Can Water-Only DNA Extraction Reduce the Logistical Footprint of Biosurveillance and Planetary Health Diagnostics? Toward a New Method.

The coronavirus disease-2019 (COVID-19) pandemic has raised the stakes for planetary health diagnostics. Because pandemics pose enormous burdens on biosurveillance and diagnostics, reduction of the logistical burdens of pandemics and ecological crises is essential. Moreover, the disruptive effects of catastrophic bioevents impact the supply chains in both highly populated urban centers and rural communities. One "upstream" focus of methodological innovation in biosurveillance is the footprint of Nucleic Acid Amplification Test (NAAT)-based assays. We report in this study a water-only DNA extraction, as an initial step in developing future protocols that may require few expendables, and with low environmental footprints, in terms of wet and solid laboratory waste. In the present work, boiling-hot distilled water was used as the main cell lysis agent for direct polymerase chain reactions (PCRs) on crude extracts. After evaluation (1) in blood and mouth swabs for human biomarker genotyping, and (2) in mouth swabs and plant tissue for generic bacterial or fungal detection, and using different combinations of extraction volume, mechanical assistance, and extract dilution, we found the method to be applicable in low-complexity samples, but not in high-complexity ones such as blood and plant tissue. In conclusion, this study examined the doability of a lean approach for template extraction in the case of NAAT-based diagnostics. Testing our approach with different biosamples, PCR settings, and instruments, including portable ones for COVID-19 or dispersed applications, warrant further research. Minimal resources analysis is a concept and practice, vital and timely for biosurveillance, integrative biology, and planetary health in the 21st century.

Cellular and Humoral Responses in Dialysis Patients after Vaccination with the BNT162b2 or mRNA-1273 Vaccines.

The outbreak of SARS-CoV-2 has raised considerable concern about the detrimental effects it can induce in public health, with the interest of the scientific community being focused on the development of preventive and therapeutic approaches. Patients with end-stage renal disease (ESRD) are amongst vulnerable populations for critical illness owing to the presence of other comorbidities, their defective immune system, and their inability of self-isolation. To date, vaccination constitutes the most promising method to manage viral dispersion. Therefore, it is particularly important to investigate the effectiveness of available vaccines against SARS-CoV-2 in this risk group. Here, we summarize initial experience regarding the humoral and cellular immune responses elicited in dialysis patients after completion of the recommended vaccination regimen, as well as after booster dose administration, with one of the two mRNA vaccines, namely, BNT162b2 and mRNA-1273. In conclusion, a significantly diminished and delayed immune pattern was observed in ESRD patients compared to healthy population, with a peak in antibody titers occurring 3-5 weeks after the second dose. A booster dose significantly augmented the immune response in dialysis patients with either mRNA-based vaccine. Variables adversely correlating with the weak immunogenicity observed in dialysis patients include immunosuppressive therapy, older age, comorbidities, longer time in hemodialysis treatment, and higher body mass index. On the contrary, previous COVID-19 infection and administration of the mRNA-1273 vaccine are deemed to induce a more favorable immune response. Further investigation is needed to thoroughly understand the efficacy of mRNA-based vaccines in hemodialysis patients and define predictive factors that can influence it.

Clinical Performance Evaluation of the NeuMoDx Flu A-B/RSV/SARS-CoV-2 Vantage Assay.

SARS-CoV-2 infections may present with various symptoms that are similar to those of other respiratory diseases. For this reason, the need for simultaneous detection of at least RSV and influenza viruses together with SARS-CoV-2 was evident from the early stages of the pandemic. In the present study, we evaluated the clinical performance of the NeuMoDx™ Flu A-B/RSV/SARS-CoV-2 Vantage Assay against the conventional low-plex PCR utilized to detect influenza A-B, RSV, and SARS-CoV-2. There were 115 known positive clinical samples and 35 negative controls obtained from asymptomatic health-care workers included in the study; 25 samples were positive for influenza viruses, 46 for RSV, and 44 for SARS-CoV-2. The sensitivity, specificity, positive predictive value, and negative predictive value of the evaluated method for influenza and SARS-CoV-2 were 100%. The Spearman correlation coefficient was 0.586 (p < 0.05) for influenza and 0.893 (p < 0.05) for SARS-CoV-2. The sensitivity of the aforementioned assay for RSV was 93.47%; the specificity and the positive predictive value were 100%, and the negative predictive value was 92.10%, while the Spearman correlation coefficient was not applicable for the RSV. Overall, the assay under evaluation was shown to be a reliable alternative for the simultaneous detection of influenza viruses, RSV and SARS-CoV-2.

Prospective evaluation of specimen pooling strategy for detection of SARS-CoV-2 using pools of five and six specimens.

The increased demand for SARS-CoV-2 molecular testing during the COVID-19 pandemic resulted in shortage of reagents and consumables. Pooling of specimens could be an alternative strategy to overcome these problems. Initial evaluation of the pooling strategy was performed using known positive specimens, previously tested individually, and their respective pools of plus four (5X), five (6X) and nine (10X) known negative specimens. Subsequently, 35 positive 5X and 35 positive 6X pools containing only one positive specimen per pool were analyzed prospectively regarding the difference in Ct values in pooled versus individual specimens. When the number of samples in the pool were five or six, the average deviation of Ct differences was < 1; therefore, this strategy was followed in the prospective study. Significant difference in Ct values was observed in positive specimens when tested individually and in 5X pools (p = 0.006), while the difference was not significant when positive specimens were tested individually and in 6X pools (p = 0.07). The difference in Ct values was not significant between the 5X and 6X pools. Testing in pools of five or six specimens is a reliable option for SARS-CoV-2 RNA detection when mass testing is needed.

Fast, Scalable, and Practical: An Alkaline DNA Extraction Pipeline for Emergency Microbiomics Biosurveillance.

Pandemics and environmental crises evident from the first two decades of the 21st century call for methods innovation in biosurveillance and early detection of risk signals in planetary ecosystems. In crises conditions, conventional methods in public health, biosecurity, and environmental surveillance do not work well. In addition, the standard laboratory amenities and procedures may become unavailable, irrelevant, or simply not feasible, for example, owing to disruptions in logistics and process supply chains. The COVID-19 pandemic has been a wakeup call in this sense to reintroduce point-of-need diagnostics with an eye to limited resource settings and biosurveillance solutions. We report here a methodology innovation, a fast, scalable, and alkaline DNA extraction pipeline for emergency microbiomics biosurveillance. We believe that the presented methodology is well poised for effective, resilient, and anticipatory responses to future pandemics and ecological crises while contributing to microbiome science and point-of-need diagnostics in nonelective emergency contexts. The alkaline DNA extraction pipeline can usefully expand the throughput in emergencies by deployment or to allow backup in case of instrumentation failure in vital facilities. The need for distributed public health genomics surveillance is increasingly evident in the 21st century. This study makes a contribution to these ends broadly, and for future pandemic preparedness in particular. We call for innovation in biosurveillance methods that remain important existentially on a planet under pressure from unchecked human growth and breach of the boundaries between human and nonhuman animal habitats.

Transmission dynamics of SARS-CoV-2 within families with children in Greece: A study of 23 clusters.

There is limited information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection clustering within families with children. We aimed to study the transmission dynamics of SARS-CoV-2 within families with children in Greece. We studied 23 family clusters of coronavirus disease 2019 (COVID-19). Infection was diagnosed by reverse-transcriptase polymerase chain reaction in respiratory specimens. The level of viral load was categorized as high, moderate, or low based on the cycle threshold values. There were 109 household members (66 adults and 43 children). The median attack rate per cluster was 60% (range: 33.4%-100%). An adult member with COVID-19 was the first case in 21 (91.3%) clusters. Transmission of infection occurred from an adult to a child in 19 clusters and/or from an adult to another adult in 12 clusters. There was no evidence of child-to-adult or child-to-child transmission. In total 68 household members (62.4%) tested positive. Children were more likely to have an asymptomatic SARS-CoV-2 infection compared to adults (40% vs 10.5%; P = .021). In contrast, adults were more likely to develop a severe clinical course compared with children (8.8% vs 0%; P = .021). In addition, infected children were significantly more likely to have a low viral load while adults were more likely to have a moderate viral load (40.7% and 18.6% vs 13.8% and 51.7%, respectively; P = .016). In conclusion, while children become infected by SARS-CoV-2, they do not appear to transmit infection to others. Furthermore, children more frequently have an asymptomatic or mild course compared to adults. Further studies are needed to elucidate the role of viral load on these findings.

Association Between Upper Respiratory Tract Viral Load, Comorbidities, Disease Severity, and Outcome of Patients With SARS-CoV-2 Infection.

BACKGROUND: There is limited information on the association between upper respiratory tract (URT) viral loads, host factors, and disease severity in SARS-CoV-2-infected patients. METHODS: We studied 1122 patients (mean age, 46 years) diagnosed by polymerase chain reaction (PCR). URT viral load, measured by PCR cycle threshold, was categorized as high, moderate, or low. RESULTS: There were 336 (29.9%) patients with comorbidities; 309 patients (27.5%) had high, 316 (28.2%) moderate, and 497 (44.3%) low viral load. In univariate analyses, compared to patients with moderate or low viral load, patients with high viral load were older, more often had comorbidities, developed Symptomatic disease (COVID-19), were intubated, and died. Patients with high viral load had longer stay in intensive care unit and longer intubation compared to patients with low viral load (P values < .05 for all comparisons). Patients with chronic cardiovascular disease, hypertension, chronic pulmonary disease, immunosuppression, obesity, and chronic neurological disease more often had high viral load (P value < .05 for all comparisons). In multivariate analysis high viral load was associated with COVID-19. Level of viral load was not associated with any other outcome. CONCLUSIONS: URT viral load could be used to identify patients at higher risk for morbidity or severe outcome.

Repeated leftover serosurvey of SARS-CoV-2 IgG antibodies, Greece, March and April 2020.

A serosurvey of IgG antibodies against severe acute respiratory coronavirus 2 (SARS-CoV-2) was performed during March and April 2020. Among 6,586 leftover sera, 24 (0.36%) were positive, with higher prevalence in females, older individuals and residents of large urban areas. Seroprevalence was estimated at 0.02% and 0.25%, respectively, in March and April, infection fatality rate at 2.66% and 0.54%. Our findings confirm low COVID-19 incidence in Greece and possibly the effectiveness of early measures.

Bloodstream Infection by Saccharomyces cerevisiae in Two COVID-19 Patients after Receiving Supplementation of Saccharomyces in the ICU.

Co-infections have an unknown impact on the morbidity and mortality of the new clinical syndrome called coronavirus disease 2019 (COVID-19). The syndrome is caused by the new pandemic coronavirus SARS-CoV-2 and it is probably connected with severe traces in the elements of the immune system. Apart from possible Aspergillus infections, particularly in patients with acute respiratory distress syndrome (ARDS), other fungal infections could occur, probably more easily, due to the immunological dysregulation and the critical condition of these patients. Probiotic preparations of Saccharomyces are broadly used for the prevention of antibiotic-associated complications, especially in the intensive care units (ICU). On the other hand, Saccharomyces organisms are reported as agents of invasive infection in immunocompromised or critically ill patients. We report two cases of bloodstream infection by Saccharomyces in two patients hospitalised in the ICU, due to severe COVID-19, after Saccharomyces supplementation.