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American tegumentary leishmaniasis (ATL) is highly endemic in the Amazon basin and occurs in all South American countries, except Chile and Uruguay. Most Brazilian ATL cases are due to Leishmania (Viannia) braziliensis, however other neglected Amazonian species are being increasingly reported. They belong to the subgenus L. (Viannia) and information on suitable models to understand immunopathology are scarce. Here, we explored the use of the golden hamster Mesocricetus auratus and its macrophages as a model for L. (Viannia) species. We also studied the interaction of parasite glycoconjugates (LPGs and GIPLs) in murine macrophages. The following strains were used: L. (V.) braziliensis (MHOM/BR/2001/BA788), L. (V.) guyanensis (MHOM/BR/85/M9945), L. (V.) shawi (MHOM/BR/96/M15789), L. (V.) lindenbergi (MHOM/BR/98/M15733) and L. (V.) naiffi (MDAS/BR/79/M5533). In vivo infections were initiated by injecting parasites into the footpad and were followed up at 20- and 40-days PI. Parasites were mixed with salivary gland extract (SGE) from wild-captured Nyssomyia neivai prior to in vivo infections. Animals were euthanized for histopathological evaluation of the footpads, spleen, and liver. The parasite burden was evaluated in the skin and draining lymph nodes. In vitro infections used resident peritoneal macrophages and THP-1 monocytes infected with all species using a MOI (1:10). For biochemical studies, glycoconjugates (LPGs and GIPLs) were extracted, purified, and biochemically characterized using fluorophore-assisted carbohydrate electrophoresis (FACE). They were functionally evaluated after incubation with macrophages from C57BL/6 mice and knockouts (TLR2-/- and TLR4-/-) for nitric oxide (NO) and cytokine/chemokine production. All species, except L. (V.) guyanensis, failed to generate evident macroscopic lesions 40 days PI. The L. (V.) guyanensis lesions were swollen but did not ulcerate and microscopically were characterized by an intense inflammatory exudate. Despite the fact the other species did not produce visible skin lesions there was no or mild pro-inflammatory infiltration at the inoculation site and parasites survived in the hamster skin/lymph nodes and even visceralized. Although none of the species caused severe disease in the hamster, they differentially infected peritoneal macrophages in vitro. LPGs and GIPLs were able to differentially trigger NO and cytokine production via TLR2/TLR4 and TLR4, respectively. The presence of a sidechain in L. (V.) lainsoni LPG (type II) may be responsible for its higher proinflammatory activity. After Principal Component analyses using all phenotypic features, the clustering of L. (V.) lainsoni was separated from all the other L. (Viannia) species. We conclude that M. auratus was a suitable in vivo model for at least four dermotropic L. (Viannia) species. However, in vitro studies using peritoneal cells are a suitable alternative for understanding interactions of the six L. (Viannia) species used here. LRV1 presence was found in L. (V.) guyanensis and L. (V.) shawi with no apparent correlation with virulence in vitro and in vivo. Finally, parasite glycoconjugates were able to functionally trigger various innate immune responses in murine macrophages via TLRs consistent with their inflammatory profile in vivo., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Soares et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Leishmania (Mundinia) enriettii is a species commonly found in the guinea pig, Cavia porcellus. Although it is a dermotropic species, there is still an uncertainty regarding its ability to visceralise during Leishmania life cycle., Objective: Here, we investigated the ability of L. enriettii (strain L88) to visceralise in lungs, trachea, spleen, and liver of C. porcellus, its natural vertebrate host., Methods: Animals were infected sub-cutaneously in the nose and followed for 12 weeks using histological (hematoxilin-eosin) and molecular tools (polymerase chain reaction-restriction fragment length polymorphism - PCR-RFLP). To isolate parasite from C. porcellus, animals were experimentally infected for viscera removal and PCR typing targeting hsp70 gene., Findings: Histological analysis revealed intense and diffuse inflammation with the presence of amastigotes in the trachea, lung, and spleen up to 12 weeks post-infection (PI). Molecular analysis of paraffin-embedded tissues detected parasite DNA in the trachea and spleen between the 4th and 8th weeks PI. At the 12th PI, no parasite DNA was detected in any of the organs. To confirm that the spleen could serve as a temporary site for L. enriettii, we performed additional in vivo experiments. During 6th week PI, the parasite was isolated from the spleen confirming previous histopathological and PCR observations., Main Conclusion: Leishmania enriettii (strain L88) was able to visceralise in the trachea, lung, and spleen of C. porcellus.

Objectives: To analyse the accuracy of American tegumentary leishmaniasis (ATL) diagnostic methods and evaluate the quality of the existing publications by means of a systematic review., Methods: Diagnostic tests evaluated in at least two studies with common reference standards were included in the sensitivity and/or specificity meta-analyses. Quality and susceptibility to bias were analysed using the QUADAS-2 and STARD tools., Results: The title and abstract of 3387 publications were evaluated after deduplication resulting from database searches. Thirty-eight studies were included in the review, and 26 of them had results inserted in meta-analyses. The diagnostic methods with the highest pooled sensitivity values were ELISA, polymerase chain reaction (PCR), indirect immunofluorescence reaction and Montenegro's intradermal reaction. Cytometry was assessed in only two studies and presented 100% sensitivity in both. Smear slide microscopy and histopathology showed low pooled values of sensitivity. For specificity, the highest pooled values were identified for PCR. High values were also identified for ELISA, except for studies in which the reference standard for defining negative participants included individuals with Chagas' disease or paracoccidioidomycosis, which also occurred for cytometry. IFR had lower specificities than ELISA. There was a predominance of case-control designs of phase 1 or 2 and only four studies were strongly recommended as evidence generators. Several reference standards were adopted, and different methods were assessed in a small number of studies., Conclusion: PCR showed the highest accuracy for the diagnosis of ATL, and its use should be encouraged in clinical practice. ELISA is recommended for the screening of suspected individuals, but the possibility of cross-reactions should be considered. New validation studies for the tests evaluated in few publications and studies of phase 3 with appropriate methods are needed., (© 2020 John Wiley & Sons Ltd.)

A procedure involving hollow fiber microporous membrane liquid-liquid extraction (HF-MMLLE) and detection by liquid chromatography with tandem mass spectrometry was developed and applied. The extraction is based on liquid-liquid microextraction with a polypropylene porous membrane as a solid support for the solvent. On the membrane walls the solvent forms a renewable liquid membrane which improves the trueness of the method and promotes the sample clean-up. The applicability of this method was evaluated through the simultaneous extraction of 18 pesticides of different classes: polar organophosphates, carbamates, neonicotinoids, amides, pyrimidines, benzimidazoles and triazoles in industrial and fresh orange juice. The parameters affecting the extraction efficiency were optimized by multivariable designs. Under optimized conditions, analytes were concentrated onto 1.5 cm long microporous membranes placed directly into the sample containing 9mL of juice at pH 7.0, 4 g of ammonium sulfate and 400 μL of toluene:ethyl acetate (85:15, v/v). The best extraction conditions were achieved at 25°C with 35 min of extraction time. The analyte desorption was carried out using 50 μL of methanol:acetone (50:50, v/v) for 2 min in an ultrasonic bath. Limits of detection ranging between 0.003-0.33 mgL(-1), 0.003-0.35 mg L(-1) and 0.003-0.15 mgL(-1) were obtained for the carton orange juice, carton light orange juice and fresh orange juice samples, respectively. Good repeatability (lower than 7.6%) was obtained for all three sample types. The method was applied to five different juice samples containing soybean extract, orange pulp, nectar, light juice and fresh orange juice. The results suggest that the proposed method represents a very simple and low-cost alternative microextraction procedure rendering adequate limits of quantification for the determination of these pesticides in juice samples., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A sensitive and precise analysis using hollow fiber renewal liquid membrane (HFRLM) extraction followed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described for determination of five sulfonamides in honey samples. In this procedure, the organic solvent introduced directly into the sample matrix extracts the sulfonamides and carries them over the polypropylene porous membrane. An organic solvent is immobilized inside the polypropylene porous membrane, leading to a homogeneous phase. The stripping phase at higher pH in the lumen of the membrane promotes the ionization of the target compounds releasing them to this phase. The most important parameters affecting the extraction efficiency were optimized by multivariable designs (pH and sample mass, pH and buffer for stripping phase, extraction temperature and time, type and volume of extractor solvent and use of salt to saturate the sample). Detection limits in the range of 5.1-27.4 μg kg(-1) and linearity coefficient of correlation higher than 0.987 were obtained for the target analytes. The results obtained for the proposed method show that HFRLM-LC-MS/MS can be used for determination of the five sulfonamides studied in honey samples with excellent precision, accuracy, practicality and short analysis time., (Copyright © 2010 Elsevier B.V. All rights reserved.)

This paper describes fundamentals and applications of multivariate statistical techniques for the optimization of chromatographic systems. The surface response methodologies: central composite design, Doehlert matrix and Box-Behnken design are discussed and applications of these techniques for optimization of sample preparation steps (extractions) and determination of experimental conditions for chromatographic separations are presented. The use of mixture design for optimization of mobile phases is also related. An optimization example involving a real separation process is exhaustively described. A discussion about model validation is presented. Some applications of other multivariate techniques for optimization of chromatographic methods are also summarized.

Chromatographic evaluations of a C18 dimethylurea phase in 150 mm x 3.9 mm HPLC columns were performed using the Tanaka and Engelhardt test mixtures. The applicability of the new C18 dimethylurea phase was also evaluated with a mixture of some herbicides and their metabolites. An artificial aging procedure was also performed by passing a potassium phosphate mobile phase buffered at pH 7.0 through C18 50 mm x 3.9 mm dimethylurea columns. The column stability was evaluated by means of the chromatographic parameters obtained for the separation of some compounds from the Neue test mixture, using apolar, polar and highly basic analytes.

New monofunctional C18 urea stationary phases with sterically protecting dimethyl and diisopropyl groups were prepared by a single step modification process. ProntoSil spherical silica (3 microm) was chemically modified with the monofunctional ethoxysilanes, [(3-octadecylurea)propyl]dimethyl and [(3-octadecylurea)propyl]diisopropyl ethoxysilanes. The phases were characterized by elemental analysis, infrared and solid-state 29Si and 13C NMR spectroscopies. Chromatographic characterizations of the new phases in 50x3.9 mm HPLC columns were performed by the separation of two different test mixtures, containing nonpolar, polar and highly basic compounds.

Chromatographic evaluations of a new C18 urea phase in 150x3.9 mm HPLC columns, involving the separation of different test mixtures, indicate good performance for both polar and basic compounds when compared with a commercial C18 reversed phase and also show promising results for the separation of some herbicides. An aging study was performed by passing a potassium phosphate mobile phase buffered at pH 7 through 50x3.9 mm HPLC columns. The column stability was evaluated by means of the chromatographic parameters obtained for the separation of some compounds of the Neue test mixture, containing apolar, polar and highly basic analytes. The applicability of the new C18 urea phase was evaluated with a herbicide mixture.

A new C18 urea stationary phase was prepared by a single-step modification process through the reaction of ProntoSil spherical silica (3 microm, Bischoff) with the trifunctional alkoxysilane (CH3CH2O)3-Si-(CH2)3-NH-C(O)-NH-(CH2)17-CH3, prepared in our laboratory. The phase was characterized by elemental analysis and solid-state 29Si and 13C nuclear mangnetic resonance spectrometry. Chromatographic evaluations of the new phase in 150 x 3.9 mm HPLC columns involving the separation of different test mixtures, indicate good performance for both polar and basic mixtures and also show promising results for further applications.