Assessment of nitrification process in a sequencing batch reactor: Modelling and genomic approach.

Nitrification of ammoniacal nitrogen (N-NH4+) to nitrate (N-NO3-) was investigated in a lab-scale sequencing batch reactor (SBR) to evaluate its efficiency. During the nitrification process the removal of N-NH4+ reached 96%, resulting in 73% formation of N-NO3-. A lineal correlation (r2 = 0.9978) was obtained between the concentration of volatile suspended solids (VSS) and the maximal N-NO3- concentration at the end of each batch cycle under stationary state. The bacterial taxons in the initial inoculum were identified, revealing a complex diverse community mainly in the two major bacterial phyla Proteobacteria and Actinobacteria. The FAPROTAX algorithm predicted the presence in the inoculum of taxa involved in relevant processes of the nitrogen metabolism, highlighting the bacterial genera Nitrospira and Nitrosomonas that are both involved in the nitrification process. A kinetic model was formulated for predicting and validating the transformation of N-NH4+, N-NO2- and N-NO3- and the removal of organic and inorganic carbon (TOC and IC, respectively). The results showed how the increase in biomass concentration slowed down the transformation to oxidised forms of nitrogen and increased denitrification in the settling and filling stages under free aeration conditions.

Integral evaluation of effective conversion of sewage sludge from WWTP into highly porous activated carbon.

Urban sewage sludge (SL) is a major concern due to the number of environmental problems it causes. Its application for different purposes is strictly regulated, limiting the possibilities of recycling and reusing this material. Thus, in this work, a complete study of a simple method to convert SL into activated carbon (AC) was carried out. The comprehensive study involves an evaluation of the main process parameters, such as the activating agent (AA) content (25 %, 33 %, 50 %), using the lowest amount of AA as novelty, different pyrolysis temperatures (600 and 800 °C), and purification conditions (6 M HCl:AC ratio, v:w). Under controlled and optimised conditions and through a single combined activation and pyrolysis step followed by acid purification, ACs with well-developed porosity can be obtained. Surface area values of around 870 m2/g and over 60 % carbon content were achieved, demonstrating that the prepared ACs could have applications in a wide variety of fields as high-value products. As an innovative aspect in this research, the gases streams and liquid effluents generated during the global process were analysed, achieving elimination of over 63 % of the concentration of the chemical elements contained in the SL during the chemical purification stage. Finally, mass, energy, and economic balances were carried out to estimate the production cost of AC derived from SL (<€ 8/kg AC).

Odor emission assessment of different WWTPs with Extended Aeration Activated Sludge and Rotating Biological Contactor technologies in the province of Cordoba (Spain).

In this study, five urban WWTPs (Wastewater Treatment Plant) with different biological treatment (Extended Aeration Activated Sludge - EAAS; Rotating Biological Contactor - RBC), wastewater type (Urban; Industrial) and size, were jointly evaluated. The aim was twofold: (1) to analyze and compare their odor emissions, and (2) to identify the main causes of its generation from the relationships between physico-chemical, respirometric and olfactometric variables. The results showed that facilities with EAAS technology were more efficient than RBC, with elimination yields of organic matter higher than 90%. In olfactometric terms, sludge managements facilities (SMFs) were found to be the critical odor source in all WWTPs compared to the Inlet point (I) or Post primary treatment (PP), and for seasonal periods with ambient temperature higher than 25 °C. Moreover, the global odor emissions quantified in all SMFs revealed that facilities with EAAS (C-WWTP, V-WWTP and Z-WWTP) had a lower odor contribution (19,345, 14,800 and 11,029 ouE/s·m2, respectively) than for those with RBC technology (P-WWTP and NC-WWTP) which accounted for 19,747 ouE/s·m2 and 80,061 ouE/s·m2, respectively. In addition, chemometric analysis helped to find groupings and differences between the WWTPs considering the wastewater (71.27% of total variance explained) and sludge management (64.52% of total variance explained) lines independently. Finally, odor emissions were adequately predicted from the physico-chemical and respirometric variables in the wastewater (r2 = 0.8738) and sludge (r2 = 0.9373) lines, being pH, volatile acidity and temperature (wastewater line), and pH, moisture, temperature, SOUR (Specific Oxygen Uptake Rate) and OD20 (Cumulative Oxygen Demand at 20 h) (sludge line) the most influential variables.

Environmental performance of an industrial biofilter: Relationship between photochemical oxidation and odorous impacts.

Biological techniques are widely used to treat gaseous streams derived from waste treatment plants. The generation of volatile organic compounds (VOCs) is one of the principal pollution sources in composting facilities from which nuisance odours are released. In addition, the generation of photochemical smog with other gases such as NOX can produce ozone at ground level due to their photochemical ozone creation potential (POCP). In this work, the performance of an industrial biofilter was evaluated from an environmental point of view. Specifically, this study evaluated the potential impact in terms of photochemical oxidation and odour emission derived from composting in a vessel under four different aeration conditions. Gas chromatography-time-of-flight mass spectrometry (GC-TOFMS) was used to perform the chemical characterisation of the gaseous streams, while dynamic olfactometry was used to carry out the sensorial analysis. A total of 95 compounds belonging to 12 different families of VOCs were selected. Principal component analysis revealed the influence of each VOC family on each impact category and explained 88% of the total variance. Multivariate regression was used to study the correlation between photochemical oxidation and odour impact, which has never been reported before. The correlations obtained (r ≥ 0.97) evidenced the direct relationship between these two impacts. Photochemical oxidation and odour emission were proven to be important environmental impacts derived from composting facilities, whose abatement might be carried out by biofiltration systems.

Sewage sludge composting under semi-permeable film at full-scale: Evaluation of odour emissions and relationships between microbiological activities and physico-chemical variables.

In the present study, physico-chemical characteristics, heavy metals content, odour emissions, microbial enumeration and enzymatic activities were analysed during industrial scale composting of sewage sludge partially pre-treated to evaluate the effect of a combined system of semi-permeable film and aeration on these parameters. The results related to physico-chemical parameters showed a decrease in total organic carbon (TOC), organic matter (OM), total carbon (TC) along the process. Volatile solids (VS) were also reduced, reaching 36% at 120 days, which is above the limit according to the current legislation. Similarly, metal content was found to be an important variable in the evolution of enzymatic activity, while lead (Pb), zinc (Zn), and nickel (Ni) were the most influential. Moreover, heavy metals were found below the limit of type B compost quality or European class 2 at the end of the process, which is suitable for agriculture soil. The odorous impact generated during the hydrolytic stage was reduced to an average value of 4 ouE/s. This suggests that, covered stage with the semi-permeable film, could be a viable solution to mitigate odour emissions. The highest temperature was reached at 10 days and it was favoured by semi-permeable film. Temperature promoted the presence of thermophilic bacteria and fungi and indicated an early biodegradation process mediated by microorganisms. Statistical analyses revealed a high correlation of physico-chemical variables with microbial activity. Thus, samples from the first 14 days were highly correlated with enzymatic activities such as ß-glucosidase (Ac-ßGlu), protease (Ac-Pr), and dehydrogenase (Ac-De), which have usually been involved in the hydrolysis of organic matter.

Full-scale composting of sewage sludge and market waste: Stability monitoring and odor dispersion modeling.

The aim of this study was to assess the odor immission derived from full-scale composting of different abundant and highly pollutant organic waste: sewage sludge with bulking agent (SL), sewage sludge pretreated through anaerobic digestion and supplemented with bulking agent (SL-AD), and market waste with olive leaves (MW-OL). The combination of dynamic olfactometry and Gaussian dispersion modeling allowed both the quantification of odor emissions from each waste and the evaluation of their global odorous impact in nearby urban areas. Wind speed, summer and winter seasons, and atmospheric conditions were considered in the dispersion model. The results revealed that high wind speed (2.6 m/s) increases the global odor immission in summer season, independently of atmospheric stability. However, the maximum odor immission concentration recommended for composting process was not exceeded in any case, which depends on each country/region. The experimental results also enable to evaluate the influence of several physico-chemical variables on odor emissions derived from composting. The removal of nitrogen and volatile solids was the main cause for odor generation. Moreover, the microbiological activity of each substrate was monitored throughout the process and different percentages of biodegradability were quantified depending on the type of substrate and pretreatment applied.

Application of ATAD technology for digesting sewage sludge in small towns: Operation and costs.

In an economic context marked by increasing energy costs and stricter legislation regarding the landfill disposal of wastewater treatment plant (WWTP) sewage sludge, and where biomethanization is difficult to implement in small WWTPs, an efficient alternative is required to manage this polluting waste. This study shows that autothermal thermophilic aerobic digestion (ATAD) is a feasible technique for treating sewage sludge in small- and medium-sized towns. The experiments were carried out at pilot scale on a cyclical basis and in continuous mode for nine months. The main results showed an optimal hydraulic retention time of 7 days, which led to an organic matter removal of 34%. The sanitized sludge meets the microbial quality standards for agronomic application set out in the proposed European sewage sludge directive. An economic assessment for the operation of ATAD technology was carried out, showing a treatment cost of €6.5/ton for dewatered sludge.

Reline aqueous solutions behaving as liquid mixtures of H-bonded co-solvents: microphase segregation and formation of co-continuous structures as indicated by Brillouin and 1H NMR spectroscopies.

Deep eutectic solvents (DESs) offer a suitable alternative to conventional solvents in terms of both performance and cost-effectiveness. Some DESs also offer certain green features, the greenness of which is notoriously enhanced when combined with water. Aqueous DES dilutions are therefore attracting great attention as a novel green medium for biotechnological processes, with the aqueous dilutions of reline - a DES composed of urea and choline chloride - being one of the most studied systems. Despite their macroscopic homogeneous appearance, both 1H NMR spectroscopic studies and molecular dynamics simulations have revealed the occurrence of certain dynamic heterogeneity at a microscopic molecular level. Ultrasonic measurements were also used with the aim of getting further insights but nonconclusive results were obtained. Herein, we have studied aqueous reline dilutions by Brillouin spectroscopy given its proved suitability for detecting local structure rearrangements in liquid mixtures of H-bonded co-solvents. Brillouin spectroscopy revealed the formation of a co-continuous structure resulting from local structure rearrangements and micro-segregation into aqueous and DES phases. Interestingly, there is agreement between 1H NMR and Brillouin spectroscopy when pointing to the DES content where microphase segregation and formation of co-continuous structures occurred.

Centralized management of sewage sludge and agro-industrial waste through co-composting.

In this research study, the co-composting process of a waste mixture containing strawberry extrudate, fish waste, sewage sludge and bulking agent (SEFW, 190:1:22:90 ratio) was carried out in a dynamic-solid respirometer at pilot scale. The aerobic biodegradability of the mixture was previously ensured in a static-liquid respirometer. The advantages and drawbacks of the SEFW co-composting process were subsequently identified through the determination of respirometric activity and the physical-chemical characterization of the waste, as well as the monitoring of odor emissions. The evolution of the physical-chemical variables showed that pH increased slightly and that the organic matter concentration, expressed as volatile solids (VS, %) or oxidable organic carbon (COXC, %), decreased by around 15% in both cases and by approximately 56% in its biodegradable form (total organic carbon, TOC, %). The low odor emission rate (OER) in the least favorable scenario (the maximum odor generation) during SEFW composting was 1.59 ouE/s, whereas this figure reached 3.52 ouE/s when only the organic fraction of municipal solid waste (OFMSW) was composted. Consequently, the co-composting of SEFW is more favorable in terms of odor emission and permits the simultaneous treatment of different types of waste.

Monitoring of pile composting process of OFMSW at full scale and evaluation of odour emission impact.

In this study, the evolution of odour concentration (ouE/m(3)STP) emitted during the pile composting of the organic fraction of municipal solid waste (OFMSW) was monitored by dynamic olfactometry. Physical-chemical variables as well as the respirometric variables were also analysed. The aim of this work was twofold. The first was to determine the relationship between odour and traditional variables to determine if dynamic olfactometry is a feasible and adequate technique for monitoring an aerobic stabilisation process (composting). Second, the composting process odour impact on surrounding areas was simulated by a dispersion model. The results showed that the decrease of odour concentration, total organic carbon and respirometric variables was similar (around 96, 96 y 98% respectively). The highest odour emission (5224 ouE/m(3)) was reached in parallel with the highest microbiological activity (SOUR and OD20 values of 25 mgO2/gVS · h and 70 mgO2/gVS, respectively). The validity of monitoring odour emissions during composting in combination with traditional and respirometric variables was demonstrated by the adequate correlation obtained between the variables. Moreover, the quantification of odour emissions by dynamic olfactometry and the subsequent application of the dispersion model permitted making an initial prediction of the impact of odorous emissions on the population. Finally, the determination of CO2 and CH4 emissions allowed the influence of composting process on carbon reservoirs and global warming to be evaluated.

Use of eutectic mixtures for preparation of monolithic carbons with CO2-adsorption and gas-separation capabilities.

With global warming becoming one of the main problems our society is facing nowadays, there is an urgent demand to develop materials suitable for CO2 storage as well as for gas separation. Within this context, hierarchical porous structures are of great interest for in-flow applications because of the desirable combination of an extensive internal reactive surface along narrow nanopores with facile molecular transport through broad "highways" leading to and from these pores. Deep eutectic solvents (DESs) have been recently used in the synthesis of carbon monoliths exhibiting a bicontinuous porous structure composed of continuous macroporous channels and a continuous carbon network that contains a certain microporosity and provides considerable surface area. In this work, we have prepared two DESs for the preparation of two hierarchical carbon monoliths with different compositions (e.g., either nitrogen-doped or not) and structure. It is worth noting that DESs played a capital role in the synthesis of hierarchical carbon monoliths not only promoting the spinodal decomposition that governs the formation of the bicontinuous porous structure but also providing the precursors required to tailor the composition and the molecular sieve structure of the resulting carbons. We have studied the performance of these two carbons for CO2, N2, and CH4 adsorption in both monolithic and powdered form. We have also studied the selective adsorption of CO2 versus CH4 in equilibrium and dynamic conditions. We found that these materials combined a high CO2-sorption capacity besides an excellent CO2/N2 and CO2/CH4 selectivity and, interestingly, this performance was preserved when processed in both monolithic and powdered form.

Usual variables and odour concentration to evaluate composting process and odour impact.

Organic waste management by means of composting produces unpleasant odours. The odour emissions of composting piles composed of the organic fraction of municipal solid waste and sewage sludge were monitored and controlled by means of dynamic olfactometry. This technique is used to determine the odour concentration expressed in European odour units per cubic metres (ou(E)/m3). To validate the technique, we analysed both respirometric variables and traditional variables such as total organic carbon and pH during composting. Finally, a dispersion model was used to evaluate the impact of the odour emissions of the wastes on neighbouring areas, not finding any differences in the cases evaluated.

Odour in composting processes at pilot scale: monitoring and biofiltration.

Although odour emissions associated with the composting process, especially during the hydrolytic stage, are widely known, their impact on surrounding areas is not easily quantifiable, For this reason, odour emissions during the first stage ofcomposting were evaluated by dynamic olfactometry at pilot scale in order to obtain results which can be extrapolated to industrial facilities. The composting was carried out in a commercial dynamic respirometer equipped with two biofilters at pilot scale filled with prunings (Populus) and mature compost obtained from the organic fraction of municipal solid waste. Given that the highest odour emissions occur in the first stage of the composting process, this stage was carried out in a closed system to better control the odour emissions, whose maximum value was estimated to be 2.78 ouF S-1 during the experiments. Odour concentration, the dynamic respiration index and temperature showed the same evolution during composting, thus indicating that odour could be a key variable in the monitoring process. Other variables such as total organic carbon (CTOC) and pH were also found to be significant in this study due to their influence over odour emissions. The efficiency of the biofilters (empty bed residence time of 86 s) was determined by quantifying the odour emissions at the inlet and outlet of both biofilters. The moisture content in the biofilters was found to be an important variable for improving odour removal efficiency, while the minimum moisture percentage to obtain successful results was found to be 55% (odour removal efficiency of 95%).

Natronococcus roseus sp. nov., a haloalkaliphilic archaeon from a hypersaline lake.

A novel halophilic archaeon, strain CG-1(T), belonging to the genus Natronococcus was isolated from sediment of the soda lake Chagannor in Inner Mongolia, China. The colonies of this strain were pink pigmented, the intensity of the colour decreased when the cells grew at salt saturation levels. The cells were non-motile cocci and strictly aerobic. Hypotonic treatment did not cause cell lysis, even in distilled water. Strain CG-1(T) grew at 15-30.0 % (w/v) NaCl and at 30-50 °C and pH 8.0-11.0, with optimal growth occurring at 25-30 % (w/v) NaCl, 37-45 °C and pH 9-9.5. MgCl(2) was not required for growth. Strain CG-1(T) was most closely related to the type strains of Natronococcus amylolyticus Ah-36(T), Natronococcus jeotgali B1(T) and Natronococcus occultus SP4(T), with which it shared 98.4 %, 96.2 and 95.7 % 16S rRNA gene sequence similarity, respectively. The polar lipids consisted of C(20)C(20) and C(20)C(25) derivatives of phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me) and minor phospholipid components. No glycolipids were detected. The DNA G+C content of strain CG-1(T) was 62.1 mol%. DNA-DNA hybridization with N. amylolyticus DSM 10524(T), phylogenetically the most closely related species, was 39 %; this value showed that strain CG-1(T) constituted a different genospecies. The comparison of 16S rRNA gene sequences, detailed phenotypic characterization, polar lipid profile and DNA-DNA hybridization studies revealed that strain CG-1(T) belongs to the genus Natronococcus and constitutes a novel species for which the name Natronococcus roseus sp. nov. is proposed. The type strain is CG-1(T) (=CECT 7984(T)=IBRC-M 10656(T)=JCM 17958(T)).

Laser-induced periodic surface structures nanofabricated on poly(trimethylene terephthalate) spin-coated films.

Here we present a precise morphological description of laser-induced periodic surface structures (LIPSS) nanofabricated on spin-coated poly(trimethylene terephthalate) (PTT) films by irradiation with 266 nm, 6 ns laser pulses and by using a broad range of fluences and number of pulses. By accomplishing real and reciprocal space measurements by means of atomic force microscopy and grazing incidence wide- and small-angle X-ray scattering respectively on LIPSS samples, the range of optimum structural order has been established. For a given fluence, an increase in the number of pulses tends to improve LIPSS in PTT. However, as the pulse doses increase above a certain limit, a distortion of the structures is observed and a droplet-like morphology appears. It is proposed that this effect could be related to a plausible decrease of the molecular weight of PTT due to laser-induced chain photo-oxidation by irradiation with a high number of pulses. A concurrent decrease in viscosity enables destabilization of LIPSS by the formation of droplets in a process similar to surface-limited dewetting.

Halorubrum aquaticum sp. nov., an archaeon isolated from hypersaline lakes.

Two halophilic archaea, strains EN-2(T) and SH-4, were isolated from the saline lakes Erliannor and Shangmatala, respectively, in Inner Mongolia, China. Cells were strictly aerobic, motile rods. Colonies were red. Strains EN-2(T) and SH-4 were able to grow at 25-50 °C (optimum 35-40 °C), with 2.5-5.0 M NaCl (optimum 3.4 M NaCl) and at pH 6.0-9.0 (optimum pH 7.5). MgCl(2) was not required for growth. Cells lysed in distilled water and the lowest NaCl concentration that prevented cell lysis was 12 % (w/v). On the basis of 16S rRNA gene sequence analysis, strains EN-2(T) and SH-4 were closely related to Halorubrum cibi B31(T) (97.9 and 98.0 % similarity, respectively), Hrr. tibetense 8W8(T) (97.3 and 97.7 %), Hrr. alkaliphilum DZ-1(T) (96.8 and 97.1 %), Hrr. luteum CGSA15(T) (96.8 and 97.0 %) and Hrr. lipolyticum 9-3(T) (96.8 and 97.0 %). DNA-DNA hybridization showed that strains EN-2(T) and SH-4 did not belong to the same species as any of these strains (≤ 45 % DNA-DNA relatedness) but that they are members of the same species (>70 % DNA-DNA relatedness). Polar lipid analysis revealed that strains EN-2(T) and SH-4 contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, sulfated diglycosyl diethers and several unidentified glycolipids. The DNA G+C content of both isolates was 62.1 mol%. It was concluded that strains EN-2(T) and SH-4 represent a novel species of the genus Halorubrum, for which the name Halorubrum aquaticum sp. nov. is proposed. The type strain is EN-2(T) ( = CECT 7174(T)  = CGMCC 1.6377(T)  = JCM 14031(T)).

[Non-drug treatments for chronic non-malignant pain: patients' perceptions].

BACKGROUND: The prevalence of chronic pain in Spain is 17%. There is a need for more scientific data on non-drug treat-ments that can be effectively used to treat chronic pain. The aim of this study is to analyze how patients with chronic non-malignant pain perceive non-drug approaches. METHOD: Mixed, descriptive and phenomenological study. Nineteen patients enrolled in a workshop on pain management and non-drug treatments that consisted of four sessions (one session a week). Each session lasted four hours. The patients then participated in a reflective writing activity about their personal experiences. Data from this activity was then ana-lyzed. Atlas.ti 8 software was used for the qualitative data analysis. RESULTS: Sixteen participants tried an alternative therapy and fourteen assessed its benefits. The participants' expecta-tions were divided into three groups of similar size: relief from physical pain, emotional pain management and tools for use in daily life. All the participants were satisfied with the workshop. Perceived personal benefits were better pain and sleep management, reduced fatigue, reduced drug consumption; a more positive approach to life, better mood, more positive energy, more motivation and improved capacity to cope. CONCLUSIONS: The participants commented that the workshop had helped them to reduce pain levels and consume to fewer analgesics, and had reduced other symptoms associated with chronic disease, thus improving their perceived health. They also expressed great satisfaction with the organization and teachers.

Structure and morphology of thin films of linear aliphatic polyesters prepared by spin-coating.

Thin films, with thicknesses from 10 to 400 nm of linear aliphatic polyesters (X, Y), based on propylenediol (X = 3) and on dicarboxylic acid of different chain length (Y = 2, 3, and 4 CH(2) units) were prepared by spin coating of CHCl(3) polymer solutions with different polymer concentrations. Morphology and structure of the spin coated thin films were investigated by atomic force microscopy (AFM) and by grazing incidence X-ray scattering techniques at small, (GISAXS) and wide angles (GIWAXS). AFM revealed a strong dewetting for all three polymers for coatings thinner than 100 nm. The polymer films are clearly semicrystalline for thicknesses higher than 50 nm. GIWAXS of the thicker films revealed their oriented crystalline nature. An edge-on-lamellae morphology is clearly shown by the AFM-phase images even for relatively thin films. SAXS with the beam parallel to the sample plane also support the presence of lamellae perpendicular to the substrate. The use of a mu-beam helped to interpret the GIWAXS patterns and allowed to obtain oriented WAXS patterns from melt solidified filaments. Thus, a crystal chain packing is proposed for the three polymers and consequently the indexing of the observed reflections. Accordingly, the polymer chains lie parallel to the substrate being the bc plane of the monoclinic crystal unit cell parallel to the substrate.

Flat-on lamellae in spin-coated, stable films of poly(propylene azelate).

Thin films (5-500 nm thick) of the linear aliphatic polyester (3,7) poly(propylene azelate) (PPAz) were prepared by spin-coating of CHCl(3) polymer solutions with different polymer concentrations. The morphology and structure of the spin-coated thin films were investigated by atomic force microscopy (AFM) and by grazing incidence wide-angle X-ray scattering (GIWAXS) techniques. AFM revealed the continuous nature of the flat, spherulitic films which are stable against dewetting even for polymer coatings as thin as 15 nm. GIWAXS patterns revealed a high crystal orientation of the films. A sharp reflection on the meridian whose spacing is related to the polymer chain unit length (c-axis) supports the presence of flat-on lamellae morphology in the whole range of film thicknesses investigated. The flat-on lamellae morphology is also supported by AFM images. A triclinic unit cell with the c*-axis perpendicular to the substrate is proposed for PPAz. The repulsion of the long aliphatic spacer by the Si-substrate is invoked as the main reason for the flat-on morphology observed.

Utility of real-time PCR for the detection of Paracoccidioides brasiliensis DNA in the diagnosis of imported paracoccidioidomycosis.

An increase in immigration from endemic regions has resulted in a number of cases of paracoccidioidomycosis (PCM) being imported into Spain. A molecular diagnostic technique based on real-time PCR was developed for the detection of Paracoccidioides brasiliensis DNA in both culture and patients' clinical samples. A Molecular Beacon probe was used, labelled with FAM and directed at the ITS1 region of ribosomic DNA. The detection limit of the technique developed was 1 fg of fungal DNA per microl of sample. This procedure proved to be very reproducible and specific. The technique was tested with cultures of 12 clinical strains and on samples from two patients with proven PCM. Real-time PCR was positive for all the culture strains, as well as those from both patients. By samples, the technique was positive in sputum and tissue biopsies but less useful on blood samples. Samples were analyzed several months after patient treatment, detecting a small amount of fungal DNA in one respiratory sample. This technique of real-time PCR is a sensitive method for rapid diagnosis of paracoccidioidomycosis and could serve to monitor patients after treatment has begun.