Toward detection of toxoplasmosis from urine in mice using hydro-gel nanoparticles concentration and parallel reaction monitoring mass spectrometry.

Diagnosis of clinical toxoplasmosis remains a challenge, thus limiting the availability of human clinical samples. Though murine models are an approximation of human response, their definitive infection status and tissue availability make them critical to the diagnostic development process. Hydrogel mesh nanoparticles were used to concentrate antigen to detectable levels for mass spectrometry. Seven Toxoplasma gondii isolates were used to develop a panel of potential peptide sequences for detection by parallel reaction monitoring (PRM) mass spectrometry. Nanoparticles were incubated with decreasing concentrations of tachyzoite lysate to explore the limits of detection of PRM. Mice whose toxoplasmosis infection status was confirmed by quantitative real-time PCR had urine tested by PRM after hydrogel mesh concentration for known T. gondii peptides. Peptides from GRA1, GRA12, ROP4, ROP5, SAG1, and SAG2A proteins were detected by PRM after nanoparticle concentration of urine, confirming detection of T. gondii antigen in the urine of an infected mouse.

Correction: Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0241600.].

Immunological detection of pyrazine-2-carboxylic acid for the detection of pyrazinamide resistance in Mycobacterium tuberculosis.

Pyrazinamide (PZA) susceptibility testing in Mycobacterium tuberculosis (Mtb) is a current area of development and PZA-resistant strains are increasingly prevalent. Previous studies have demonstrated that the detection of pyrazinoic acid (POA), the metabolite produced by the deamidation of PZA, is a good predictor for PZA resistance since a resistant strain would not convert PZA into POA at a critical required rate, whereas a susceptible strain will do, expelling POA to the extracellular environment at a certain rate, and allowing for quantification of this accumulated analyte. In order to quantify POA, an indirect competitive ELISA (icELISA) test using hyperimmune polyclonal rabbit serum against POA was developed: for this purpose, pure POA was first covalently linked to the highly immunogenic Keyhole Limpet Hemocyanine, and inoculated in rabbits. A construct made of bovine serum albumin (BSA) linked to pure POA and fixed at the bottom of wells was used as a competitor against spiked samples and liquid Mtb culture supernatants. When spiked samples (commercial POA alone) were analyzed, the half maximal inhibitory concentration (IC50) was 1.16 mg/mL, the limit of detection 200 µg/mL and the assay was specific (it did not detect PZA, IC50 > 20 mg/mL). However, culture supernatants (7H9-OADC-PANTA medium) disrupted the competition and a proper icELISA curve was not obtainable. We consider that, although we have shown that it is feasible to induce antibodies against POA, matrix effects could damage its analytical usefulness; multiple, upcoming ways to solve this obstacle are suggested.

Evaluation of microbiological variants of sputum processing and concentration of mycobacteria to optimize the microscopic and imaging diagnosis of tuberculosis.

BACKGROUND: Direct sputum smear is still the first-choice tool for screening of tuberculosis worldwide. Variants of this technique, to improve the sensitivity are desired. METHODS: Two microbiological variants of the standard sputum smear ("pellet" and "diluted-pellet") for both Ziehl-Neelsen (ZN) and auramine fluorescence (AF) staining were evaluated. In addition, two methods for concentration of mycobacteria in sputum, using positive and negative pressure filtration, were tested and compared. The evaluation of the microbiological variants was performed on 98 culture positive sputum samples from different TB patients. The diagnostics sensitivity and the level of detritus in the processed sputum were determined. Bacilli load in the smear variants was determined by microscopic observation and by manual inspection of microscopic digital images. The comparison of the mycobacteria filtration methods was performed on 76 smear positive sputum samples. Filters retaining the concentrated mycobacteria were stained with AF and compared with the direct smear. Bacilli load, detritus level, filtered volume, filtration time and background noise level, were determined. RESULTS: The sensitivity of microscopy with the microbiological variants was 7.1% and 2% higher in ZN and AF respectively, compared to direct smear. The sensitivity of AF in diluted pellet was significantly higher than all ZN variants (P < 0.05). Detritus level observed in slides was significantly lower in the diluted pellet than the pellet and direct smear in ZN and AF (P < 0.001). A significant increase in the bacilli load in microscopic observation and digital images analysis was observed in pellet and diluted pellet than the direct method (P <0.0001). The concentration of mycobacteria using positive-pressure filtration showed a trend to produce a higher bacilli load compared to the negative-pressure filtration and direct smear, although it was not significant. Detritus levels were significantly higher in both variants of filtration (P < 0.0001). Filtered volumes were higher in positive-pressure compared to negative-pressure filtration. Filtration times were significantly higher in negative-pressure compared to positive-pressure filtration (P < 0.0001). CONCLUSION: The proposed variants improved the performance of the standard sputum smear, making it an important test for settings with high rates of smear-negative TB cases.

Residents at high altitude show a lower glucose profile than sea-level residents throughout 12-hour blood continuous monitoring.

It is known that residents at high altitude (HA) have a lower basal glycemia than residents at sea level (SL). However, whether such a difference is maintained throughout the full day remains unknown. We compared 12-h blood glucose profiles from 10 healthy males native residents at HA (3250 m) and 8 male residents at SL. Glucose profile at HA was lower throughout the glucose monitoring than that at SL (mean profile: 50.6 +/- 3.7 and 73.4 +/- 4.0 mg/dL, respectively; p < 0.001). Basal and postprandial insulin and triacylglycerol values were similar in both groups. In conclusion, HA natives resident have a lower blood glucose profile than SL residents throughout 12-h continuous monitoring.

Respuesta hipófiso gonadal a la infusión pulsátil de GnRH en la altura / Hypophyso-gonadal response to GnRH pulsatile infusion at high altitude

Introducción: Se conoce que la capacidad fértil del sujeto nativo de altura es similar a la del sujeto nativo de nivel del mar. Objetivos: Evaluar la reserva hipófiso testicular, utilizando una metodología de infusión pulsátil con dosis bajas de GnRH. Diseño: Estudio experimental, básico. Lugar: Instituto de Investigaciones Clínicas, Universidad Nacional Mayor de San Marcos, Lima, Perú; Laboratorio de Ciencias Biológicas, Universidad San Antonio Abad, Cusco, Perú. Participantes: Sujetos normales nativos de nivel del mar y de altura. Intervenciones: Se evaluó 12 sujetos de sexo masculino, habitantes de la altura (Cusco, 3395 m.s.n.m.) y un grupo similar de sujetos habitantes de nivel del mar (Lima, 150 m.s.n.m.). Fueron sometidos a una infusión pulsátil intravenosa de GnRH por 24 horas. Se midió las concentraciones plasmáticas de LH, FSH y testosterona. Principales medidas de resultados: Concentraciones de LH, FSH y testosterona. Resultados: Las concentraciones basales de LH, FSH y testosterona fueron similares en ambos grupos. Asimismo, se observó elevaciones significativas de LH sobre su basal, desde las tres hasta las 24 horas, en ambos grupos. Conclusiones: No se observó diferencias significativas en las concentraciones promedio de FSH y testosterona durante la infusión entre ambos grupos; solo los habitantes de la altura mostraron elevación significativa de FSH, en comparación con su valor basal, lo cual podría sugerir que los habitantes de la altura tendrían una mayor reserva hipofisaria.

Introduction: It is known that fertility of normal high altitude dwellers is similar to that at sea level. Objectives: To determine the hypophyso- gonadal reserve using a pulsatile methodology with low doses of GnRH. Design: Experimental, basic study. Setting: Instituto de Investigaciones Clinicas, Universidad Nacional Mayor de San Marcos, Lima, and Laboratorio de Ciencias Biologicas, Universidad San Antonio Abad, Cusco, Peru. Participants: Sea level and high altitude normal subjects. Interventions: Twelve normal male high altitude dwellers (Cusco, 3395 m.a.s.l.) and 12 subjects living at sea level (Lima, 150 m.a.s.l.) were submitted to an intermittent intravenous infusion of GnRH for 24 hours. Serum LH, FSH and testosterone concentrations were measured using RIA techniques. Main outcome measures: LH, FSH y testosterone concentrations. Results: LH, FSH and testosterone basal concentrations were similar in both groups. Significant LH increases from basal concentrations was observed from 3 to 24 hours in both groups. Conclusions: Although no significant differences in serum FSH and testosterone mean concentrations were found during infusion to both groups, only high altitude dwellers presented significant FSH elevation from their basal values which might suggest that the normal high altitude dwellers have a higher hypophysis reserve.