RNA was isolated from -infected hamsters or murine peritoneal macrophages and lesional biopsies from patients with post kala-azar dermal leishmaniasis (PKDL). Following cDNA conversion, EvaGreen-based ddPCR was performed using specific primers for or and parasite load expressed in copies per L. Assay was optimized and the specificity of amastigote-specific and was confirmed. In hepatic and splenic tissues of -infected hamsters and peritoneal macrophages, ddPCR demonstrated a greater abundance of than . Treatment of -infected peritoneal macrophages with conventional anti-leishmanials, miltefosine and amphotericin B translated into a dose-dependent reduction in copies per L of and , and the extrapolated IC was comparable with results obtained by counting LD bodies in Giemsa-stained macrophages. Similarly, in dermal biopsies of patients with PKDL, and were detected. Overall, monitoring of by ddPCR can be an objective measure of parasite burden and potentially adaptable into a high throughput approach necessary for drug development and monitoring disease progression when the causative species is .
Topics: Amphotericin B; Animals; Humans; Leishmania donovani; Leishmaniasis, Cutaneous; Leishmaniasis, Visceral; Mice; Parasite Load
PubMed: 35535469
DOI: 10.1017/S0031182022000610 -
Microbiology Spectrum Oct 2021The high morbidity and mortality of cryptococcal meningitis is due to the limited range of therapeutic options: only three classes of antifungal drugs are available...
The high morbidity and mortality of cryptococcal meningitis is due to the limited range of therapeutic options: only three classes of antifungal drugs are available (polyenes [amphotericin B], azoles [fluconazole], and pyrimidine analogues [flucytosine]). Fluconazole is the most widely used antifungal drug in sub-Saharan Africa, where cryptococcal meningitis is a major cause of death in patients infected with HIV. In this study, we found that exposure to fluconazole, even for short times (48 h) at subinhibitory concentrations, drove rapid adaptation of Cryptococcus neoformans serotype A strain H99 via the acquisition of different aneuploid chromosomes. These aneuploidies conferred heteroresistance to fluconazole. Importantly, most of the adaptors were cross-tolerant to flucytosine. Some of the aneuploid adaptors were not heteroresistant to fluconazole but were tolerant to amphotericin B. Thus, exposure to one antifungal drug class can promote adaptation to two antifungal drug classes, highlighting the plasticity of the C. neoformans genome and raising concerns about the rapid reduction in the range of treatment options for cryptococcal infections. Cryptococcosis is a globally distributed invasive fungal infection caused by infections with Cryptococcus neoformans or Cryptococcus gattii. Only three classes of therapeutic drugs are clinically available for treating cryptococcosis: polyenes (amphotericin B), azoles (fluconazole), and pyrimidine analogues (flucytosine). Fluconazole is the primary drug available in resource-limited countries. Aneuploidy is a genomic state due to the gain or loss of chromosomes. We found that C. neoformans rapidly adapted to fluconazole by acquiring diverse aneuploidies and that specific aneuploidies enabled improved growth of isolates susceptible (tolerance) to amphotericin B and/or cross-tolerance to both fluconazole and flucytosine. Therefore, aneuploidy is an underlying mechanism of drug tolerance that not only arises rapidly during growth in fluconazole but can also confer tolerance to other antifungal drugs without prior exposure to those drugs. Resistant isolates have high MICs, and all cells grow similarly in medium with the drug, while tolerant isolates test as susceptible and grow slowly at drug concentrations above the MIC.
Topics: Adaptation, Physiological; Amphotericin B; Aneuploidy; Antifungal Agents; Cryptococcosis; Cryptococcus gattii; Cryptococcus neoformans; Drug Resistance, Fungal; Fluconazole; Flucytosine; Humans; Microbial Sensitivity Tests; Serogroup
PubMed: 34585947
DOI: 10.1128/Spectrum.00723-21 -
Journal of Investigative Medicine High... 2020Coccidioidomycosis is an infection caused by inhalation of arthroconidia produced by dimorphic fungi in the genus . Forty percent of patients will develop an...
Coccidioidomycosis is an infection caused by inhalation of arthroconidia produced by dimorphic fungi in the genus . Forty percent of patients will develop an influenza-like illness with symptoms suggestive of a mild and self-limited respiratory infection; however, 5% of these individuals will develop extrapulmonary disseminated disease. An immunocompromised patient presented with right upper quadrant pain, ultrasound with pericholecystic fluid, in which a percutaneous cholecystostomy contained biliary fluid that grew the fungus . Patient was initiated on intravenous amphotericin therapy and was followed closely with postoperative bile drainage with eventual laparoscopic cholecystectomy. We present a very rare case of disseminated coccidioidomycosis to the gallbladder.
Topics: Administration, Intravenous; Amphotericin B; Cholecystectomy, Laparoscopic; Cholecystitis; Coccidioides; Coccidioidomycosis; Drainage; Humans; Immunocompromised Host; Male; Middle Aged; Spores, Fungal; Treatment Outcome
PubMed: 32131637
DOI: 10.1177/2324709620910636 -
Viruses Sep 2022Several flaviviruses such as Hepatitis C virus, West Nile virus, Dengue virus and Japanese Encephalitis virus exploit the raft platform to enter host cells whereas the...
Several flaviviruses such as Hepatitis C virus, West Nile virus, Dengue virus and Japanese Encephalitis virus exploit the raft platform to enter host cells whereas the involvement of lipid rafts in Zika virus-host cell interaction has not yet been demonstrated. Zika virus disease is caused by a flavivirus transmitted by spp. Mosquitoes, although other mechanisms such as blood transfusion, sexual and maternal-fetal transmission have been demonstrated. Symptoms are generally mild, such as fever, rash, joint pain and conjunctivitis, but neurological complications, including Guillain-Barré syndrome, have been associated to this viral infection. During pregnancy, it can cause microcephaly and other congenital abnormalities in the fetus, as well as pregnancy complications, representing a serious health threat. In this study, we show for the first time that Zika virus employs cell membrane lipid rafts as a portal of entry into Vero cells. We previously demonstrated that the antifungal drug Amphotericin B (AmphB) hampers a microbe-host cell interaction through the disruption of lipid raft architecture. Here, we found that Amphotericin B by the same mechanism of action inhibits both Zika virus cell entry and replication. These data encourage further studies on the off-label use of Amphotericin B in Zika virus infections as a new and alternate antiviral therapy.
Topics: Amphotericin B; Animals; Antifungal Agents; Antiviral Agents; Chlorocebus aethiops; Female; Flavivirus; Humans; Membrane Lipids; Membrane Microdomains; Pregnancy; Vero Cells; Zika Virus; Zika Virus Infection
PubMed: 36146865
DOI: 10.3390/v14092059 -
International Journal For Parasitology.... Dec 2019The development of an effective amphotericin B (AmB) topical formulation to replace the systemically toxic injections currently used in cutaneous leishmaniasis (CL)...
The development of an effective amphotericin B (AmB) topical formulation to replace the systemically toxic injections currently used in cutaneous leishmaniasis (CL) treatment is challenging due to poor absorption through the skin. Aiming at an effective local chemotherapy, we designed PLGA (poly(lactide-co-glycolide acid) microparticles loaded with deoxycholate amphotericin B (d-AmB) for both macrophage intracellular targeting and sustained extracellular release. For that, d-AmB/PLGA microparticles with sizes ranging from 0.5 μm to 20 μm were synthesized and tested both in vitro and in vivo. In vitro, d-AmB/PLGA was more selectively active against intracellular amastigotes of Leishmania amazonensis than free d-AmB (selectivity index = 50 and 25, respectively). In vivo, the efficacy of a single intralesional (i.l) injection with d-AmB/PLGA was determined in early and established BALB/c mouse ear lesions. In early lesions, a single injection given on day 10 of infection was more effective in controlling parasite growth than eight i.l. injections with free d-AmB, as measured on day 120. Such d-AmB/PLGA injection was also effective in established lesions (day 30), leading to 97% parasite burden reduction, as compared with d-AmB or liposomal AmB (Ambisome®) i.l. injection containing the same AmB dose. Pharmacokinetic studies showed that following d-AmB/PLGA injection, AmB leaked slower from non-infected than infected ears, yet remaining in the ear tissue for as long as 30 days. Of interest, AmB was not detectable in the circulating plasma for at least two weeks of d-AmB/PLGA injection, contrasting with the rapid and durable (2 days) detection after free d-AmB injection. Despite the transient ear swelling and local cell infiltration, no alterations in AST, ALT and creatinine serum levels was induced by d-AmB/PLGA. For its approved components, local efficacy, and single-dose applicability, this novel and safe AmB microparticle depot formulation has strong potential as a new therapy for human CL.
Topics: Amphotericin B; Animals; Antiprotozoal Agents; Deoxycholic Acid; Drug Combinations; Drug Delivery Systems; Ear; Leishmaniasis, Cutaneous; Male; Mice; Mice, Inbred BALB C; Nanoparticles; Polyglactin 910
PubMed: 31331828
DOI: 10.1016/j.ijpddr.2019.06.001 -
PloS One 2022The resistance and the birth of new intrinsic and multidrug-resistant pathogenic species like C. auris is creating great concern in the antifungal world. Given the...
The resistance and the birth of new intrinsic and multidrug-resistant pathogenic species like C. auris is creating great concern in the antifungal world. Given the limited drug arsenal and the lack of effectiveness of the available compounds, there is an urgent need for innovative approaches. The murine mAb 2G8 was humanized and engineered in silico to develop a single-chain fragment variable (hscFv) antibody against β-1,3-glucans which was then expressed in E. coli. Among the recombinant proteins developed, a soluble candidate with high stability and affinity was obtained. This selected protein is VL-linker-VH oriented, and it is characterized by the presence of two ubiquitin monomers at the N-terminus and a His tag at the C-terminus. This construct, Ub2-hscFv-His, guaranteed stability, solubility, efficient purification and satisfactory recovery of the recombinant product. HscFv can bind β-1,3-glucans both as coated antigens and on C. auris and C. albicans cells similarly to its murine parental and showed long stability and retention of binding ability when stored at 4°, -20° and -80° C. Furthermore, it was efficient in enhancing the antifungal activity of drugs caspofungin and amphotericin B against C. auris. The use of biological drugs as antifungals is limited; here we present a promising hscFv which has the potential to be useful in combination with currently available antifungal drugs.
Topics: Mice; Animals; Antifungal Agents; Escherichia coli; Amphotericin B; Mycoses; Glucans; Microbial Sensitivity Tests
PubMed: 36315567
DOI: 10.1371/journal.pone.0276786 -
Journal of Clinical Microbiology Aug 2021The global incidence of mucormycosis has increased in recent years owing to higher numbers of individuals at risk for these infections. The diagnosis and treatment of...
The global incidence of mucormycosis has increased in recent years owing to higher numbers of individuals at risk for these infections. The diagnosis and treatment of this aggressive fungal infection are of clinical concern due to differences in species distribution in different geographic areas and susceptibility profiles between different species that are capable of causing highly aggressive infections. The purpose of this study was to evaluate the epidemiology and susceptibility profiles of Mucorales isolates in the United States over a 52-month period. Species identification was performed by combined phenotypic characteristics and DNA sequence analysis, and antifungal susceptibility testing was performed by CLSI M38 broth microdilution for amphotericin B, isavuconazole, itraconazole, and posaconazole. During this time frame, 854 isolates were included, representing 11 different genera and over 26 species, of which (58.6%) was the predominant genus, followed by (19.6%). The majority of isolates were cultured from the upper and lower respiratory tracts (55%). Amphotericin B demonstrated the most potent activity, with geometric mean (GM) MICs of ≤0.25 μg/ml against all genera with the exception of species (GM MIC of 1.30 μg/ml). In head-to-head comparisons, the most active azole was posaconazole, followed by isavuconazole. Differences in azole and amphotericin B susceptibility patterns were observed between the genera with the greatest variability observed with isavuconazole. Awareness of the epidemiology of Mucorales isolates and differences in antifungal susceptibility patterns in the United States may aide clinicians in choosing antifungal treatment regimens. Further studies are warranted to correlate these findings with clinical outcomes.
Topics: Amphotericin B; Antifungal Agents; Fungi; Humans; Itraconazole; Microbial Sensitivity Tests; Mucorales; Mucormycosis; United States
PubMed: 34232068
DOI: 10.1128/JCM.01230-21 -
Scientific Reports Dec 2022In the present work, single wall carbon nanotubes (SWCNT) were successively functionalized with phospholipid DSPE-PEG carboxylic acid, and then, with ethylenediamine...
In the present work, single wall carbon nanotubes (SWCNT) were successively functionalized with phospholipid DSPE-PEG carboxylic acid, and then, with ethylenediamine (EDA), to obtain double functionalized single wall carbon nanotube (DFSWCNT). Then, DFSWCNT was applied as a carrier for delivering amphotericin B (Amb) and EGFP plasmid. FSWCNT's concentration obtained via UV-visible analysis was 0.99 mg/mL. The TGA analysis results provided the lost weights of DSPE-PEG-COOH, EDA, Amb and SWCNT impurities. XPS results showed that carbon atoms' percentage decreased during the functionalization processes from 97.2% (SWCNT) to 76.4% (FSWCNT) and 69.9% (DFSWNCT). Additionally, the oxygen atoms' percentage increased from 2.3% (SWCNT) to 21% and 22.5% for FSWCNT and DFSWCNT, respectively. New bonds such as C-N and N-C=O appeared in the synthesized nanocarrier. The I/I ratio in Raman analysis decreased from 7.15 (SWCNT) to 4.08 (FSWCNT). The amount of Amb released to phosphate buffer saline medium was about 33% at pH = 5.5 and 75% at pH = 7.4 after 48 h. CCK8 results confirmed that the toxicity of functionalized SWCNT had decreased. In a 2:1 ratio of DFSWCNT/EGFP plasmid, the cell viability (87%) and live transfected cells (56%) were at their maximum values. The results indicate that carbon nanotubes have the potential to be applied as drug/gene delivery systems with outstanding properties such as high loading capacity and easy penetration to cell membrane.
Topics: Nanotubes, Carbon; Amphotericin B
PubMed: 36476955
DOI: 10.1038/s41598-022-25222-1 -
Molecules (Basel, Switzerland) Jun 2023During the COVID-19 pandemic, the treatment of pulmonary fungal infection faced noteworthy challenges. Amphotericin B has shown promising therapeutic effects as an...
During the COVID-19 pandemic, the treatment of pulmonary fungal infection faced noteworthy challenges. Amphotericin B has shown promising therapeutic effects as an inhalation treatment for pulmonary fungal infections, especially those associated with the COVID-19 virus, due to its rare resistance. However, because the drug frequently produces renal toxicity, its effective dose is limited in clinical use. In this work, the DPPC/DPPG mixed monolayer was used as the pulmonary surfactant monolayer to study the interaction between amphotericin B and the pulmonary surfactant monolayer during inhalation therapy using the Langmuir technique and atomic force microscopy. The effects of different molar ratios of AmB on the thermodynamic properties and surface morphology of the pulmonary surfactant monolayer at different surface pressures was evaluated. The results showed that when the molar ratio of AmB to lipids in the pulmonary surfactant was less than 1:1, the main intermolecular force was attractive at a surface pressure greater than 10 mN/m. This drug had little effect on the phase transition point of the DPPC/DPPG monolayer, but decreased the height of the monolayer at 15 mN/m and 25 mN/m. When the molar ratio of AmB to lipids was greater than 1:1, the intermolecular force was mainly repulsive at a surface pressure greater than 15 mN/m, and AmB increased the height of the DPPC/DPPG monolayer at both 15 mN/m and 25 mN/m. These results are helpful in understanding the interaction between the pulmonary surfactant model monolayer and different doses of drugs at various surface tensions during respiration.
Topics: Humans; Pulmonary Surfactants; Amphotericin B; 1,2-Dipalmitoylphosphatidylcholine; Pandemics; COVID-19; Respiration; Surface Properties
PubMed: 37375395
DOI: 10.3390/molecules28124840 -
PLoS Neglected Tropical Diseases Aug 2019Amphotericin B provides improved therapy for visceral leishmaniasis (VL) caused by Leishmania donovani, with single dose liposomal-encapsulated Ambisome providing the... (Comparative Study)
Comparative Study
Amphotericin B provides improved therapy for visceral leishmaniasis (VL) caused by Leishmania donovani, with single dose liposomal-encapsulated Ambisome providing the best cure rates. The VL elimination program aims to reduce the incidence rate in the Indian subcontinent to <1/10,000 population/year. Ability to predict which asymptomatic individuals (e.g. anti-leishmanial IgG and/or Leishmania-specific modified Quantiferon positive) will progress to clinical VL would help in monitoring disease outbreaks. Here we examined whole blood transcriptional profiles associated with asymptomatic infection, active disease, and in treated cases. Two independent microarray experiments were performed, with analysis focussed primarily on differentially expressed genes (DEGs) concordant across both experiments. No DEGs were identified for IgG or Quantiferon positive asymptomatic groups compared to negative healthy endemic controls. We therefore concentrated on comparing concordant DEGs from active cases with all healthy controls, and in examining differences in the transcriptome following different regimens of drug treatment. In these comparisons 6 major themes emerged: (i) expression of genes and enrichment of gene sets associated with erythrocyte function in active cases; (ii) strong evidence for enrichment of gene sets involved in cell cycle in comparing active cases with healthy controls; (iii) identification of IFNG encoding interferon-γ as the major hub gene in concordant gene expression patterns across experiments comparing active cases with healthy controls or with treated cases; (iv) enrichment for interleukin signalling (IL-1/3/4/6/7/8) and a prominent role for CXCL10/9/11 and chemokine signalling pathways in comparing active cases with treated cases; (v) the novel identification of Aryl Hydrocarbon Receptor signalling as a significant canonical pathway when comparing active cases with healthy controls or with treated cases; and (vi) global expression profiling support for more effective cure at day 30 post-treatment with a single dose of liposomal encapsulated amphotericin B compared to multi-dose non-liposomal amphotericin B treatment over 30 days. (296 words; 300 words allowed).
Topics: Adolescent; Adult; Aged; Amphotericin B; Antiprotozoal Agents; Asymptomatic Diseases; Biomarkers; Blood Cells; Child; Female; Gene Expression Profiling; Humans; India; Leishmaniasis, Visceral; Male; Microarray Analysis; Middle Aged; Transcriptome; Young Adult
PubMed: 31419223
DOI: 10.1371/journal.pntd.0007673