Rapid Salivary IgG Antibody Screening for Hepatitis A.

Hepatitis A virus (HAV) is a common infection that is transmitted through the fecal-oral route, shed in the stool of infected individuals, and spread either by direct contact or by ingesting contaminated food or water. Each year, approximately 1.4 million acute cases are reported globally with a major risk factor for exposure being low household socioeconomic status. Recent trends show a decrease in anti-HAV antibodies in the general population, with concomitant increases in the numbers of HAV outbreaks. In line with a recreational water study, this effort aims to assess the prevalence of salivary IgG antibodies against HAV and subsequent incident infections (or immunoconversions) in visitors to a tropical beach impacted by a publicly owned treatment works (POTW). We applied a multiplex immunoassay to serially collected saliva samples gathered from study participants who recreated at Boquerón Beach, Puerto Rico. Analysis of assay results revealed an immunoprevalence rate of 16.17% for HAV with 1.43% of the cohort immunoconverting to HAV. Among those who immunoconverted, 10% reported chronic gastrointestinal symptoms and none experienced diarrhea. Tests on water samples indicated good water quality with low levels of fecal indicator bacteria; however, the collection and analysis of saliva samples afforded the ability to detect HAV infections in beachgoers. This rapid assay serves as a cost-effective tool for examining exposure to environmental pathogens and can provide critical information to policy makers, water quality experts, and risk assessment professionals seeking to improve and protect recreational water and public health.

Interlaboratory comparison of real-time PCR protocols for quantification of general fecal indicator bacteria.

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol.

Visitors to a Tropical Marine Beach Show Evidence of Immunoconversions to Multiple Waterborne Pathogens.

Determining infections from environmental exposures, particularly from waterborne pathogens is a challenging proposition. The study design must be rigorous and account for numerous factors including study population selection, sample collection, storage, and processing, as well as data processing and analysis. These challenges are magnified when it is suspected that individuals may potentially be infected by multiple pathogens at the same time. Previous work demonstrated the effectiveness of a salivary antibody multiplex immunoassay in detecting the prevalence of immunoglobulin G (IgG) antibodies to multiple waterborne pathogens and helped identify asymptomatic norovirus infections in visitors to Boquerón Beach, Puerto Rico. In this study, we applied the immunoassay to three serially collected samples from study participants within the same population to assess immunoconversions (incident infections) to six waterborne pathogens: Helicobacter pylori, Campylobacter jejuni, Toxoplasma gondii, hepatitis A virus, and noroviruses GI. I and GII.4. Further, we examined the impact of sampling on the detection of immunoconversions by comparing the traditional immunoconversion definition based on two samples to criteria developed to capture trends in three sequential samples collected from study participants. The expansion to three samples makes it possible to capture the IgG antibody responses within the survey population to more accurately assess the frequency of immunoconversions to target pathogens. Based on the criteria developed, results showed that when only two samples from each participant were used in the analysis, 25.9% of the beachgoers immunoconverted to at least one pathogen; however, the addition of the third sample reduced immunoconversions to 6.5%. Of these incident infections, the highest levels were to noroviruses followed by T. gondii. Moreover, many individuals displayed evidence of immunoconversions to multiple pathogens. This study suggests that detection of simultaneous infections is possible, with far reaching consequences for the population. The results may lead to further studies to understand the complex interactions that occur within the body as the immune system attempts to ward off these infections. Such an approach is critical to our understanding of medically important synergistic or antagonistic interactions and may provide valuable and critical information to public health officials, water treatment personnel, and environmental managers.

The development of point-of-use water filters as sampling devices in bioforensics: extent of microbial sorption and elution.

The foundational idea for this project is that household faucet-mounted water filters may be used as bioforensic sampling devices to detect the extent of a potential bioagent release in domestic water supplies. An optimized eluent solution was determined experimentally by quantifying recoveries of microorganisms from point-of-use (POU) drinking water filters. The optimized extraction protocol was then used in mock bioagent release experiments to determine the feasibility of POU filters as bioforensic sampling devices. Bacillus atrophaeus spores, Escherichia coli and PP7 virus were exposed to filters and the number of attached organisms was determined by enumerating the unattached organisms on selective agar media. Subsequently, the filters were eluted and the percent of extracted organisms was determined based on the number of attached organisms. Two popular brands of carbon block filters retained 92%-99% of representative virus, spore and vegetative bacteria. In back-flush elutions of single filters, the most efficient eluent was identified as a combination of 1% peptone and 1% Tween-80, and extraction recovered 25.4% (+/-17.5%) of attached E. coli, 20.4% (+/-3.6%) of B. atrophaeus spores, and 9.4% (+/-5.2%) of PP7 virions (+/- standard deviations). In bioagent release studies in which filters were challenged with 100 agents mL(-1), greater than 99% of the spores were retained by the filters, and the percent of attached spores that were recovered ranged from 10.4% at day 0 to 4.3% five days after the release event (averaged from five separate experiments). In contrast, E. coli, Salmonella typhimurium and PP7 virus were rapidly inactivated in the chlorinated tap water, indicating their improbable survival in chlorinated water supplies. It is therefore concluded that household water filters can be used as microbial sampling devices for bioforensic applications in the event of a bioagent release in domestic drinking water supplies.

Asymptomatic norovirus infection associated with swimming at a tropical beach: A prospective cohort study.

BACKGROUND: Swimming in fecally-contaminated waterbodies can result in gastrointestinal infections. However, the pathogenic microorganisms responsible are not well understood because sporadic cases of illness are not reported completely, exposure information is often not collected, and epidemiology studies rely on self-reported symptoms. Noroviruses are considered a likely cause because they are found in high densities in sewage, resistant to wastewater treatment and survive in the environment. In this study, saliva samples were collected from subjects at a beach in Puerto Rico and tested for evidence of norovirus-specific IgG responses as an indicator of incident norovirus infection. METHODS: Saliva samples were collected from 1298 participants using an oral swab. Samples were collected on the day of the beach visit (S1); after 10-12 days (S2); and after three weeks (S3). Saliva was tested for IgG responses to GI.1 and GII.4 noroviruses using a microsphere based multiplex salivary immunoassay. Immunoconversion was defined as a four-fold increase in median fluorescence intensity (MFI) from S1 to S2 with the S3 sample at least three times above the S1 MFI. RESULTS: Thirty-four subjects (2.6%) immunoconverted to GI.1 or GII.4 norovirus. Swimmers who immersed their head in water had a higher rate of immunoconversion (3.4%), compared to either non-swimmers (0.0%, p = 0.003) or waders and non-swimmers combined (0.4%, Odds Ratio: 5.07, 95% Confidence Interval:1.48-17.00). Immunoconversion was not associated with gastrointestinal symptoms. CONCLUSIONS: This is the first study to demonstrate an association between swimming at a beach impacted by fecal contamination and asymptomatic norovirus infection. The findings implicate recreational water as potentially important transmission pathway for norovirus infection.

Immunoprevalence to Six Waterborne Pathogens in Beachgoers at Boquerón Beach, Puerto Rico: Application of a Microsphere-Based Salivary Antibody Multiplex Immunoassay.

Waterborne infectious diseases are a major public health concern worldwide. Few methods have been established that are capable of measuring human exposure to multiple waterborne pathogens simultaneously using non-invasive samples such as saliva. Most current methods measure exposure to only one pathogen at a time, require large volumes of individual samples collected using invasive procedures, and are very labor intensive. In this article, we applied a multiplex bead-based immunoassay capable of measuring IgG antibody responses to six waterborne pathogens simultaneously in human saliva to estimate immunoprevalence in beachgoers at Boquerón Beach, Puerto Rico. Further, we present approaches for determining cutoff points to assess immunoprevalence to the pathogens in the assay. For the six pathogens studied, our results show that IgG antibodies against antigens from noroviruses GI.I and GII.4 were more prevalent (60 and 51.6%, respectively) than Helicobacter pylori (21.4%), hepatitis A virus (20.2%), Campylobacter jejuni (8.7%), and Toxoplasma gondii (8%) in the saliva of the study participants. The salivary antibody multiplex immunoassay can be used to examine immunoprevalence of specific pathogens in human populations.

Multi-laboratory survey of qPCR enterococci analysis method performance in U.S. coastal and inland surface waters.

Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are several methodological options. Here we evaluated how three method permutations, type of mastermix, sample extract dilution and use of controls in results calculation, affect method reliability among multiple laboratories with respect to sample interference. Multiple samples from each of 22 sites representing an array of habitat types were analyzed using EPA Method 1611 and 1609 reagents with full strength and five-fold diluted extracts. The presence of interference was assessed three ways: using sample processing and PCR amplifications controls; consistency of results across extract dilutions; and relative recovery of target genes from spiked enterococci in water sample compared to control matrices with acceptable recovery defined as 50 to 200%. Method 1609, which is based on an environmental mastermix, was found to be superior to Method 1611, which is based on a universal mastermix. Method 1611 had over a 40% control assay failure rate with undiluted extracts and a 6% failure rate with diluted extracts. Method 1609 failed in only 11% and 3% of undiluted and diluted extracts analyses. Use of sample processing control assay results in the delta-delta Ct method for calculating relative target gene recoveries increased the number of acceptable recovery results. Delta-delta tended to bias recoveries from apparent partially inhibitory samples on the high side which could help in avoiding potential underestimates of enterococci--an important consideration in a public health context. Control assay and delta-delta recovery results were largely consistent across the range of habitats sampled, and among laboratories. The methodological option that best balanced acceptable estimated target gene recoveries with method sensitivity and avoidance of underestimated enterococci densities was Method 1609 without extract dilution and using the delta-delta calculation method. The applicability of this method can be extended by the analysis of diluted extracts to sites where interference is indicated but, particularly in these instances, should be confirmed by augmenting the control assays with analyses for target gene recoveries from spiked target organisms.

Statistical approaches to developing a multiplex immunoassay for determining human exposure to environmental pathogens.

There are numerous pathogens that can be transmitted through water. Identifying and understanding the routes and magnitude of exposure or infection to these microbial contaminants are critical to assessing and mitigating risk. Conventional approaches of studying immunological responses to exposure or infection such as Enzyme-Linked Immunosorbent Assays (ELISAs) and other monoplex antibody-based immunoassays can be very costly, laborious, and consume large quantities of patient sample. A major limitation of these approaches is that they can only be used to measure one analyte at a time. Multiplex immunoassays provide the ability to study multiple pathogens simultaneously in microliter volumes of samples. However, there are several challenges that must be addressed when developing these multiplex immunoassays such as selection of specific antigens and antibodies, cross-reactivity, calibration, protein-reagent interferences, and the need for rigorous optimization of protein concentrations. In this study, a Design of Experiments (DOE) approach was used to optimize reagent concentrations for coupling selected antigens to Luminex™ xMAP microspheres for use in an indirect capture, multiplex immunoassay to detect human exposure or infection from pathogens that are potentially transmitted through water. Results from Helicobacter pylori, Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella typhimurium singleplexes were used to determine the mean concentrations that would be applied to the multiplex assay. Cut-offs to differentiate between exposed and non-exposed individuals were determined using finite mixed modeling (FMM). The statistical approaches developed facilitated the detection of Immunoglobulin G (IgG) antibodies to H. pylori, C. jejuni, Toxoplasma gondii, hepatitis A virus, rotavirus and noroviruses (VA387 and Norwalk strains) in fifty-four diagnostically characterized plasma samples. Of the characterized samples, the detection rate was 87.5% for H. pylori, and 100% for T. gondii assays and 89% for HAV. Further, the optimized multiplex assay revealed exposure/infection to several other environmental pathogens previously uncharacterized in the samples.

Comparison of 2 ultrafiltration systems for the concentration of seeded viruses from environmental waters.

The use of ultrafiltration as a concentration method to recover viruses from environmental waters was investigated. Two ultrafiltration systems (hollow fiber and tangential flow) in a large- (100 L) and small-scale (2 L) configuration were able to recover greater than 50% of multiple viruses (bacteriophage PP7 and T1 and poliovirus type 2) from varying water turbidities (10-157 nephelometric turbidity units (NTU)) simultaneously. Mean recoveries (n = 3) in ground and surface water by the large-scale hollow fiber ultrafiltration system (100 L) were comparable to recoveries observed in the small-scale system (2 L). Recovery of seeded viruses in highly turbid waters from small-scale tangential flow (2 L) (screen and open channel) and hollow fiber ultrafilters (2 L) (small pilot) were greater than 70%. Clogging occurred in the hollow fiber pencil module and when particulate concentrations exceeded 1.6 g/L and 5.5 g/L (dry mass) in the screen and open channel filters, respectively. The small pilot module was able to filter all concentrates without clogging. The small pilot hollow fiber ultrafilter was used to test recovery of seeded viruses from surface waters from different geographical regions in 10-L volumes. Recoveries >70% were observed from all locations.

Hollow-fiber ultrafiltration of Cryptosporidium parvum oocysts from a wide variety of 10-L surface water samples.

An optimized hollow-fiber ultrafiltration system (50 000 MWCO) was developed to concentrate Cryptosporidium oocysts from 10-L samples of environmental water. Seeded experiments were conducted using a number of surface-water samples from the southwestern U.S.A. and source water from four water districts with histories of poor oocyst recovery. Ultrafiltration produced a mean recovery of 47.9% from 19 water samples (55.3% from 39 individual tests). We also compared oocyst recoveries using the hollow-fiber ultrafiltration system with those using the Envirochek filter. In limited comparison tests, the hollow-fiber ultrafiltration system produced recoveries similar to those of the Envirochek filter (hollow fiber, 74.1% (SD = 2.8); Envirochek, 71.9% (SD = 5.2)) in low-turbidity (3.9 NTU) samples and performed better than the Envirochek filter in high-turbidity (159.0 NTU) samples (hollow fiber, 27.5%; Envirochek, 0.4%). These results indicate that hollow-fiber ultrafiltration can efficiently recover oocysts from a wide variety of surface waters and may be a cost-effective alternative for concentrating Cryptosporidium from water, given the reusable nature of the filter.

Occurrence of Cryptosporidium and Giardia in wild ducks along the Rio Grande River valley in southern New Mexico.

Fecal samples were taken from wild ducks on the lower Rio Grande River around Las Cruces, N. Mex., from September 2000 to January 2001. Giardia cysts and Cryptosporidium oocysts were purified from 69 samples by sucrose enrichment followed by cesium chloride (CsCl) gradient centrifugation and were viewed via fluorescent-antibody (FA) staining. For some samples, recovered cysts and oocysts were further screened via PCR to determine the presence of Giardia lamblia and Crytosporidium parvum. The results of this study indicate that 49% of the ducks were carriers of Cryptosporidium, and the Cryptosporidium oocyst concentrations ranged from 0 to 2,182 oocysts per g of feces (mean +/- standard deviation, 47.53 +/- 270.3 oocysts per g); also, 28% of the ducks were positive for Giardia, and the Giardia cyst concentrations ranged from 0 to 29,293 cysts per g of feces (mean +/- standard deviation, 436 +/- 3,525.4 cysts per g). Of the 69 samples, only 14 had (oo)cyst concentrations that were above the PCR detection limit. Samples did test positive for Cryptosporidium sp. However, C. parvum and G. lamblia were not detected in any of the 14 samples tested by PCR. Ducks on their southern migration through southern New Mexico were positive for Cryptosporidium and Giardia as determined by FA staining, but C. parvum and G. lamblia were not detected.

Identification of Cryptosporidium parvum oocysts by an artificial neural network approach.

Microscopic detection of Cryptosporidium parvum oocysts is time-consuming, requires trained analysts, and is frequently subject to significant human errors. Artificial neural networks (ANN) were developed to help identify immunofluorescently labeled C. parvum oocysts. A total of 525 digitized images of immunofluorescently labeled oocysts, fluorescent microspheres, and other miscellaneous nonoocyst images were employed in the training of the ANN. The images were cropped to a 36- by 36-pixel image, and the cropped images were placed into two categories, oocyst and nonoocyst images. The images were converted to grayscale and processed into a histogram of gray color pixel intensity. Commercially available software was used to develop and train the ANN. The networks were optimized by varying the number of training images, number of hidden neurons, and a combination of these two parameters. The network performance was then evaluated using a set of 362 unique testing images which the network had never "seen" before. Under optimized conditions, the correct identification of authentic oocyst images ranged from 81 to 97%, and the correct identification of nonoocyst images ranged from 78 to 82%, depending on the type of fluorescent antibody that was employed. The results indicate that the ANN developed were able to generalize the training images and subsequently discern previously unseen oocyst images efficiently and reproducibly. Thus, ANN can be used to reduce human errors associated with the microscopic detection of Cryptosporidium oocysts.

Effects of pH and magnetic material on immunomagnetic separation of Cryptosporidium oocysts from concentrated water samples.

In this study, we examined the effect that magnetic materials and pH have on the recoveries of Cryptosporidium oocysts by immunomagnetic separation (IMS). We determined that particles that were concentrated on a magnet during bead separation have no influence on oocyst recovery; however, removal of these particles did influence pH values. The optimal pH of the IMS was determined to be 7.0. The numbers of oocysts recovered from deionized water at pH 7.0 were 26.3% higher than those recovered from samples that were not at optimal pH. The results indicate that the buffers in the IMS kit did not adequately maintain an optimum pH in some water samples. By adjusting the pH of concentrated environmental water samples to 7.0, recoveries of oocysts increased by 26.4% compared to recoveries from samples where the pH was not adjusted.

Optimization of a reusable hollow-fiber ultrafilter for simultaneous concentration of enteric bacteria, protozoa, and viruses from water.

The detection and identification of pathogens from water samples remain challenging due to variations in recovery rates and the cost of procedures. Ultrafiltration offers the possibility to concentrate viral, bacterial, and protozoan organisms in a single process by using size-exclusion-based filtration. In this study, two hollow-fiber ultrafilters with 50,000-molecular-weight cutoffs were evaluated to concentrate microorganisms from 2- and 10-liter water samples. When known quantities (10(5) to 10(6) CFU/liter) of two species of enteric bacteria were introduced and concentrated from 2 liters of sterile water, the addition of 0.1% Tween 80 increased Escherichia coli strain K-12 recoveries from 70 to 84% and Salmonella enterica serovar Enteritidis recoveries from 36 to 72%. An E. coli antibiotic-resistant strain, XL1-Blue, was recovered at a level (87%) similar to that for strain K-12 (96%) from 10 liters of sterile water. When E. coli XL1-Blue was introduced into 10 liters of nonsterile Rio Grande water with higher turbidity levels (23 to 29 nephelometric turbidity units) at two inoculum levels (9 x 10(5) and 2.4 x 10(3) per liter), the recovery efficiencies were 89 and 92%, respectively. The simultaneous addition of E. coli XL1-Blue (9 x 10(5) CFU/liter), Cryptosporidium parvum oocysts (10 oocysts/liter), phage T1 (10(5) PFU/liter), and phage PP7 (10(5) PFU/liter) to 10 liters of Rio Grande surface water resulted in mean recoveries of 96, 54, 59, and 46%, respectively. Using a variety of surface waters from around the United States, we obtained recovery efficiencies for bacteria and viruses that were similar to those observed with the Rio Grande samples, but recovery of Cryptosporidium oocysts was decreased, averaging 32% (the site of collection of these samples had previously been identified as problematic for oocyst recovery). Results indicate that the use of ultrafiltration for simultaneous recovery of bacterial, viral, and protozoan pathogens from variable surface waters is ready for field deployment.