Epidemiological and etiological investigations of hand, foot, and mouth disease in Jiashan, northeastern Zhejiang Province, China, during 2016 to 2022.

Background: Hand, foot, and mouth disease (HFMD) is a common infectious disease in children. Enterovirus A71 (EV71) and coxsackievirus A16 (CA16) have been identified as the predominant pathogens for several decades. In recent years, coxsackievirus A6 (CA6) and coxsackievirus A10 (CA10) have played increasingly important roles in a series of HFMD outbreaks. We performed a retrospective analysis of the epidemiology of HFMD and the spectrum of different viral serotypes, to elucidate the genetic and phylogenetic characteristics of the main serotypes in the Jiashan area during 2016 to 2022. Methods: Descriptive epidemiological methods were used to analyze the time and population distribution of HFMD in Jiashan during 2016 to 2022 based on surveillance data. Molecular diagnostic methods were performed to identify the viral serotypes and etiological characteristics of HFMD. Phylogenetic analyses was based on VP1 region of CA16 and CA6. Results: The average annual incidence rate of HFMD fluctuated from 2016 to 2022. Children aged 1-5 years accounted for 81.65% of cases and boys were more frequently affected than girls. Except when HFMD was affected by the COVID-19 epidemic in 2020 and 2022, epidemics usually peak in June to July, followed by a small secondary peak from October to December and a decline in February. Urban areas had a high average incidence and rural areas had the lowest. Among 560 sample collected in Jiashan, 472 (84.29%) were positive for enterovirus. The most frequently identified serotypes were CA6 (296, 52.86%), CA16 (102, 18.21%), EV71 (16, 2.86%), CA10 (14, 2.50%) and other enteroviruses (44, 7.86%). There were 71 and 142 VP1 sequences from CA16 and CA6, respectively. Substitution of N218D, A220L and V251I was detected in CA16 and may have been related to viral infectivity. Phylogenetic analysis showed that CA16 could be assigned to two genogroups, B1a and B1b, while all the CA6 sequences belonged to the D3a genogroup. Conclusion: CA6 and CA16 were the two major serotypes of enteroviruses circulating in the Jiashan area during 2016 to 2022. Continuous and comprehensive surveillance for HFMD is needed to better understand and evaluate the prevalence and evolution of the associated pathogens.

Surveillance To Track Progress Toward Polio Eradication - Worldwide, 2022-2023.

The reliable and timely detection of poliovirus cases through surveillance for acute flaccid paralysis (AFP), supplemented by environmental surveillance of sewage samples, is a critical component of the polio eradication program. Since 1988, the number of polio cases caused by wild poliovirus (WPV) has declined by >99.9%, and eradication of WPV serotypes 2 and 3 has been certified; only serotype 1 (WPV1) continues to circulate, and transmission remains endemic in Afghanistan and Pakistan. This surveillance update evaluated indicators from AFP surveillance, environmental surveillance for polioviruses, and Global Polio Laboratory Network performance data provided by 28 priority countries for the program during 2022-2023. No WPV1 cases have been detected outside of Afghanistan and Pakistan since August 2022, when an importation into Malawi and Mozambique resulted in an outbreak during 2021-2022. During 2022-2023, among 28 priority countries, 20 (71.4%) met national AFP surveillance indicator targets, and the number of environmental surveillance sites increased. However, low national rates of reported AFP cases in priority countries in 2023 might have resulted from surveillance reporting lags; substantial national and subnational AFP surveillance gaps persist. Maintaining high-quality surveillance is critical to achieving the goal of global polio eradication. Monitoring surveillance indicators is important to identifying gaps and guiding surveillance-strengthening activities, particularly in countries at high risk for poliovirus circulation.

Enterovirus A71 infection-induced dry eye-like symptoms by damaging the lacrimal glands.

Purpose: To determine the effects of EV-A71 (Enterovirus A71) infection on ocular surface and its mechanism. Methods: AG6 mice aged two to three weeks were randomly divided into control and EV-A71 infected groups. Slit-lamp observation, fluorescein staining, and phenol red thread test were used to assess symptoms of ocular surface at 4 dpi (days post infection). The pathological changes of cornea and lacrimal gland were observed by H&E staining, PAS staining, TUNEL assay, IHC staining and qRT-PCR. Corneas and lacrimal glands from mice were obtained and processed for RNA sequencing analysis. Newly diagnosed HFMD patients caused by EV-A71 were recruited and ensured they met the inclusion criteria. Ocular surface parameters (TMH and NIKBUT) were measured using the OCULUS Keratograph 5M. Tear samples were taken to examine Cxcl1 and IL-6 levels through the ELISA method. Results: Mice studies revealed that EV-A71 infection caused tear film instability, decreased tear secretions, decreased in lacrimal gland size, and distinct goblet cell loss. It also resulted in increased large vacuoles within acinar cells and structural damage in lacrimal gland. Apart from minor damage to the epidermis, there was no obvious inflammatory changes or apoptosis in the cornea. However, there were significant inflammatory injury and apoptosis in the lacrimal gland. RNA-seq analysis showed IL-17 and NF-κB signaling pathways were activated in the lacrimal glands of mice infected with EV-A71. In HFMD patients, the THM was in a low range and NITBUT was significantly shorter than the control group by Oculus Keratograph 5M. ELISA assay showed a higher tear Cxcl1 and IL-6 level in them. Conclusion: EV-A71 infection affected lacrimal gland structure and function and induced dry eye-like symptoms.

Enterovirus virus-like-particle and inactivated poliovirus vaccines do not elicit substantive cross-reactive antibody responses.

Human enteroviruses are the most common human pathogen with over 300 distinct genotypes. Previous work with poliovirus has suggested that it is possible to generate antibody responses in humans and animals that can recognize members of multiple enterovirus species. However, cross protective immunity across multiple enteroviruses is not observed epidemiologically in humans. Here we investigated whether immunization of mice or baboons with inactivated poliovirus or enterovirus virus-like-particles (VLPs) vaccines generates antibody responses that can recognize enterovirus D68 or A71. We found that mice only generated antibodies specific for the antigen they were immunized with, and repeated immunization failed to generate cross-reactive antibody responses as measured by both ELISA and neutralization assay. Immunization of baboons with IPV failed to generate neutralizing antibody responses against enterovirus D68 or A71. These results suggest that a multivalent approach to enterovirus vaccination is necessary to protect against enterovirus disease in vulnerable populations.

Poliomyelitis and COVID-19: a repeated history.

The measurement of the enterovirus and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage water is relevant in the early detection of the introduction or disappearance of these viruses in the ecosystem. We evaluated the co-circulation of the enteroviruses and SARS-CoV-2 in 81 sewage water samples collected between September 2021 and April 2023 from different regions of north and southeast Romania, at the border with Ukraine. We used, for the molecular detection of the pathogens, the multiplex real-time polymerase chain reaction (PCR) assay produced for respiratory samples and the Respiratory 2.1 Plus panel Biofire Film array. The isolation of enteroviruses was performed on cell culture lines, in accordance with the World Health Organization (WHO) recommendations. By molecular investigations, we detected the SARS-CoV-2 in 22 (27%) samples, and the human rhinovirus/enterovirus in 64 (79%) samples. By isolation on cell culture lines, 27 samples (33,33%) were positive for non-polio enteroviruses, and no poliovirus strains were isolated, proving the maintenance of the polio-free status in Romania. In an emergency situation, the molecular detection of the pathogens in sewage water using a PCR system integrating sample preparation, amplification, detection, and analysis in 1 h could be implemented.

Global landscape of coxsackieviruses in human health.

Coxsackieviruses-induced infections, particularly in infants and young children, are one of the most important public health issues in low- and middle-income countries, where the surveillance system varies substantially, and these manifestations have been disregarded. They are widespread throughout the world and are responsible for a broad spectrum of human diseases, from mildly symptomatic conditions to severe acute and chronic disorders. Coxsackieviruses (CV) have been found to have 27 identified genotypes, with overlaps in clinical phenotypes between genotypes. In this review, we present a concise overview of the most recent studies and findings of coxsackieviruses-associated disorders, along with epidemiological data that provides comprehensive details on the distribution, variability, and clinical manifestations of different CV types. We also highlight the significant roles that CV infections play in the emergence of neurodegenerative illnesses and their effects on neurocognition. The current role of CVs in oncolytic virotherapy is also mentioned. This review provides readers with a better understanding of coxsackieviruses-associated disorders and pointing the impact that CV infections can have on different organs with variable pathogenicity. A deeper knowledge of these infections could have implications in designing current surveillance and prevention strategies related to severe CVs-caused infections, as well as encourage studies to identify the emergence of more pathogenic types and the etiology of the most common and most severe disorders associated with coxsackievirus infection.

The multiple roles of viral 3Dpol protein in picornavirus infections.

The Picornaviridae are a large group of positive-sense, single-stranded RNA viruses, and most research has focused on the Enterovirus genus, given they present a severe health risk to humans. Other picornaviruses, such as foot-and-mouth disease virus (FMDV) and senecavirus A (SVA), affect agricultural production with high animal mortality to cause huge economic losses. The 3Dpol protein of picornaviruses is widely known to be used for genome replication; however, a growing number of studies have demonstrated its non-polymerase roles, including modulation of host cell biological processes, viral replication complex assembly and localization, autophagy, and innate immune responses. Currently, there is no effective vaccine to control picornavirus diseases widely, and clinical therapeutic strategies have limited efficiency in combating infections. Many efforts have been made to develop different types of drugs to prohibit virus survival; the most important target for drug development is the virus polymerase, a necessary element for virus replication. For picornaviruses, there are also active efforts in targeted 3Dpol drug development. This paper reviews the interaction of 3Dpol proteins with the host and the progress of drug development targeting 3Dpol.

Identification and genetic characterization of a recently identified enterovirus C116 in China.

Enterovirus C116 (EV-C116) is a new member of the enterovirus C group which is closely associated with several infectious diseases. Although sporadic studies have detected EV-C116 in clinical samples worldwide, there is currently limited information available. In this study, two EV-C-positive fecal specimens were detected in apparently healthy children, which harbored low abundance, through meta-transcriptome sequencing. Based on the prototypes of several EV-Cs, two lineages were observed. Lineage 1 included many types that could not cause EV-like cytopathic effect in cell culture. Three genogroups of EV-C116 were divided in the maximum likelihood tree, and the two strains in this study (XZ2 and XZ113) formed two different lineages, suggesting that EV-C116 still diffuses worldwide. Obvious inter-type recombination events were observed in the XZ2 strain, with CVA22 identified as a minor donor. However, another strain (XZ113) underwent different recombination situations, highlighting the importance of recombination in the formation of EV-Cs biodiversity. The EV-C116 strains could propagate in rhabdomyosarcoma cell cultures at low titer; however, EV-like cytopathic effects were not observed. HEp-2, L20B, VERO, and 293T cell lines did not provide an appropriate environment for EV-C116 growth. These results challenge the traditional recognition of the uncultured nature of EV-C116 strains and explain the difficulty of clinical detection.

Modulation of nucleotide metabolism by picornaviruses.

Viruses actively reprogram the metabolism of the host to ensure the availability of sufficient building blocks for virus replication and spreading. However, relatively little is known about how picornaviruses-a large family of small, non-enveloped positive-strand RNA viruses-modulate cellular metabolism for their own benefit. Here, we studied the modulation of host metabolism by coxsackievirus B3 (CVB3), a member of the enterovirus genus, and encephalomyocarditis virus (EMCV), a member of the cardiovirus genus, using steady-state as well as 13C-glucose tracing metabolomics. We demonstrate that both CVB3 and EMCV increase the levels of pyrimidine and purine metabolites and provide evidence that this increase is mediated through degradation of nucleic acids and nucleotide recycling, rather than upregulation of de novo synthesis. Finally, by integrating our metabolomics data with a previously acquired phosphoproteomics dataset of CVB3-infected cells, we identify alterations in phosphorylation status of key enzymes involved in nucleotide metabolism, providing insight into the regulation of nucleotide metabolism during infection.

Diversity of enteroviruses in cerebrospinal fluid specimens collected from hospitalised patients in the private and public sector in South Africa.

Enteroviruses cause a wide range of neurological illnesses such as encephalitis, meningitis, and acute flaccid paralysis. Two types of enteroviruses, echovirus E4 and E9, have recently been detected in South Africa and are known to be associated with meningitis and encephalitis. The objective of this study was to characterize enterovirus strains detected in cerebrospinal fluid specimens of hospitalized patients in the private and public sector to identify genotypes associated with meningitis and encephalitis. From January 2019 to June 2021 enterovirus positive nucleic acid samples were obtained from a private (n = 116) and a public sector (n = 101) laboratory. These enteroviruses were typed using a nested set of primers targeting the VP1 region of the enterovirus genome, followed by Sanger sequencing and BLASTn analysis. Forty-two percent (91/217) of the strains could be genotyped. Enterovirus B species was the major species detected in 95% (86/91) of the specimens, followed by species C in 3% (3/91) and species A in 2% (2/91) of the specimens. Echovirus E4 and E9 were the two major types identified in this study and were detected in 70% (64/91) and in 10% (9/91) of specimens, respectively. Echovirus E11 has previously been identified in sewage samples from South Africa, but this study is the first to report Echovirus E11 in cerebrospinal fluid specimens from South African patients. The genotypes identified during this study are known to be associated with encephalitis and meningitis. The predominant detection of echovirus E4 followed by E9 corresponds with other studies conducted in South Africa.

Increased circulation of echovirus 11 in the general population and hospital patients as elicited by the non-polio enterovirus laboratory-based sentinel surveillance in northern Italy, 2023.

OBJECTIVES: Following the alert of echovirus 11 (E-11) infection in neonates in EU/EEA Member States, we conducted an investigation of E-11 circulation by gathering data from community and hospital surveillance of enterovirus (EV) in northern Italy from 01 August 2021 to 30 June 2023. METHODS: Virological results of EVs were obtained from the regional sentinel surveillance database for influenza-like illness (ILI) in outpatients, and from the laboratory database of ten hospitals for inpatients with either respiratory or neurological symptoms. Molecular characterization of EVs was performed by sequence analysis of the VP1 gene. RESULTS: In our ILI series, the rate of EV-positive specimens showed an upward trend from the end of May 2023, culminating at the end of June, coinciding with an increase in EV-positive hospital cases. The E-11 identified belonged to the D5 genogroup and the majority (83%) were closely associated with the novel E-11 variant, first identified in severe neonatal infections in France since 2022. E-11 was identified sporadically in community cases until February 2023, when it was also found in hospitalized cases with a range of clinical manifestations. All E-11 cases were children, with 14 out of 24 cases identified through hospital surveillance. Of these cases, 60% were neonates, and 71% had severe clinical manifestations. CONCLUSION: Baseline epidemiological data collected since 2021 through EV laboratory-based surveillance have rapidly tracked the E-11 variant since November 2022, alongside its transmission during the late spring of 2023.

Occurrence of poliovirus and non-polio enterovirus among children with acute flaccid paralysis in Cameroon from 2015 to 2020.

INTRODUCTION: Poliovirus (PV) and non-polio enteroviruses (NPEV) belong to the Picornaviridae family. They are found worldwide and are responsible for a wide range of diseases such as acute flaccid paralysis (AFP). This study aimed to evaluate the detection rate of PV and NPEV in stool samples from children under fifteen years of age presenting with AFP in Cameroon and their distribution over time. METHODOLOGY: Stool samples were collected as part of poliovirus surveillance throughout Cameroon from 2015 to 2020. Virus isolation was performed using RD and L20B cells maintained in culture. Molecular methods such as intratypic differentiation were used to identify PVs serotypes and analysis of the VP1 genome was performed. RESULTS: A total of 12,354 stool samples were analyzed. The EV detection rate by virus isolation was 11.42% (1411/12354). This rate varied from year to year with a mean distribution of 11.41 with a 95% confidence interval [11.37; 11.44]. Of the viruses detected, suspected poliovirus accounted for 31.3% (442/1411) and NPEV 68.67% (969/1411). No wild poliovirus (WPV) was isolated. Sabin types 1 and 3 were continuously isolated. Surprisingly, from February 2020, vaccine-derived PV type 2 (VDPV2) was detected in 19% of cases, indicating its resurgence. CONCLUSIONS: This study strongly supports the successful elimination of WPV in Cameroon and the resurgence of VDPV2. However, as long as VDPV outbreaks continue to be detected in Africa, it remains essential to monitor how they spread.

Validation of improved automated nucleic acid extraction methods for direct detection of polioviruses for global polio eradication.

Polioviruses (PV), the main causative agent of acute flaccid paralysis (AFP), are positive-sense single-stranded RNA viruses of the family Picornaviridae. As we approach polio eradication, accurate and timely detection of poliovirus in stool from AFP cases becomes vital to success for the eradication efforts. Direct detection of PV from clinical diagnostic samples using nucleic acid (NA) extraction and real-time reverse transcriptase polymerase chain reaction (rRT-PCR) instead of the current standard method of virus isolation in culture, eliminates the long turn-around time to diagnosis and the need for high viral titer amplification in laboratories. An essential component of direct detection of PV from AFP surveillance samples is the efficient extraction of NA. Potential supply chain issues and lack of vendor presence in certain areas of the world necessitates the validation of multiple NA extraction methods. Using retrospective PV-positive surveillance samples (n=104), two extraction kits were compared to the previously validated Zymo Research Quick-RNA™ Viral Kit. The Roche High Pure Viral RNA Kit, a column-based manual extraction method, and the MagMaX™ Pathogen RNA/DNA kit used in the automated Kingfisher Flex system were both non-inferior to the Zymo kit, with similar rates of PV detection in pivotal rRT-PCR assays, such as pan-poliovirus (PanPV), poliovirus serotype 2 (PV2), and wild poliovirus serotype 1 (WPV1). These important assays allow the identification and differentiation of PV genotypes and serotypes and are fundamental to the GPLN program. Validation of two additional kits provides feasible alternatives to the current piloted method of NA extraction for poliovirus rRT-PCR assays.

Effectiveness of Enterovirus 71 inactivated vaccines against hand, foot, and mouth disease: A test-negative case-control study.

The Enterovirus A71 (EV-A71) vaccine was introduced in China in December 2015 as a preventive measure against hand, foot, and mouth disease (HFMD) caused by EV-A71. However, the effectiveness of the vaccine (VE) in real-world settings needs to be evaluated. We conducted a test-negative case-control study to assess the effectiveness of EV-A71 vaccines in preventing EV-A71-associated HFMD. Children aged 6-71 months with HFMD were enrolled as participants. The case group comprised those who tested positive for EV-A71, while the control group comprised those who tested negative for EV-A71. To estimate VE, a logistic regression model was employed, adjusting for potential confounders including age, gender, and clinical severity. In total, 3223 children aged 6 to 71 months were included in the study, with 162 in the case group and 3061 in the control group. The proportion of children who received EV-A71 vaccination was significantly lower in the case group compared to the control group (p < .001). The overall VEadj was estimated to be 90.8%. The VEadj estimates for partially and fully vaccinated children were 90.1% and 90.9%, respectively. Stratified by age group, the VEadj estimates were 88.7% for 6 to 35-month-olds and 95.5% for 36 to 71-month-olds. Regarding disease severity, the VEadj estimates were 86.3% for mild cases and 100% for severe cases. Sensitivity analysis showed minimal changes in the VE point estimates, with most changing by no more than 1% point. Our study demonstrates a high level of vaccine effectiveness against EV-A71-HFMD, especially in severe cases. Active promotion of EV-A71 vaccination is an effective strategy in preventing EV-A71 infections.

Patterns of Non-influenza Respiratory Viruses Among Severe Acute Respiratory Infection Cases in Burkina Faso: A Surveillance Study.

BACKGROUND: Although influenza viruses cause only one-fifth of severe acute respiratory infections (SARI) in Burkina Faso, the other viral causes of SARI remain poorly investigated to inform clinical and preventive decision making. METHODS: Between 2016 and 2019, we prospectively enrolled inpatients meeting the World Health Organization (WHO) case definition of SARI in Burkina Faso. Results of viral etiologies among inpatients tested negative for influenza using the Fast Track Diagnostics Respiratory Kits (FTD-33) were reported. RESULTS: Of 1541 specimens tested, at least one respiratory virus was detected in 76.1% of the 1231 specimens negative for influenza virus. Human rhinoviruses (hRVs) were the most detected pathogens (476; 38.7%), followed by human adenoviruses (hAdV) (17.1%, 210/1231), human respiratory syncytial virus (hRSV) (15.4%, 189/1231), enterovirus (EnV) (11.2%, 138/1231), human bocavirus (hBoV) (7.9%, 97/1231), parainfluenza 3 (hPIV3) (6.1%, 75/1231), human metapneumovirus (hMPV) (6.0%,74/1321), parainfluenza 4 (hPIV4) (4.1%, 51/1231), human coronavirus OC43 (hCoV-OC43) (3.4%, 42/1231), human coronavirus HKU1(hCoV-HKU1) (2.7%, 33/1231), human coronavirus NL63 (hCoV-NL63) (2.5%, 31/1231), parainfluenza 1 (hPIV1) (2.0%, 25/1231), parainfluenza 2 (hPIV2) (1.8%, 22/1231), human parechovirus (PeV) (1.1%, 14/1231), and human coronavirus 229E (hCoV-229E) (0.9%, 11/1231). Among SARI cases, infants aged 1-4 years were mostly affected (50.7%; 622/1231), followed by those <1 year of age (35.7%; 438/1231). Most detected pathogens had year-long circulation patterns, with seasonal peaks mainly observed during the cold and dry seasons. CONCLUSION: Several non-influenza viruses are cause of SARI in Burkina Faso. The integration of the most common pathogens into the routine influenza surveillance system might be beneficial.

Age-time-specific transmission of hand-foot-and-mouth disease enterovirus serotypes in Vietnam: A catalytic model with maternal immunity.

Hand, foot and mouth disease (HFMD) is highly prevalent in the Asia Pacific region, particularly in Vietnam. To develop effective interventions and efficient vaccination programs, we inferred the age-time-specific transmission patterns of HFMD serotypes enterovirus A71 (EV-A71), coxsackievirus A6 (CV-A6), coxsackievirus A10 (CV-A10), coxsackievirus A16 (CV-A16) in Ho Chi Minh City, Vietnam from a case data collected during 2013-2018 and a serological survey data collected in 2015 and 2017. We proposed a catalytic model framework with good adaptability to incorporate maternal immunity using various mathematical functions. Our results indicate the high-level transmission of CV-A6 and CV-A10 which is not obvious in the case data, due to the variation of disease severity across serotypes. Our results provide statistical evidence supporting the strong association between severe illness and CV-A6 and EV-A71 infections. The HFMD dynamic pattern presents a cyclical pattern with large outbreaks followed by a decline in subsequent years. Additionally, we identify the age group with highest risk of infection as 1-2 years and emphasise the risk of future outbreaks as over 50% of children aged 6-7 years were estimated to be susceptible to CV-A16 and EV-A71. Our study highlights the importance of multivalent vaccines and active surveillance for different serotypes, supports early vaccination prior to 1 year old, and points out the potential utility for vaccinating children older than 5 years old in Vietnam.

Effectiveness of enterovirus A71 vaccine against pediatric HFMD and disease profile of post-vaccination infection.

BACKGROUND: Vaccination has been proven effective against infection with enterovirus A71 (EV-A71) in clinical trials, but vaccine effectiveness in real-world situations remains incompletely understood. Furthermore, it is not clear whether previous vaccination will result in symptom attenuation among post-vaccinated cases. METHODS: Based on long-term data extracted from the only designed referral hospital for infectious diseases, we used a test-negative case-control design and multivariate logistic regression models to analyze the effectiveness of EV-A71 vaccine against hand, foot and mouth disease (HFMD). And then, generalized linear regression models were used to evaluate the associations between prior vaccination and disease profiles. RESULTS: We selected 4883 inpatients for vaccine efficacy estimations and 2188 inpatients for disease profile comparisons. Vaccine effectiveness against EV-A71-induced HFMD for complete vaccination was 63.4 % and 51.7 % for partial vaccination. The vaccine effectiveness was higher among cases received the first dose within 12 months. No protection was observed against coxsackievirus (CV) A6-, CV-A10- or CV-A16-associated HFMD among children regardless of vaccination status. Completely vaccinated cases had shorter hospital stay and disease course compared to unvaccinated cases (P < 0.05). CONCLUSIONS: These findings reiterate the need to continue the development of a multivalent vaccine or combined vaccines, and have implications for introducing optimized vaccination strategies.

Efficacy of sample collection without virus transport medium in suspected enteroviral infections for molecular diagnosis.

Clinical swabs with suspected viral infection are usually transported in virus transport medium (VMT). During epidemics/pandemics, tampons without VTM would be more suitable for saving space and cost. This study was conducted to verify the applicability of throat swabs without VTM in the diagnosis/screening of enteroviral infections by polymerase chain reaction (PCR) in a volunteer study group. Three different swab types were used in 40 volunteers: swabs with two different tips (cotton- or synthetic-tipped) without VTM and standard synthetic tips with VTM. The swabs were processed immediately or after 12 days of storage at either -80°C or +4°C. The molecular analysis included viral RNA extraction, and combination of reverse transcriptase PCR and nested PCR. Enteroviral RNA was detected in 15% (6/40) of the studied volunteers. When processed immediately, the results for all three swab types were compatible. Swabs without VTM may be used for collection of clinical samples in the diagnosis of suspected enteroviral infections or as potential screening tools for enteroviruses (Tab. 2, Ref. 15). Keywords: enterovirus infection, swab, transport medium, PCR, molecular diagnostics.

Assessing the nucleic acid decay of human wastewater markers and enteric viruses in estuarine waters in Sydney, Australia.

This research investigated the in-situ decay rates of four human wastewater-associated markers (Bacteroides HF183 (HF183), Lachnospiraceae Lachno3 (Lachno3), cross-assembling phage (crAssphage), pepper mild mottle virus (PMMoV) and three enteric viruses (human adenovirus 40/41 (HAdV 40/41), enterovirus (EV) and human norovirus GII (HNoV GII) in two estuarine water environments (Davidson Park (DP) and Hen and Chicken Bay (HCB) in temperate Sydney, NSW, Australia, employing qPCR and RT-qPCR assays. The study also aimed to compare decay rates observed in mesocosms with previously published laboratory microcosms, providing insights into the persistence of markers and viruses in estuarine environments. Results indicated varying decay rates between DP and HCB mesocosms, with HF183 exhibiting relatively faster decay rates compared to other markers and enteric viruses in sunlight and dark mesocosms. In DP mesocosms, HF183 decayed the fastest, contrasting with PMMoV, which exhibited the slowest. Sunlight induced higher decay rates for all markers and viruses in DP mesocosms. In HCB sunlight mesocosms, HF183 nucleic acid decayed most rapidly compared to other markers and enteric viruses. In dark mesocosms, crAssphage showed the fastest decay, while PMMoV decayed at the slowest rate in both sunlight and dark mesocosms. Comparisons with laboratory microcosms revealed faster decay of markers and enteric viruses in laboratory microcosms than the mesocosms, except for crAssphage and HAdV 40/41 in dark, and PMMoV in sunlight mesocosms. The study concludes that decay rates of markers and enteric viruses vary between estuarine mesocosms, emphasizing the impact of sunlight exposure, which was potentially influenced by the elevated turbidity at HCB estuarine waters. The generated decay rates contribute valuable insights for establishing site-specific risk-based thresholds of human wastewater-associated markers.