Use of a Disposable Water Filter for Prevention of False-Positive Results due to Nontuberculosis Mycobacteria in a Clinical Laboratory Performing Routine Acid-Fast Staining for Tuberculosis

A point-of-use 0.2-μm filter was evaluated for elimination of nontuberculosis mycobacteria in laboratory water to reduce false-positive acid-fast bacillus staining results. Use of the point-of-use filter can significantly reduce the false-positive rate to 1.2% compared to samples treated with tap water (10.7%) and deionized water (8.7%).     

Reducing false-positive prediction of minimotifs with a genetic interaction filter

Background

Minimotifs are short contiguous peptide sequences in proteins that have known functions. At its simplest level, the minimotif sequence is present in a source protein and has an activity relationship with a target, most of which are proteins. While many scientists routinely investigate new minimotif functions in proteins, the major web-based discovery tools have a high rate of false-positive prediction. Any new approach that reduces false-positives will be of great help to biologists.

Methods and Findings

We have built three filters that use genetic interactions to reduce false-positive minimotif predictions. The basic filter identifies those minimotifs where the source/target protein pairs have a known genetic interaction. The HomoloGene genetic interaction filter extends these predictions to predicted genetic interactions of orthologous proteins and the node-based filter identifies those minimotifs where proteins that have a genetic interaction with the source or target have a genetic interaction. Each filter was evaluated with a test data set containing thousands of true and false-positives. Based on sensitivity and selectivity performance metrics, the basic filter had the best discrimination for true positives, whereas the node-based filter had the highest sensitivity. We have implemented these genetic interaction filters on the Minimotif Miner 2.3 website. The genetic interaction filter is particularly useful for improving predictions of posttranslational modifications such as phosphorylation and proteolytic cleavage sites.

Conclusions

Genetic interaction data sets can be used to reduce false-positive minimotif predictions. Minimotif prediction in known genetic interactions can help to refine the mechanisms behind the functional connection between genes revealed by genetic experimentation and screens.     

Evaluation of Enterolert for enumeration of enterococci in recreational waters.

Enterolert (IDEXX Laboratories Inc., Westbrook, Maine), a semiautomated, most probable number method for enumeration of enterococci, was compared with the standard membrane filter method by parallel testing of 138 marine and freshwater recreational bathing water samples. No statistically significant difference and a strong linear correlation were found between methods. Culturing of 501 Enterolert test wells resulted in false-positive and false-negative rates of 5.1 and 0.4%, respectively. Less time for setup, incubation (24 versus 48 h), and reading of Enterolert permits more efficient monitoring of recreational bathing areas.     

Effects of Temperature and pH on Reduction of Bacteria in a Point-of-Use Drinking Water Treatment Product for Emergency Relief

The effects of temperature and pH on the water treatment performance of a point-of-use (POU) coagulant/disinfectant product were evaluated. Cold temperatures (∼5°C) reduced the bactericidal efficiency of the product with regard to Escherichia coli and total coliform log₁₀ reductions.     

Effect of point-of-use disinfection, flocculation and combined flocculation-disinfection on drinking water quality in western Kenya

AIMS: Point-of-use drinking water disinfection with sodium hypochlorite has been shown to improve water quality and reduce diarrhoeal disease. However, the chlorine demand of highly turbid water may render sodium hypochlorite less effective. METHODS AND RESULTS: We evaluated a novel combined flocculant-disinfectant point-of-use water treatment product and compared its effect on drinking water quality with existing technologies in western Kenya. In water from 30 sources, combined flocculant-disinfectant reduced Escherichia coli concentrations to <1 CFU100 ml(-1) for 29 (97%) and reduced turbidity to <5 nephelometric turbidity units (NTU) for 26 (87%). By contrast, water from 30 sources treated with sodium hypochlorite reduced E. coli concentrations to <1 CFU 100 ml(-1) for 25 (83%) and turbidity to <5 NTU for 5 (17%). CONCLUSIONS: For source waters over a range of turbidities in western Kenya, combined flocculant-disinfectant product effectively reduces turbidity to <5 NTU and reduces E. coli concentrations to <1 CFU 100 ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: The novel flocculant-disinfectant product may be acceptable to consumers and may be effective in reducing diarrhoeal disease in settings where source water is highly turbid.     

What Point-of-Use Water Treatment Products Do Consumers Use? Evidence from a Randomized Controlled Trial among the Urban Poor in Bangladesh

Background

There is evidence that household point-of-use (POU) water treatment products can reduce the enormous burden of water-borne illness. Nevertheless, adoption among the global poor is very low, and little evidence exists on why.

Methods

We gave 600 households in poor communities in Dhaka, Bangladesh randomly-ordered two-month free trials of four water treatment products: dilute liquid chlorine (sodium hypochlorite solution, marketed locally as Water Guard), sodium dichloroisocyanurate tablets (branded as Aquatabs), a combined flocculant-disinfectant powdered mixture (the PUR Purifier of Water), and a silver-coated ceramic siphon filter. Consumers also received education on the dangers of untreated drinking water. We measured which products consumers used with self-reports, observation (for the filter), and chlorine tests (for the other products). We also measured drinking water''s contamination with E. coli (compared to 200 control households).

Findings

Households reported highest usage of the filter, although no product had even 30% usage. E. coli concentrations in stored drinking water were generally lowest when households had Water Guard. Households that self-reported product usage had large reductions in E. coli concentrations with any product as compared to controls.

Conclusion

Traditional arguments for the low adoption of POU products focus on affordability, consumers'' lack of information about germs and the dangers of unsafe water, and specific products not meshing with a household''s preferences. In this study we provided free trials, repeated informational messages explaining the dangers of untreated water, and a variety of product designs. The low usage of all products despite such efforts makes clear that important barriers exist beyond cost, information, and variation among these four product designs. Without a better understanding of the choices and aspirations of the target end-users, household-based water treatment is unlikely to reduce morbidity and mortality substantially in urban Bangladesh and similar populations.     

New medium for the simultaneous detection of total coliforms and Escherichia coli in water.

A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities. TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound that fluoresced under longwave UV light (366 nm), while E. coli produced beta-glucuronidase, which cleaved indoxyl-beta-D-glucuronide to form a blue color. The new medium TC and E. coli recoveries were compared with those of mEndo agar and two E. coli media, mTEC agar and nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide, using natural water samples and spiked drinking water samples. On average, the new medium recovered 1.8 times as many TC as mEndo agar, with greatly reduced background counts (< or = 7%). These differences were statistically significant (significance level, 0.05). Although the overall analysis revealed no statistically significant difference between the E. coli recoveries on MI agar and mTEC agar, the new medium recovered more E. coli in 16 of 23 samples (69.6%). Both MI agar and mTEC agar recovered significantly more E. coli than nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Specificities for E. coli, TC, and noncoliforms on MI agar were 95.7% (66 of 69 samples), 93.1% (161 of 173 samples), and 93.8% (61 of 65 samples), respectively. The E. coli false-positive and false-negative rates were both 4.3%. This selective and specific medium, which employs familiar membrane filter technology [corrected] to analyze several types of water samples, is less expensive than the liquid chromogen and fluorogen media and may be useful for compliance monitoring of drinking water.     

Comparison of two methods for detection of Giardia cysts and Cryptosporidium oocysts in water.

The steps of two immunofluorescent-antibody-based detection methods were evaluated for their efficiencies in detecting Giardia cysts and Cryptosporidium oocysts. The two methods evaluated were the American Society for Testing and Materials proposed test method for Giardia cysts and Cryptosporidium oocysts in low-turbidity water and a procedure employing sampling by membrane filtration, Percoll-Percoll step gradient, and immunofluorescent staining. The membrane filter sampling method was characterized by higher recovery rates in all three types of waters tested: raw surface water, partially treated water from a flocculation basin, and filtered water. Cyst and oocyst recovery efficiencies decreased with increasing water turbidity regardless of the method used. Recoveries of seeded Giardia cysts exceeded those of Cryptosporidium oocysts in all types of water sampled. The sampling step in both methods resulted in the highest loss of seeded cysts and oocysts. Furthermore, much higher recovery efficiencies were obtained when the flotation step was avoided. The membrane filter method, using smaller tubes for flotation, was less time-consuming and cheaper. A serious disadvantage of this method was the lack of confirmation of presumptive cysts and oocysts, leaving the potential for false-positive Giardia and Cryptosporidium counts when cross-reacting algae are present in water samples.     

Persistence of Nontuberculous Mycobacteria in a Drinking Water System after Addition of Filtration Treatment

There is evidence that drinking water may be a source of infections with pathogenic nontuberculous mycobacteria (NTM) in humans. One method by which NTM are believed to enter drinking water distribution systems is by their intracellular colonization of protozoa. Our goal was to determine whether we could detect a reduction in the prevalence of NTM recovered from an unfiltered surface drinking water system after the addition of ozonation and filtration treatment and to characterize NTM isolates by using molecular methods. We sampled water from two initially unfiltered surface drinking water treatment plants over a 29-month period. One plant received the addition of filtration and ozonation after 6 months of sampling. Sample sites included those at treatment plant effluents, distributed water, and cold water taps (point-of-use [POU] sites) in public or commercial buildings located within each distribution system. NTM were recovered from 27% of the sites. POU sites yielded the majority of NTM, with >50% recovery despite the addition of ozonation and filtration. Closely related electrophoretic groups of Mycobacterium avium were found to persist at POU sites for up to 26 months. Water collected from POU cold water outlets was persistently colonized with NTM despite the addition of ozonation and filtration to a drinking water system. This suggests that cold water POU outlets need to be considered as a potential source of chronic human exposure to NTM.     

Effect of point-of-use, activated carbon filters on the bacteriological quality of rural groundwater supplies.

The water quality of 24 rural, domestic groundwater supplies treated with point-of-use, powdered activated carbon (PAC) filters was monitored to determine how such treatment might impact the bacteriological quality of private, residential drinking water supplies. Heterotrophic-plate-count (HPC) and total coliform analyses were performed on raw, PAC-treated, and overnight or stagnant (first-draw) PAC-treated water samples. Densities of HPC bacteria were elevated by 0.86 and 0.20 orders of magnitude for spring and well water systems, respectively, in PAC-treated effluents following overnight stagnation compared with levels in untreated treated effluents. Densities of HPC bacteria in PAC-treated effluents were significantly reduced (P < 0.01) below influent levels, however, after the point-of-use device was flushed for 2 min. While PAC significantly reduced the number of coliforms in product waters (P < 0.01), these indicator organisms were still detected in some effluents. Seasonal variations were evident in microbial counts from spring but not well water systems. It appears that aside from periods following stagnant-water use, such as overnight, PAC treatment does not compromise the bacteriological quality of drinking water obtained from underground sources.     

Evaluation of a Semiautomated System for Direct Fluorescent Antibody Detection of Salmonellae

A semi-automatic system under development by Aerojet Medical and Biological Systems for the direct fluorescent antibody detection of salmonellae was evaluated with various food, feed, and environmental samples. All samples were simultaneously examined by Automated Bioassay System (ABS), manual direct fluorescent antibody procedures and cultural procedures. The ABS gave satisfactory results with the processed samples. It detected all of the culturally positive powdered egg and candy samples with no false negative results and gave only 6.6 and 5.3% false positive rates, respectively. With meatmeal samples the ABS failed to detect one culturally positive specimen that was also positive by manual fluorescent antibody and gave one (1.1%) false-positive result. A high rate of false-negative results was obtained by ABS on unprocessed samples of creek water, poultry, and sausage. Adding another enrichment step to the protocol reduced the false-negative rate considerably but severely increased the false-positive rate. The instruments worked reasonably well, but research is needed to improve enrichment procedures for samples to be processed by the system.     

Synergistic False-Positive Coliform Reaction on M-Endo MF Medium

The reported occurrence of synergistic false-positive coliform reactions on M-Endo MF medium was investigated. The objectives of the study were (i) to isolate populations of bacteria which produced such false-positive reactions, (ii) to determine whether false-positive sheen reactions are a result of synergistic interaction, and (iii) to determine the metabolic intermediates involved in false-positive sheen production. Samples of river water were subjected to coliform analysis by the membrane filter technique with M-Endo MF medium. Suspect sheen-forming colonies were analyzed to determine purity and identity of cultures. Mixed cultures were separated, and individual isolates were examined for sheen production. The isolates not producing a sheen were recombined and tested further for sheen production. Those mixtures reproducing the sheen were characterized biochemically and tested to determine the metabolic intermediates involved. Chromatographic analysis of the metabolites showed that individual isolates produced an assortment of neutral organic compounds including 2-butanone, 2,3-butanedione, formaldehyde, and butyraldehyde, whereas acetaldehyde or propionaldehyde was produced only by the mixed cultures. Tests showed that both propionaldehyde and acetaldehyde could react to produce a sheen on M-Endo medium. The conclusion was reached that synergistic false-positive coliform reactions do indeed occur on M-Endo MF medium.     

Persistence of nontuberculous mycobacteria in a drinking water system after addition of filtration treatment

There is evidence that drinking water may be a source of infections with pathogenic nontuberculous mycobacteria (NTM) in humans. One method by which NTM are believed to enter drinking water distribution systems is by their intracellular colonization of protozoa. Our goal was to determine whether we could detect a reduction in the prevalence of NTM recovered from an unfiltered surface drinking water system after the addition of ozonation and filtration treatment and to characterize NTM isolates by using molecular methods. We sampled water from two initially unfiltered surface drinking water treatment plants over a 29-month period. One plant received the addition of filtration and ozonation after 6 months of sampling. Sample sites included those at treatment plant effluents, distributed water, and cold water taps (point-of-use [POU] sites) in public or commercial buildings located within each distribution system. NTM were recovered from 27% of the sites. POU sites yielded the majority of NTM, with >50% recovery despite the addition of ozonation and filtration. Closely related electrophoretic groups of Mycobacterium avium were found to persist at POU sites for up to 26 months. Water collected from POU cold water outlets was persistently colonized with NTM despite the addition of ozonation and filtration to a drinking water system. This suggests that cold water POU outlets need to be considered as a potential source of chronic human exposure to NTM.     

Direct counting of Cryptosporidium parvum oocysts using fluorescence in situ hybridization on a membrane filter

This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8+/-0.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe.     

Evaluation of MK filters for recovery of enteroviruses from tap water.

The MK filter is an electropositively charged filter that can be used to concentrate enteroviruses from large volumes (400 to 1,000 liters) of water. This filter is less expensive than the commonly used 1MDS electropositive filter. In this study, we compared the recovery of poliovirus 1 (PV1) and that of coxsackievirus B3 (CB3) from 378 liters of tap water, using both the MK and the 1MDS filters. Viruses were eluted from the filters with 3% beef extract buffered with 0.05 M glycine (pH 9.5) and reconcentrated via organic flocculation. At high virus inputs (approximately 10(6) PFU), the overall recovery (after elution and reconcentration) of PV1 and CB3 from tap water with the MK filter was less than that achieved with the 1MDS filter (P < 0.05). The recoveries of PV1 from tap water with the MK and 1MDS filters were 73.2% +/- 26% (n = 5 trials) and 90.2% +/- 5.9% (n = 5 trials), respectively. The recoveries of CB3 from tap water with the MK and 1MDS filters were 32.8% +/- 34.5% (n = 4 trials) and 95.8% +/- 12.0% (n = 4 trials), respectively. This study indicated that the MK filter consistently provided lower recovery, with wider variability, of PV1 and CB3 from tap water than the 1MDS filter.     

Expression of RNA isolated from the water-shunting complex of a sap-sucking insect increases the membrane permeability for water in Xenopus oocytes.

The highly specialized membranes of the filter chamber found in the digestive tract of some homopteran insects could represent a favorable material for characterizing water channels. In order to demonstrate that membrane proteins of this epithelial complex serve as water channels, we have investigated the membrane permeability for water in Xenopus oocytes injected with RNA isolated from the filter chamber. Volumes of oocytes injected with filter chamber RNA were increased by 15% following a 16-min osmotic shock, while volumes of oocytes injected with RNA from midgut not of filter chamber or with water were increased only by 8.5 and 10%, respectively. This significant difference in oocyte swelling leads us to conclude that RNA isolated from the filter chamber contains mRNA coding for water channel proteins.     

Effects of activated carbon and bacteriostatic filters on microbiological quality of drinking water

Three activated carbon filters for point-of-use water treatment were tested in laboratory and field studies for chemical removal and microbiological effects on water. All removed free available chlorine in municipally treated water to below the limit of detection, but removed only about 50 to 70% of the total available chlorine and 4 to 33% of the total organic carbon. Standard plate count bacteria in the effluent increased steadily with time for 3 weeks and remained elevated over the 8-week period of the study. Total coliform bacteria were found to persist and proliferate on the filters for several days after transient contamination of the influent water. Silver-containing activated carbon filters suppressed total coliform but not total bacterial growth. Pseudomonas aeruginosa was recovered from the effluents of all filters at some time during the tests.     

Effects of activated carbon and bacteriostatic filters on microbiological quality of drinking water.

Three activated carbon filters for point-of-use water treatment were tested in laboratory and field studies for chemical removal and microbiological effects on water. All removed free available chlorine in municipally treated water to below the limit of detection, but removed only about 50 to 70% of the total available chlorine and 4 to 33% of the total organic carbon. Standard plate count bacteria in the effluent increased steadily with time for 3 weeks and remained elevated over the 8-week period of the study. Total coliform bacteria were found to persist and proliferate on the filters for several days after transient contamination of the influent water. Silver-containing activated carbon filters suppressed total coliform but not total bacterial growth. Pseudomonas aeruginosa was recovered from the effluents of all filters at some time during the tests.     

Quantitative procedure for enumeration of bifidobacteria.

A membrane filter technique has been developed for the enumeration of bifidobacteria in natural aquatic environments. The technique is quantitative, selective, and differential. The medium (YN-6) contains: yeast extract, 2.0 g; agar, 1.5 g; polypeptone peptone, 1.0 g; vitamin-free Casamino Acids, 0.8 g; sodium chloride, 0.32 g; and L-cysteine hydrochloride, 0.003 g; in 100 ml of deionized water. The medium is adjusted to pH 7.0 before autoclaving. Nalidixic acid (80 micrograms/ml), neomycin sulfate (2.5 micrograms/ml), and bromcresol green (300 micrograms/ml) are included as selective and differential agents. After incubation for 48 h at 37 degrees C in an anaerobic environment, Gram-stained smears from green, glistening, smooth entire colonies are examined microscopically for typical bifidobacterial morphology. No significant difference in recoveries was observed when YN-6 was compared with reinforced clostridial agar, using bifidobacteria freshly isolated from feces and raw sewage. Using this technique with aquatic and fecal samples, less than 9% false-positive and 8% false-negative isolates were observed. These results indicated that the medium was able to satisfactorily recover organisms from a variety of situations.     

Presence and partial characterization of internal acid protease of Aspergillus oryzae.

A membrane filter procedure is described for the enumeration of Candida albicans in natural waters. Several hundred milliliters of sample can be examined by filtration through 1.2-micrometer membranes. Selectivity is achieved by the use of a defined (yeast-nitrogen base plus maltos-) agar medium inclusion of the antimicrobial agents chloramphenicol and cycloheximide, and incubation at 37 degrees C. C. albicans colonies are differentiated primarily through color by use of a bismuth salt indicator system. Average recovery of various strains of C. albicans stressed in seawater at 4 degrees C was 82%, compared with those of spread plate controls on a noninhibitory medium. With river water and raw sewage, 90% of typical C. albicans colonies were confirmed as such in a simplified germ tube test. Atypical colonies verified as C. albicans were infrequent (3%). C. tropicalis and Torulopsis candida were the most common false-positive colonies.