Qualitative detection of E. coli in distributed drinking water using real-time reverse transcription PCR targeting 16S rRNA: Validation and practical experiences.

Escherichia coli (E. coli) plays a central role as an indicator for fecal contamination to predict the possible presence of microbial pathogens in drinking water. Current detection methods for E. coli are based on time-consuming culture-based techniques. There is a strong need for methods to detect fecal contamination rapidly in distributed drinking water to prevent outbreaks of waterborne disease and support water utilities to efficiently manage their operations like actions to repair or maintain distribution pipes, to minimize impact on consumers. This study describes the validation and application of a qualitative real time reverse transcription PCR (RT-PCR) method targeting 16S ribosomal RNA (rRNA) for rapid detection of E. coli in distributed drinking water. The RT-PCR assay targets 16S rRNA, a highly abundant RNA in viable cells, enabling robust detection at the required sensitivity of 1 CFU/100 ml. The validation was performed by comparing the RT-PCR method with the culture-based chromogenic reference method (CCA) using the protocol and criteria described in ISO 16,140-2:2016. The validation demonstrated that this RT-PCR method can be used to specifically detect E. coli in a broad range of drinking water samples with at least the same limit of detection as the culture method (Relative Limit Of Detection = 0.75, range 0.43-1.43). The inclusivity study showed that the RT-PCR method was able to detect a broad range of E. coli strains derived from different sources and geographic areas, including pathogenic serotype O157 strains that are not detected with the culture method. The exclusivity study determined that other bacterial genera are not detected with this RT-PCR. However, Escherichia fergusonii was detected and, based on "in silico" analysis, it is expected that also E. albertii and E. marmotae and Shigella species will be detectable using this RT-PCR. An interlaboratory study confirmed that the RT-PCR and culture method have comparable sensitivities when tested by different participants at different laboratories. The application of RT-PCR to confirm the hygienic quality of distributed drinking water after actions to repair or maintain distribution pipes was compared with the culture method on 8076 routine samples, analyzed by the drinking water laboratories in the Netherlands. This comparison study showed a 96.4 % agreement between RT-PCR and culture. In 3.3 % of the samples E. coli was detected with RT-PCR and not with the culture method and in 0.1 % of the samples E. coli was only detected by culture confirming either a higher sensitivity for RT-PCR or the detection of RNA from uncultivable cells. Finally, the application of RT-PCR was highlighted during a contamination event in Belgium where we demonstrate the potency of RT-PCR as a tool to rapidly monitor the spread of microbial contamination and to monitor the effect of measures to remove the contamination This is the first fully validated rapid nucleic based method for detection of E. coli in distributed drinking water. These results demonstrate that this RT-PCR method can be used as a rapid alternative to the culture method to monitor E. coli in distributed drinking water. However, it should be emphasized that nucleic acid based detection methods rely on highly different detection principles (detection of captured nucleic acids present in a sample) than culture base methods (presence of cells cultivable on a selective medium) resulting in occasional different analysis results. Varying treatment and disinfection steps (UV, chlorine, monochloramine, Ozone) or environmental factors (decay) can influence the results and cause differences between RT-PCR and culture methods.

Effect of four hydroxy fatty acids on lipid accumulation in the 3T3-L1 cells: A comparative study.

Notwithstanding the several investigations of the hydroxy fatty acids (hFAs)' physiological functions, studies focusing on their anti-obesity effects are limited. This study investigated the anti-obesity effects of four hFAs, 10-hydroxy stearic acid (10-hSA), 12-hydroxy stearic acid (12-hSA), 9,12-hydroxy stearic acid (9,12-dhSA), and 12-hydroxy oleic acid (12-hOA), on the 3T3-L1 cells. All hFAs suppressed lipid accumulation, with 10-hSA and 12-hOA exhibiting the strongest suppression, followed by 12-hSA and 9, 12-hSA. This trend was similar to that observed for the glycerol-3-phosphate dehydrogenase (GPDH) activity degree. Contrastingly, only 9,12-dhSA suppressed cell viability. The mRNA levels of HK1 and Aldoa were markedly suppressed by 10-hSA and 12-hSA compared to the control. Additionally, mRNA expression of Gyk was considerably suppressed by 12-hSA. Thus, all hFAs suppressed lipid accumulation by suppressing GPDH activity, although their molecular mechanisms were different. These findings will aid the application of hFAs in the food and medical industries.

A one-step low-cost molecular test for SARS-CoV-2 detection suitable for community testing using minimally processed saliva.

The gold standard for coronavirus disease 2019 diagnostic testing relies on RNA extraction from naso/oropharyngeal swab followed by amplification through reverse transcription-polymerase chain reaction (RT-PCR) with fluorogenic probes. While the test is extremely sensitive and specific, its high cost and the potential discomfort associated with specimen collection made it suboptimal for public health screening purposes. In this study, we developed an equally reliable, but cheaper and less invasive alternative test based on a one-step RT-PCR with the DNA-intercalating dye SYBR Green, which enables the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from saliva samples or RNA isolated from nasopharyngeal (NP) swabs. Importantly, we found that this type of testing can be fine-tuned to discriminate SARS-CoV-2 variants of concern. The saliva RT-PCR SYBR Green test was successfully used in a mass-screening initiative targeting nearly 4500 asymptomatic children under the age of 12. Testing was performed at a reasonable cost, and in some cases, the saliva test outperformed NP rapid antigen tests in identifying infected children. Whole genome sequencing revealed that the antigen testing failure could not be attributed to a specific lineage of SARS-CoV-2. Overall, this work strongly supports the view that RT-PCR saliva tests based on DNA-intercalating dyes represent a powerful strategy for community screening of SARS-CoV-2. The tests can be easily applied to other infectious agents and, therefore, constitute a powerful resource for an effective response to future pandemics.

Time to SARS-CoV-2 clearance in African, Caucasian, and Asian ethnic groups.

BACKGROUND: COVID-19 may become a seasonal disease. SARS-CoV-2 active circulation coupled with vaccination efforts has undoubtedly modified the virus dynamic. It is therefore important investigate SARS-CoV-2 dynamic in different groups of population following the course of spatiotemporal variance and immunization. METHODS: To investigate SARS-CoV-2 clearance in different ethnic groups and the impact of immunization, we recruited 777 SARS-CoV-2-positive patients (570 Africans, 156 Caucasians, and 51 Asians). Participants were followed and regularly tested for 2 months until they had two negative tests. RESULTS: The vaccination rate was 64.6%. African individuals were less symptomatic (2%), Caucasians (41%) and Asians (36.6%). On average, viral clearance occurred after 10.5 days. Viral load at diagnosis was inversely correlated with viral clearance (p < 0.0001). The time of SARS-CoV-2 clearance was higher in Africans and Caucasians than in Asians (Dunn's test p < 0.0001 and p < 0.05, respectively). On average, viral clearance occurred within 9.5 days during the second semester (higher rate of vaccination and SARS-CoV-2 exposition), whereas it took 13.6 days during the first semester (lower rate of vaccination and SARS-CoV-2 exposition) (Mann-Whitney t-test p < 0.0001). CONCLUSION: In conclusion, ethnicity and spatiotemporal changes including SARS-CoV-2 exposition and immunization affect SARS-CoV-2 clearance.

A novel method for high-sensitivity detection of SARS-CoV-2 using dual double-quenched fluorescence probes.

SARS-CoV-2 has infected many people around the world, fast and accurate detection of the virus can help control the spread of the virus. RT-PCR is the gold standard method for SARS-CoV-2 detection. In this study, we improved the RT-PCR by proposing a novel method using dual double-quenched fluorescence probes. We used the improved probes to detect the plasmid DNA and RNA reference materials of SARS-CoV-2 respectively. When the improved probes were used, the background fluorescence intensity has reduced by 50%, the fluorescence increment has increased to 2.8 folds, and the Ct value has significantly reduced by 3 or more, indicating that the detection sensitivity has increased at least eight times. In addition, we also demonstrated that the improved probes have well performance in detecting SARS-CoV-2 with the minimum concentration of 6.2 copies/µL. This study will help biological companies develop better products for SARS-CoV-2 and other clinical pathogen infection.

Mixed infection of two mandariviruses identified by high-throughput sequencing in Kinnow mandarin and development of their specific detection using duplex RT-PCR.

In the current study, high-throughput sequencing (HTS) was used to identify viruses associated with the Kinnow mandarin (Citrus reticulata) plants exhibiting yellow vein clearing, mottling, and chlorosis symptoms at experimental farm of ICAR-Indian Agricultural Research Institute, New Delhi, India. During November 2022, leaf samples of symptomatic and asymptomatic Kinnow mandarin trees were collected, subjected to HTS and one of the representative symptomatic samples was subjected to leaf-dip electron microscopy (EM). In the EM results, flexuous virus particles typical of mandarivirus were observed. Ribosomal RNA was depleted from total RNA of pooled samples and RNA sequencing was done using NovaSeq 6000. Host unaligned reads were de novo assembled into contigs, which were annotated through BLASTn using database of plant viruses/viroids reference genomes (NCBI). Results of assembled contigs revealed near-complete genomes of two mandariviruses, i.e., citrus yellow vein clearing virus (CYVCV) and citrus yellow mottle-associated virus (CiYMaV). The values of fragments per kilo base transcript length per million fragments mapped estimation indicated the dominance of CYVCV in HTS data and it was also confirmed through krona plot distribution of viruses in the pooled samples. A rapid and reliable duplex RT-PCR assay was also developed and standardized for the simultaneous detection of both CYVCV and CiYMaV in a pooled Kinnow mandarin sample. The developed duplex RT-PCR was then validated for the presence of these viruses in individual Kinnow mandarin samples. The specificity and sensitivity results confirmed that primers were highly specific to their targets and able to detect viruses up to 10-2 dilutions of RNA in standard and duplex RT-PCR. Therefore, the developed rapid duplex RT-PCR can be used for virus indexing and production of virus-free Kinnow mandarin plants for certification programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04011-9.

A Brief Update on Inter-lab Quality Control (ILQC) Activities for COVID-19 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) by All India Institute of Medical Sciences (AIIMS) Nagpur, India.

Inter-lab quality control (ILQC) is vital for ensuring reliable test results, especially when laboratories are using assays authorized for newly emerging pathogens. The Indian Council of Medical Research (ICMR), New Delhi, had developed a network of laboratories to assess the quality of real-time reverse transcription (RT) polymerase chain reaction (PCR) assays used in India to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In a three-tier ILQC lab structure, All India Institute of Medical Sciences (AIIMS) Nagpur, an institute of national importance & a tertiary care hospital, was designated as a state quality control (QC) lab for the region of Maharashtra. ILQC activities were planned biannually. The ICMR had assigned 22 government and 19 private SARS-CoV-2 RT-PCR testing laboratories, under the Department of Microbiology, AIIMS Nagpur. AIIMS Nagpur had conducted four ILQC activities during 2020-2021. The finding of the ILQC assessment (cumulative includes all four ILQC) conducted by AIIMS Nagpur revealed that the results of 77% of laboratories were 100% concordant, the results of 14% of laboratories were 90%, and very few laboratories (i.e. 9%) showed <90% concordant.

The Role of Circulating MicroRNAs in the Prediction of Response to Neoadjuvant Chemotherapy in Locally Advanced Breast Cancer in the Indian Population.

INTRODUCTION: MicroRNAs (miRNAs) are known to play an important role in cancer cell proliferation, susceptibility of cancer cells to chemotherapy, and patient survival. Identifying miRNAs that can predict response to chemotherapy in locally advanced breast cancer (LABC), the most common variant, can help to choose appropriate drug regimens to suit the epigenetic profile of individual patients. OBJECTIVE: To investigate the expression of the differentially expressed miRNAs identified by next-generation sequencing from a pilot study involving cases and controls, in peripheral blood mononuclear cells (PBMC) of patients with LABC during the course of neoadjuvant chemotherapy (NAC) and determine their role in response to chemotherapy. METHODS: This study included 30 newly diagnosed LABC patients. Peripheral blood from every participant was collected before the start of chemotherapy, at the end of the third cycle, and at the end of the seventh cycle of NAC. Based on the results of a pilot study in a similar population with suitable controls, four differentially expressed miRNAs namely miR-24-2, miR-192-5p, miR-3609, and miR-664b-3p were considered to be validated in this study. The expression of these four miRNAs was examined by qRT-PCR, and their association with response to chemotherapy was analyzed. RESULT:  A significant change in the expression of miR-192-5p was found in responders (p = 0.001) over a period of seven cycles and the difference between the expression of miR-24-2 from baseline to the seventh cycle of NAC was higher in responders while compared to the non-responders (p < 0.05). CONCLUSION: miR-192-5p and miR-24-2 were identified as predictive biomarkers for response to NAC in south Indian patients with LABC.

Dataset of acute respiratory infections: Epidemiological and etiological data from a cohort of 801 Moroccan children.

Acute respiratory infections remain a leading cause of death among young children in low- and middle-income countries. The etiological diagnosis of these infections is challenging due to the similarity in clinical presentations and overlapping symptoms caused by various pathogens. This database provides comprehensive epidemiological, clinical, paraclinical, and biological data on 801 Moroccan children admitted to the Children's Hospital of Rabat for the management of Clinical Severe Pneumonia. Identification of the pathogens responsible of respiratory infections was carried out using blood samples for hemoculture, standard bacterial culture and multiplex RT-PCR using the TrueScience RespiFinder Pathogen Identification Panel (Applied Biosystems).

Molecular characterization of influenza virus circulating in Nepal in the year 2019.

Influenza (sometimes referred to as "flu") is a contagious viral infection of the airways in the lungs that affects a significant portion of the world's population. Clinical symptoms of influenza virus infections can range widely, from severe pneumonia to moderate or even asymptomatic sickness. If left untreated, influenza can have more severe effects on the heart, brain, and lungs than on the respiratory tract and can necessitate hospitalization. This study was aimed to investigate and characterize all types of influenza cases prevailing in Nepal and to analyze seasonal occurrence of Influenza in Nepal in the year 2019. A cross sectional, retrospective and descriptive study was carried out at National Influenza Center (NIC), National Public Health Laboratory Kathmandu Nepal for the period of one year (Jan-Dec 2019). A total of 3606 throat swab samples from various age groups and sexes were processed at the NIC. The specimens were primarily stored at 4 °C and processed using ABI 7500 RT PCR system for the identification of Influenza virus types and subtypes. Data accessed for research purpose were retrieved from National Influenza Centre (NIC) on 1st Jan 2020. Of the total 3606 patients suspected of having influenza infection, influenza viruses were isolated from 1213 (33.6%) patients with male predominance. The highest number of infection was caused by Influenza A/Pdm09 strain 739 (60.9%) followed by Influenza B 304 (25.1%) and Influenza A/H3 169 (13.9%) and most remarkable finding of this study was the detection of H5N1 in human which is the first ever case of such infection in human from Nepal. Similar to other tropical nations, influenza viruses were detected year-round in various geographical locations of Nepal. The influenza virus type and subtypes that were in circulation in Nepal were comparable to vaccine candidate viruses, which the currently available influenza vaccine may prevent.

Isolation of Human Progenitor Epidermal Cells on Collagen Type IV and Analysis of Their Markers with Flow Cytometry and PCR Methods.

Epidermal stem cells, located in the skin, together with keratinocytes are transplanted in regenerative therapies, e.g., for the treatment of burns or other wounds. Here, we describe the protocol of their enzymatic isolation from human skin. It includes separation of the epidermis form the dermis by incubation with dispase followed by cell isolation for epidermis by digestion with trypsin. Cell isolated with this method can be seeded on collagen IV-coated dishes. The methods of analysis of epidermal stem cells markers (e.g., CD71, CD29) with flow cytometry and RT-PCR are also included.

Investigating the Clinico-Demographic Characteristics of Dengue Fever and Its Seroprevalence at a Tertiary Care Hospital in Northern India.

Background and objective Dengue virus (DENV) is a major global health threat, causing over 50,000 deaths annually. The state of Uttar Pradesh (UP) in India faces significant challenges due to the increasing number of dengue cases detected. This study aimed to assess DENV seropositivity in the Raebareli district of UP, to offer crucial insights into the region's effective control and management strategies. Materials and methods This study, after obtaining approval from the ethics committee, analyzed blood samples of individuals suspected of having dengue at a teaching hospital in rural UP between January and December 2022. To determine the disease's seroprevalence, both dengue NS1 antigen ELISA and dengue IgM Microlisa were conducted. Furthermore, RT-PCR was performed on NS1-positive samples to confirm the serotypes. The collected data were analyzed using Epi Info 7.0. Results Of the 589 suspected dengue cases, 86 (14.60%) tested positive for dengue NS1 and/or IgM. Our findings showed that males (n=330, 56.03%) and adolescents and young adults (n=301, 51.1%) from rural areas (n=523, 88.4%) were predominantly affected. Cases peaked post-monsoon, and platelet levels were notably low in NS1-positive cases. Dengue serotype 2 (DEN-2) was found in all RT-PCR-positive samples. Our results revealed a dengue seroprevalence of 14.60% (n=86), which peaked in post-monsoon months. The higher incidence among males and young adults from rural areas attending the outpatient department highlights the importance of targeted interventions and community surveillance. RT-PCR confirmed the circulation of a single serotype in the region. Conclusions This study contributes crucial insights into dengue's epidemiology and clinical profile and its findings are all the more significant now as India prepares for phase 3 trials of a quadrivalent dengue-virus vaccine in 2024. Adolescent and young adult males have an increased likelihood of acquiring the virus, and this demographic can be prioritized for vaccine trials.

Broad-spectrum dengue virus detection using the commercial RealStar dengue RT-PCR kit 3.0 (Altona) and an in-house combined real-time RT-PCR assay.

In endemic areas, the genetic diversity among co-circulating dengue virus (DENV) strains is considerable and new, highly divergent strains are identified on a regular basis. It is thus critical to ensure that molecular diagnostic tools effectively detect virus genomes even in case of important genetic variation. Here, we tested both the pan-DENV detection capacity and the limit of detection of two real-time RT-PCR assays: (i) the commercial RealStar Altona 3.0 system and (ii) a laboratory developed test (LDT) combining two RT-PCR systems in a single reaction tube (DenAllDUO). We used a panel of DENV strains representative of the genetic diversity within DENV species, combined with three in vitro transcribed RNAs as surrogates for unavailable strains corresponding to recently discovered strains with substantial genetic divergence: DENV serotype 1 (DENV-1) Brun2014, DENV-2 QML22 and DENV-4 DKE121. Both systems (i) targeted the genome 3' untranslated region, (ii) displayed a broad detection spectrum, encompassing most of DENV species diversity, and (iii) detected the three aforementioned divergent strains. DenAllDUO detected all the strains tested, whereas the RealStar system failed to detect strains from DENV-4 genotype III. Altogether, our findings support the value of these two RT-PCR systems as part of the Dengue diagnostic arsenal.

Original article: novelty of Canadian manufacture nasopharyngeal swabs for collection of samples being tested for SARS-CoV-2 in a pandemic setting.

Objectives: The COVID-19 pandemic caused a global shortage of nasopharyngeal (NP) swabs, required for RT-PCR testing. Canadian manufacturers were contacted to share NP swab innovations. The primary objective was to determine whether novel NP test swabs were comparable to commercially available swabs regarding user characteristics, ability to collect a specimen, and diagnostic performance using RT-PCR testing. Methods: Participants were randomized by swab (test/control) and nostril (left/right). A calculated positive percent agreement ≥90% was considered successful. Mean Ct values of viral genes and housekeeping gene (RNase P) were considered similar if a Ct difference ≤ 2 between control and test group was obtained. There also was a qualitative assessment of swabs usability. Results: 647 participants were enrolled from Huaycan Hospital in Lima, Peru, distributed over 8 NP swabs brands. Seven brands agreed to share their results. There were no statistically significant differences between the test swabs of these 7 brands and control swabs. Conclusion: All the seven brands are comparable to the commercially available flocked swabs used for SARS-CoV-2 regarding test results agreement, ability to collect a specimen, and user characteristics.

Molecular Detection and Serological Investigation of Newcastle Disease in Intensive, Semi-Intensive, and Backyard Production Systems in Central and Southwestern Areas of Ethiopia.

Purpose: The purpose of this research is to detect Newcastle disease virus and to assess the seropositivity among backyard, semi-intensive, and intensive farms located in central and southwestern areas of Ethiopia. Material and Methods: A total of 239 oropharyngeal and cloacal swab samples were collected from symptomatic birds found in Holeta, Burayu, Jimma towns as well as Seka Chekorsa and Nadhigibe woredas of Jimma Zone. In addition, ninety blood samples were collected from wing veins of unvaccinated birds found in the study areas of Jimma zone. Side-by-side information related to risk factors estimated to contribute to the susceptibility of the disease was collected by interviewing owners of sampled birds. Reverse transcription polymerase-chain reaction (RT-PCR) was conducted to detect NDV. Likewise, Enzyme-linked immunosorbent assay (ELISA) was performed to determine the seropositivity of ND. Results: The proportion of samples where NDV was detected was 24.6%. Similarly, 68.9% of the sampled birds were seropositive. It was observed that adult birds were more likely to encounter the disease than youngs (OR = 11.6; 95% CI: 4.0-33.3; P = 0.000). Birds owned by respondents who leave diseased birds in the flock were more likely infected (OR = 6.2; 95% CI: 1.8-21.2; P=0.004) as compared to those isolated and mode of disposal of dead chicken significantly affect exposure (OR = 0.13; 95% CI: 0.10-4.88; P = 0.044). Likewise, access to veterinary services highly likely reduces susceptibility to the disease (OR = 12.4; 95% CI: 3.2-46.9; P = 0.000). It was also found that birds farmed intensively were the most at risk (OR = 2.8; 95% CI: 0.58-13.71; P = 0.199). Conclusion: Detection of ND from a significant proportion of sampled birds and their high seropositivity percentage revealed the circulation of the virus in the study areas.

Analysis of VEGF, IGF1/2 and the Long Noncoding RNA (lncRNA) H19 Expression in Polish Women with Endometriosis.

The coordinated action of VEGF, IGF1/2 and H19 factors influences the development of endometriosis. The aim of this study was to analyze the expression level of these genes in patients with endometriosis. The study group consisted of 100 patients who were diagnosed with endometriosis on laparoscopic and pathological examination. The control group consisted of 100 patients who were found to be free of endometriosis during the surgical procedure and whose eutopic endometrium wasnormal on histopathological examination. These patients were operated on for uterine fibroids. Gene expression was determined by RT-PCR. The expression of the VEGF gene was significantly higher in the samples classified as clinical stage 1-2 compared to the control material (p < 0.05). There was also a statistically significant difference between the samples studied at clinical stages 1-2 and 3-4 (p < 0.01). The expression of the VEGF gene in the group classified as 1-2 was significantly higher. IGF1 gene expression was significantly lower both in the group of samples classified as clinical stages 1-2 and 3-4 compared to the control group (p < 0.05 in both cases). The expression of the H19 gene was significantly lower in the group of samples classified as clinical stage 3-4 compared to the control group (p < 0.01). The reported studies suggest significant roles of VEGF, IGF and H19 expression in the pathogenesis of endometriosis.

Reliability of the rapid antigen test for diagnosis of SARS-CoV-2 in Tunisia.

Background: Early and accurate diagnosis is crucial for preventing the spread of SARS-CoV-2 infection. The rapid antigen test was developed for testing infection, and it was necessary to assess its performance before widespread use in Tunisia. Aim: To evaluate the effectiveness of a rapid antigen test for the detection of SARS-CoV-2 in nasopharyngeal swabs in Tunisia. Methods: Nasopharyngeal samples were taken from COVID-19 suspected cases between October and December 2020 and tested using the Standard Q COVID-19 Ag test (SD-Biosensor, Republic of Korea) and real-time reverse transcription polymerase chain reaction (RT­PCR). Results: Overall, 4539 patients were tested. Of the total study population (N = 4539), 82.5% of positive samples remained positive with the rapid antigen test, while 20.2% (470/2321) of samples that were negative with rapid antigen test were confirmed positive with RT-PCR, giving a negative predictive value of 79.8% for the rapid antigen test. The sensitivity and negative predictive value of the rapid antigen test were 70.2% and 65.8%, respectively. These results improved to 96.4% and 92.8%, respectively, when considering the cycle threshold value by RT-PCR below 25. Conclusion: Although the rapid antigen test was less sensitive than RT-PCR, its ability to rapidly detect individuals with high viral loads makes it suitable for use during an epidemic.

Analytical and clinical validation of a novel, laboratory-developed, modular multiplex-PCR panel for fully automated high-throughput detection of 16 respiratory viruses.

BACKGROUND: Viral respiratory Infections pose a health risk, especially to vulnerable patient populations. Effective testing programs can detect and differentiate these infections at an early stage, which is particularly important for high-risk clinical departments. The objective of this study was to develop and validate a multiplex PCR-panel for 16 different respiratory viruses on a fully-automated high-throughput platform. METHODS: Three multiplex-PCR assays were designed to run on the cobas5800/6800/8800 systems, consolidating 16 viral targets: RESP1: SARS-CoV-2, influenza-A/B, RSV; RESP2: hMPV, hBoV, hAdV, rhino-/ENV; RESP3: HPIV-1-4, hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1. Analytic performance was evaluated using digital-PCR based standards and international reference material. Clinical performance was determined by comparing results from clinical samples with reference assays. RESULTS: Analytical sensitivity (i.e. lower limit of detection (LoD), 95 % probability of detection) was determined as follows: SARS-CoV-2: 29.3 IU/ml, influenza-A: 179.9 cp/ml, influenza-B: 333.9 cp/ml and RSV: 283.1 cp/ml. LoDs of other pathogens ranged between 9.4 cp/ml (hCoV-NL63) and 21,419 cp/ml (HPIV-2). Linearity was verified over 4-7 log-steps with pooled standard differentials (SD) ranging between 0.18-0.70ct. Inter-/intra-run variability (precision) was assessed for all targets over 3 days. SDs ranged between 0.13-0.74ct. Positive agreement in clinical samples was 99.4 % and 95 % for SARS-CoV-2 and influenza-A respectively. Other targets were in the 80-100 % range. Negative agreement varied between 96.3-100 %. DISCUSSION: Lab-developed tests are a key factor for effective clinical diagnostics. The multiplex panel presented in this study demonstrated high performance and provides an easily scalable high-throughput solution for respiratory virus testing, e.g. for testing in high-risk patient populations.

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.

A comprehensive meta-analysis and systematic review of breath analysis in detection of COVID-19 through Volatile organic compounds.

BACKGROUND: The COVID-19 pandemic had profound global impacts on daily lives, economic stability, and healthcare systems. Diagnosis of COVID-19 infection via RT-PCR was crucial in reducing spread of disease and informing treatment management. While RT-PCR is a key diagnostic test, there is room for improvement in the development of diagnostic criteria. Identification of volatile organic compounds (VOCs) in exhaled breath provides a fast, reliable, and economically favorable alternative for disease detection. METHODS: This meta-analysis analyzed the diagnostic performance of VOC-based breath analysis in detection of COVID-19 infection. A systematic review of twenty-nine papers using the grading criteria from Newcastle-Ottawa Scale (NOS) and PRISMA guidelines was conducted. RESULTS: The cumulative results showed a sensitivity of 0.92 (95 % CI, 90 %-95 %) and a specificity of 0.90 (95 % CI 87 %-93 %). Subgroup analysis by variant demonstrated strong sensitivity to the original strain compared to the Omicron and Delta variant in detection of SARS-CoV-2 infection. An additional subgroup analysis of detection methods showed eNose technology had the highest sensitivity when compared to GC-MS, GC-IMS, and high sensitivity-MS. CONCLUSION: Overall, these results support the use of breath analysis as a new detection method of COVID-19 infection.