An international trial of quantitative PCR for monitoring Legionella in artificial water systems

Aims: To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae. Methods and Results: Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l^?1) and culture (CFU l^?1) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l^?1 always exceeded CFU l^?1, and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring. Conclusions: Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required. Significance and Impact of the Study: This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection.     

Effects of the fungus Aspergillus penicillioides on the house dust mite Dermatophagoides pteronyss'mus: an experimental re-evaluation

Abstract. In this report the widely-held view that house dust mites benefit from fungal contamination of the dietary substratum is re-examined. The performance of Dermatophagoides pteronyssinus (Acari: Pyroglyphidae) is documented over two successive generations in the presence or absence of the xerophilic fungus Aspergillus penicillioides (Hyphomycetales: Moniliaceae). This fungus reduced survival, development rate, adult length and fecundity of D.pteronyssinus. Detrimental effects of A.penicillioides were proportional to the fungal density. Despite the antagonistic effects of A.penicillioides, a requirement for the fungus was indicated by the poor performance of fungus-free mites in the second generation; sustained culture of D.pteronyssinus in the absence of fungi is probably not possible. It is suggested that fungi may alter the particulate nature of the substratum to the detriment of house dust mites, but also provide micronutrients deficient in the diet.     

Real-time PCR detection of environmental mycobacteria in house dust

Analysing the number and species of microbes in indoor dust is needed for assessment of human exposure to microbes in dwellings. Environmental mycobacteria are common heterotrophic bacteria in both natural and man-made environments and potential inducers of human immune system. Culture of mycobacteria from samples rich with other microbes is difficult due to the slow growth rate of mycobacteria and this has hampered the studies on their role in indoor human exposure. A quantitative, real-time 5'-nuclease (TaqMan) PCR assay was developed to detect environmental mycobacteria in indoor dust samples. The specificity of the primers and the probe targeting the 16S rDNA of mycobacteria, tested with 26 mycobacterial and 10 non-mycobacterial but related species, proved to be high. When tested on 20 indoor dust samples collected from five homes, the assay gave counts varying between 4.8 × 10⁴ and 7.2 × 10⁶ cell/g, being on average 1.1 × 10³ times higher than culture. Seasonal variation in the dust counts of mycobacteria was observed by both culture and qPCR. Total of 140 isolates considered as mycobacteria by Ziehl-Neelsen staining and GLC-analyses were subjected to PCR analysis with the mycobacterial primers, and 39 isolates to partial 16S rDNA sequencing. All proved to be mycobacteria and revealed high diversity of mycobacterial species in the dust samples. Majority of the sequences were related to M. terrae and M. avium complexes.     

Effectiveness of benzyl benzoate in elimination of house dust mites

The effect of elimination treatment with benzyl benzoate was examined in 30 adults with asthma caused by sensitivity to house dust mites. The patients were randomised into a control and an active group, who treated their mattresses with benzyl benzoate (Acarosan^®). The study lasted 12 months and the effect of the treatment was monitored by monthly dust sampling, analyzed for major allergens fromDermatophagoides pteronyssinus andfarinae by the ELISA method and for guanine by the Acarex^® method. The clinical effect was assessed by measuring lung function, daily peak flows, symptoms and medicine consumption as well as skin prick tests, and specific IgE to mite allergens. For both groups, a significant decrease was observed in house dust mite allergens, but there was no significant difference between the groups. No considerable differences were observed in clinical parameters within or between the groups. A good correlation was observed between ELISA and Acarex^®. but the latter showed a major variation in the different classes. In conclusion. treatment of mattresses with benzyl benzoate had no effect in a group of patients with asthma due to house dust mite allergy. Regular vacuum cleaning of the bed may reduce house dust mite allergen exposure.     

Occurrence of a human-associated microbial source tracking marker and its relationship with faecal indicator bacteria in an urban estuary

One of the main impacts of urban sprawl in rapidly growing countries has been contamination of coastal environments by waterborne pathogens, posing a critical risk to ecosystem and human health. Microbial source tracking (MST) has been a robust tool to identify the origin of these pathogens globally. This study compared the occurrence of a human-associated Bacteroides marker (BT-α) with faecal indicator bacteria (FIB) in an urban estuary (Golden Horn, Istanbul, Turkey). Faecal coliform (culture method), enterococci (both culture and qPCR method) concentrations and physicochemical variables were compared with the BT-α concentrations in monthly collected samples for a year (n = 108). Enterococci concentrations detected by culture and qPCR were positively correlated (r = 0·86, P < 0·01) suggesting that qPCR can be an alternative method for monitoring. BT-α marker was positive for 30% of the samples and positively correlated with enterococci (r = 0·61 and r = 0·64 for culture and qPCR methods respectively, P < 0·01). Rainfall had a moderate positive correlation with all faecal/MST indicators suggesting combined sewer overflows also severely impacted estuarine water quality. The high FIB and BT-α concentrations at upper estuary suggested that faecal pollution mainly originated from the peri-urban settlements around two creeks entering the estuary.     

The colonisation of new houses by house dust mites (Acari: Pyroglyphidae)

House dust mites,Dermatophagoides species (Acari: Pyroglyphidae), produce allergens, known for the provocation of asthma and other allergic reactions. To determine the time needed for complete colonisation of a new house by house dust mites, dust samples were collected from carpets of houses varying from 2 weeks to 2 years in age. In contrast to the expectation, no relation was found between age of the houses on the one hand and average levels of mite-allergensDer pI andDer pII and mite numbers on the other. However, presence of dogs appeared to be positively related to allergen levels. Furthermore, carpets in bedrooms appeared to contain more allergens than carpets in living-rooms. Finally, the age of the mattress was not related to allergen levels of bedroom floors.     

Isolation and identification ofNeosartorya species from house dust as hazardous indoor pollutants

Isolation of ascomycetous microfungi from 58 house dust samples from detached and apartment dwellings around Kobe City revealed that species ofTalaromyces, Eurotium andNeosartorya were common ascomycetous propagules in the house dust.Neosartorya species accounted for 8.3% of the total identified isolates, of whichN. pseudofischeri was the main constituent, indicating that it may be a common potential fungal pathogen in the dwelling environment. Based on the specimens collected,N. pseudofischeri is described and illustrated as new to Japan.     

The fauna and distribution of house dust mites in residential homes of Bandar Abbas District,Southern Iran

This study was designed to determine the occurrence, distribution and abundance of house dust mites (HDM) in residential homes in Bandar Abbas (Hormozgan Province), because of numerous complaints of allergies in this oriental city. The study area was divided in five sampling zones based on population density and geographical distribution. In each sampling zone 10 houses were randomly selected. A total of 50 home dust samples were collected using a portable vacuum cleaner for 2 min from 1 m² of the surface of mattresses, carpets, sofas and furniture in residential houses. After collection, samples were immediately frozen. Mite species were identified and counted using standard methods and keys. Of the sampled houses 88% (44 houses) were contaminated with at least one HDM species. Three species were identified: Dermatophagoides pteronyssinus (63.1%), D. farinae (32.8%) and D. evansi (4.1%) (Pyroglyphidae). Our findings indicate a relationship between HDM density and moisture and temperature of residential places. The high contamination rate of residential houses (88%) and the favourable environmental conditions for these arthropods stress that they should be considered as important allergic causing agents.     

Seasonal prevalence of allergenic mites in house dust of Kolkata Metropolis,India

During the past few decades, house dust mites have attracted worldwide interest among medical entomologists and acarologists because of their importance in causing nasobronchial allergic disorders in human beings. House dust mites are present throughout the year; however, their relative densities differ in different seasons and habitats. Because the prevalence of house dust mite allergen is important epidemiologically and clinically, detailed knowledge on the seasonal abundance of important allergenic mites is of great importance for better understanding of the pathogenesis of the disease. In view of this, a systematic survey was carried out on the prevalence of total mites and four common allergenic mites in the city of Kolkata for two consecutive years. Both bed and bedroom floor dust were collected separately from homes inhabited by asthmatic patients situated in different corners of the city on monthly basis from January 2004 to December 2005. The population levels of total mites and four common allergenic mites, namely Dermatophagoides pteronyssinus, D. farinae, Austroglycyphagus geniculatus, and Blomia tropicalis separately, were highest during the pre-monsoon period (March–May), irrespective of habitat, whereas densities were low in all cases during winter (December–February). The study indicates that season had the most significant effect on the relative abundance of house dust mites except Dermatophagoides farinae, irrespective of habitat.     

Interference in foraging behaviour of European and American house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) by catmint, Nepeta cataria (Lamiaceae)

The European and American house dust mites, Dermatophagoides pteronyssinus and D. farinae, have a huge impact upon human health worldwide due to being the most important indoor trigger of atopic diseases such as asthma, rhinitis and atopic dermatitis. Preceding studies have shown that the behavioural response of house dust mites towards volatile chemicals from food sources can be assessed using a Y-tube olfactometer assay. In the current study, we used this assay to investigate, for the first time, the ability of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), known to repel other ectoparasites affecting human and animal health, to interfere with the attraction of D. pteronyssinus and D. farinae towards a standard food source (fish flakes). Two distinct chemotypes (A and B), enriched in the iridoid compounds (4aS,7S,7aR)-nepetalactone and (4aS,7S,7aS)-nepetalactone, and the sesquiterpene (E)-(1R,9S)-caryophyllene, were used. Initial assays with a hexane extract of fish flakes (FF extract) confirmed attraction of mites to this positive control (P < 0.001 and P < 0.05 for D. pteronyssinus and D. farinae respectively), but when presented in combination with either N. cataria chemotype, tested across a range of doses (10, 1, 0.1 and 0.01 μg), decreasing attraction of mites to their food source was observed as the dose augmented. Our study shows that N. cataria, enriched in iridoid nepetalactones and (E)-(1R,9S)-caryophyllene, exhibits potent repellent activity for house dust mites, and has the potential for deployment in control programmes based on interference with normal house dust mite behaviour.     

How relevant are house dust mite-fungal interactions in laboratory culture to the natural dust system?

Both house dust and house dust mitesDermatophagoides pteronyssinus contained a wider range of fungi than laboratory mite cultures. In total, nine species of fungi were isolated fromD. pteronyssinus in house dust, and these included three xerophilic species (Eurotium amstelodami, Aspergillus penicillioides andWallemia sebi) commonly found in laboratory cultures ofD. pteronyssinus. It is concluded that mites do interact with a similar range of fungi in natural dust and in laboratory culture, but that the diversity of fungal species in the laboratory is reduced and the density of individual fungal species in culture exceeds that of house dust. In a second experiment, dust samples were incubated at room temperature with 75% relative humidity. The diversity of fungi invariably declined from up to 13 genera to the few species recorded in laboratory culture. This suggests that the dominance of xerophilic fungi in laboratory mite rearings is mediated primarily by low relative humidity, and the exclusion of air-borne spores.     

Quick and accurate detection of Sclerotinia sclerotiorum and Phomopsis spp. in soybean seeds using qPCR and seed‐soaking method

Seed‐borne pathogenic fungi can cause serious damage to soybean crops by reducing the germination, vigour and emergence of the seeds. Special attention should be paid to pathogen detection in seeds to prevent its introduction in disease‐free areas. Considering the importance of rapid and successful diagnosis of seed‐borne pathogenic fungi in soybeans, this study evaluated a method to detect Sclerotinia sclerotiorum and Phomopsis spp. in seeds using quantitative polymerase chain reaction (qPCR). Naturally infested samples were subjected to detection using qPCR and blotter test, and the findings were compared. Using soybean seeds soaked in water, both pathogens were detected at an infestation level up a 0.0625% (one infected seed out of 1,599 healthy seeds) by qPCR. This technique allowed the detection of 300 fg of S. sclerotiorum and 30 fg of Phomopsis spp. DNA in the seed samples. Phomopsis spp. was detected in 40.7% of the evaluated seed batches (81 batches) and S. sclerotiorum was detected in 32.1% of the evaluated batches, although most of the seeds had low infestation levels. It was up to 28.5 times more efficient to use qPCR rather than blotter test to detect pathogens with a low incidence of occurrence in soybean seeds. If routinely used to test healthy seeds, qPCR would contribute to reducing soybean losses due to diseases as well as decreasing the costs required to control those diseases.     

Detection of <Emphasis Type="Italic">Legionella</Emphasis> by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment

Background

Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool for risk assessment.

Results

In water collected from the apartments Legionella spp were detected by qPCR in the concentration range from LOQ to 9.6*10⁵GU/L while L. pneumophila were detected in a range from LOQ to 6.8*10⁵ GU/L. By culturing, the legionellae were detected in the range from below detection limit (> 10 CFU/L) to 1.6*10⁶ CFU/L. In circulating water and in first flush water from shower hoses, culture and qPCR showed the same tendencies. The overall correlation between the bacteria number detected by culture and the two developed qPCR assays (L. spp and L. pneumophila) was relatively poor (r² = 0.31 for culture and Legionella spp. assay, r² = 0.20 for culture and L. pneumophila assay).

Conclusion

Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc is dubious. However, the rapid detection by qPCR of high concentrations of Legionella - especially Legionella pneumophila - is valuable as an indicator of risk, although it may be false positive compared to culture results. On the other hand, the detection of a low number of bacteria by qPCR is a strong indication for the absence of risk.

     

Age structure and dynamics of house dust mite populations

Age structure—the relative numbers of eggs, immatures and adults—in populations of the house dust mitesDermatophagoides pteronyssinus andEuroglyphus maynei was investigated in four sequential monthly samples taken from mattresses in each of eight homes in Glasgow, Scotland. Additionally, age structure ofD. pteronyssinus was determined in samples taken bimonthly for 6 months from nine quadrats of a double mattress. It was found that although age structure varied considerably with time, forD. pteronyssinus in different homes the most common structure was one in which immatures were dominant, then eggs and then adults (31% of samples). Immatures or eggs were dominant in 75% of samples. ForE. maynei the age structure was quite different: the most common structure was one in which adults were dominant, then immatures and then eggs (69% of samples). In different quadrats of a double mattress, mean age structure ofD. pteronyssinus underwent a shift towards higher proportions of immatures and then eggs during the sampling period, which reflected the increase in population density detected during this period.Life and fecundity tables were constructed forD. pteronyssinus andE. maynei using previously-available in vitro data on fecundity and survivorship rates and hypothetical values based on means derived from a number of studies. From the tables the stable age distributions were calculated and compared with the age structures of the natural populations. It was found that mean age structure of natural populations ofD. pteronyssinus was fairly close to the predicted stable age distribution, but those ofE. maynei indicated the populations were in decline during the sampling period, a fact confirmed by abundance data. The concept that the rate of increase of house dust mite populations can be estimated by determining age structure of mites isolated from dust samples was explored using the hypothetical population parameters ofD. pteronyssinus. It was predicted that quite large differences in fecundity and mortality would not drastically alter the proportions of eggs, immatures and adults in stable populations.Eggs as components of the house dust mite population are considered seriously for the first time. Those ofD. pteronyssinus andE. maynei were identified and differentiated by allometry. It is stressed that forD. pteronyssinus, during the sampling period, half or more of the mites in a dust sample may be represented as eggs, and to ignore them is to deliberately make a less accurate estimate of population density than could be otherwise achieved.     

Characterization of lipopolysaccharides present in settled house dust

The 3-hydroxy fatty acids (3-OHFAs) in lipopolysaccharides (LPS) play an important role in determining endotoxin activity, and childhood exposure to endotoxin has recently been associated with reduced risk of atopic diseases. To characterize the 3-OHFAs in house dust (HD), we used gas chromatography-mass spectrometry to assay 190 HD samples. Dust from beds, bedroom floors, family rooms, and kitchen floors was collected as part of a birth cohort study of childhood asthma (study 1) and a longitudinal study of home allergen and endotoxin (study 2). We also measured endotoxin activity with a Limulus assay and computed specific activity (endotoxin activity per nanomole of LPS). Longer-chain (C(16:0) and C(18:0)) 3-OHFAs were predominant in HD compared with short-chain (C(10:0), C(12:0), and C(14:0)) acids. Endotoxin activity was positively correlated with short-chain 3-OHFAs in both studies. In study 2, 3-OH C(16:0) was negatively correlated and 3-OH C(18:0) was not correlated with endotoxin activity, consistent with previous findings that the Limulus assay responds preferentially to LPS containing short-chain 3-OHFAs. Kitchen dust contained the highest concentrations of 3-OH C(10:0), the highest endotoxin activities, and the highest specific activities (P < 0.03). Bed dust contained the largest amounts of long-chain 3-OHFAs, the highest concentrations of LPS, and the lowest specific activities. Apartments had significantly different types of LPS (P = 0.03) compared with single-family homes in study 2. These data suggest that the Limulus assay may underestimate exposure to certain types of LPS. Because nontoxic LPS may have immune modulating effects, analysis of 3-OHFAs may be useful in epidemiologic studies.     

Quantitative real‐time PCR detection of a harmful unarmoured dinoflagellate,Karlodinium australe (Dinophyceae)

We investigated a harmful algal bloom (HAB) associated with the massive fish kills in Johor Strait, Malaysia, which recurred a year after the first incident in 2014. This incident has urged for the need to have a rapid and precise method in HAB monitoring. In this study, we develop a SYBR green‐based real‐time PCR (qPCR) to detect the culpable dinoflagellate species, Karlodinium australe. Species‐specific qPCR primers were designed in the gene region of the second internal transcribed spacer of the ribosomal RNA gene (rDNA). The species specificity of the primers designed was evaluated by screening on the non‐target species (Karlodinium veneficum, Takayama spp., and Karenia spp.) and no cross‐detection was observed. The extractable gene copies per cell of K. australe determined in this study were 19 998 ± 505 (P < 0.0001). Estimation of cell densities by qPCR in the experimental spiked samples showed high correlation with data determined microscopically (R² = 0.93). Using the qPCR assay developed in this study, we successfully detected the 2015 bloom species as K. australe. Single‐cell PCR and rDNA sequencing from the field samples further confirmed the finding. With the sensitivity as low as five cells, the qPCR assay developed in this study could effectively and rapidly detect cells of K. australe in the environmental samples for monitoring purpose.     

Specific Measurement of a Major Mite Allergen,Der f II,by an Enzyme-linked Immunosorbent Assay System Using Monoclonal Anti-Der II Antibodies

An enzyme linked immunosorbent assay system using a monoclonal antibody, 15E11, specific for a major allergen Der f II in house dust mite, was developed. This system detected only Der f II in the presence of Der p II and other allergens. The Der f II contents in several house dust samples significantly correlated with the numbers of the mites in the same house dust samples (n = 14, r = 0.88, p < 0.001). These data showed that this system was useful for specific measurement of Der f II in house dusts.     

Improving PCR and qPCR detection of hydrogenase A (<Emphasis Type="Italic">hyd</Emphasis>A) associated with Clostridia in pure cultures and environmental sludges using bovine serum albumin

Detection of hydA genes of Clostridia spp. using degenerative and species specific primers for C. butyricum were optimized by the addition of bovine serum albumin (BSA) to polymerase chain reaction (PCR) and quantitative PCR (qPCR) reactions. BSA concentrations ranging from 100 to 400 ng/μl were examined using pure cultures and a variety of environmental samples as test targets. A BSA concentration of 100 ng/μl, which is lower than previously reported in the literature, was found to be most effective in improving the detection limit. The brightness of amplicons with 100 ng/μl BSA increased in ethidium bromide-treated gels, the minimum detection limit with BSA was at least one log greater, and cycle threshold (C _T) values were lower than without BSA in qPCR indicating improved detection of target deoxyribonucleic acid for most samples tested. Although amplicon visualization was improved at BSA concentrations greater than or equal to 100 ng/μl, gene copy numbers detected by qPCR were less, C_T values were increased, and T _m values were altered. SYBR Green dissociation curves of qPCR products of DNA from pure culture or sludge samples showed that BSA at 100 ng/μl reduced the variability of peak areas and T _m values.