Development of a Methodology for Estimating the Ergosterol in Meat Product-Borne Toxigenic Moulds to Evaluate Antifungal Agents.

Antifungal agents are commonly used in the meat industry to prevent the growth of unwanted moulds, such as toxigenic ones, on dry-cured meat products. For enhancing the application of antifungals, their mode of action must be evaluated. Their effect on the mould ergosterol content is one of the most studied ones, since it is the target site of some commercialised antifungals or of those that are in development. The aim of this study was to develop a methodology for determining how the antifungal agents used in the meat industry work. A method for analysing ergosterol was firstly developed using high-performance liquid chromatography with fluorescence detection coupled to a diode array detector (HPLC-FLD/DAD). The chromatographically optimised conditions (gradient and mobile phases) allowed us to reduce the time per analysis with respect to previously published methods up to 22 min. Withing the six checked extraction methods, method 5, showing the best mean recovery values (99.51%), the shortest retention time (15.8 min), and the lowest standard deviation values (9.92) and working temperature (60 °C), was selected. The limit of detection and limit of quantification were 0.03 and 0.1 µg/mL, respectively. All the validation parameters corroborated the method's suitability. Finally, its feasibility for evaluating the effect of a commercial antifungal preparation (AP) and different herbs that are frequently added to meat products on the ergosterol content of several toxigenic moulds was studied. Differences at the strain level were obtained in the presence of AP. Moreover, the addition of herbs significantly reduced the ergosterol content in Penicillium nordicum up to 83.91%. The developed methodology is thus suitable for screening the antifungals' role in altering mould ergosterol biosynthesis before their application in real meat products.

Selection and Evaluation of Staphylococcus xylosus as a Biocontrol Agent against Toxigenic Moulds in a Dry-Cured Ham Model System.

Toxigenic moulds can develop on the surface of dry-cured meat products during ripening due to their ecological conditions, which constitutes a risk for consumers. A promising strategy to control this hazard is the use of antifungal microorganisms usually found in these foods. However, to date, the effectiveness of gram-positive catalase-positive cocci (GCC+) has not been explored. The aim of this work was to select GCC+ isolates with antifungal activity to study its effectiveness in a dry-cured ham model system at the environmental conditions reached during the ripening. Forty-five strains of GCC+ were evaluated and the isolate Staphylococcus xylosus Sx8 was selected to assess its efficacy at two different concentrations (106 and 104 cfu/mL) against Penicillium nordicum, Aspergillus flavus, Aspergillus parasiticus, and Penicillium griseofulvum at 15, 20, and 25 °C. The results showed that the inoculation of 106 cfu/mL of S. xylosus completely inhibited the growth of most fungi. In addition, in the presence of this strain at 104 cfu/mL, a significant reduction in fungal growth and mycotoxins production was observed at the three temperatures studied. In conclusion, S. xylosus Sx8 possesses great potential as a biological agent to control toxigenic moulds in dry-cured meat products.

Efficacy of the Combined Protective Cultures of Penicillium chrysogenum and Debaryomyces hansenii for the Control of Ochratoxin A Hazards in Dry-Cured Ham.

The ecological conditions during the ripening of dry-cured ham favour the development of moulds on its surface, being frequently the presence of Penicillium nordicum, a producer of ochratoxin A (OTA). Biocontrol using moulds and yeasts usually found in dry-cured ham is a promising strategy to minimize this hazard. The aim of this work is to evaluate the effect of previously selected Debaryomyces hansenii and Penicillium chrysogenum strains on growth, OTA production, and relative expression of genes involved in the OTA biosynthesis by P. nordicum. P. nordicum was inoculated against the protective cultures individually and combined on dry-cured ham for 21 days at 20 °C. None of the treatments reduced the growth of P. nordicum, but all of them decreased OTA concentration. The lower production of OTA could be related to significant repression of the relative expression of otapksPN and otanpsPN genes of P. nordicum. The efficacy of the combined protective cultures was tested in 24 dry-cured hams in industrial ripening (an 8 month-long production). OTA was detected in nine of the 12 dry-cured hams in the batch inoculated only with P. nordicum. However, in the batch inoculated with both P. nordicum and the combined protective culture, a considerable reduction of OTA contamination was observed. In conclusion, although the efficacy of individual use P. chrysogenum is great, the combination with D. hansenii enhances its antifungal activity and could be proposed as a mixed protective culture to control the hazard of the presence of OTA in dry-cured ham.

QuantiFERON-TB Gold In-Tube as a Confirmatory Test for Tuberculin Skin Test in Tuberculosis Contact Tracing: A Noninferiority Clinical Trial.

Background: Screening strategies based on interferon-γ release assays in tuberculosis contact tracing may reduce the need for preventive therapy without increasing subsequent active disease. Methods: We conducted an open-label, randomized trial to test the noninferiority of a 2-step strategy with the tuberculin skin test (TST) followed by QuantiFERON-TB Gold In-Tube (QFT-GIT) as a confirmatory test (the TST/QFT arm) to the standard TST-alone strategy (TST arm) for targeting preventive therapy in household contacts of patients with tuberculosis. Participants were followed for 24 months after randomization. The primary endpoint was the development of tuberculosis, with a noninferiority margin of 1.5 percentage points. Results: A total of 871 contacts were randomized. Four contacts in the TST arm and 2 in the TST/QFT arm developed tuberculosis. In the modified intention-to-treat analysis, this accounted for 0.99% in the TST arm and 0.51% in the TST/QFT arm (-0.48% difference; 97.5% confidence interval [CI], -1.86% to 0.90%); in the per-protocol analysis, the corresponding rates were 1.67% and 0.82% in the TST and TST/QFT arms, respectively (-0.85% difference; 97.5% CI, -3.14% to 1.43%). Of the 792 contacts analyzed, 65.3% in the TST arm and 42.2% in the TST/QFT arm were diagnosed with tuberculosis infection (23.1% difference; 95% CI, 16.4% to 30.0%). Conclusions: In low-incidence settings, screening household contacts with the TST and using QFT-GIT as a confirmatory test is not inferior to TST-alone for preventing active tuberculosis, allowing a safe reduction of preventive treatments. Clinical Trials Registration: NCT01223534.

Growth inhibition and stability of PgAFP from Penicillium chrysogenum against fungi common on dry-ripened meat products.

Dry-ripened foods favor the development of a superficial fungal population that may include toxigenic molds. To combat unwanted molds, an antifungal protein from Penicillium chrysogenum (PgAFP) can be useful. The aim of the present work was to study the antimicrobial activity of PgAFP against microorganisms common in dry-ripened foods, and to evaluate its sensitivity to proteolytic enzymes and heat treatments that may be applied to foods, as well as to different pH values. The inhibitory effect of the purified protein on 38 microbial strains grown in culture medium was determined. PgAFP sensitivity to various proteases, heat treatments, and preincubation at different pH values was tested by means of the residual activity on selected reference strains. Inhibitory activity of PgAFP against unwanted molds was tested in a dry-fermented sausage. This protein exhibited potent inhibitory activity against unwanted molds, including the main mycotoxin-producing species of Aspergillus and Penicillium of concern for dry-ripened foods. PgAFP withstood most proteases, intense heat and a wide range of pH values. PgAFP efficiently reduced counts of A. flavus and P. restrictum inoculated on a dry-fermented sausage. This protein can be of interest to control hazardous molds in dry-ripened foods.

Quantification of viable Escherichia coli O157:H7 in meat products by duplex real-time PCR assays.

Rapid and specific detection of viable Escherichia coli O157:H7 cells in ready-to-eat (RTE) meat products, by duplex quantitative PCR (qPCR) procedures with mRNA and SYBR Green and TaqMan methodologies were developed. Specific primers and probes were designed based on the serotype of E. coli O157:H7, fliCh7 and rfbE genes. No cross-reactivity with other microorganisms was observed. The detection limit of the assays was 10(1) or 10(2)CFU/g for artificially contaminated meat products, and after a 4h enrichment period at 37 °C, the detection limit decreased to about 1 CFU/g. Time-to completion of the assay was approximately 8h. Thus, these qPCR methods offer a useful, rapid and efficient tool for screening viable E. coli O157:H7 in RTE meat products. This tool could also be proposed for monitoring these foodborne pathogens in HACCP programs.

Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods.

A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1-4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain.

Actitudes y prácticas de los médicos de atención primaria ante el diagnósticode la infección por virus de la inmunodeficiencia humana / Primary care doctors attitudes and practices in the diagnosis of HIV infection

Objetivo: Explorar las actitudes y prácticas de los médicos del primer nivel asistencial del Servicio Madrileño de Salud respecto al diagnóstico de la infección por VIH según los protocolos actuales y el grado de aceptación de la introducción de el diagnóstico de la infección VIH de forma simplificada (sin un documento aparte de consentimiento informado ni el interrogatorio acerca de las prácticas de riesgo).Material y métodos Estudio observacional descriptivo transversal realizado en atención primaria de salud de la Comunidad Autónoma de Madrid. La recogida de datos se hizo mediante encuestas telefónicas durante 2009.ResultadosSe consultó a 210 médicos. El 21% ya realizaba el diagnóstico de la infección VIH de forma simplificada y el 28,6% manifestó una actitud favorable hacia las nuevas recomendaciones. El 71,4% no pedía consentimiento informado por escrito aparte y un 42% no manifestó dificultades de comunicación. Una gran mayoría opinó que la excepcionalidad en el manejo del diagnóstico de la infección por VIH, comparándola con otras infecciones de similar forma de transmisión, podría contribuir a mantener el estigma. Para un 75,2% la falta de tiempo en la consulta no representaba un problema y un 97,1% tenía la autopercepción de ser un agente esencial en el diagnóstico de la infección por el VIH. Conclusiones La simplificación del diagnóstico de VIH es aceptable para un porcentaje elevado de los encuestados y uno de cada cinco médicos ya lo está realizando en las consultas de atención primaria (AU)

Objective: To explore the attitudes and practices of Primary Health Care professionals in the diagnosis of HIV infection according to current protocols and the degree of acceptance of simplified HIV testing(without a separate written consent and without asking about risk practices).Material and methods: An observational cross-sectional descriptive study conducted in Primary Care Centres of the Madrid Public Health Service. Data were collected by telephone surveys during 2009.Results: A total of 210 doctors were interviewed. Twenty one percent were already performing simplified HIV testing (and 28.6% expressed a favourable attitude towards the new recommendations). The majority(71.4% did not use a separate written consent for HIV testing, and 42% did not report any communication difficulties. Most of them considered that comparing HIV with other similar ways of transmission infections, making HIV testing exceptual may lead to stigma. Lack of time was not a problem for 75.2%, and97.1% considered they had an essential role in controlling the HIV epidemic. Conclusions: The acceptance of simplified HIV testing is high and is already being performed by 1 out of5 Primary Care Doctors in the Madrid Public Health Service (AU)

[Primary care doctors attitudes and practices in the diagnosis of HIV infection]. / Actitudes y prácticas de los médicos de atención primaria ante el diagnóstico de la infección por virus de la inmunodeficiencia humana.

OBJECTIVE: To explore the attitudes and practices of Primary Health Care professionals in the diagnosis of HIV infection according to current protocols and the degree of acceptance of simplified HIV testing (without a separate written consent and without asking about risk practices). MATERIAL AND METHODS: An observational cross-sectional descriptive study conducted in Primary Care Centres of the Madrid Public Health Service. Data were collected by telephone surveys during 2009. RESULTS: A total of 210 doctors were interviewed. Twenty one percent were already performing simplified HIV testing (and 28.6% expressed a favourable attitude towards the new recommendations). The majority (71.4% did not use a separate written consent for HIV testing, and 42% did not report any communication difficulties. Most of them considered that comparing HIV with other similar ways of transmission infections, making HIV testing exceptual may lead to stigma. Lack of time was not a problem for 75.2%, and 97.1% considered they had an essential role in controlling the HIV epidemic. CONCLUSIONS: The acceptance of simplified HIV testing is high and is already being performed by 1 out of 5 Primary Care Doctors in the Madrid Public Health Service.

Production of secondary metabolites by some terverticillate penicillia on carbohydrate-rich and meat substrates.

Most terverticillate penicillia isolated from dry-cured meat products are toxigenic, but their ability to produce hazardous metabolites on meat-based substrates is not well known. The production of extrolites by selected terverticillate penicillia isolated from dry-cured ham has been studied on carbohydrate-rich media (malt extract agar, Czapek yeast autolysate agar, rice extract agar, and rice), meat extract triolein salt agar, and ham slices. Chloroform extracts from the selected strains grown on malt extract agar were toxic for the brine shrimp (Artemia salina) larvae and VERO cells at a concentration of 2 mg/ml, but 0.02 mg/ml produced no toxic effect. Analysis by high-pressure liquid chromatography (HPLC) coupled with photodiode array detection (DAD) or with mass spectrometry (MS) and an atmospheric pressure chemical ionization (APCI) source revealed different biologically active metabolites: cyclopiazonic acid and rugulovasine A from Penicillium commune; verrucosidin, anacine, puberuline, verrucofortine, and viridicatols from Penicillium polonicum; arisugacin and viridicatols from Penicillium echinulatum; and compactin and viridicatols from Penicillium solitum. Most of these metabolites, including the amino acid-derived compounds, were produced in the media containing high levels of carbohydrates. High concentrations of nitrogen compounds in the medium does not imply a greater production of the metabolites studied, not even those derived from the amino acids. However, molds growing on dry-cured ham are able to synthesize limited amounts of some secondary metabolites, a fact not previously reported. The combination of HPLC coupled with DAD and MS-APCI was useful for identification of closely related terverticillate Penicillium species from dry-cured ham. These techniques could be used to characterize the risk associated with the potential production of secondary metabolites in cured meats.

Overcoming resistance: virologic response to a salvage regimen with the combination of ritonavir plus indinavir.

PURPOSE: To explore the possibility of overcoming resistance to protease inhibitors (PIs) and to determine the resistance cutoff values that continue to predict treatment failure with a dual PI regimen. METHOD: We performed a prospective study of 53 patients who had failed in several PIs and who were included in a ritonavir (RTV) plus indinavir (IDV) salvage regimen. Median HIV RNA level decrease was evaluated according to resistance assays and indinavir trough levels. RESULTS: Eighty-seven percent of patients had previously failed on an IDV-containing regimen. Overall, median HIV RNA decrease was -1.25 log(10) copies/mL after 3 months on therapy. A significant blunted virologic response was observed only in isolates with more than 12 substitutions including the V82A (-0.75 vs. -1.3 log(10) copies/mL; p =.04), or in isolates with more than 30 fold-increase in the IC(50) (-0.43 vs. -1.2 log(10) copies/mL). Higher drug levels were observed in patients with resistant isolates who achieved an HIV RNA decrease greater than 1 log (1742 vs. 1100 ng/mL). CONCLUSION: Our preliminary data suggest the possibility of overcoming resistance with the combination of RTV plus IDV. They also suggest the need for establishing new resistance cutoff values when using PIs in combination.

Production of cyclopiazonic acid by Penicillium commune isolated from dry-cured ham on a meat extract-based substrate.

Penicillium commune, a mold frequently found on dry-cured meat products, is able to synthesize the mycotoxin cyclopiazonic acid (CPA). To evaluate the hazard due to CPA on such foods, the ability of P. commune to grow and produce CPA at water activities (a(w)) in the range of 0.99 to 0.90 with a meat extract-based medium from 12 to 30 degrees C was determined. CPA was quantified by high-pressure liquid chromatography and mass spectrometry. P. commune was able to grow at every a(w) and temperature tested. The optimal environmental conditions for growth were 20 to 25 degrees C, at 0.97 to 0.96 a(w), but the highest amount of CPA was produced at 30 degrees C, 0.96 a(w). No direct correlation between growth rate and CPA production was assessed. Temperature seems to be the most important factor influencing CPA production. However, there was an interaction between temperature and a(w) that significantly (P < 0.001) affected growth and CPA production. An a(w) of 0.90 had a marked effect, depressing growth and CPA production. Meat extract-based medium proved to be an appropriate substrate for CPA biosynthesis by P. commune under a wide range of conditions.