Internally controlled PCR system for detection of airborne microorganisms

Recently, we reported the outcomes of feasibility studies of a technological approach allowing rapid detection of a wide range of bioaerosols by combining a personal bioaerosol sampler with a real-time PCR technology. The protocol was found suitable for detection of targeted microorganisms within relatively short time periods. Considering the crucial importance of the PCR procedure quality control, the current paper reports the results of the development of an internally controlled PCR system for utilization by the above technology. The suggested strategy is based on utilization of only two fluorescent dyes, which are used respectively for target and internal amplification control (IAC) DNA amplification. A bacteriophage T4 and recombinant phage fd (M13) were used in this research as target and IAC, respectively. The constructed IAC was added directly to the collection liquid of the personal bioaerosol sampler enabling quality control to be present throughout the entire sampling-analysis procedures. For performance evaluation, serial ten-fold dilutions of T4 phage were aerosolized and sampled over a 10 minutes time period. The results showed that T4 phage could be reliably detected at the concentration of around 200 PFU per litre of air over the 10 minutes sampling period. The developed PCR assay demonstrated high specificity and no cross reaction. It is concluded that the recombinant phage fd is suitable for utilization as an internal control enabling to significantly minimize false negative results for bioaerosol detection procedures.     

Evaluation of interference to conventional and real-time PCR for detection and quantification of fungi in dust

Advances in polymerase chain reaction (PCR) have permitted accurate, rapid and quantitative identification of microorganisms in pure cultures regardless of viability or culturability. In this study, a simple sample processing method was investigated for rapid identification and quantification of fungal spores from dust samples using both conventional and real-time PCR. The proposed method was evaluated for susceptibility to interference from environmental dust samples. Stachybotrys chartarum and Aspergillus fumigatus were used as test organisms. The sensitivity of detection in pure culture was 0.1 spore DNA equivalents per PCR reaction corresponding to 20 spores ml(-1) in the sample. However, 1 spore DNA equivalent per PCR reaction corresponding to 200 spores ml(-1) in the sample was the lowest amount of spores tested without interference in dust samples spiked with spores of either fungal species. The extent of inhibition was calculated using conventional and real-time PCR reactions containing fungal spores, specific primers, specific probes (for real-time PCR) and various amounts of dust. The results indicate that the extent of inhibition by dust on PCR varies with the type and amount of dust, and number of spores. No interference in the analysis of spiked samples was detected from 0.2 mg ml(-1) of four real-life dust samples at p-value >0.05 using 2 x 10(4) spores for conventional PCR and 2 x 10(5) spores for real-time PCR. However, samples containing >0.2 mg ml(-1) real-life dust compromised the PCR assay. These results suggest the potential usefulness of a simple sample processing method in conjunction with PCR for monitoring the fungal content of aerosols collected from indoor environments.     

Application of PCR and probe hybridization techniques in detection of airborne fungal spores in environmental samples

Specific PCR amplification and probe hybridization techniques were applied to examine the compositions of airborne fungi in samples from three different environments. The results from microscopic and CFU counting were compared to those of the molecular-based detections. The detection sensitivity for PCR amplifications was 9 to 73 spores and 1.3 to 19.3 CFUs per PCR reaction. The hybridization detection limit was 2 to 4 spores and 0.2 to 1.2 CFU. The hybridization method was more sensitive than PCR amplification and showed less variation among samples. Using specific PCR primers and probes we identified the presence of several fungal groups and species in the air samples. Specific detections through probe hybridization to PCR products amplified with universal or group-specific fungal primers have promising applications in the examination of air samples for environmental monitoring.     

Real-time PCR detection of toxigenic Fusarium in airborne and settled grain dust and associations with trichothecene mycotoxins

Inhalation of immunomodulating mycotoxins produced by Fusarium spp. that are commonly found in grain dust may imply health risks for grain farmers. Airborne Fusarium and mycotoxin exposure levels are mainly unknown due to difficulties in identifying Fusarium and mycotoxins in personal aerosol samples. We used a novel real-time PCR method to quantify the fungal trichodiene synthase gene (tri5) and DNA specific to F. langsethiae and F. avenaceum in airborne and settled grain dust, determined the personal inhalant exposure level to toxigenic Fusarium during various activities, and evaluated whether quantitative measurements of Fusarium-DNA could predict trichothecene levels in grain dust. Airborne Fusarium-DNA was detected in personal samples even from short tasks (10-60 min). The median Fusarium-DNA level was significantly higher in settled than in airborne grain dust (p < 0.001), and only the F. langsethiae-DNA levels correlated significantly in settled and airborne dust (r(s) = 0.20, p = 0.003). Both F. langsethiae-DNA and tri5-DNA were associated with HT-2 and T-2 toxins (r(s) = 0.24-0.71, p < 0.05 to p < 00.01) in settled dust, and could thus be suitable as indicators for HT-2 and T-2. The median personal inhalant exposure to specific toxigenic Fusarium spp. was less than 1 genome m(-3), but the exposure ranged from 0-10(5) genomes m(-3). This study is the first to apply real-time PCR on personal samples of inhalable grain dust for the quantification of tri5 and species-specific Fusarium-DNA, which may have potential for risk assessments of inhaled trichothecenes.     

Real-time PCR for detection of the Aspergillus genus

Aspergillus is a genus of mold that has strong indoor sources, including several species capable of acting as opportunistic pathogens. Previous studies suggest that Aspergillus could serve as an indicator for abnormal mold growth or moisture, making it an important genus for environmental monitoring. Here, a quantitative polymerase chain reaction (qPCR, or real-time PCR) assay is presented for Aspergillus. The assay shows good specificity for the genus, detecting all Aspergillus species tested, although a few non-Aspergillus species are also amplified. Sensitivity testing demonstrates that DNA representing one conidium can be detected. A validation study compared qPCR results against direct microscopy counts using A. fumigatus conidia aerosolized into a laboratory chamber. The assay was then used to quantify Aspergillus in indoor air samples, demonstrating its utility for environmental monitoring. Analysis of a small number of clinical sputum samples showed complete agreement with culturing results.     

The statistical investigation on airborne fungi and pollen grains of atmosphere in Izmir-Turkey

This study aims to investigate the differences in the concentrations of airborne fungi and pollens between the towns located in the province of Izmir and to determine the factors contributing to these differences. Five stations in each of four towns (Buca, Konak, Bornova, and Karsiyaka) were randomly selected as the research areas. Fungus (cfu/m³) and pollen counts (cm²/pollen count) in the air samples collected from each station between June 2003 and May 2004 were measured. The results revealed that whereas Karsiyaka had the highest fungus concentration (521.33 ± 777.1), Buca and Bornova had the lowest concentration (482.67 ± 308.44). The mean fungus concentration in the province of İzmir was 501.5 ± 486.7. Pollen concentration was the highest in Konak (486.67 ± 839.06) and the lowest in Bornova (369.83 ± 551.13). Fungus and pollen concentrations revealed no difference between the towns (p > 0.05). The relationship between pollen-fungus concentrations and temperature-dust-humidity-sulphurdioxide was investigated but it was found statistically insignificant (p > 0.05). As a result of regression analysis, it was determined that correlation of atmospheric parameters had no effects on pollen and fungus concentrations (p > 0.05).     

Evaluation of airborne lead in the welding working environment

Manual and automatic welding machines (which use leaded alloys) are considered to be important sources of the emission of lead fumes into the general air of the working environment. Three workplaces at a television factory were selected for the present study, to determine the control class of the working unit. The concentrations of conventional measurements ("A" sampling points) were lower than the administrative control level (statutory standard of lead, 150 microg m(-3)), whereas the maximum concentration of 264.1 microg m(-3) ("B" sampling point) was higher at one working unit than the administrative control level. However, the control classes varied between class III (bad) and class I (good).     

Monitoring and assessment of airborne fungi in Kolkata, India, by viable and non-viable air sampling methods

The composition and variability of airborne fungal spores were studied using two complementary sampling methods in an outdoor environment in Kolkata suburb for 2 years, from November 2002 to October 2004. For monitoring the total fungal spore burden in the air, Burkard 7-day volumetric sampler was used, whereas Andersen two-sage viable sampler was used for isolating the cultivable airborne fungi. Among the 37 fungal spore types identified in the air samples, the predominant ones were Cladosporium, unidentified ascospores, unidentified basidiospores, Aspergilli/Penicilli, Nigrospora, Periconia, Chaetomium, Drechslera, Alternaria, Coprinus, Ganoderma, Pithomyces, and rust spores. Only six fungal spore types (Alternaria, Aspergilli/Penicilli, Cladosporium, Curvularia, Drechslera, and Nigrospora) were recovered in common by the two samplers. For Aspergilli/Penicilli, Drechslera, and Nigrospora, the spore concentration was underestimated in the non-viable sampling method (Burkard sampler). In general, higher spore count was recorded in winter. The highest fungal species variability was observed in early monsoon (June). Relative humidity could significantly predict the seasonal periodicity of the maximum number of airborne spores. The total airborne fungi concentration recorded in the study (15-16?×?10(3) spores m(-3) of air) was lower than the proposed threshold limit value for clinical significance, suggesting apparently no or less airborne-fungi-exposure-related health risk in the sampling area. Cladosporium cladosporioides was recorded beyond the proposed threshold limit value in January 2003 and March 2004; Aspergillus fumigatus and Aspergillus nidulans in winter that might have posed considerable health risk to sensitized individuals.     

Quantitative PCR analysis of fungi and bacteria in building materials and comparison to culture-based analysis

Prolonged moisture on building materials can lead to microbial growth on them. Microbes can emit spores, metabolites and structural parts into the indoor air and thus, cause adverse health effects of people living and working in these buildings. So far, culture methods have been used for assessment of microbial contamination of building materials. In this work, we used quantitative PCR (qPCR) for the detection of selected fungal and bacterial groups in 184 building materials of different types and compared the results with culture-based analysis. Nine either commonly found species, genera or groups of fungi, or those considered as moisture damage indicators, and one bacterial genus, Streptomyces, were determined using qPCR. Fungi and mesophilic actinomycetes were also cultivated using standard media and conditions of the routine analysis. The bacterial genus Streptomyces and the fungal group Penicillium/Aspergillus/Paecilomyces were the most prevalent microbial groups in all building material types, followed by Stachybotrys chartarum and Trichoderma viride/atroviride/koningii. The highest prevalences, concentrations and species diversity was observed on wooden materials. In general, the results of the two methods did not correlate well, since concentrations of fungi and streptomycetes were higher and their occurrence more prevalent when determined by qPCR compared to culture-based results. However, with increasing concentrations, the correlation generally increased. The qPCR assay did not detect Aspergillus versicolor and Acremonium strictum as often as culture.     

Quantitative PCR analysis of house dust can reveal abnormal mold conditions

Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean : RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotium group, and Cladosporium sphaerospermum) were >1 (Group I). Logistic regression analysis of the sum of the logs of the concentrations of Group I species resulted in a 95% probability for separating MH from RH. These results suggest that it may be possible to evaluate whether a home has an abnormal mold condition by quantifying a limited number of mold species in a dust sample. Also, four common species of Aspergillus were quantified by standard culturing procedures and their concentrations compared to QPCR results. Culturing underestimated the concentrations of these four species by 2 to 3 orders of magnitude compared to QPCR.     

Comparative study of fluorogenic and chromogenic media for specific detection of environmental isolates of thermotolerant Escherichia coli

In a field study 78 water samples were analysed employingFluorocult Brilla Broth (BB) and its performance was comparedwith standard MPN procedure. Out of 78 water samples analysed 56(71.7%) samples yielded positive reactions in BB whereas, 50(64.1%) samples were positive by standard fecal coliform test.A comparative study of fluorogenic and chromogenic mediacontaining substrate -D glucuronide for specificdetection of environmental isolates of 313 thermotolerant E.coli has been undertaken. Five fluorogenic media wereused: Fluorocult MacConkey agar (MCA), Fluorocult ECD agar(ECD), Fluorocult VRB agar (VRB), Fluorocult E. coli0157:H7 agar (ECH7) and Fluorocult Brilla Broth (BB) andChromogenic Chromocult agar (CCA). BB and CCA were found to behighly specific and sensitive media to detect E. coli asall E. coli yielded positive reaction on them. On ECH7 andECD agar 67.5 and 64.9 of E. coli isolates gave positivereaction, respectively. Low sensitivity was observed in case ofMCA and VRB agar in detecting E. coli. The performance ofBB appears to be better when compared with standard MPNprocedure employing MacConkey broth/Brilliant green bile brothin detecting E. coli in drinking water.     

Development of monitoring technology for airborne particulate matter

Particulate matter suspended in the air has adverse effects onhuman health. Its level of concentration is an important parameter in evaluating the degree of hazard it poses to the atmosphere. Conventional methods used in measuring particulatematter are often filter-based, which indicates some disadvantagesbecause such a base requires labor and time. In this study, to achieve real-time measurements, a new electrical method was developed for measuring PM10 and PM2.5 concentrations. The basicprinciple is to electrically charge particles passing through thePM inlet using a corona charger and measure the currents createdby charged particles to obtain the number concentration of particulate matter. A new type inlet based on the particle cupimpactor configuration was designed and its performance was evaluated. A unipolar diffusion charger was developed and thecharger's efficiency was determined experimentally in terms ofPn, which represents the penetration through the charger,P, times the average charge number acquired by a particle,n, for different particle sizes. The correlation was constructed between the PM10 (or the PM2.5) mass concentrationsand the electrical currents due to particles, which were chargedby the diffusion charger.     

Recovery of Francisella tularensis from soil samples by filtration and detection by real-time PCR and cELISA

The aim of this study was to develop a specific and highly sensitive method able to detect very low concentrations of Francisella tularensis in soil samples by real-time PCR (qPCR) with SYBR Green I. tul4 gene, which encodes the 17-kDa protein (TUL4) in F. tularensis strains, was amplified using a LightCycler (LC) device. We achieved a detection limit of 0.69 fg of genomic DNA from F. tularensis subp. holarctica live vaccine strain (LVS), corresponding to a value less than 3.4 genome equivalents per reaction. The qPCR was shown to be specific, highly sensitive and reproducible. In addition, we evaluated 2 new methods for recovering bacteria from soil based on 1-step filtration using glass fiber filters and PVDF filters. These filtration methods enabled us to recover F. tularensis efficiently from soil samples. As few as 50 CFU per 0.5 g of soil were detected by qPCR. Capture enzyme-linked immunosorbent assay (cELISA) allowed us to detect and quantify the amount of bacteria recovered from soil by an immunological method. Although qPCR was more sensitive than cELISA, we did not observe substantial differences in the amount of bacteria quantified by both methods.     

Observations of urban airborne particle number concentrations during rush-hour conditions: analysis of the number based size distributions and modal parameters

A summertime study of the number concentration and the size distribution of combustion derived nanometre sized particles (termed nanoparticles) from diesel and spark-ignition (SI) engine emissions were made under rush-hour and free-flow traffic conditions at an urban roadside location in Leeds, UK in July 2003. The measured total particle number concentrations (N(TOTAL)) were of the order 1.8 x 10(4) to 3.4 x 10(4) cm(-3), and tended to follow the diurnal traffic flow patterns. The N(TOTAL) was dominated by particles < or =100 nm in diameter which accounted for between 89-93% of the measured particle number. By use of a log-normal fitting procedure, the modal parameters of the number based particle size distribution of urban airborne particulates were derived from the roadside measurements. Four component modes were identified. Two nucleation modes were found, with a smaller, more minor, mode composed principally of sub-11 nm particles, believed to be derived from particles formed from the nucleation of gaseous species in the atmosphere. A second mode, much larger in terms of number, was composed of particles within the size range of 10-20 nm. This second mode was believed to be principally derived from the condensation of the unburned fuel and lube oil (the solvent organic fraction or SOF) as it cooled on leaving the engine exhaust. Third and fourth modes were noted within the size ranges of 28-65 nm and 100-160 nm, respectively. The third mode was believed to be representative of internally mixed Aitken mode particles composed of a soot/ash core with an adsorbed layer of readily volatilisable material. The fourth mode was believed to be composed of chemically aged, secondary particles. The larger nucleation and Aitken modes accounted for between 80-90% of the measured N(TOTAL), and the particles in these modes were believed to be derived from SI and diesel engine emissions. The overall size distribution, particularly in modes II-IV, was observed to be strongly related to the number of primary particle emissions, with larger count median diameters observed under conditions where low numbers of primary soot based particles were present.     

Evaluation of airborne lead levels in storage battery workshops and some welding environments in Kumasi metropolis in Ghana

Airborne lead levels were assessed in nine workshops, three each from battery, electronic repair, and welding sources within the Kumasi Metropolis in Ghana. Samples were collected at 0, 2.5, and 5.0 m away from the emission source at the workshops during working hours and another at 5.0 m during break hours. Airborne lead particulates were collected and analyzed using the filter membrane technique and flame atomic absorption spectrophotometry, respectively. There were significant differences (p ≤ 0.05) among the air lead levels from the workshops. Workshop 3b produced the highest significant values of air lead concentrations of 2,820.31 ± 53.89, 2,406.74 ± 71.87, 754.55 ± 72.52, and 549.01 ± 67.30 μg/m³ at distances of 0, 2.5, 5.0, and 5.0 m (break-time measurement), respectively, while workshop 1w significantly produced the lowest air lead concentration values of 261.06 ± 21.60, 190.92 ± 36.90, 86.43 ± 16.26, and 61.05 ± 3.88 μg/m³ at distances of 0, 2.5, 5.0, and 5.0 m (break-time measurement), respectively. The air lead levels reduced with distance from emission source at the workshops. At all the distances of measurement at working hours, the airborne lead levels were higher than the World Health Organization standard of 50 μg/m³ and exceeded the threshold limit values of 100 to 150 μg/m³ recommended in most jurisdictions. Workers and people in the immediate environs were exposed to air lead levels that were too high by most international standards, thus posing a serious threat to their health.     

Evaluation of exposure to the airborne asbestos in an asbestos cement sheet manufacturing industry in Iran

Iran imports nearly 55,000 tons of Chrysotile asbestos per year and asbestos cement (AC) plants contribute nearly 94% of the total national usage. In the present study, airborne asbestos concentrations during AC sheet manufacturing were measured. The fiber type and its chemical composition were also evaluated by scanning electron microscopy (SEM), with energy-dispersive X-ray analysis. Airborne total fiber concentrations of 45 personal samples were analyzed by phase contrast microscopy. The results have highlighted that 15.5% of samples exceed the threshold limit value (TLV) established the American Conference of Governmental Industrial Hygienists, which is 0.1 fiber per milliliter (f/ml). Personal monitoring of asbestos fiber levels indicated a ranged from 0.02?±?0.01 to 0.16?±?0.03 f/ml. The geometrical mean was 0.05?±?1.36 f/ml, which is considerably lower than the TLV. SEM data demonstrate that the fibrous particles consisted, approximately, of Chrysotile (55.89%) and amphiboles (44.11%). We conclude that the industrial consumption of imported Chrysotile asbestos is responsible for the high airborne amphibole asbestos levels in the AC sheet industry. More research is needed to improve characterization of occupational exposures by fiber size and concentration in a variety of industries.     

Repetitive extragenic palindromic PCR (REP-PCR) as a method used for bulking process detection in activated sludge

Bulking of activated sludge is a world-widely prevalent problem and can lead to loss of bio-oxidation, further deterioration of effluent quality, and even to a complete breakdown of the entire treatment process. Most common reasons of bulking are bacterial community changes, especially excessive growth of filamentous bacteria or excess of biopolymers on surface of non-filamentous microbes. Because of complex nature of the bulking phenomenon, the successful bulking control strategy finding is still a very important need awaiting new options and advices. The repetitive extragenic palindromic PCR (REP-PCR) fingerprinting method has been applied to distinguish bacterial community in non-bulking and bulking activated sludge. The characteristic REP-PCR fingerprinting patterns, using the Ward??s clustering method, have been analyzed to determine homology/similarity relation between particular non-bulking and bulking sludge sampling. The received clustering results were in high concordance with activated sludge typing done based on physicochemical sludge analysis. The choice and application of molecular typing method in sludge analysis will depend upon the needs, skill level, and resources of the laboratory. The proposed REP-PCR method and statistical analysis of fingerprinting patterns seems to be simple, rapid, and effective methods to show differences between population in non-bulking and bulking activated sludge. It is easy to implement, and it may be useful for routinely activated sludge monitoring as well as may be helpful in early detection of bulking process.     

Development of a PCR protocol for the detection of Escherichia coli O157:H7 and Salmonella spp. in surface water

Escherichia coli O157:H7 and Salmonella are pathogenic microorganisms that can cause severe gastrointestinal illness in humans. These pathogens may be transmitted in a variety of ways, including food and water. The presence of Salmonella and E. coli O157:H7 in surface waters constitutes a potential threat to human health when used for either drinking or recreation. As with most waterborne pathogens, Salmonella and E. coli O157:H7 are difficult to detect and enumerate with accuracy in surface waters due to methodological limitations. The aim of this study was to develop a protocol for the detection of Salmonella spp., E. coli O157:H7 and E. coli virulence genes (stx ₁, stx ₂ and eae) in water using a single enrichment step and PCR. In spiked water samples, PCR results showed high sensitivity (<3 CFU/L) for both microorganisms. The protocol developed in this study has been applied in different surface waters in association with microbiological and physical analysis. The frequency of PCR positive samples was 33% for Salmonella and 2% for E. coli O157:H7 producing intimin (eae) and Shiga-like toxin I (stx ₁). Moreover, the finding of amplicons corresponding to eae and stx ₁ genes in the absence of E. coli O157:H7 suggested the possible presence of other pathogenic bacteria that carry these genes (e.g. EHEC, Shigella strains). The results obtained showed that the developed protocol could be applied as a routine analysis of surface water for the evaluation of microbiological risks.     

Evaluation of simple microbiol tests for detection of fecal coliforms directly at 44.5 degrees C

Simple microbial test comprising H₂S paper strip test,presence-absence (PA) test, and fluorogenic brila broth (BB)test performed directly at 44.5 °C were evaluated andcompared with the standard most probable number (MPN) method fordetection of fecal coliforms in 173 drinking water sources. BBand PA test were comparable with standard MPN method, whereas,poor compliance was noted for H₂S test. PA test whencompared with standard MPN test only 15%; disagreement wasdetected, whereas, highest disagreement of 40%; was observed incase of H₂S test. BB test was found to be highly sensitiveas only 7.8% disagreement with that of standard MPN test wasfound. Three hundred cultures obtained from positive tests wereidentified in order to evaluate the specificities of test usedin detection of fecal indicator Escherichia coli. BB testwas also found highly specific in detection of indicatororganism as compared to PA and H₂S test. Among theorganisms isolated from BB test 84.4%; of them were identifiedas E. coli as compared to 43.4 and 33.3 in PA and H₂Stest, respectively. The low incidence of recovery of E.coli (18.1%) for the standard MPN method places doubt on thevalidity of its application in tropical areas. The result ofthis investigation suggest that BB performed directly at 44.5 °C could be suitable cost effective test to assess themicrobiological quality of drinking water in India and other tropical countries.