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The red-rimmed melania or Malaysian Trumpet Snail, Melanoides tuberculata, is a common exotic freshwater snail that has been invading an increasing number of water bodies in North America. As a well-known host for trematodes causing human and animal diseases, the pattern of invasion and parasitic infection for this species is of great concern. Snail specimens were collected from an urban stream in Los Angeles that drains into a fragile, protected wetland ecosystem. Molecular analysis identified four trematode species: Haplorchis pumilio, Fasciola jacksoni, Parorchis sp. TH-2019, and an unclassified trematode species, Trematoda distomecercaria WN-2016. H. pumilio is responsible for haplorchiasis infections, previously considered endemic to Asia. F. jacksoni infection is a significant cause of mortality in Asian elephants. This study represents the first occurrence of F. jacksoni in North America and a novel occurrence of that trematode in association with M. tuberculata. This study also represents new occurrences of Parorchis sp. TH-2019 and an unclassified trematode species, Trematoda distomecercaria WN-2016, within California and North America. Parorchis sp. TH-2019 has previously only been documented in a marine whelk. This identification of these trematodes in association with M. tuberculata further exemplifies the need for ongoing monitoring and detection, especially considering the significance of H. pumilio and haplorchiasis to public health.

Multiple pathways of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission have been examined, and the role of contaminated foods as a source of SARS-CoV-2 exposure has been suggested. As many cases of SARS-CoV-2 have been linked to meat processing plants, it may be that conditions in live animal markets and slaughterhouses or meat processing plant procedures transfer viral particles to meat, poultry, and seafood during animal slaughter, processing, storage, or transport. Because of the potential for contamination of foods such as beef, chicken, pork, or fish, the goal of this study was to evaluate the survival of a lipid enveloped RNA bacteriophage, phi 6, as well as two animal coronaviruses, murine hepatitis virus (MHV) and transmissible gastroenteritis virus (TGEV), as SARS-CoV-2 surrogates for their survival under various meat and fish cold-storage conditions over 30 days. Viral surrogates differed in survival, depending on food product and temperature, but overall, viruses survived for extended periods of time at high concentrations at both refrigerated and frozen temperatures. The ability of SARS-CoV-2 viral surrogates like Phi 6 and animal coronaviruses to survive for varying extents on some meat and fish products when stored refrigerated or frozen is a significant and concerning finding. Continued efforts are needed to prevent contamination of foods and food processing surfaces, worker hands, and food processing utensils such as knives, and there is a need to better address the lack of or inadequate disinfection of these foods prior to meat packaging.

The ability of SARS-CoV-2 viral surrogates like Phi 6 and animal coronaviruses to survive for long periods on meat and fish products at cold temperatures emphasizes the need for rigorous and sustained food sanitation and hygiene in the harvest, transport, processing, and distribution of these foods.

BackgroundIn a year-long pneumonia etiology study conducted June 2017 to May 2018 in Sarawak, Malaysia, 599 patients’ nasopharyngeal swab specimens were studied with real-time polymerase chain reaction (rPCR)/ reverse-transcription (rRT-PCR) assays for respiratory pathogens known to contribute to the high burden of lower respiratory tract infections. The study team sought to compare real-time assay results with panspecies conventional molecular diagnostics to compare sensitivities and learn if novel viruses had been missed.MethodsSpecimens were studied for evidence of adenovirus (AdV), enterovirus (EV) and coronavirus (CoV) with panspecies gel-based nested PCR/RT-PCR assays. Gene sequences of specimens positive by panspecies assays were sequenced and studied with the NCBI Basic Local Alignment Search Tool software.ResultsThere was considerable discordance between real-time and conventional molecular methods. The real-time AdV assay found a positivity of 10.4%; however, the AdV panspecies assay detected a positivity of 12.4% and the conventional AdV-Hexon assay detected a positivity of 19.6%. The CoV and EV panspecies assays similarly detected more positive specimens than the real-time assays, with a positivity of 7.8% by the CoV panspecies assay versus 4.2% by rRT-PCR, and 8.0% by the EV panspecies assay versus 1.0% by rRT-PCR. We were not able to ascertain virus viability in this setting. While most discordance was likely due to assay sensitivity for previously described human viruses, two novel, possible zoonotic AdV were detected.ConclusionsThe observed differences in the two modes of amplification suggest that where a problem with sensitivity is suspected, real-time assay results might be supplemented with panspecies conventional PCR/RT-PCR assays.

It is estimated that 780 million people do not have access to improved drinking water sources and approximately 2 billion people use fecally contaminated drinking water. Effective point-of-use water treatment systems (POU) can provide water with sufficiently reduced concentrations of pathogenic enteric microorganisms to not pose significant health risks to consumers. Household water treatment (HWT) systems utilize various technologies that physically remove and/or inactivate pathogens. A limited number of governmental and other institutional entities have developed testing protocols to evaluate the performance of POU water treatment systems. Such testing protocols are essential to documenting effective performance because inferior and ineffective POU treatment technologies are thought to be in widespread use. This critical review examines specific practices, procedures and specification of widely available POU system evaluation protocols. Testing protocols should provide standardized and detailed instructions yet be sufficiently flexible to deal with different treatment technologies, test microbe priorities and choices, testing facility capabilities and public health needs. Appropriate infectivity or culture assays should be used to quantify test enteric bacteria, viruses and protozoan parasites, or other appropriate surrogates or substitutes for them, although processes based on physical removal can be tested by methods that detect microbes as particles. Recommendations include further research of stock microbe production and handling methods to consistently yield test microbes in a realistic state of aggregation and, in the case of bacteria, appropriately physiologically stressed. Bacterial quantification methods should address the phenomenon of bacterial injury and repair in order to maximally recover those that are culturable and potentially infectious. It is only with harmonized national and international testing protocols and performance targets that independent and unbiased testing can be done to assure consumers that POU treatment technologies are able to produce water of high microbial quality and low health risk.

In 2018, our team collected aerosols samples from five poultry farms in Malaysia. Influenza D virus was detected in 14% of samples. One sample had an 86.3% identity score similar to NCBI accession numberMH785020.1. This is the first molecular sequence of influenza D virus detected in Southeast Asia from a bioaerosol sample. Our findings indicate that further study of role of IDV in poultry is necessary.

Despite close contact between humans and animals on large scale farms, little to no infectious disease research is conducted at this interface. Our goal in this preliminary study was to explore if we could detect swine pathogens using a non-invasive, indirect approach through the study of swine slurry. From April to November 2018, 105 swine slurry samples were collected by farm personnel from waste pits at two sites on a swine farm in North Carolina. These samples were tested for DNA and RNA viruses using a real-time PCR and RT-PCR. Statistical analyses were performed to measure association between virus positive outcomes and potential predictors such as date of sample collection, weight of pigs, number of pigs in barn, temperature, and weather conditions. Overall, 86% of the samples had evidence of at least one of the targeted viruses. Ultimately, this study demonstrated the utility of conducting noninvasive surveillance for swine pathogens through the study of swine slurry. Such swine slurry surveillance may supplant the need to handle, restrain, and collect specimens directly from pigs thus providing an approach to emerging pathogen detection that appeals to the swine industry.

Disease outbreaks can readily threaten swine production operations sometimes resulting in large economic losses. Pathogen surveillance within swine farms can be an effective approach for the early identification of new disease threats and the mitigation of transmission before broad dissemination among a herd occurs. Non-invasive environmental bioaerosol sampling could be an effective and affordable approach for conducting routine surveillance in farms, providing an additional tool for farmers to protect their animals and themselves from new disease threats. In this pilot study, we implemented a non-invasive, prospective bioaerosol sampling strategy in a swine farm located in the United States to detect economically important swine pathogens. Farm personnel collected air samples from two swine barns for 23 weeks between July and December 2017. Samples were then tested within 24 hours of collection by molecular techniques for a number of economically important swine pathogens. Of the 86 bioaerosol samples collected, 4 (4.7%) were positive for influenza A, 1 (1.2%) was positive for influenza D, 13 (15.1%) were positive for PCV2, and 13 (15.1%) were positive for PCV3. Overall, this pilot study showed that our bioaerosol surveillance strategy was feasible and able to generate data that could be quickly disseminated back to the farm stakeholders (within 24 hours). We were also able to identify PCV2, PCV3, and influenza A virus in air samples as clinical disease became apparent in the pigs, strongly suggesting that bioaerosol sampling can be used as an effective non-invasive surveillance approach for the detection of multiple pathogens in this and likely other animal production environments.

Treated wastewater is increasingly of interest for either nonpotable purposes, such as agriculture and industrial use, or as source water for drinking water supplies; however, this type of advanced treatment for water supply is not always possible for many low resource settings. As an alternative, multiple barriers of physical, chemical and biological treatment with lower cost and simpler operation and maintenance have been proposed as more globally applicable. One such water reclamation system for both non-potable and potable reuse, is that approved by the State of North Carolina "for Type 2" reclaimed water (NCT2RW). NC Type 2 potable reuse systems consist of a sequence of tertiary treatment to produce well oxidized reclaimed water that is then then further treated by two steps of disinfection, typically UV radiation and chlorination. In this case study, the log10 microbial reduction performance of NCT2RW producing water reclamation facilities is evaluated. Based on the results presented here, NCT2RW consistently achieved high (6 for bacteria, 4 for virus and 4 for protozoan parasite surrogates) log10 reductions using the NC proposed treatment methods. Additionally, lower but significant log10 reduction performance was also documented for protozoan parasites and human enteric viruses.

This year, in 2018, we mark 100 years since the 1918 influenza pandemic. In the last 100 years, we have expanded our knowledge of public health and increased our ability to detect and prevent influenza; however, we still face challenges resulting from these continually evolving viruses. Today, it is clear that influenza viruses have multiple animal reservoirs (domestic and wild), making infection prevention in humans especially difficult to achieve. With this report, we summarize new knowledge regarding influenza A, B, C and D viruses and their control. We also introduce how a multi-disciplinary One Health approach is necessary to mitigate these threats.

Burkholderia pseudomallei infections are prevalent in Southeast Asia and northern Australia and often misdiagnosed. Diagnostics are often neither sensitive nor rapid, contributing up to 50% mortality rate. In this 2018 pilot study, we enrolled 100 patients aged 6 months–79 years from Kapit Hospital in Sarawak, Malaysia, with symptoms of B. pseudomallei infection. We used three different methods for the detection of B. pseudomallei: a real-time polymerase chain reaction (PCR) assay, a rapid lateral flow immunoassay, and the standard-of-care bacterial culture—the gold standard. Among the 100 participants, 24 (24%) were positive for B. pseudomallei by one or more of the detection methods. Comparing the two individual diagnostic methods against the gold standard—bacterial culture—of any positive test, there was low sensitivity for each test (25–44%) but high specificity (93–98%). It seems clear that more sensitive diagnostics or a sensitive screening diagnostic followed by specific confirmatory diagnostic is needed for this disease.

Summary A hospital outbreak of human parainfluenza virus type 3 (HPIV-3) in haematologic oncology patients is described in 12 patients over a four-week period. Exposure histories and molecular analysis of HPIV-3 isolates suggest that both community-acquired and nosocomially transmitted infections occurred during this outbreak. Molecular analysis of HPIV-3 isolates indicated that a chain of transmission occurred among multiple patients in an oncology ward. This transmission was later determined to be associated with the movement of fomites, visitors, and activities in the unit. The infection prevention team stopped nosocomial spread of HPIV-3 through interventions including advanced cleaning procedures.

Background Although pneumonia is a known cause of morbidity and mortality in Sarawak, Malaysia, the etiology and epidemiology of pneumonia are not well described in this equatorial region. Routine clinical diagnostics for pneumonia etiology at government hospitals in Sarawak had historically involved only bacterial diagnostics. Viral diagnostics were only obtained through outside consultations. Methods From June 15, 2017 to May 14, 2018, we collected nasopharyngeal swabs from 600 patients of all ages older than 1 month hospitalized with pneumonia at Sibu and Kapit Hospitals. Specimens were examined at our collaborating institutions with a panel of molecular assays for viral pathogens including influenza A (IAV), IBV, ICV, and IDV, human adenovirus (AdV), human enterovirus (EV), human coronavirus (CoV), respiratory syncytial virus subtype A (RSV-A) or RSV-B, and parainfluenza virus (PIV) types 1–4. Results Of 599 samples examined, 288 (48%) had molecular evidence of 1 or more respiratory viruses. Overall, the most prevalent virus detected was RSV-A (14.2%) followed by AdV (10.4%) and IAV (10.4%), then RSV-B (6.2%), EV (4.2%), IBV (2.2%), PIV-3 (1.7%), CoV (1.0%), PIV-1 (1.0%), PIV-4 (0.7%), and PIV-2 (0.2%). No specimens were confirmed positive for ICV or IDV. Conclusions The high prevalence of viruses detected in this study suggest that respiratory viruses may be responsible for considerable morbidity in equatorial regions such as Sarawak. Access to viral diagnostics are very necessary for medical staff to determine appropriate pneumonia treatments.

Evidence of exposure to enteric pathogens through the air and associated risk of infection is scarce in the literature outside of animal- or human-waste handling settings. Cities with poor sanitation are important locations to investigate this aerial exposure pathway as their rapid growth will pose unprecedented challenges in waste management. To address this issue, simple surveillance methods are needed. Therefore, the objectives of this study were to optimize a community exposure bioaerosol surveillance strategy for urban outdoor locations with poor sanitation, and to determine which bioaerosols could contribute to exposure. Passive and active bioaerosol sampling methods were used to characterize the fate and transport of sanitation-related bioaerosols during the rainy and dry seasons in La Paz, Bolivia. Median coliform bacteria fluxes were 71 CFU/(m2 × h) during the rainy season and 64 CFU/(m2 × h) during the dry season, with 38% of the dry season samples testing positive for E. coli. Wind speed, relative humidity and UVB irradiance were identified as significant covariates to consider in bioaerosol transport models in La Paz. Active sampling yielded one positive sample (10%) for human adenovirus (HadV) and one sample (10%) for influenza A virus during the rainy season. HadV was detected at the site with the highest bacterial flux. Four samples (8%) were positive for influenza A virus in the dry season. These findings suggest that aerosols can contribute to community exposure to potentially pathogenic microorganisms in cities with poor sanitation. The use of passive sampling, despite its limitations, can provide quantitative data on microorganisms' viability within realistic timeframes of personal exposure., Graphical abstract Unlabelled Image, Highlights • Bioaerosols were characterized near a river in a city with poor sanitation. • Passive and active sampling methods were used to detect fecal pathogens. • Coliforms, E. coli, Influenza A virus and Adenovirus were detected. • A Gaussian plume model could be used to estimate transport from the source. • The sampling strategy can be optimized for bioaerosol surveillance in urban centers.

Highlights • The MERS, SARS and COVID-19 epidemics have demonstrated that coronavirus (CoVs) have an affinity for interspecies transmission and causing epidemics. • We developed a diagnostic to detect all CoVs from the four main genera. • This assay can detect and identify all previously recognized CoVs and any future related CoVs that may emerge. • The assay was highly specific and sensitive in detecting CoVs, and performed well on different sample types. • Sustained surveillance for the emergence of novel CoVs is greatly needed at the human-animal interface. This diagnostic may help., Background During the past two decades, three novel coronaviruses (CoVs) have emerged to cause international human epidemics with severe morbidity. CoVs have also emerged to cause severe epidemics in animals. A better understanding of the natural hosts and genetic diversity of CoVs are needed to help mitigate these threats. Objective To design and evaluate a molecular diagnostic tool for detection and identification of all currently recognized and potentially future emergent CoVs from the Orthocoronavirinae subfamily. Study design and Results We designed a semi-nested, reverse transcription RT-PCR assay based upon 38 published genome sequences of human and animal CoVs. We evaluated this assay with 14 human and animal CoVs and 11 other non-CoV respiratory viruses. Through sequencing the assay's target amplicon, the assay correctly identified each of the CoVs; no cross-reactivity with the 11 common respiratory viruses was observed. The limits of detection ranged from 4 to 4 × 102 copies/reaction, depending on the CoV species tested. To assess the assay's clinical performance, we tested a large panel of previously studied specimens: 192 human respiratory specimens from pneumonia patients, 5 clinical specimens from COVID-19 patients, 81 poultry oral secretion specimens, 109 pig slurry specimens, and 31 aerosol samples from a live bird market. The amplicons of all RT-PCR-positive samples were confirmed by Sanger sequencing. Our assay performed well with all tested specimens across all sample types. Conclusions This assay can be used for detection and identification of all previously recognized CoVs, including SARS-CoV-2, and potentially any emergent CoVs in the Orthocoronavirinae subfamily.

Severe acute respiratory illness (SARI) is a major cause of death and morbidity in low- and middle-income countries, however, the etiologic agents are often undetermined due to the lack of molecular diagnostics in hospitals and clinics. To examine evidence for select viral infections among patients with SARI in northern Vietnam, we studied 348 nasopharyngeal samples from military and civilian patients admitted to 4 hospitals in the greater Hanoi area from 2017-2019. Initial screening for human respiratory viral pathogens was performed in Hanoi, Vietnam at the National Institute of Hygiene and Epidemiology (NIHE) or the Military Institute of Preventative Medicine (MIPM), and an aliquot was shipped to Duke-NUS Medical School in Singapore for validation. Patient demographics were recorded and used to epidemiologically describe the infections. Among military and civilian cases of SARI, 184 (52.9%) tested positive for one or more respiratory viruses. Influenza A virus was the most prevalent virus detected (64.7%), followed by influenza B virus (29.3%), enterovirus (3.8%), adenovirus (1.1%), and coronavirus (1.1%). Risk factor analyses demonstrated an increased risk of influenza A virus detection among military hospital patients (adjusted OR, 2.0; 95% CI, 1.2-3.2), and an increased risk of influenza B virus detection among patients enrolled in year 2017 (adjusted OR, 7.9; 95% CI, 2.7-22.9). As influenza A and B viruses were commonly associated with SARI and are treatable, SARI patients entering these hospitals would benefit if the hospitals were able to adapt onsite molecular diagnostics.

AIMS The purpose of this study was to evaluate the effects of temperature, mixing and sunlight exposure on the 5-day survival of Escherichia coli, Enterococcus sp., F+/male-specific coliphages, somatic coliphages and Clostridium perfringens spores in an 80/20 blend of surface water and reclaimed water approved for potable reuse in North Carolina. METHODS AND RESULTS Grab samples of tertiary treated, dual disinfected North Carolina 'Type 2' reclaimed water were collected and mixed with ambient surface waters to create the 80/20 mix and then spiked with naturally occurring organisms present in the blended water or organisms isolated from sewage. Organism survival over the 5-day period was evaluated at 4 and 20°C, 0, 60 and 120 rev min-1 mixing speeds and exposure to sunlight or darkness. The log10 survival ratio was then calculated for each organism at each condition. CONCLUSIONS There were measurable differences between the log10 survival ratios at 5 days for most organisms; indicating that storage can decrease microbial concentrations. Mixing conditions were not a significant factor in microbe survival over the 5-day storage period. Sunlight was the most effective treatment factor to decrease log10 survival during 5-day storage. SIGNIFICANCE AND IMPACT OF THE STUDY No previous studies have evaluated the survival of micro-organisms in the NC approved 80/20 blend of surface and reclaimed water over the 5-day storage. This study provides the first results on the survival of regulated faecal indicator organisms stored for 5 days in blended water under different environmental conditions.

Recognizing that crowded, high-traffic airports and airplanes have been implicated in respiratory disease transmission, we partnered with administrators of Raleigh Durham International Airport (RDU) in conducting a pilot study of aerosol surveillance for respiratory viruses at RDU. From January to March 2018 we used NIOSH 2-stage samplers to collect 150 min aerosol samples in crowded areas at RDU. Four (17%) of the 24 samples were positive for known respiratory pathogens including influenza D virus and adenovirus. These results suggest the feasibility of employing bioaerosol surveillance techniques in public transportation areas, such as airports, as a noninvasive way to detect and characterize novel respiratory viruses.

Summary During the last 10 years, scientists have grown increasingly aware that emerging respiratory viruses are often zoonotic in their origin. These infections can originate from or be amplified in livestock. Less commonly recognized are instances when humans have transmitted their respiratory pathogens to animals (reverse zoonoses). Even with this knowledge of viral exchange at the human–livestock interface, few studies have been conducted to understand this cross‐over. In this pilot study, we examined persons with influenza‐like illness at an outpatient clinic for evidence of infection with novel zoonotic respiratory pathogens in rural North Carolina where there are dense swine and poultry farming. Environmental air sampling was also conducted. From July 2016 to March 2017, a total of 14 human subjects were enrolled and sampled, and 192 bioaerosol samples were collected. Of the 14 human subject samples molecularly tested, three (21.4%) were positive for influenza A, one (7.1%) for influenza B and one (7.1%) for human enterovirus. Of the 192 bioaerosol samples collected and tested by real‐time RT‐PCR or PCR, three (1.6%) were positive for influenza A and two (1.0%) for adenovirus. No evidence was found for novel zoonotic respiratory viruses.