Kelly A. Reynolds

Risk of waterborne illness via drinking water in the United States.

The quality of drinking water in the United States is among the best in the world; however, waterborne disease outbreaks continue to occur, and many more cases of endemic illness are estimated. Documented waterborne disease outbreaks are primarily the result of technological failures or failure to treat the water (Craun et al. 2006). Current federal regulations require that all surface waters used for a drinking water supply be treated to reduce the level of pathogens so as to reduce the risk of infection to 1:10,000 per year (Regli et al. 1991). To achieve this goal, water treatment must, at a minimum, reduce infectious viruses by 99.99% and protozoan parasites by 99.9% (Regli et al. 2003). If Cryptosporidium concentrations exceed a certain level in the source water, additional reductions are required. This degree of treatment is usually achieved by a combination of physical processes (coagulation, sedimentation, and filtration) and disinfection (chlorination, ozonation). Filtration is essential for the removal of protozoan parasites due to their resistance to chlorination and ozonation at doses normally used in drinking water treatment (Barbeau et al. 2000; Korich et al. 1990; Rennecker et al. 1999). A variance from filtration is allowed in some cases if the watershed is protected and carefully monitored for protozoan pathogens.

Occurrence of bacteria and biochemical markers on public surfaces.

From 1999 – 2003, the hygiene of 1061 environmental surfaces from shopping, daycare, and office environments, personal items, and miscellaneous activities (i.e., gymnasiums, airports, movie theaters, restaurants, etc.), in four US cities, was monitored. Samples were analyzed for fecal and total coliform bacteria, protein, and biochemical markers. Biochemical markers, i.e., hemoglobin (blood marker), amylase (mucus, saliva, sweat, and urine marker), and urea (urine and sweat marker) were detected on 3% (26/801); 15% (120/801), and 6% (48/801) of the surfaces, respectively. Protein (general hygiene marker) levels ⩾ 200 μg/10 cm2 were present on 26% (200/801) of the surfaces tested. Surfaces from childrens playground equipment and daycare centers were the most frequently contaminated (biochemical markers on 36%; 15/42 and 46%; 25/54, respectively). Surfaces from the shopping, miscellaneous activities, and office environments were positive for biochemical markers with a frequency of 21% (69/333), 21% (66/308), and 11% (12/105), respectively). Sixty samples were analyzed for biochemical markers and bacteria. Total and fecal coliforms were detected on 20% (12/60) and 7% (4/60) of the surfaces, respectively. Half and one-third of the sites positive for biochemical markers were also positive for total and fecal coliforms, respectively. Artificial contamination of public surfaces with an invisible fluorescent tracer showed that contamination from outside surfaces was transferred to 86% (30/35) of exposed individuals hands and 82% (29/35) tracked the tracer to their home or personal belongings hours later. Results provide information on the relative hygiene of commonly encountered public surfaces and aid in the identification of priority environments where contaminant occurrence and risk of exposure may be greatest. Childrens playground equipment is identified as a priority surface for additional research on the occurrence of and potential exposure to infectious disease causing agents.

ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples

This study applied the integrated cell culture/polymerase chain reaction methodology (ICC/PCR) for rapid and specific detection of both cytopathogenic and noncytopathogenic viruses. Results of this study showed that the use of direct RT-PCR or conventional cell culture alone may yield erroneous results with the analysis of environmental samples. The purpose of this study was to compare cultural, molecular, and combined assays for the most effective method of virus detection in variable environmental samples. Using ICC/PCR, stock enterovirus inocula of > or =10 PFU were PCR positive in at least 4/5 replicate flasks after only 5 h of incubation in cell culture, and in all flasks after > or =10 h. An inoculum of one PFU was detected by PCR after 20 h of cell culture incubation while for concentrations of virus below one PFU, 25 h of incubation was sufficient. Similarly, hepatitis A virus (HAV) inocula of 100 MPN/flask, produced indeterminate CPE in cell culture, but were clearly detected by ICC/PCR following 48 h of incubation. Lower levels of HAV, 1 and 10 MPN, were detected by ICC/PCR after 96 to 72 h of incubation, respectively. Cell culture lysates from 11 environmental sample concentrates of sewage, marine water, and surface drinking water sources, were positive for enteroviruses by ICC/PCR compared to 3 positive by direct RT-PCR alone. Results from ICC/PCR eventually agreed with cell culture but required < or =48 h of incubation, compared to as long as 3 weeks for CPE following incubation with BGM and FRhK cells.

Detection of Giardia and Cryptosporidium in marine waters

Raw sewage disposal in marine waters is a common practice in many countries. This practice raises health risk concerns of possible transmission of Giardia and Cryptosporidium . Both of these protozoa have been shown to be transmitted by recreational swimming. To date no studies have determined the efficiency of their detection and concentration in marine waters. This study evaluated the efficiency of their detection in tap water and from marine waters in Hawaii with two different filter types. This study compared a polypropylene fiber cartridge filter, DPPPY (1.0 μm nominal porosity) (Cuno, Meriden, CT) which is typically used for parasite detection and the Filterite negatively charged filter (0.45μm) (Filtemp Sales, Inc., Phoenix, AZ). The latter would allow for both viruses and parasites to be concentrated simultaneously. The organisms were removed from the filter by passing the eluent through the filters in the opposite direction of collection and detected by indirect immunofluorescence antibody staining specific for Giardia and Cryptosporidium . Processing was simpler and faster with the Filterite filter and the overall efficiency for both Giardia and Cryptosporidium detection was greater. These methods are currently being used for the detection of the oocysts and cysts at bathing beaches in Hawaii impacted by marine sewage discharge.

Use of integrated cell culture-PCR to evaluate the effectiveness of poliovirus inactivation by chlorine.

ABSTRACT Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.

Integrated Cell Culture/PCR for Detection of Enteric Viruses in Environmental Samples

Recently, an integrated cell culture/polymerase chain reaction (ICC/PCR) technique has been developed for the detection of viruses in environmental samples providing a reliable method for practical analysis and direct monitoring of environmental samples for viral pathogens. CC/PCR allows for detection of infectious viruses in hours to days compared with the days or weeks necessary with cell culture alone. Bacterial indicator organisms are commonly used to evaluate environmental samples with respect to fecal contamination and potential public health impacts. These organisms do not correlate well with the presence of viruses, but a rapid, reliable method was not previously available for direct virus testing. Using ICC/PCR, environmental samples may be directly surveyed for pathogenic viruses, in a timely manner. Direct virus analysis will lead to better assessment of the presence and risk of human enteric viruses in the environment, so that control measures may be developed with true virus occurrence data. The ICC/PCR approach combines two previously applied virus detection methods, conventional cell culture and PCR amplification, utilizing the major advantages and overcoming the major limitations of each methodology when used alone. Cell culture assay is the standard method for the detection of viable human viruses (i.e., poliovirus, coxsackievirus, echovirus, adenovirus, hepatitis A virus, reovirus, and rotavirus) in environmental samples, serving as the method against which all newer technologies are evaluated. Although cell culture is theoretically capable of detecting a single viable virus in relatively large volumes of sample, the time required for confirmed results with conventional cell culture makes it an impractical method for routine monitoring of environmental samples. Furthermore, cell culture does not detect noncytopathogenic viruses (viruses that are viable, infecting cells, and continually spreading to neighboring cells but that do not cause a visible cytopathogenic effect [CPE] on the cell monolayer). Rotavirus and most wild-type hepatitis A viruses (HAV) are infectious to cell cultures but do not produce a clear CPE.

Opto-electrophoretic detection of bio-molecules using conducting chalcogenide glass sensors

Novel telluride glasses with high electrical conductivity, wide infrared transparency and good resistance to crystallization are used to design an opto-electrophoretic sensor for detection and identification of hazardous microorganisms. The sensor is based on an attenuated total reflectance element made of Ge-As-Te glass that serves as both an optical sensing zone and an electrode for driving the migration of bio-molecules within the evanescent wave of the sensor. An electric field is applied between the optical element and a counter electrode in order to induce the migration of bio-molecules carrying surface charges. The effect of concentration and applied voltage is tested and the migration effect is shown to be reversible upon switching the electric field. The collected signal is of high quality and can be used to identify different bacterial genus through statistical spectral analysis. This technique therefore provides the ability to detect hazardous microorganisms with high specificity and high sensitivity in aqueous environments. This has great potential for online monitoring of water quality.

Detection of enteroviruses in marine waters by direct RT-PCR and cell culture

Sewage outfalls and storm water runoff introduces pathogenic human enteric viruses into marine coastal waters, which may pose a potential public health risk. Although members of the enterovirus group have been suggested as possible indicators of sewage pollution in marine waters, the lack of rapid, sensitive and cost effective methods have prevented routine monitoring in the United States. This study compared traditional cell culture and direct RT-PCR (reverse transcriptase-polymerase chain reaction) amplification for detection of an enterovirus. Poliovirus could be recovered from 100 L of artificial seawater with an average efficiency of 77%, using adsorption and elution from electronegative filters. Viruses were eluted from the filters with 1.5% beef extract for viruses (BEV) adjusted to pH 9.5 and reconcentrated by organic flocculation to a volume of 30 mL. Substances which interfered with detection by RT-PCR were removed by treatment of the concentrates with sephadex and chelex resins. Direct RT-PCR could detect 2.5 and 0.025 PFU (plaque forming units) for single (25 cycles) and double PCR (2 × 25 cycles) in 10 μL of pure culture poliovirus samples, respectively. These methods are currently being applied to assess the occurrence of enteroviruses at marine bathing beaches influenced by sewage discharges.

Multimodal Imaging and Lighting Bias Correction for Improved μPAD-based Water Quality Monitoring via Smartphones.

Smartphone image-based sensing of microfluidic paper analytical devices (μPADs) offers low-cost and mobile evaluation of water quality. However, consistent quantification is a challenge due to variable environmental, paper, and lighting conditions, especially across large multi-target μPADs. Compensations must be made for variations between images to achieve reproducible results without a separate lighting enclosure. We thus developed a simple method using triple-reference point normalization and a fast-Fourier transform (FFT)-based pre-processing scheme to quantify consistent reflected light intensity signals under variable lighting and channel conditions. This technique was evaluated using various light sources, lighting angles, imaging backgrounds, and imaging heights. Further testing evaluated its handle of absorbance, quenching, and relative scattering intensity measurements from assays detecting four water contaminants – Cr(VI), total chlorine, caffeine, and E. coli K12 – at similar wavelengths using the green channel of RGB images. Between assays, this algorithm reduced error from μPAD surface inconsistencies and cross-image lighting gradients. Although the algorithm could not completely remove the anomalies arising from point shadows within channels or some non-uniform background reflections, it still afforded order-of-magnitude quantification and stable assay specificity under these conditions, offering one route toward improving smartphone quantification of μPAD assays for in-field water quality monitoring.

Assessment of swimmer behaviors on pool water ingestion.

Enteric pathogens in pool water can be unintentionally ingested during swimming, increasing the likelihood of acute gastrointestinal illness (AGI). AGI cases in outbreaks are more likely to submerge heads than non-cases, but an association is unknown since outbreak data are self-reported and prone to bias. In the present study, head submersion frequency and duration were observed and analyzed for associations with pool water ingestion measured using ultra high pressure liquid chromatography - tandem mass spectrometry. Frequency of splashes to the face was also quantified. Reliable tools that assess activities associated with pool water ingestion are needed to identify ingestion risk factors and at-risk populations. Objectives were to determine if the observed activities were associated with ingestion, and to test environmental sensor and videography assessment tools. Greater frequency and duration of head submersion were not associated with ingestion, but frequency of splashes to the face, leisurely swimming, and being ≤18 were. Videography was validated for assessing swimmer head submersion frequency. Results demonstrate ingestion risk factors can be identified using videography and urine analysis techniques. Expanding surveys to include questions on leisure swimming participation and frequency of splashes to the face is recommended to improve exposure assessment during outbreak investigations.