Ex-vivo RNA expression analysis of vaccine candidate genes in COPD sputum samples.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a lung disease characterised by airflow-limiting inflammation and mucus production. Acute exacerbations are a major cause of COPD-related morbidity and mortality and are mostly associated with bacterial or viral infections. A vaccine targeting non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat), the main bacteria associated with exacerbations, was tested in a Phase 2 trial. We assessed "ex-vivo" expression of vaccine candidate and housekeeping genes pd, pe, pilA, gapA, ompP6 of NTHi, and uspA2, parE, polA of Mcat in sputum samples of COPD patients and determined whether expression of the vaccine candidate genes pd, pe, pilA (NTHi) and uspA2 (Mcat) differed between stable and exacerbation samples. METHODS: A single-centre, prospective, observational cohort study was conducted where 123 COPD patients were seen on enrolment, followed monthly for 2 years, and reviewed after onset of acute exacerbations. We selected 69 patients with sputum samples positive for NTHi or Mcat by PCR during at least one stable and one exacerbation visit. mRNA was isolated from the sputum, and expression of NTHi and Mcat genes was analysed with RT-PCR. Statistical analyses compared mRNA concentrations between stable and exacerbation samples and in relationship to COPD severity and exacerbation frequency. RESULTS: The vaccine candidate genes were variably expressed in sputum samples, suggesting they are expressed in the lung. Absolute and relative expression of all NTHi vaccine candidate genes and Mcat uspA2 were similar between exacerbation and stable samples. Expression of pd and pilA was slightly associated with the number of exacerbations in the year before enrolment, and uspA2 with the disease severity status at enrolment. CONCLUSIONS: The NTHi-Mcat vaccine candidate genes were expressed in sputum samples, and each gene had a specific level of expression. No statistically significant differences in gene expression were detectable between stable and exacerbation samples. However, the history of COPD exacerbations was slightly associated with the expression of pd, pilA and uspA2. Trial registration NCT01360398 ( https://www. CLINICALTRIALS: gov ).

Examining the persistence of human Coronavirus 229E on fresh produce.

Human coronaviruses (HCoVs) are mainly associated with respiratory infections. However, there is evidence that highly pathogenic HCoVs, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome (MERS-CoV), infect the gastrointestinal (GI) tract and are shed in the fecal matter of the infected individuals. These observations have raised questions regarding the possibility of fecal-oral route as well as foodborne transmission of SARS-CoV-2 and MERS-CoV. Studies regarding the survival of HCoVs on inanimate surfaces demonstrate that these viruses can remain infectious for hours to days, however, there is limited data regarding the viral survival on fresh produce, which is usually consumed raw or with minimal heat processing. To address this knowledge gap, we examined the persistence of HCoV-229E, as a surrogate for highly pathogenic HCoVs, on the surface of commonly consumed fresh produce, including: apples, tomatoes, cucumbers and lettuce. Herein, we demonstrated that viral infectivity declines within a few hours post-inoculation (p.i) on apples and tomatoes, and no infectious virus was detected at 24h p.i, while the virus persists in infectious form for 72h p.i on cucumbers and lettuce. The stability of viral RNA was examined by droplet-digital RT-PCR (ddRT-PCR), and it was observed that there is no considerable reduction in viral RNA within 72h p.i.

Evaluation of porcine gastric mucin assay for detection and quantification of human norovirus in fresh herbs and leafy vegetables.

Leafy vegetables and fresh herbs are important parts of a healthy diet, however, they can be common vehicles of norovirus (NoV) infection and lead to serious health and economic concerns globally. NoV is highly infectious and persistent in the food and the environment, while being resistant to conventional food decontamination practices. Herbs and leafy greens are often consumed raw, and if contaminated with NoV, they may cause illness. Consequently, for outbreak prevention and surveillance purposes, sensitive and rapid methods are required to detect the presence of infectious NoV in naturally contaminated produce during its shelf life. Herein, we compared the extraction efficiency of the ISO/TS 15216-1:2017 method with the porcine gastric mucin coated magnetic beads (PGM-MB) assay, combined with heat-denaturation for RNA extraction, for detection of human NoV in artificially contaminated fresh green seaweed, basil, mint, and baby spinach. Droplet-digital RT-PCR was used to quantify the extracted genome by both methods. Our data demonstrated that while the PGM-MB assay takes considerably less time, it yields significantly higher recovery rates compared with the ISO/TS 15216-1:2017. Furthermore, since this method has the ability to be adapted in high-throughput and automated systems, it can be further modified to be employed by the food industry to reduce the number of NoV illnesses and outbreaks at the source of distribution.

Droplet Digital RT-PCR (dd RT-PCR) Detection of SARS-CoV-2 in Honey Bees and Honey Collected in Apiaries across the Campania Region.

Coronaviruses (CoVs), a subfamily of Orthocoronavirinae, are viruses that sometimes present a zoonotic character. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for the recent outbreak of COVID-19, which, since its outbreak in 2019, has caused about 774,593,066 confirmed cases and 7,028,881 deaths. Aereosols are the main route of transmission among people; however, viral droplets can contaminate surfaces and fomites as well as particulate matter (PM) in suspensions of natural and human origin. Honey bees are well known bioindicators of the presence of pollutants and PMs in the environment as they can collect a great variety of substances during their foraging activities. The aim of this study was to evaluate the possible role of honey bees as bioindicators of the prevalence SARS-CoV-2. In this regard, 91 samples of honey bees and 6 of honey were collected from different apiaries of Campania region (Southern Italy) in four time periods from September 2020 to June 2022 and were analyzed with Droplet Digital RT-PCR for SARS-CoV-2 target genes Orf1b and N. The screening revealed the presence of SARS-CoV-2 in 12/91 in honey bee samples and in 2/6 honey samples. These results suggest that honey bees could also be used as indicators of outbreaks of airborne pathogens such as SARS-CoV-2.

Long-term monitoring of SARS-CoV-2 variants in wastewater using a coordinated workflow of droplet digital PCR and nanopore sequencing.

Quantitative polymerase chain reaction (PCR) and genome sequencing are important methods for wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The reverse transcription-droplet digital PCR (RT-ddPCR) is a highly sensitive method for quantifying SARS-CoV-2 RNA in wastewater samples to track the trends of viral activity levels but cannot identify new variants. It also takes time to develop new PCR-based assays targeting variants of interest. Whole genome sequencing (WGS) can be used to monitor known and new SARS-CoV-2 variants, but it is generally not quantitative. Several short-read sequencing techniques can be expensive and might experience delayed turnaround times when outsourced due to inadequate in-house resources. Recently, a portable nanopore sequencing system offers an affordable and real-time method for sequencing SARS-CoV-2 variants in wastewater. This technology has the potential to enable swift response to disease outbreaks without relying on clinical sequencing results. In addressing concerns related to rapid turnaround time and accurate variant analysis, both RT-ddPCR and nanopore sequencing methods were employed to monitor the emergence of SARS-CoV-2 variants in wastewater. This surveillance was conducted at 23 sewer maintenance hole sites and five wastewater treatment plants in Michigan from 2020 to 2022. In 2020, the wastewater samples were dominated by the parental variants (20A, 20C and 20 G), followed by 20I (Alpha, B.1.1.7) in early 2021 and the Delta variant of concern (VOC) in late 2021. For the year 2022, Omicron variants dominated. Nanopore sequencing has the potential to validate suspected variant cases that were initially undetermined by RT-ddPCR assays. The concordance rate between nanopore sequencing and RT-ddPCR assays in identifying SARS-CoV-2 variants to the clade-level was 76.9%. Notably, instances of disagreement between the two methods were most prominent in the identification of the parental and Omicron variants. We also showed that sequencing wastewater samples with SARS-CoV-2 N gene concentrations of >104 GC/100 ml as measured by RT-ddPCR improve genome recovery and coverage depth using MinION device. RT-ddPCR was better at detecting key spike protein mutations A67V, del69-70, K417N, L452R, N501Y, N679K, and R408S (p-value <0.05) as compared to nanopore sequencing. It is suggested that RT-ddPCR and nanopore sequencing should be coordinated in wastewater surveillance where RT-ddPCR can be used as a preliminary quantification method and nanopore sequencing as the confirmatory method for the detection of variants or identification of new variants. The RT-ddPCR and nanopore sequencing methods reported here can be adopted as a reliable in-house analysis of SARS-CoV-2 in wastewater for rapid community level surveillance and public health response.

In vitro fitness of SARS-CoV-2 variants as assessed by competition experiments followed by ddRT-PCR and whole genome sequencing.

OBJECTIVE: To develop a new method for reliable and rapid determination of the fitness of SARS-CoV-2 variants of concern. METHODS: Competition experiments between two SARS-CoV-2 variants were performed in cells of the upper (nasal human airway epithelium) and lower (Calu-3 cells) respiratory tracts followed by quantification of variant ratios by droplet digital reverse transcription (ddRT)-PCR. RESULTS: In competition experiments, the delta variant outcompeted the alpha variant in both cells of the upper and lower respiratory tracts. A 50/50% mixture of delta and omicron variants indicated a predominance of omicron in the upper respiratory tract whereas delta predominated in the lower respiratory tract. There was no evidence of recombination events between variants in competition as assessed by whole gene sequencing. CONCLUSION: Differential replication kinetics were shown between variants of concern which may explain, at least partly, the emergence and disease severity associated with new SARS-CoV-2 variants.

SARS-CoV-2 detection in nasopharyngeal swabs: Performance characteristics of a real-time RT-qPCR and a droplet digital RT-PCR assay based on the exonuclease region (ORF1b, nsp 14).

The emergence and spread of SARS-CoV-2 has led to a compelling request for accurate diagnostic tests. The aim of this study was assessing the performance of a real-time RT-qPCR (rt RT-qPCR) assay and of a droplet digital RT-PCR (dd RT-PCR) targeting the nsp14 genome region for the detection of SARS-CoV-2 in nasopharyngeal swabs. A total of 258 nasopharyngeal swabs were analyzed with the nsp14 assays and, for comparison, with a reference assay targeting the RdRp and E genes. Conflicting results were further investigated by two additional protocols, the Centers for Disease Control and Prevention (CDC) real-time targeting N1/N2, and a nested RT-PCR for the spike region. Agreement of results was achieved on 226 samples (156 positive and 70 negative), 8 samples were positive in the reference assay and in the nsp14 rt RT-qPCR but negative with the dd RT-PCR, and 24 samples provided different combinations of results with the three assays. Sensitivity, specificity and accuracy (95 %C.I.) of the nsp14 assays were: 100.0 % (97.4-100.0), 98.7 % (92.1-100.0), and 99.6 % (97.5-100.0) for the rt RT-qPCR; 92.4 % (87.4-95.6), 100.0 % (94.2-100.0), and 94.7 % (91.1-97.0) for the dd RT-PCR. The results of the study support the use of the nsp14 real-time RT-qPCR and ddPCR for the detection of SARS-CoV-2 in nasopharyngeal swabs.

Development of an RNA Extraction Protocol for Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR.

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster's digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

Evaluation of High-Pressure Processing in Inactivation of the Hepatitis E Virus.

Hepatitis E virus (HEV) causes acute hepatitis with approximately 20 million cases per year globally. Based on genetic diversity, HEV is classified into different genotypes, with genotype 3 (HEV-3) being most prevalent in Europe and North America. The transmission of HEV-3 has been shown to be zoonotic and mainly associated with the consumption of raw or undercooked pork products. Herein, we investigated the efficacy of high-pressure processing (HPP) in inactivation of HEV-3 using a cell culture system. HPP has been indicated as a promising non-thermal pathogen inactivation strategy for treatment of certain high-risk food commodities, without any noticeable changes in their nature. For this purpose, we treated HEV-3 in media with different conditions of HPP: 400 MPa for 1 and 5 min, as well as 600 MPa for 1 and 5 min, at ambient temperature. All four HPP treatments of HEV in media were observed to result in a 2-log reduction in HEV load, as determined by the amounts of extracellular HEV RNA produced at 14-day post-infection, using the A549/D3 cell culture system. However, application of the same treatments to artificially contaminated pork pâté resulted in 0.5 log reduction in viral load. These results indicate that the efficacy of HPP treatment in the inactivation of HEV-3 is matrix-dependent, and independent of maximum pressure between 400 and 600 MPa and hold time between 1 and 5 min. Based on the obtained results, although the HPP treatment of pork pâté reduces the HEV-3 load, it might not be sufficient to fully mitigate the risk.

Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples.

Bluetongue (BT) is a major Office International des Epizooties (OIE)-listed disease of wild and domestic ruminants caused by several serotypes of Bluetongue virus (BTV), a virus with a segmented dsRNA genome belonging to the family Reoviridae, genus Orbivirus. BTV is transmitted through the bites of Culicoides midges. The aim of this study was to develop a new method for quantification of BTV Seg-10 by droplet digital RT-PCR (RTdd-PCR), using nucleic acids purified from complex matrices such as blood, tissues, and midges, that notoriously contain strong PCR inhibitors. First, RTdd-PCR was optimized by using RNAs purified from serially 10-fold dilutions of a BTV-1 isolate (105.43TCID50/ml up to 10-0.57 TCID50/ml) and from the same dilutions spiked into fresh ovine EDTA-blood and spleen homogenate. The method showed a good degree of linearity (R 2 ≥ 0.995). The limit of detection (LoD) and the limit of quantification (LoQ) established were 10-0.67TCID50/ml (0.72 copies/µl) and 100.03TCID50/ml (3.05 copies/µl) of BTV-1, respectively. Second, the newly developed test was compared, using the same set of biological samples, to the quantitative RT-PCR (RT-qPCR) detecting Seg-10 assay widely used for the molecular diagnosis of BTV from field samples. Results showed a difference mean of 0.30 log between the two assays with these samples (p < 0.05). Anyway, the analysis of correlation demonstrated that both assays provided similar measurements with a very close agreement between the systems.

Implications of and lessons learned from external assurance of eight influenza diagnostics in China.

This study evaluated the ability of laboratories in the Chinese mainland to conduct molecular detection of seasonal A(H1N1), A(H1N1)pdm09, A(H3N2), A(H5N1), A(H7N9), A(H9N2), B(Victoria), and B(Yamagata). Based on a genetically engineered system of virus-like particles (VLPs), the National Center for Clinical Laboratories of China (NCCLs) developed an external quality assessment (EQA) panel. The panel was distributed to 35 laboratories in mainland China to investigate the proficiency of the 16 assays for influenza molecular detection. Using genetic engineering technology, VLPs encapsulating the 37 target genes of 8 influenza viruses were generated. After verification and quantification, 26 influenza virus surrogates with different concentrations were prepared for EQA. Among the 35 participating laboratories, 319 datasets were returned to the NCCLs. Overall, 95.6% (305/319) of datasets correctly reported all 30 samples, while 2.2% (7/319) of datasets with more than one incorrect result were considered as "improvable". A total of 16 misdiagnosed and 18 undiagnosed results were reported. The data analyzed in this study showed good reproducibility in China, but improvements are needed to decrease misdiagnosed and undiagnosed cases, particularly for the A(H9N2) NA gene. Moreover, VLPs are a good alternative specimen type for assay training and proficiency testing purposes.