Definition of a concentration and RNA extraction protocol for optimal whole genome sequencing of SARS-CoV-2 in wastewater (ANRS0160).

Monitoring the presence of RNA from emerging pathogenic viruses, such as SARS-CoV-2, in wastewater (WW) samples requires suitable methods to ensure an effective response. Genome sequencing of WW is one of the crucial methods, but it requires high-quality RNA in sufficient quantities, especially for monitoring emerging variants. Consequently, methods for viral concentration and RNA extraction from WW samples have to be optimized before sequencing. The purpose of this study was to achieve high coverage (≥ 90 %) and sequencing depth (at least ≥200×) even for low initial RNA concentrations (< 105 genome copies (GC)/L) in WW. A further objective was to determine the range of SARS-CoV-2 RNA concentrations that allow high-quality sequencing, and the optimal sample volume for analysis. Ultrafiltration (UF) methods were used to concentrate viral particles from large influent samples (up to 500 mL). An RNA extraction protocol using silica beads, neutral phenol-chloroform treatment, and a PCR inhibitor removal kit was chosen for its effectiveness in extracting RNA and eliminating PCR inhibitors, as well as its adaptability for use with large influent samples. Recovery rates ranged from 24 % to 63 % (N = 17) for SARS-CoV-2 naturally present in WW samples. 200 mL WW samples can be enough for UF concentration, as they showed high quality sequencing analyses with between 5 × 104 GC/L and 6 × 103 GC/L. Below 6 × 103 GC/L, high-quality sequencing was also achieved for ∼40 % of the samples using 500 mL of WW. Sequencing analysis for variant detection was performed on 200 mL WW samples with coverage of >95 % and sequencing depth of >1000×. Analyses revealed the predominance of variant EG.5, known as Eris (66 %-100 %). The use of UF methods in combination with a suitable RNA extraction protocol appear promising for sequencing enveloped viruses in WW in a context of viral emergence.

Detection of Human Adenovirus and Rotavirus in Wastewater in Lusaka, Zambia: Demonstrating the Utility of Environmental Surveillance for the Community.

Enteric infections due to viral pathogens are a major public health concern. Detecting the risk areas requires a strong surveillance system for pathogenic viruses in sources such as wastewater. Towards building an environmental surveillance system in Zambia, we aimed to identify group A rotavirus (RVA) and human adenovirus (HAdV) in wastewater. Convenient sampling was conducted at four study sites every Tuesday for five consecutive weeks. The research team focused on three different methods of viral concentration to determine the suitability in terms of cost and applicability for a regular surveillance system: the bag-mediated filtration system (BMFS), polyethylene glycol-based (PEG) precipitation, and skimmed milk (SM) flocculation. We screened 20 wastewater samples for HAdV and RVA using quantitative polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR). Of the 20 samples tested using qPCR, 18/20 (90%) tested positive for HAdV and 14/20 (70%) tested positive for RVA. For the genetic sequencing, qPCR positives were subjected to cPCR, of which 12 positives were successfully amplified. The human adenovirus was identified with a nucleotide identity range of 98.48% to 99.53% compared with the reference genome from GenBank. The BMFS and SM flocculation were the most consistent viral concentration methods for HAdV and RVA, respectively. A statistical analysis of the positives showed that viral positivity differed by site (p < 0.001). SM and PEG may be the most appropriate options in resource-limited settings such as Zambia due to the lower costs associated with these concentration methods. The demonstration of HAdV and RVA detection in wastewater suggests the presence of the pathogens in the communities under study and the need to establish a routine wastewater surveillance system for the identification of pathogens.

Assessing infectivity of emerging enveloped viruses in wastewater and sewage sludge: Relevance and procedures.

The emergence of SARS-CoV-2 has heightened the need to evaluate the detection of enveloped viruses in the environment, particularly in wastewater, within the context of wastewater-based epidemiology. The studies published over the past 80 years focused primarily on non-enveloped viruses due to their ability to survive longer in environmental matrices such as wastewater or sludge compared to enveloped viruses. However, different enveloped viruses survive in the environment for different lengths of time. Therefore, it is crucial to be prepared to assess the potential infectious risk that may arise from future emerging enveloped viruses. This will require appropriate tools, notably suitable viral concentration methods that do not compromise virus infectivity. This review has a dual purpose: first, to gather all the available literature on the survival of infectious enveloped viruses, specifically at different pH and temperature conditions, and in contact with detergents; second, to select suitable concentration methods for evaluating the infectivity of these viruses in wastewater and sludge. The methodology used in this data collection review followed the systematic approach outlined in the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) guidelines. Concentration methods cited in the data gathered are more tailored towards detecting the enveloped viruses' genome. There is a lack of suitable methods for detecting infectious enveloped viruses in wastewater and sludge. Ultrafiltration, ultracentrifugation, and polyethylene glycol precipitation methods, under specific/defined conditions, appear to be relevant approaches. Further studies are necessary to validate reliable concentration methods for detecting infectious enveloped viruses. The choice of culture system is also crucial for detection sensitivity. The data also show that the survival of infectious enveloped viruses, though lower than that of non-enveloped ones, may enable environmental transmission. Experimental data on a wide range of enveloped viruses is required due to the variability in virus persistence in the environment.

Comparative assessment of Nanotrap and polyethylene glycol-based virus concentration in wastewater samples.

Wastewater-based epidemiology is now widely used in many countries for the routine monitoring of SARS-CoV-2 and other viruses at a community level. However, efficient sample processing technologies are still under investigation. In this study, we compared the performance of the novel Nanotrap® Microbiome Particles (NMP) concentration method to the commonly used polyethylene glycol (PEG) precipitation method for concentrating viruses from wastewater and their subsequent quantification and sequencing. For this, we first spiked wastewater with SARS-CoV-2, influenza and measles viruses and norovirus and found that the NMP method recovered 0.4%-21% of them depending on virus type, providing consistent and reproducible results. Using the NMP and PEG methods, we monitored SARS-CoV-2, influenza A and B viruses, RSV, enteroviruses and norovirus GI and GII and crAssphage in wastewater using quantitative PCR (qPCR)-based methods and next-generation sequencing. Good viral recoveries were observed for highly abundant viruses using both methods; however, PEG precipitation was more successful in the recovery of low-abundance viruses present in wastewater. Furthermore, samples processed with PEG precipitation were more successfully sequenced for SARS-CoV-2 than those processed with the NMP method. Virus recoveries were enhanced by high sample volumes when PEG precipitation was applied. Overall, our results suggest that the NMP concentration method is a rapid and easy virus concentration method for viral targets that are abundant in wastewater, whereas PEG precipitation may be more suited to the recovery and analysis of low-abundance viruses and for next generation sequencing.

Evaluation of concentration procedures, sample pre-treatment, and storage condition for the detection of SARS-CoV-2 in wastewater.

Crucial information on the pandemic's spread has been gathered by monitoring the trend of SARS-CoV-2 in wastewater. This surveillance has highlighted that the initial concentration is a critical step of the analytical procedure due to the low viral titer that may be present in this matrix. This paper presents the results of the evaluation of two different wastewater concentration protocols to determine the most efficient and cost-effective. The two methods tested were the following: (a) a biphasic separation system with PEG-dextran and (b) a PEG/NaCl precipitation protocol. Other aspects of the detection method were also investigated including the influence of storage temperature on virus recovery and the heat treatment of pasteurization, which aims to make samples safer for operators and the environment. The PEG/NaCl precipitation method was found to perform better than the biphasic separation system, allowing for more sensitive identification of the presence of the virus and the detection of a higher viral titer than that identified with the biphasic separation in all results. Storage of the samples at 4.3±0.2°C for up to 3 weeks did not adversely affect the virus titer and the pasteurization pre-treatment increases operator safety and maintains the identification of the viral concentration.

Detecting SARS-CoV-2 in sludge samples: A systematic review.

AIMS: This paper aims to review the main sludge concentration methods used for SARS-CoV-2 detection in sewage sludge samples, discussing the main methods and sample volume related to increased viral load. In addition, we aim to evaluate the countries associated with increased positivity rates for SARS-CoV-2 in sludge samples. METHODS: This systematic methodology was registered in PROSPERO and followed the PRISMA guidelines. The search was carried out in the SciELO, PubMed/MEDLINE, Lilacs, and Google Scholar databases in January-March 2022. Quantitative studies with conclusive results were included in this review. Concentration methods (polyethylene glycol (PEG), PEG + NaCl, gravity thickening, skimmed milk flocculation, ultrafiltration, filtration using charged filters, primary sedimentation, and anaerobic digestion), as well as detection methods (RTqPCR and reverse transcription droplet digital PCR assay) were evaluated in this review. The SPSS v23 software program was used for statistical analysis. RESULTS: PEG (with or without NaCl addition) and gravity thickening were the most used sludge concentration methods to detect SARS-CoV-2. The main method associated with increased viral load (>2,02 × 10^4 copies/mL) was PEG + NaCl (p < 0.05, Mann-Whitney test). The average positivity rate for SARS-CoV-2 in sludge samples was 61 %, and a correlation was found between the sludge volume and the viral load (ro 0.559, p = 0.03, Spearman correlation). CONCLUSION: The sludge volume may influence the SARS-CoV-2 load since the virus can adhere to solid particles in these samples. Other factors may be associated with SARS-CoV-2 load, including the methods used; especially PEG + NaCl may result in a high viral load detected in sludge, and may provide a suitable pH for SARS-CoV-2 recovery.

Comparative analysis of Adsorption-Extraction (AE) and Nanotrap® Magnetic Virus Particles (NMVP) workflows for the recovery of endogenous enveloped and non-enveloped viruses in wastewater.

In this study, two virus concentration methods, namely Adsorption-Extraction (AE) and Nanotrap® Magnetic Virus Particles (NMVP) along with commercially available extraction kits were used to quantify endogenous pepper mild mottle virus (PMMoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in nucleic acid extracted from 48 wastewater samples collected over six events from eight wastewater treatment plants (WWTPs). The main aim was to determine which workflow (i.e., concentration and extraction methods) produces greater concentrations of endogenous PMMoV and SARS-CoV-2 gene copies (GC) in comparison with each other. Turbidity and total suspended solids (TSS) of wastewater samples within and among the eight WWTPs were highly variable (41-385 NTU and 77-668 mg/L TSS). In 58 % of individual wastewater samples, the log10 GC concentrations of PMMoV were greater by NMVP workflow compared to AE workflow. Paired measurements of PMMoV GC/10 mL from AE and NMVP across all 48 wastewater samples were weakly correlated (r = 0.455, p = 0.001) and demonstrated a poor linear relationship (r2 = 0.207). The log10 GC concentrations of SARS-CoV-2 in 69 % of individual samples were greater by AE workflow compared to NMVP workflow. In contrast to PMMoV, the AE and NMVP derived SARS-CoV-2 GC counts were strongly correlated (r = 0.859, p < 0.001) and demonstrated a strong linear relationship (r2 = 0.738). In general, the PMMoV GC achieved by the NMVP workflow decreased with increasing turbidity, but the PMMoV GC by the AE workflow did not appear to be as sensitive to either turbidity or TSS levels. These findings suggest that wastewater sample turbidity or suspended solids concentration, and the intended target for analysis should be considered when validating an optimal workflow for wastewater surveillance of viruses.

Concentration techniques tailored for the detection of SARS-CoV-2 genetic material in domestic wastewater and treatment plant sludge: A review.

Upon the outbreak of COVID-19 pandemic, detection and quantification of SARS-CoV-2 genetic material in domestic wastewater have led to an increase in the efforts to define and implement the wastewater-based epidemiology (WBE). This application provides valuable information to define local contamination monitoring, emergence of COVID-19 and its variants and many other aspects to cope with and control the pandemic. WBE surveillance, however, requires several consecutive steps such as sampling, pretreatment and concentration of samples, and detection and quantification of SARS-CoV-2 genetic material in wastewater. In this review paper, the literature regarding to all these applications reviewed considering their advantages, disadvantages as well as their applicability. A specific emphasis was placed on the last step, detection and quantification since it covers the most critical procedure for concentrating the virus before measurement. Evaluation of the existing data indicating ultrafiltration, polyethylene glycol (PEG) precipitation and electronegative membrane filtration (ENMF) were the most promising techniques for concentration. The ongoing studies are proposed to be continued within the context of standard methods. Future research needs are delineated and suggestions are made for details.

Quantitative detection of human adenovirus from river water by monolithic adsorption filtration and quantitative PCR.

Water contaminated with fecally derived viruses, also known as enteric viruses, represents a particularly high risk for human health. However, they have not been included in water quality regulations yet. The detection of these viruses is often more expensive and time-consuming compared to the analysis of conventional fecal indicator organisms. In addition, most methods are not sensitive enough to detect small viral loads that may already cause serious health issues if present in water. In this study, we established a workflow for the successful and direct enrichment of human adenovirus (HAdV) from artificially contaminated river water based on monolithic adsorption filtration (MAF) and quantitative polymerase reaction (qPCR). With a clear focus on efficiency, we used targeted synthetic DNA fragments as standard for the quantification of HAdV by qPCR, leading to accurate and robust results with a qPCR efficiency of 95 %, a broad working range over 6 orders of magnitude and an LOD of 1 GU/µL. We carried out a cascade of spiking experiments, enhancing the complexity of the spiking matrix with each step to progressively evaluate MAF for the direct concentration of HAdV. We found that negatively charged MAF using monoliths with hydroxyl groups (MAF-OH) showed a better reproducibility and a significantly faster turnaround time than skimmed milk flocculation (SMF) when concentrating HAdV35 from artificially contaminated, acidified mineral water. We then validated positively charged MAF using monoliths with diethyl aminoethyl groups (MAF-DEAE) for the direct concentration of HAdV5 without pre-conditioning of water samples using tap water as spiking matrix with a less defined and controlled water chemistry. Finally, we evaluated MAF-DEAE for the direct concentration of HAdV5 from surface water using river water as representative matrix with an undefined water chemistry. We found, that MAF-DEAE achieved reproducible recoveries of HAdV5, independently of the spiked concentration level or sample volume. Furthermore, we showed, that MAF-DEAE drastically reduced the limit of detection (LOD) of HAdV5 by a factor of 115 from 6.0 ∙ 103 GU/mL before to 5.2 ∙ 101 GU/mL after MAF-DEAE. We identified that recoveries increased for smaller processing volumes with a peak at 0.5 L of 84.0 % and showed that recovery efficiency depends on sample volume and matrix type. The here presented workflow based on MAF-DEAE and qPCR offers an easy-to-implement and highly efficient alternative to existing approaches and allows for a fast detection of HAdV in water.

Epidemiological surveillance of SARS-CoV-2 by genome quantification in wastewater applied to a city in the northeast of France: Comparison of ultrafiltration- and protein precipitation-based methods.

The aim of the present study was to develop a simple, sensitive, and specific approach to quantifying the SARS-CoV-2 genome in wastewater and to evaluate this approach as a means of epidemiological surveillance. Twelve wastewater samples were collected from a metropolitan area in north-eastern France during April and May 2020. In addition to the quantification of the SARS-CoV-2 genome, F-specific RNA phages of genogroup II (FRNAPH GGII), naturally present in wastewater, were used as an internal process control for the viral concentration and processing of RT-PCR inhibitors. A concentration method was required to allow the quantification of the SARS-CoV-2 genome over the longest possible period. A procedure combining ultrafiltration, phenol-chloroform-isoamyl alcohol purification, and the additional purification of the RNA extracts was chosen for the quantification of the SARS-CoV-2 genome in 100-mL wastewater samples. At the same time, the COVID-19 outbreak was evaluated through patients from the neighbouring University Hospital of Nancy, France. A regular decrease in the concentration of the SARS-CoV-2 genome from ~104 gc/L to ~102 gc/L of wastewater was observed over the eight weeks of the study, during which the population was placed under lockdown. The SARS-CoV-2 genome was even undetectable during one week in the second half of May and present but non-quantifiable in the last sample (28 May). A concordant circulation in the human community was highlighted by virological diagnosis using respiratory samples, which showed a decrease in the number of COVID-19 cases from 677 to 52 per week over the same period. The environmental surveillance of COVID-19 using a reliable viral quantification procedure to test wastewater is a key approach. The real-time detection of viral genomes can allow us to predict and monitor the circulation of SARS-CoV-2 in clinical settings and survey the entire urban human population.

Detection and evaluation of rotavirus surveillance methods as viral indicator in the aquatic environments.

Group A rotaviruses (RVAs) have been introduced as the most important causative agents of acute gastroenteritis in the young children. One of every 260 children born globally will die due to rotavirus (RV) before 5 years old. The RV is widely known as a viral indicator for health (fecal contamination) because this pathogen has a high treatment resistance nature, which has been listed as a relevant waterborne pathogen by the World Health Organization (WHO). Therefore, monitoring of environmental is important, and RV is one of the best-known indicators for monitoring. It has been proved that common standards for microbiological water quality do not guarantee the absence of viruses. On the other hand, in order to recover and determine RV quantity within water, standard methods are scarce. Therefore, dependable prediction of RV quantities in water sample is crucial to be able to improve supervision efficiency of the treatment procedure, precise quantitative evaluation of the microbial risks as well as microbiological water safety. Hence, this study aimed to introduce approaches to detecting and controlling RV in environmental waters, and discussed the challenges faced to enable a clear perception on the ubiquity of the RV within different types of water across the world.

Intestinal Parasitoses among Chepang and Musahar Community People of Makwanpur and Nawalparasi Districts of Nepal.

BACKGROUND: Intestinal parasitosis is one of the commonly perceived serious problems often observed in children leading to high mortality. The objective of the study was to identify the intestinal parasites and study their prevalence in the two mostly disadvantaged communities (Musahar and Chepang) of Nepal. METHODS: This was a cross-sectional study conducted in the Musahar and Chepang communities of Nepal from April to October 2019. A total of 205 random stool samples were collected in dry, clean and screw-capped plastic containers and mixed with 2.5% potassium dichromate solution. A pre-structured questionnaire was used to collect data on predisposing factors. The laboratory examination of the stool samples was done by direct microscopy and further confirmed by concentration methods (formalin ether sedimentation technique and flotation technique using Sheather's sugar solution), and modified acid-fast staining. Detection of eggs of Enterobius vermicularis was done by cellophane tape method. RESULTS: The overall prevalence of intestinal parasitic infection was found to be 36.6%, with a similar prevalence in the Chepangs (39.8%) and in the Musahars (33.3%) (P > 0.05). The most predominant helminth was Ascaris lumbricoides (15.6%), while the most prevalent protozoan was Entamoeba histolytica/dispar (5.4%). The study also assessed a significant association between the prevalence of parasites with socio-demographic factors, types of drinking water consumption and sanitation habits of the people (P < 0.05). CONCLUSION: The findings of the study suggest a need for formulating effective preventive and control strategies against intestinal parasitic infections along with the continuity of mass deworming program.

Decay of SARS-CoV-2 RNA along the wastewater treatment outfitted with Upflow Anaerobic Sludge Blanket (UASB) system evaluated through two sample concentration techniques.

For the first time, we present, i) an account of decay in the genetic material loading of SARS-CoV-2 during Upflow Anaerobic Sludge Blanket (UASB) treatment of wastewater, and ii) comparative evaluation of polyethylene glycol (PEG), and ultrafiltration as virus concentration methods from wastewater for the quantification of SARS-CoV-2 genes. The objectives were achieved through tracking of SARS-CoV-2 genetic loadings i.e. ORF1ab, N and S protein genes on 8th and 27th May 2020 along the wastewater treatment plant (106000 m3 million liters per day) equipped with UASB system in Ahmedabad, India. PEG method performed better in removing materials inhibiting RT-qPCR for SARS-CoV-2 gene detection from the samples, as evident from constant and lower CT values of control (MS2). Using the PEG method, we found a reduction >1.3 log10 reduction in SARS-CoV-2 RNA abundance during UASB treatment, and the RNA was not detected at all in the final effluent. The study implies that i) conventional wastewater treatment systems is effective in SARS-CoV-2 RNA removal, and ii) UASB system significantly reduces SARS-CoV-2 genetic loadings. Finally, PEG method is recommended for better sensitivity and inhibition removal during SARS-CoV-2 RNA quantification in wastewater.

Evaluation of viral concentration methods for SARS-CoV-2 recovery from wastewaters.

Wastewater-based epidemiology (WBE) is a useful tool that has the potential to act as a complementary approach to monitor the presence of SARS-CoV-2 in the community and as an early alarm system for COVID-19 outbreak. Many studies reported low concentrations of SARS-CoV-2 in sewage and also revealed the need for methodological validation for enveloped viruses concentration in wastewater. The aim of this study was to evaluate different methodologies for the concentration of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. A total of 11 concentration techniques based on different principles were evaluated: adsorption-elution protocols with negatively charged membranes followed by polyethylene glycol (PEG) precipitation (Methods 1-2), PEG precipitation (Methods 3-7), aluminum polychloride (PAC) flocculation (Method 8), ultrafiltration (Method 9), skim milk flocculation (Method 10) and adsorption-elution with negatively charged membrane followed by ultrafiltration (Method 11). To evaluate the performance of these concentration techniques, feline calicivirus (FCV) was used as a process control in order to avoid the risk associated with handling SARS-CoV-2. Two protocols, one based on PEG precipitation and the other on PAC flocculation, showed high efficiency for FCV recovery from wastewater (62.2% and 45.0%, respectively). These two methods were then tested for the specific recovery of SARS-CoV-2. Both techniques could recover SARS-CoV-2 from wastewater, PAC flocculation showed a lower limit of detection (4.3 × 102 GC/mL) than PEG precipitation (4.3 × 103 GC/mL). This work provides a critical overview of current methods used for virus concentration in wastewaters and the analysis of sensitivity for the specific recovery of SARS-CoV-2 in sewage. The data obtained here highlights the viability of WBE for the surveillance of COVID-19 infections in the community.

Concentration methods for the quantification of coronavirus and other potentially pandemic enveloped virus from wastewater.

As the novel SARS-CoV-2 was detected in faeces, environmental researchers have been using centrifugal ultrafiltration, polyethylene glycol precipitation and aluminium hydroxide flocculation to describe its presence in wastewater samples. High recoveries (up to 65%) are described with electronegative filtration when using surrogate viruses, but few literature reports recovery efficiencies using accurate quantification of enveloped viruses. Considering that every single virus will have a different behaviour during viral concentration, it is recommended to use an enveloped virus, and if possible, a betacoronaviruses as murine hepatitis virus, as a surrogate. In this review, we show new data from a newly available technology that provides a quick ultrafiltration protocol for SARS-CoV-2. Wastewater surveillance is an efficient system for the evaluation of the relative prevalence of SARS-CoV-2 infections in a community, and there is the need of using reliable concentration methods for an accurate and sensitive quantification of the virus in water.

Comparison of viral elution-concentration methods for recovering noroviruses from deli meats.

This study aimed to assess viral elution-concentration methods for recovering noroviruses from deli meats. Spiking experiments were conducted to evaluate the recovery success rates and recovery efficiencies of human norovirus (NoV) GI and GII and murine norovirus 1 (MNV-1) using polyethylene glycol (PEG 6000) precipitation, skimmed milk flocculation (SMF), TRIzol® reagent, and a combination of PEG/TRIzol® and SMF/TRIzol® methods. Molecular analysis using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) revealed TRIzol® as the best method to be used for viral recovery from ham with medium recovery rates of 37.6% for NoV GI and 50.1% for NoV GII. Viral recovery from turkey meat showed medium recovery rates of 14.4% for NoV GI and 8.9% for NoV GII. For MNV-1, the rates varied from 0.5% to 80.8% not only according to the matrix but also with the associated virus and its inoculum (NoV GI or GII). The monitoring of commercial samples obtained in the Great Metropolitan region of Rio de Janeiro in order to demonstrate the occurrence of NoV GI and GII contamination in both matrices was also performed in 60 samples. NoV GI or GII were not detected in any samples, while MNV-1 used as the sample process control viruswas successfully recovered in 100% of samples.

Development of a fast and efficient method for hepatitis A virus concentration from green onion.

Hepatitis A virus (HAV) can cause serious liver disease and even death. HAV outbreaks are associated with the consumption of raw or minimally processed produce, making it a major public health concern. Infections have occurred despite the fact that effective HAV vaccine has been available. Development of a rapid and sensitive HAV detection method is necessary for an investigation of an HAV outbreak. Detection of HAV is complicated by the lack of a reliable culture method. In addition, due to the low infectious dose of HAV, these methods must be very sensitive. Current methods rely on efficient sample preparation and concentration steps followed by sensitive molecular detection techniques. Using green onions which was involved in most recent HAV outbreaks as a representative produce, a method of capturing virus particles was developed using carboxyl-derivatized magnetic beads in this study. Carboxyl beads, like antibody-coated beads or cationic beads, detect HAV at a level as low as 100 pfu/25g of green onions. RNA from virus concentrated in this manner can be released by heat-shock (98°C 5min) for molecular detection without sacrificing sensitivity. Bypassing the RNA extraction procedure saves time and removes multiple manipulation steps, which makes large scale HAV screening possible. In addition, the inclusion of beef extract and pectinase rather than NP40 in the elution buffer improved the HAV liberation from the food matrix over current methods by nearly 10 fold. The method proposed in this study provides a promising tool to improve food risk assessment and protect public health.

A method for the improved detection of aerosolized influenza viruses and the male-specific (F+) RNA coliphage MS2.

The detection of aerosolized viruses can serve as an important surveillance and control tool in agriculture, human health, and environmental settings. Here, we adapted an anion exchange resin-based method, initially developed to concentrate negatively charged viruses from water, to liquid impingement-based bioaerosol sampling. In this method, aerosolized viruses are collected in a 20ml liquid sample contained within widely used impingers, BioSamplers (SKC Inc., Eighty Four, PA), and further concentrated via adsorption to an anion exchange resin that is suspended within this liquid. Viral nucleic acids are then extracted from the resin to facilitate molecular analyses through a reduction in the effective sample volume. For this study, various quantities of two negatively charged viruses, type A and type B influenza viruses (FluMist Quadrivalent vaccine) and the male-specific (F+) RNA coliphage MS2 (MS2), were nebulized into a custom-built bioaerosolization chamber, and sampled using BioSamplers with and without anion exchange resin. Compared to direct testing of the BioSampler liquid, detection was improved by 6.77× and 3.33× for type A and type B influenza viruses, respectively, by using the anion exchange resin. For MS2, the anion exchange resin method allowed for an average improvement in detection of 8.26×.

Comparing concentration methods: parasitrap® versus Kato-Katz for studying the prevalence of Helminths in Bengo province, Angola.

BACKGROUND: Helminth intestinal parasitoses are responsible for high levels of child mortality and morbidity. Hence, the capacity to diagnose these parasitoses and consequently ensure due treatment represents a factor of great importance. OBJECTIVES: The main objective of this study involves comparing two methods of concentration, parasitrap and Kato-Katz, for the diagnosis of intestinal parasitoses in faecal samples. METHODS: Sample processing made recourse to two different concentration methods: the commercial parasitrap® method and the Kato-Katz method. RESULTS: We correspondingly collected a total of 610 stool samples from pre-school and school age children. The results demonstrate the incidence of helminth parasites in 32.8% or 32.3% of the sample collected depending on whether the concentration method applied was either the parasitrap method or the Kato-Katz method. We detected a relatively high percentage of samples testing positive for two or more species of helminth parasites. We would highlight that in searching for larvae the Kato-Katz method does not prove as appropriate as the parasitrap method. CONCLUSION: Both techniques prove easily applicable even in field working conditions and returning mutually agreeing results. This study concludes in favour of the need for deworming programs and greater public awareness among the rural populations of Angola.

Comparison of concentration methods for rapid detection of hookworm ova in wastewater matrices using quantitative PCR.

Hookworm infection contributes around 700 million infections worldwide especially in developing nations due to increased use of wastewater for crop production. The effective recovery of hookworm ova from wastewater matrices is difficult due to their low concentrations and heterogeneous distribution. In this study, we compared the recovery rates of (i) four rapid hookworm ova concentration methods from municipal wastewater, and (ii) two concentration methods from sludge samples. Ancylostoma caninum ova were used as surrogate for human hookworm (Ancylostoma duodenale and Necator americanus). Known concentration of A. caninum hookworm ova were seeded into wastewater (treated and raw) and sludge samples collected from two wastewater treatment plants (WWTPs) in Brisbane and Perth, Australia. The A. caninum ova were concentrated from treated and raw wastewater samples using centrifugation (Method A), hollow fiber ultrafiltration (HFUF) (Method B), filtration (Method C) and flotation (Method D) methods. For sludge samples, flotation (Method E) and direct DNA extraction (Method F) methods were used. Among the four methods tested, filtration (Method C) method was able to recover higher concentrations of A. caninum ova consistently from treated wastewater (39-50%) and raw wastewater (7.1-12%) samples collected from both WWTPs. The remaining methods (Methods A, B and D) yielded variable recovery rate ranging from 0.2 to 40% for treated and raw wastewater samples. The recovery rates for sludge samples were poor (0.02-4.7), although, Method F (direct DNA extraction) provided 1-2 orders of magnitude higher recovery rate than Method E (flotation). Based on our results it can be concluded that the recovery rates of hookworm ova from wastewater matrices, especially sludge samples, can be poor and highly variable. Therefore, choice of concentration method is vital for the sensitive detection of hookworm ova in wastewater matrices.