Development and evaluation of a ligation-free sequence-independent, single-primer amplification (LF-SISPA) assay for whole genome characterization of viruses.

Molecular identification and characterization of novel or re-emerging infectious pathogens is critical for disease surveillance and outbreak investigations. Next generation sequencing (NGS) using Sequence-Independent, Single-Primer Amplification (SISPA) is being used extensively in sequencing of viral genomes but it requires an expensive library preparation step. We developed a simple, low-cost method that enriches nucleic acids followed by a ligation-free (LF) 2-step Polymerase Chain Reaction (PCR) procedure for library preparation.

Virus Removal and Inactivation Mechanisms during Iron Electrocoagulation: Capsid and Genome Damages and Electro-Fenton Reactions.

Virus destabilization and inactivation are critical considerations in providing safe drinking water. We demonstrate that iron electrocoagulation simultaneously removed (via sweep flocculation) and inactivated a non-enveloped virus surrogate (MS2 bacteriophage) under slightly acidic conditions, resulting in highly effective virus control (e.g.

Removal and Inactivation of an Enveloped Virus Surrogate by Iron Conventional Coagulation and Electrocoagulation.

It is imperative to understand the behavior of enveloped viruses during water treatment to better protect public health, especially in the light of evidence of detection of coronaviruses in wastewater. We report bench-scale experiments evaluating the extent and mechanisms of removal and/or inactivation of a coronavirus surrogate (ϕ6 bacteriophage) in water by conventional FeCl coagulation and Fe(0) electrocoagulation. Both coagulation methods achieved ∼5-log removal/inactivation of ϕ6 in 20 min.

CrAssphage as a Novel Tool to Detect Human Fecal Contamination on Environmental Surfaces and Hands.

CrAssphage is a recently discovered human gut-associated bacteriophage. To validate the potential use of crAssphage for detecting human fecal contamination on environmental surfaces and hands, we tested stool samples (n = 60), hand samples (n = 30), and environmental swab samples (n = 201) from 17 norovirus outbreaks for crAssphage by real-time PCR. In addition, we tested stool samples from healthy persons (n = 173), respiratory samples (n = 113), and animal fecal specimens (n = 68) and further sequenced positive samples.

A new solid matrix for preservation of viral nucleic acid from clinical specimens at ambient temperature.

Stabilizing paper matrix methods for retaining nucleic acid from inactivated clinical specimens offer a solution for molecular diagnostics when specimens may be stored or shipped at ambient temperature. We developed cellulose disks (UNEXP) saturated with a total nucleic acid extraction buffer (UNEX) modified from a previously developed lysis buffer for multiple enteric pathogens. Infectivity of hepatitis A virus, adenovirus and poliovirus was destroyed after 2-3 h incubation at room temperature on the UNEXP disks.

A Randomized Controlled Trial to Assess the Impact of Ceramic Water Filters on Prevention of Diarrhea and Cryptosporidiosis in Infants and Young Children-Western Kenya, 2013.

is a leading cause of diarrhea among Kenyan infants. Ceramic water filters (CWFs) are used for household water treatment. We assessed the impact of CWFs on diarrhea, cryptosporidiosis prevention, and water quality in rural western Kenya.

Pool water quality and prevalence of microbes in filter backwash from metro-Atlanta swimming pools.

During the 2012 summer swim season, aquatic venue data and filter backwash samples were collected from 127 metro-Atlanta pools. Last-recorded water chemistry measures indicated 98% (157/161) of samples were from pools with ≥1 mg/L residual chlorine without stabilized chlorine or ≥2 mg/L with stabilized chlorine and 89% (144/161) had pH readings 7.2-7.

Evaluation of an Ultrafiltration-Based Procedure for Simultaneous Recovery of Diverse Microbes in Source Waters.

In this study, hollow-fiber ultrafiltration (UF) was assessed for recovery of , spores, oocysts, echovirus 1, and bacteriophages MS2 and ΦX174 from ground and surface waters. Microbes were seeded into twenty-two 50-L water samples that were collected from the Southeastern United States and concentrated to ∼500 mL by UF. Secondary concentration was performed for by centrifugation followed by immunomagnetic separation.

Development of a nucleic Acid extraction procedure for simultaneous recovery of DNA and RNA from diverse microbes in water.

Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance.

Real-time PCR and sequencing assays for rapid detection and identification of avian schistosomes in environmental samples.

Cercarial dermatitis, also known as swimmer's itch, is an allergenic skin reaction followed by intense itching caused by schistosome cercariae penetrating human skin. Cercarial dermatitis outbreaks occur globally and are frequently associated with freshwater lakes and are occasionally associated with marine or estuarine waters where birds reside year-round or where migratory birds reside. In this study, a broadly reactive TaqMan assay targeting 18S rRNA gene (ribosomal DNA [rDNA]) sequences that was based on a genetically diverse panel of schistosome isolates representing 13 genera and 20 species (the 18S rDNA TaqMan assay) was developed.

The first association of a primary amebic meningoencephalitis death with culturable Naegleria fowleri in tap water from a US treated public drinking water system.

Background: Naegleria fowleri is a climate-sensitive, thermophilic ameba found in warm, freshwater lakes and rivers. Primary amebic meningoencephalitis (PAM), which is almost universally fatal, occurs when N. fowleri-containing water enters the nose, typically during swimming, and migrates to the brain via the olfactory nerve.

Draft Genome Sequence of Raoultella planticola, Isolated from River Water.

We isolated Raoultella planticola from a river water sample, which was phenotypically indistinguishable from Escherichia coli on MI agar. The genome sequence of R. planticola was determined to gain information about its metabolic functions contributing to its false positive appearance of E.

Draft Genome Sequence of Buttiauxella agrestis, Isolated from Surface Water.

MI agar is routinely used for quantifying Escherichia coli in drinking water. A suspect E. coli colony isolated from a water sample was identified as Buttiauxella agrestis.

Microbial and chemical contamination during and after flooding in the Ohio River-Kentucky, 2011.

Surface water contaminants in Kentucky during and after 2011 flooding were characterized. Surface water samples were collected during flood stage (May 2-4, 2011; n = 15) and after (July 25-26, 2011; n = 8) from four different cities along the Ohio River and were analyzed for the presence of microbial indicators, pathogens, metals, and chemical contaminants. Contaminant concentrations during and after flooding were compared using linear and logistic regression.

Visual endpoint detection of Escherichia coli O157:H7 using isothermal Genome Exponential Amplification Reaction (GEAR) assay and malachite green.

Rapid and specific detection methods for bacterial agents in drinking water are important for disease prevention and responding to suspected contamination events. In this study, an isothermal Genome Exponential Amplification Reaction (GEAR) assay for Escherichia coli O157:H7 was designed specifically to recognize a 199-bp fragment of the lipopolysaccharide gene (rfbE) for rapid testing of water samples. The GEAR assay was found to be specific for E.

Improved Method for the Detection and Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic Separation and Real-Time PCR.

Primary amebic meningoencephalitis (PAM) is a rare and typically fatal infection caused by the thermophilic free-living ameba, Naegleria fowleri. In 2010, the first confirmed case of PAM acquired in Minnesota highlighted the need for improved detection and quantification methods in order to study the changing ecology of N. fowleri and to evaluate potential risk factors for increased exposure.

Molecular diagnosis of malaria by photo-induced electron transfer fluorogenic primers: PET-PCR.

There is a critical need for developing new malaria diagnostic tools that are sensitive, cost effective and capable of performing large scale diagnosis. The real-time PCR methods are particularly robust for large scale screening and they can be used in malaria control and elimination programs. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P.

Real-time loop-mediated isothermal amplification (RealAmp) for the species-specific identification of Plasmodium vivax.

Plasmodium vivax infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries.

Primary amebic meningoencephalitis deaths associated with sinus irrigation using contaminated tap water.

Background: Naegleria fowleri is a climate-sensitive, thermophilic ameba found in the environment, including warm, freshwater lakes and rivers. Primary amebic meningoencephalitis (PAM), which is almost universally fatal, occurs when N. fowleri-containing water enters the nose, typically during swimming, and N.

Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E.

Hollow-fiber ultrafiltration for simultaneous recovery of viruses, bacteria and parasites from reclaimed water.

Hollow-fiber ultrafiltration (UF) is a technique that has been reported to be effective for recovering a diverse array of microbes from water, and may also be potentially useful for microbial monitoring of effluent from water reclamation facilities. However, few data are available to indicate the potential limitations and efficacy of the UF technique for treated wastewater. In this study, recovery efficiencies were determined for various options available for performing the tangential-flow UF technique, including hollow-fiber ultrafilter (i.

Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria.

Background: Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms.

Evaluation of a molecular beacon real-time PCR assay for detection of Baylisascaris procyonis in different soil types and water samples.

Baylisascaris procyonis is a helminth parasite commonly found in North American raccoons (Procyon lotor) that is a cause of clinical neural, ocular, and visceral larva migrans in humans when infective eggs are ingested. Rapid detection of B. procyonis eggs in contaminated soil and water would assist public health analysts in evaluating risks associated with public exposure to areas of known raccoon activity.

Design of FRET-TaqMan probes for multiplex real-time PCR using an internal positive control.

The multiplexing capabilities with different fluorescent dyes are limited in real-time PCR instruments equipped with one excitation source. Considering this limitation, a design was developed to create a triple-labeled probe as an internal positive control (IPC) that utilizes a combination of the fluorescence resonance energy transfer (FRET) and TaqMan techniques. The IPC probe, labeled with FAM and Cy5.