Development, confirmation, and application of a seeded Escherichia coli process control organism to validate Salmonella enterica serovar Typhi environmental surveillance methods

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of Typhoid fever. Blood culture is the gold standard for clinical diagnosis, but this is often difficult to employ in resource limited settings. Environmental surveillance of waste-impacted waters is a promising supplement to clinical surveillance, however validating methods is challenging in regions where S. Typhi concentrations are low. To evaluate existing S. Typhi environmental surveillance methods, a novel process control organism (PCO) was created as a biosafe surrogate. Using a previous described qPCR assay, a modified PCR amplicon for the staG gene was cloned into E. coli. We developed a target region that was recognized by the Typhoid primers in addition to a non-coding internal probe sequence. A multiplex qPCR reaction was developed that differentiates between the typhoid and control targets, with no cross-reactivity or inhibition of the two probes. The PCO was shown to mimic S. Typhi in lab-based experiments with concentration methods using primary wastewater: filter cartridge, recirculating Moore swabs, membrane filtration, and differential centrifugation. Across all methods, the PCO seeded at 10 CFU/mL and 100 CFU/mL was detected in 100% of replicates. The PCO is detected at similar quantification cycle (Cq) values across all methods at 10 CFU/mL (Average = 32.4, STDEV = 1.62). The PCO was also seeded into wastewater at collection sites in Vellore (India) and Blantyre (Malawi) where S. Typhi is endemic. All methods tested in both countries were positive for the seeded PCO. The PCO is an effective way to validate performance of environmental surveillance methods targeting S. Typhi in surface water

Evaluation of micro-well collector for capture and analysis of aerosolized Bacillus subtilis spores.

Bioaerosol sampling and identification are vital for the assessment and control of airborne pathogens, allergens, and toxins. In-situ analysis of chemical and biological particulate matter can significantly reduce the costs associated with sample preservation, transport, and analysis. The analysis of conventional filters is challenging, due to dilute samples in large collection regions. A low-cost cartridge for collection and analysis of aerosols is developed for use in epidemiological studies and personal exposure assessments. The cartridge collects aerosol samples in a micro-well which reduces particles losses due to the bounce and does not require any coating. The confined particle collection area (dwell~1.4 mm) allows reducing the elution volume for subsequent analysis. The performance of the cartridge is validated in laboratory studies using aerosolized bacterial spores (Bacillus subtilis). Colony forming unit analysis is used for bacterial spore enumeration. Cartridge collection efficiency is evaluated by comparison with the reference filters and found to be consistent with tested flow rates. Sample recovery for the pipette elution is ~80%. Due to the high density of the collected sample, the cartridge is compatible with in-situ spectroscopic analysis and sample elution into the 10-20 μl liquid volume providing a significant increase in sample concentration for subsequent analysis

Method Development for Enteric Virus Recovery from Primary Sludge

Enteric viruses, such as poliovirus, are a leading cause of gastroenteritis, which causes 2–3 million deaths annually. Environmental surveillance of wastewater supplements clinical surveillance for monitoring enteric virus circulation. However, while many environmental surveillance methods require liquid samples, some at-risk locations utilize pit latrines with waste characterized by high solids content. This study’s objective was to develop and evaluate enteric virus concentration protocols for high solids content samples. Two existing protocols were modified and tested using poliovirus type 1 (PV1) seeded into primary sludge. Method 1 (M1) utilized acid adsorption, followed by 2 or 3 elutions (glycine/sodium chloride and/or threonine/sodium chloride), and skimmed milk flocculation. Method 2 (M2) began with centrifugation. The liquid fraction was filtered through a ViroCap filter and eluted (beef extract/glycine). The solid fraction was eluted (beef extract/disodium hydrogen phosphate/citric acid) and concentrated by skimmed milk flocculation. Recovery was enumerated by plaque assay. M1 yielded higher PV1 recovery than M2, though this result was not statistically significant (26.1% and 15.9%, respectively). M1 was further optimized, resulting in significantly greater PV1 recovery when compared to the original protocol (p < 0.05). This method can be used to improve understanding of enteric virus presence in communities without liquid waste streams

The size distribution of airborne particles.

<p>The size distribution of airborne particles.</p

<i>B</i>. <i>subtilis</i> collection efficiencies in μ-well inertial impactors as a function of the collector flow rate.

<p><i>B</i>. <i>subtilis</i> collection efficiencies in μ-well inertial impactors as a function of the collector flow rate.</p

The measured pressure drop of the collection cartridge at different flow rates.

<p>The measured pressure drop of the collection cartridge at different flow rates.</p

Evaluation of micro-well collector for capture and analysis of aerosolized <i>Bacillus subtilis</i> spores

<div><p>Bioaerosol sampling and identification are vital for the assessment and control of airborne pathogens, allergens, and toxins. In-situ analysis of chemical and biological particulate matter can significantly reduce the costs associated with sample preservation, transport, and analysis. The analysis of conventional filters is challenging, due to dilute samples in large collection regions. A low-cost cartridge for collection and analysis of aerosols is developed for use in epidemiological studies and personal exposure assessments. The cartridge collects aerosol samples in a micro-well which reduces particles losses due to the bounce and does not require any coating. The confined particle collection area (d_well~1.4 mm) allows reducing the elution volume for subsequent analysis. The performance of the cartridge is validated in laboratory studies using aerosolized bacterial spores (<i>Bacillus subtilis</i>). Colony forming unit analysis is used for bacterial spore enumeration. Cartridge collection efficiency is evaluated by comparison with the reference filters and found to be consistent with tested flow rates. Sample recovery for the pipette elution is ~80%. Due to the high density of the collected sample, the cartridge is compatible with in-situ spectroscopic analysis and sample elution into the 10–20 μl liquid volume providing a significant increase in sample concentration for subsequent analysis.</p></div

Evaluation of micro-well collector for capture and analysis of aerosolized <i>Bacillus subtilis</i> spores - Fig 1

<p>(a) The photograph of the assembled μ-well aerosol collection cartridge and (b) the cartridge in a disassembled state.</p

The configuration of the aerosol chamber experiment.

<p>The configuration of the aerosol chamber experiment.</p

Detection of potentially pathogenic enteric viruses in environmental samples from Kenya using the bag-mediated filtration system

Enteric virus environmental surveillance via a highly sensitive method is critical, as many enteric viruses have low infectious doses and can persist in the environment for extended periods. This study determined the potential of the novel bag-mediated filtration system (BMFS) to recover human enteric viruses and pepper mild mottle virus (PMMoV) from wastewater and wastewater-impacted surface waters, examined PMMoV use as a fecal contamination indicator in Kenya, and identified potential BMFS process controls. From April 2015 to April 2016, BMFS samples were collected from seven sites in Kenya (n = 59). Enteroviruses and PMMoV were detected in 100% of samples, and human adenovirus, human astrovirus, hepatitis A virus, norovirus GI, norovirus GII, sapovirus, and human rotavirus were detected in the majority of samples. The consistent detection of enteroviruses and PMMoV suggests that these viruses could be used as indicators in similarly fecally contaminated sites and BMFS process controls. As contamination of surface water sources remains a global issue, enteric virus environmental surveillance is necessary. This study demonstrates an effective way to sample large volumes of wastewater and wastewater-impacted surface waters for the detection of multiple enteric viruses simultaneously.The Paul G. Allen Family Foundation (NPT.1938-603689), with management by the Bill and Melinda Gates Foundation, the UW NIEHS sponsored Biostatistics, Epidemiologic and Bioinformatic Training in Environmental Health (BEBTEH) Training Grant (NIEHS T32ES015459) and the National Science Foundation (OCE-1566562).http://wst.iwaponline.comhj2019Medical Virolog