Microbial biosensors for recreational and source waters.

Biosensors are finding new places in science, and the growth of this technology will lead to dramatic improvements in the ability to detect microorganisms in recreational and source waters for the protection of public health. Much of the improvement in biosensors has followed developments in molecular biology processes and coupling these with advances in engineering. Progress in the fields of nano-engineering and materials science have opened many new avenues for biosensors. The adaptation of these diverse technological fields into sensors has been driven by the need to develop more rapid sensors that are highly accurate, sensitive and specific, and have other desirable properties, such as robust deployment capability, unattended operations, and remote data transfer. The primary challenges to the adoption of biosensors in recreational and source water applications are cost of ownership, particularly operations and maintenance costs, problems caused by false positive rates, and to a lesser extent false negative rates, legacy technologies, policies and practices which will change as biosensors improve to the point of replacing more traditional methods for detecting organisms in environmental samples.

Participation in water-exercising long-term after breast cancer surgery: Experiences of significant factors for continuing exercising as a part of cancer rehabilitation.

Although physical exercising has great benefits, little is known regarding factors of significance for cancer survivors to continue exercising within their rehabilitation. The objective was to describe factors experienced to be of significance for cancer survivors to continue with water-exercising long-term after breast cancer surgery. Women (n = 29) who had undergone breast cancer surgery (mastectomy 79%, axillary surgery 86%, and radiotherapy 86%) for median (md) 13 (25th-75th percentile 3-21.5) was followed up regarding their rehabilitation, arm function Disabilities of Arm Shoulder and Hand (md 14, IQR 7-32), EQ-5D score (md 0.8, IQR 0.73-1.0) and quality of life EQ health barometer (md 80, IQR 64-95). We performed qualitative focus-group interviews regarding the women's views (n = 24). The women had participated in water-exercising 1-46 semesters, md 8 (25th-75th percentile 3-21.5) semesters. Nearly all, 97%, participated in the water-exercising group every week, and 21 (72%) had participated in the water-exercising group at least half of the time since their breast cancer surgery, without complications. The women experienced that factors of significance to continue with water-exercising were the convenience of easily modified weightless exercising in the water, social interaction, and access to a private dressing room. These factors would be important to consider to encourage continuing in exercising.

Multigeneration Cross-Contamination of Mail with Bacillus anthracis Spores.

The release of biological agents, including those which could be used in biowarfare or bioterrorism in large urban areas, has been a concern for governments for nearly three decades. Previous incidents from Sverdlosk and the postal anthrax attack of 2001 have raised questions on the mechanism of spread of Bacillus anthracis spores as an aerosol or contaminant. Prior studies have demonstrated that Bacillus atrophaeus is easily transferred through simulated mail handing, but no reports have demonstrated this ability with Bacillus anthracis spores, which have morphological differences that may affect adhesion properties between spore and formite. In this study, equipment developed to simulate interactions across three generations of envelopes subjected to tumbling and mixing was used to evaluate the potential for cross-contamination of B. anthracis spores in simulated mail handling. In these experiments, we found that the potential for cross-contamination through letter tumbling from one generation to the next varied between generations while the presence of a fluidizer had no statistical impact on the transfer of material. Likewise, the presence or absence of a fluidizer had no statistically significant impact on cross-contamination levels or reaerosolization from letter opening.

WATER EXERCISE COMPARED TO LAND EXERCISE OR STANDARD CARE IN FEMALE CANCER SURVIVORS WITH SECONDARY LYMPHEDEMA.

There are few studies showing that physical exercise can improve secondary lymphedema. We hypothesized that water exercise would be more effective than land exercise in reducing limb volume. Secondary objectives were joint movement, BMI, daily function, well-being, and body image. Limb volume was measured with circumference or was volumetric. Well-being and body image were measured with a study-specific questionnaire and daily function with DASH and HOOS questionnaires. Eighty-eight eligible patients with secondary lymphedema after breast or gynecological cancer participated in this controlled clinical intervention study. There was a higher proportion of women who participated in water exercises who reduced their secondary arm limb volume (p = 0.029), and there were also significant differences for BMI (p = 0.047) and self-reported frequency of swelling (p = 0.031) in the water exercise group after intervention. Women with arm lymphedema in the land exercise group improved DASH scores (p = 0.047) and outer rotation in the shoulder (p = 0.001). Our results suggest that to reduce objective and self-reported swelling, lymphedema patients may be offered water exercise training while to improve daily shoulder function, land exercises are preferred. To guide female cancer survivors with lymphedema to effective exercise resulting in reduced limb volume and improved function, adequate evidenced-based programs are needed.

A Method Detection Limit for Bacillus anthracis Spores in Water Using an Automated Waterborne Pathogen Concentrator.

The method detection limit (MDL, 99% chance of detecting a positive result in a single replicate), as per the United States Code of Federal Regulations, was determined for a protocol using an ultrafiltration based automated waterborne pathogen concentration device. Bacillus anthracis Sterne strain spores were seeded at low levels into 100 L reagent water samples. Suspect colonies were confirmed through morphological, chemical, and genetic tests. Samples of 100 L (n=14) of reagent water were seeded with five B. anthracis CFUs each. To confirm the estimated detection limit, a second set (n=19) of 100 L reagent water samples were seeded at a higher level (7 CFUs). The second estimate of the MDL could not be pooled with the first, due to significant difference in variance. A third trial (n=7) seeded with 10 CFUs produced an estimate of the MDL that could be pooled with the higher previous estimate. Another trial consisting of eight 100 L samples of tap water were seeded with approximately 7 CFUs. Recovery in these samples was not significantly different from the pooled MDL. Theoretically a concentration of 4.6 spores/100 L would be required for detection 95% of the time, based on a Poisson distribution. The calculated pooled MDL, based on experimental data was approximately 6 B. anthracis CFU/100 L (95% confidence interval 4.8 to 8.4). Detection at this level was achieved in municipal water samples.

Assessment of relative potential for Legionella species or surrogates inhalation exposure from common water uses.

The Legionella species have been identified as important waterborne pathogens in terms of disease morbidity and mortality. Microbial exposure assessment is a tool that can be utilized to assess the potential of Legionella species inhalation exposure from common water uses. The screening-level exposure assessment presented in this paper developed emission factors to model aerosolization, quantitatively assessed inhalation exposures of aerosolized Legionella species or Legionella species surrogates while evaluating two generalized levels of assumed water concentrations, and developed a relative ranking of six common in-home uses of water for potential Legionella species inhalation exposure. Considerable variability in the calculated exposure dose was identified between the six identified exposure pathways, with the doses differing by over five orders of magnitude in each of the evaluated exposure scenarios. The assessment of exposure pathways that have been epidemiologically associated with legionellosis transmission (ultrasonic and cool mist humidifiers) produced higher estimated inhalation exposure doses than pathways where epidemiological evidence of transmission has been less strong (faucet and shower) or absent (toilets and therapy pool). With consideration of the large uncertainties inherent in the exposure assessment process used, a relative ranking of exposure pathways from highest to lowest exposure doses was produced using culture-based measurement data and the assumption of constant water concentration across exposure pathways. In this ranking, the ultrasonic and cool mist humidifier exposure pathways were estimated to produce the highest exposure doses, followed by the shower and faucet exposure pathways, and then the toilet and therapy pool exposure pathways.

Comparison of filters for concentrating microbial indicators and pathogens in lake water samples.

Bacterial indicators are used to indicate increased health risk from pathogens and to make beach closure and advisory decisions; however, beaches are seldom monitored for the pathogens themselves. Studies of sources and types of pathogens at beaches are needed to improve estimates of swimming-associated health risks. It would be advantageous and cost-effective, especially for studies conducted on a regional scale, to use a method that can simultaneously filter and concentrate all classes of pathogens from the large volumes of water needed to detect pathogens. In seven recovery experiments, stock cultures of viruses and protozoa were seeded into 10-liter lake water samples, and concentrations of naturally occurring bacterial indicators were used to determine recoveries. For the five filtration methods tested, the highest median recoveries were as follows: glass wool for adenovirus (4.7%); NanoCeram for enterovirus (14.5%) and MS2 coliphage (84%); continuous-flow centrifugation (CFC) plus Virocap (CFC+ViroCap) for Escherichia coli (68.3%) and Cryptosporidium (54%); automatic ultrafiltration (UF) for norovirus GII (2.4%); and dead-end UF for Enterococcus faecalis (80.5%), avian influenza virus (0.02%), and Giardia (57%). In evaluating filter performance in terms of both recovery and variability, the automatic UF resulted in the highest recovery while maintaining low variability for all nine microorganisms. The automatic UF was used to demonstrate that filtration can be scaled up to field deployment and the collection of 200-liter lake water samples.

Intragenomic sequence variation of the ITS-1 region within a single flow-cytometry-counted Cyclospora cayetanensis oocysts.

Cyclospora cayetanensis, a protozoan of emerging concern, causes self-limiting gastroenteritis in immune-competent hosts. It has been established that sequence variability exists in the first internal transcribed spacer region (ITS-1) of the ribosomal DNA operon from collections of oocysts obtained from individual or pooled fecal samples. To determine if single oocysts also exhibited ITS-1 sequence variability, DNA was extracted from individually flow-cytometry-counted oocysts. We determined that ITS-1 sequence variability exists at an individual-genome level for C. cayetanensis and approached or exceeded the variability exhibited among oocyst collections. ITS-1 variability, at the genome level, reduces this region's utility for inferring relationships between strains.

Multigeneration cross contamination of mail with Bacillus species spores by tumbling.

In 2001, envelopes loaded with Bacillus anthracis spores were mailed to Senators Daschle and Leahy as well as to the New York Post and NBC News buildings. Additional letters may have been mailed to other news agencies because there was confirmed anthrax infection of employees at these locations. These events heightened the awareness of the lack of understanding of the mechanism(s) by which objects contaminated with a biological agent might spread disease. This understanding is crucial for the estimation of the potential for exposure to ensure the appropriate response in the event of future attacks. In this study, equipment to simulate interactions between envelopes and procedures to analyze the spread of spores from a "payload" envelope (i.e., loaded internally with a powdered spore preparation) onto neighboring envelopes were developed. Another process to determine whether an aerosol could be generated by opening contaminated envelopes was developed. Subsequent generations of contaminated envelopes originating from a single payload envelope showed a consistent two-log decrease in the number of spores transferred from one generation to the next. Opening a tertiary contaminated envelope resulted in an aerosol containing 10(3) B. anthracis spores. We developed a procedure for sampling contaminated letters by a nondestructive method aimed at providing information useful for consequence management while preserving the integrity of objects contaminated during the incident and preserving evidence for law enforcement agencies.

Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizers.

Toxoplasma gondii oocysts are highly resistant to many chemical sanitizers. Methods used to determine oocyst infectivity have relied primarily on mouse, chicken, and feline bioassays. Although considered gold standards, they only provide a qualitative assessment of oocyst viability. In this study, two alternative approaches were developed to quantitate viable T. gondii oocysts following treatment with several common sanitizers. The first is a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay targeting the ACT1 and SporoSAG genes to enumerate viable T. gondii oocysts. RT-qPCR C(T) values between Wescodyne(R), acidified ethanol, or heat treated oocysts were not significantly different as compared with untreated controls. By contrast, treatment with formalin or Clorox(R) resulted in a 2-log(10) reduction in C(T) values. An in vitro T. gondii oocyst plaque assay (TOP-assay) was also developed to measure oocyst viability. This assay used a combination of bead milling and bile digestion, followed by culturing the excysted sporozoites in a confluent fibroblast cell monolayer. Results showed that no significant reduction in sporozoite viability was detected in acidified ethanol or Wescodyne(R) treated oocysts while at least a 2-log(10) reduction in plaques formed was observed with Clorox(R) treated oocysts. Moreover, formalin or heat treatment of oocysts resulted in at least a 5-log(10) reduction in plaques formed. This study demonstrates that an mRNA-based PCR viability assay targeting the ACT1 or SporoSAG genes is a relatively rapid technique compared to in vitro and in vivo assays. In addition, the TOP-assay proved very effective and sensitive at quantifying oocyst viability when compared with animal bioassays.

Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR.

AIMS: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. METHODS AND RESULTS: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. CONCLUSIONS: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms.

Concentrating Toxoplasma gondii and Cyclospora cayetanensis from surface water and drinking water by continuous separation channel centrifugation.

AIMS: To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters. METHODS AND RESULTS: Ready-to-seed vials with known quantities of T. gondii and C. cayetanensis oocysts were prepared by flow cytometry. Oocysts were seeded at densities ranging from 1 to 1000 oocysts l(-1) into 10 to 100 l test volumes of finished drinking water, water with manipulated turbidity, and the source waters from nine drinking water utilities. Oocysts were recovered using continuous separation channel centrifugation and counted on membrane filters using epifluorescent microscopy. Recovery efficiencies of both parasites were > or =84% in 10 l volumes of drinking water. In source waters, recoveries ranged from 64% to 100%, with the lowest recoveries in the most turbid waters. Method precision was between 10% and 20% coefficient of variation. CONCLUSION: Toxoplasma gondii and C. cayetanensis are effectively concentrated from various water matrices by continuous separation channel centrifugation. SIGNIFICANCE AND IMPACT OF THE STUDY: Waterborne transmission of T. gondii and C. cayetanensis presents another challenge in producing clean drinking water and protecting public health. Detection of these parasites relies on effectively concentrating oocysts from ambient water, otherwise false negatives may result. Validation data specific to T. gondii and C. cayetanensis concentration methods are limited. Continuous separation channel centrifugation recovers oocysts with high efficiency and precision, the method attributes required to accurately assess the risk of waterborne transmission.

Comparison of traditional and molecular analytical methods for detecting biological agents in raw and drinking water following ultrafiltration.

AIMS: To compare the performance of traditional methods to quantitative polymerase chain reaction (qPCR) for detecting five biological agents in large-volume drinking-water samples concentrated by ultrafiltration (UF). METHODS AND RESULTS: Drinking-water samples (100 l) were seeded with Bacillus anthracis, Cryptospordium parvum, Francisella tularensis, Salmonella Typhi, and Vibrio cholerae and concentrated by UF. Recoveries by traditional methods were variable between samples and between some replicates; recoveries were not determined by qPCR. Francisella tularensis and V. cholerae were detected in all 14 samples after UF, B. anthracis was detected in 13, and C. parvum was detected in 9 out of 14 samples. Numbers found by qPCR after UF were significantly or nearly related to those found by traditional methods for all organisms except for C. parvum. A qPCR assay for S. Typhi was not available. CONCLUSIONS: qPCR can be used to rapidly detect biological agents after UF as well as traditional methods, but additional work is needed to improve qPCR assays for several biological agents, determine recoveries by qPCR, and expand the study to other areas. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study to compare the use of traditional and qPCR methods to detect biological agents in large-volume drinking-water samples.

Recovery efficiency and limit of detection of aerosolized Bacillus anthracis Sterne from environmental surface samples.

After the 2001 anthrax incidents, surface sampling techniques for biological agents were found to be inadequately validated, especially at low surface loadings. We aerosolized Bacillus anthracis Sterne spores within a chamber to achieve very low surface loading (ca. 3, 30, and 200 CFU per 100 cm(2)). Steel and carpet coupons seeded in the chamber were sampled with swab (103 cm(2)) or wipe or vacuum (929 cm(2)) surface sampling methods and analyzed at three laboratories. Agar settle plates (60 cm(2)) were the reference for determining recovery efficiency (RE). The minimum estimated surface concentrations to achieve a 95% response rate based on probit regression were 190, 15, and 44 CFU/100 cm(2) for sampling steel surfaces and 40, 9.2, and 28 CFU/100 cm(2) for sampling carpet surfaces with swab, wipe, and vacuum methods, respectively; however, these results should be cautiously interpreted because of high observed variability. Mean REs at the highest surface loading were 5.0%, 18%, and 3.7% on steel and 12%, 23%, and 4.7% on carpet for the swab, wipe, and vacuum methods, respectively. Precision (coefficient of variation) was poor at the lower surface concentrations but improved with increasing surface concentration. The best precision was obtained with wipe samples on carpet, achieving 38% at the highest surface concentration. The wipe sampling method detected B. anthracis at lower estimated surface concentrations and had higher RE and better precision than the other methods. These results may guide investigators to more meaningfully conduct environmental sampling, quantify contamination levels, and conduct risk assessment for humans.

Detection of Toxoplasma gondii oocysts in water sample concentrates by real-time PCR.

PCR techniques in combination with conventional parasite concentration procedures have potential for the sensitive and specific detection of Toxoplasma gondii oocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were analyzed for the detection of T. gondii tachyzoites and oocysts. Lower sensitivity and specificity were obtained with the B1 gene-based PCR than with the 529-bp repeat-based PCR. New procedures for the real-time PCR detection of T. gondii oocysts in concentrates of surface water were developed and tested in conjunction with a method for the direct extraction of inhibitor-free DNA from water. This technique detected as few as one oocyst seeded to 0.5 ml of packed pellets from water samples concentrated by Envirocheck filters. Thus, this real-time PCR may provide a detection method alternative to the traditional mouse assay and microscopy.

Evaluation of ultrafiltration cartridges for a water sampling apparatus.

AIMS: To determine the efficiency of various ultrafiltration cartridges (UFC) in concentrating test micro-organisms from drinking water. METHODS AND RESULTS: Replicate drinking water samples from three potable water supplies were dosed with Bacillus anthracis Sterne, Francisella tularensis LVS, Yersinia pestis CO92, bacteriophages MS2 and phi-X174, and Cryptosporidium parvum. The test micro-organisms were dosed together in 100 l of water, which was then recirculated through one of five different UFC until the retentate volume was reduced to c. 500 ml. The micro-organisms were assayed before and after ultrafiltration concentration and per cent recoveries were calculated. There were nine statistically significant differences among pairs of filters out of a possible 180 different combinations of UFC, test micro-organisms, and water types. CONCLUSIONS: No filter consistently performed better or worse than the others for each test micro-organism in all water samples tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides performance data on the ability of several different UFC to concentrate a panel of test micro-organisms from three sources of potable water. Water utilities and first responders may use these data when selecting UFC for use in emergency response protocols. This study also provides additional data as to the efficacy of ultrafiltration for recovering bacteria, virus-like particles, and protozoan oocysts from water samples.

Evaluation of rayon swab surface sample collection method for Bacillus spores from nonporous surfaces.

AIM: To evaluate US Centers for Disease Control and Prevention recommended swab surface sample collection method for recovery efficiency and limit of detection for powdered Bacillus spores from nonporous surfaces. METHODS AND RESULTS: Stainless steel and painted wallboard surface coupons were seeded with dry aerosolized Bacillus atrophaeus spores and surface concentrations determined. The observed mean rayon swab recovery efficiency from stainless steel was 0.41 with a standard deviation (SD) of +/-0.17 and for painted wallboard was 0.41 with an SD of +/-0.23. Evaluation of a sonication extraction method for the rayon swabs produced a mean extraction efficiency of 0.76 with an SD of +/-0.12. Swab recovery quantitative limits of detection were estimated at 25 colony forming units (CFU) per sample area for both stainless steel and painted wallboard. CONCLUSIONS: The swab sample collection method may be appropriate for small area sampling (10 -25 cm2) with a high agent concentration, but has limited value for large surface areas with a low agent concentration. The results of this study provide information necessary for the interpretation of swab environmental sample collection data, that is, positive swab samples are indicative of high surface concentrations and may imply a potential for exposure, whereas negative swab samples do not assure that organisms are absent from the surfaces sampled and may not assure the absence of the potential for exposure. SIGNIFICANCE AND IMPACT OF THE STUDY: It is critical from a public health perspective that the information obtained is accurate and reproducible. The consequence of an inappropriate public health response founded on information gathered using an ineffective or unreliable sample collection method has the potential for undesired social and economic impact.

Using ultrafiltration to concentrate and detect Bacillus anthracis, Bacillus atrophaeus subspecies globigii, and Cryptosporidium parvum in 100-liter water samples.

A strategy that uses ultrafiltration (UF) to concentrate microorganisms from water samples has been developed and tested. This strategy was tested using 100-liter water samples with volume reduction achieved through ultrafiltration and recycling the microorganisms of interest through a retentate vessel, rather than returning them to the sample container, where they might pose an incremental hazard to sample takers or the environment. Three protocols based on this strategy were tested. The first protocol entailed sample volume reduction and collection of the final reduced sample. The second and third protocols both incorporated pretreatment of the filter and fluid lines with a solution to prevent microorganisms from adhering. In the second protocol, the filter was back flushed with a surfactant solution to recover microorganisms. The third protocol used recirculation of a surfactant solution to recover microorganisms. Tests were undertaken using 100-liter water samples spiked with approximately 100 or 1000 microorganisms (1 or 10 per liter). Test microorganisms included Bacillus anthracis Sterne strain, Bacillus atrophaeus subsp. globigii, and Cryptosporidium parvum. The first protocol had significantly lower recovery than the other two. Back flushing resulted in higher recovery than forward flushing, but the difference was not statistically significant.

Enhanced concentration and isolation of Cyclospora cayetanensis oocysts from human fecal samples.

Cyclospora cayetanensis is the causative agent of cyclosporiasis, an emerging infectious disease. We present a new method for the purification of C. cayetanensis oocysts from feces using a modified detachment solution and Renocal-sucrose gradient sedimentation. This method yields oocysts free from adherent fecal debris and amenable to processing using flow cytometry.