Efficient Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from Exhaled Breath.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is transmitted through airborne particles in exhaled breath, causing severe respiratory disease, coronavirus disease-2019 (COVID-19), in some patients. Samples for SARS-CoV-2 testing are typically collected by nasopharyngeal swab, with the virus detected by PCR; however, patients can test positive for 3 months after infection. Without the capacity to assay SARS-CoV-2 in breath, it is not possible to understand the risk for transmission from infected individuals. To detect virus in breath, the Bubbler-a breathalyzer that reverse-transcribes RNA from SARS-CoV-2 particles into a sample-specific barcoded cDNA-was developed. In a study of 70 hospitalized patients, the Bubbler was both more predictive of lower respiratory tract involvement (abnormal chest X-ray) and less invasive than alternatives. Samples tested using the Bubbler were threefold more enriched for SARS-CoV-2 RNA than were samples from tongue swabs, implying that virus particles were being directly sampled. The barcode-enabled Bubbler was used for simultaneous diagnosis in large batches of pooled samples at a lower limit of detection of 334 genomic copies per sample. Diagnosis by sequencing can provide additional information, such as viral load and strain identity. The Bubbler was configured to sample nucleic acids in water droplets circulating in air, demonstrating its potential in environmental monitoring and the protective effect of adequate ventilation.

Cytologic findings in effusions from patients with SARS-CoV-2 infection.

INTRODUCTION: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with "flu-like" upper respiratory tract symptoms and pneumonia. Body cavity effusions develop in a subset of patients with advanced disease. Although SARS-CoV-2 is known to be present in certain body fluids (eg, blood) of COVID patients, it remains unclear if body cavity fluids are sites of infection. Our aim was to characterize the cytologic and clinical findings in COVID-19 patients with effusions. MATERIALS AND METHODS: A record search for all cases of body cavity effusion cytology in SARS-CoV-2 positive patients from March 1, 2020, to September 1, 2020, was performed. Clinical history, fluid chemical analysis, cytologic findings, and patient outcomes were recorded. All cytology slides were reviewed. In situ hybridization (ISH) targeting SARS-CoV-2 spike protein transcript (V-nCoV2019-S) was performed on cell block material in all cases. RESULTS: A total of 17 effusion cytology cases were identified among 15 COVID patients, including 13 pleural, 2 pericardial, and 2 peritoneal. Most (13 of 15) patients were hospitalized for COVID complications. Eight patients died during hospitalization, 7 from COVID complications. All fluids were transudative by protein criteria. Lymphocytic or histiocytic inflammation predominated in 12 of 17 cases. Five exhibited hemophagocytosis. No viral cytopathic changes or extra-medullary megakaryocytes were seen. Viral RNA was not detected in any case by ISH. CONCLUSIONS: Body cavity effusion is an ominous finding in patients with advanced COVID-19 disease. Such effusions tend to be transudative with lymphohistiocytic inflammation, and commonly exhibit hemophagocytosis, an otherwise rare finding in effusion cytologies. No direct infection of cellular elements by SARS-CoV-2 was identified by ISH.

A cohort study of men infected with COVID-19 for presence of SARS-CoV-2 virus in their semen.

INTRODUCTION: Whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected in semen and transmitted sexually is a vital question that has, thus far, been inconclusive. Prior studies, with limited numbers, have included men in various stages of infection with most in the recovery phase of the illness. The timing of test results and severity of illness has made recruiting study participants a significant challenge. Our pilot study will examine semen from men with a recent diagnosis of COVID-19 as well as those in the convalescent phase to determine if SARS-CoV-2 can be detected and its relationship, if any, with the severity of the disease. METHODS: Eighteen men with a median age of 32 (range, 24-57) who tested positive for COVID-19 by rt-PCR analysis were enrolled and provided a semen sample. The study group demonstrated symptoms of COVID-19 ranging from asymptomatic to moderate and none required hospitalization. Samples were subjected to viral RNA extraction and then processed by real-time RT-PCR using the US Centers for Disease Control and Prevention (CDC, USA) panel of 2019-Novel Coronavirus (2019-nCoV) primers and probes to detect the presence of SARS-CoV-2 RNA. RESULTS: Length of time from diagnosis to providing a specimen ranged from 1 to 28 days (median, 6 days). Fifteen participants were symptomatic and three were asymptomatic, including recovering men, at the time of semen collection. No SARS-CoV-2 was detected in any of the semen samples. CONCLUSION: Based on these preliminary results and consistent with prior findings, we suggest SARS-CoV-2 is not present in semen during the acute or convalescent phase of COVID-19.

A review on human body fluids for the diagnosis of viral infections: scope for rapid detection of COVID-19.

Introduction: The unprecedented outbreaks of corona virus disease of 2019 (COVID-19) have highlighted the necessity of readily available, reliable, precise, and faster techniques for its detection. Nasopharyngeal swab has been the gold standard for the diagnosis of COVID-19. However, it is not an ideal screening procedure for massive screening as it implicates the patient's stay in the hospital or at home until diagnosis, thus causing crowding of the specimen at the diagnostic centers. Present study deal with the exploration of potential application of different body fluids using certain highly objective techniques (Optical and e-Nose) for faster detection of molecular markers thereby diagnosing viral infections.Areas covered: This report presents an evaluation of different body fluids, and their advantages for the rapid detection of COVID-19, coupled with highly sensitive optical techniques for the detection of molecular biomarkers.Expert opinion: Tears, saliva, and breath samples can provide valuable information about viral infections. Our brief review strongly recommends the application of saliva/tears and exhaled breath as clinical samples using technics such as high-performance liquid chromatography-laser-induced fluorescence, photoacoustic spectroscopy, and e-Nose, respectively, for the fast diagnosis of viral infections.

Extended Lifetime of Respiratory Droplets in a Turbulent Vapor Puff and Its Implications on Airborne Disease Transmission.

To quantify the fate of respiratory droplets under different ambient relative humidities, direct numerical simulations of a typical respiratory event are performed. We found that, because small droplets (with initial diameter of 10 µm) are swept by turbulent eddies in the expelled humid puff, their lifetime gets extended by a factor of more than 30 times as compared to what is suggested by the classical picture by Wells, for 50% relative humidity. With increasing ambient relative humidity the extension of the lifetimes of the small droplets further increases and goes up to around 150 times for 90% relative humidity, implying more than 2 m advection range of the respiratory droplets within 1 sec. Employing Lagrangian statistics, we demonstrate that the turbulent humid respiratory puff engulfs the small droplets, leading to many orders of magnitude increase in their lifetimes, implying that they can be transported much further during the respiratory events than the large ones. Our findings provide the starting points for larger parameter studies and may be instructive for developing strategies on optimizing ventilation and indoor humidity control. Such strategies are key in mitigating the COVID-19 pandemic in the present autumn and upcoming winter.

Adolescents' and caregivers' perceptions of caregiver-provided testing and HIV self-testing using oral mucosal transudate tests in Zimbabwe: a short report.

Uptake of HIV testing remains lower among children and adolescents compared to adults. This study explored adolescents' perceptions of HIV self-testing (HIVST) and caregivers' perceptions of testing their children using an oral mucosal transudate (OMT) rapid HIV test (caregiver-provided testing). We conducted 31 interviews with adolescents aged 16-18 years and caregivers of children aged 2-15 years who received an OMT test. Participants described barriers to HIV testing including lack of privacy and the potential for discrimination by community members towards children and adolescents who received an HIV test. Most participants felt caregiver-provided testing and HIVST could address these barriers through increased privacy. Some participants expressed worry about their ability to correctly perform the OMT and their anxious reactions to a positive result. Counseling and assistance from health care workers were viewed as ways to alleviate concerns. Concerns shaped participants' preferences for facility-based HIVST and caregiver-provided testing. Findings demonstrate HIVST performed by adolescents and caregiver-provided testing could increase the uptake of HIV testing. Concerns related to being able to test correctly and the availability of post-test counseling must be addressed in any future delivery mechanisms.

Inflammatory Mediator Profiles in Secretory Otitis Media in Relationship to Viable Bacterial Pathogens and Bacterial and Viral Nucleic Acids.

Secretory otitis media (SOM) is characterized by persistence of fluid in the middle ear, often following an episode of acute otitis media. Our hypothesis is that failure to eliminate bacterial or viral pathogens may result in persistent low-grade inflammation. In this study, we analyzed inflammatory mediators in middle ear fluids from 67 children with SOM. This was combined with determinations of viable bacteria by culture along with detection of bacterial and viral genetic material by real-time polymerase chain reaction (PCR). The inflammatory mediators found at the highest concentrations (>30 ng/mL) were stem cell growth factor-ß (median 110 ng/mL), CXCL1, IL-16, IL-8, migration inhibitory factor, CXCL10, and CXCL9. Among bacterial pathogens, Moraxella catarrhalis and Haemophilus influenzae dominated, regardless of detection methods, while rhinovirus dominated among viral pathogens. Middle ear fluid levels of interleukin (IL)-1α, IL-17, IL-1ß, fibroblast growth factor basic, and tumor necrosis factor correlated strongly with presence of bacteria detected either by culture or PCR, while IL-1RA, IL-3, IL-6, IL-8, CCL3, CCL4, and granulocyte-colony stimulating factor correlated significantly with real-time PCR values. CXCL10, CXCL9, CCL2, and TRAIL correlated significantly with viral nucleic acid levels. To conclude, persistence of viral and bacterial pathogens may fuel persistent inflammation in SOM. Bacteria caused a broad inflammatory response, while viruses chiefly elicited the interferon-induced chemokines CXCL9 and CXCL10.

Persistent Detection and Infectious Potential of SARS-CoV-2 Virus in Clinical Specimens from COVID-19 Patients.

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) that emerged in December 2019 as the causative agent of Coronavirus 2019 (COVID-19) and was declared a pandemic by the World Health Organization in March 2020 has several distinctive features, including extensive multiorgan involvement with a robust systemic inflammatory response, significant associated morbidity and mortality, and prolonged persistence of viral RNA in the clinical specimens of infected individuals as detected by Reverse Transcription Polymerase Chain Reaction (RT-PCR) amplification. This review begins with an overview of SARS-CoV-2 morphology and replication and summarizes what is known to date about the detection of the virus in nasal, oropharyngeal, and fecal specimens of patients who have recovered from COVID-19, with a focus on the factors thought to contribute to prolonged detection. This review also provides a discussion on the infective potential of this material from asymptomatic, pre-symptomatic, and convalescing individuals, to include a discussion of the relative persistence and infectious potential of virus in clinical specimens recovered from pediatric COVID-19 patients.

Occupational Exposure to Blood and Body Fluids and Knowledge of HIV Post-Exposure Prophylaxis among Medical and Allied Health Students in Northern Nigeria.

BACKGROUND: Clinical students are at increased risk of exposure to blood-borne pathogens. However, little has been documented about their exposure to blood and body fluids and their knowledge of post-exposure prophylaxis (PEP) in high-HIV burden settings, such as Nigeria. OBJECTIVE: To determine the prevalence and predictors of BBF exposure and knowledge about PEP among medical and allied health students in northern Nigeria. METHODS: In a cross-sectional study, 273 clinical students were asked to complete structured questionnaires. The prevalence of BBF exposure was determined. Binary logistic regression was used to determine the independent predictors of BBF exposure. RESULTS: The majority of the respondents (98.2%) had heard about PEP; 26.0% (n=71) had adequate knowledge about PEP. 76 (27.8%) of the 273 respondents reported accidental exposure to HIV. 230 (84.2%) respondents had positive attitude toward HIV PEP. Of those who had had accidental exposure to HIV (n=76), only 13% (n=10) received PEP. The level of knowledge about PEP was predicted by previous training (aOR 0.43, 95% CI 0.23 to 0.80 ["no" vs "yes"]), year of training (aOR 4.10, 95% CI 1.60 to 10.47 [6thvs 4th year]), course of study (aOR 4.69, 95% CI 2.06 to 10.68 ["allied health" vs "clinical medicine"]) and religion (aOR 5.39, 95% CI 1.40 to 20.71 ["non-Muslim" vs "Muslim"]). Similarly, accidental exposure was independently predicted by respondents' sex (aOR 2.55, 95% CI1.36 to 4.75 ["female" vs "male"]), age (aOR 2.54, 95% CI 1.06 to 6.15 ["25-29" vs "20-24" years]), ethnicity (aOR 2.15, 95% CI1.10 to 5.14 ["others" vs "Hausa/Fulani"]), course of study (aOR 0.06, 95% CI 0.01 to 0.38 ["allied health" vs "clinical medicine"]), and previous PEP training (aOR 0.39, 95% CI 0.20 to 0.78 ["no" vs "yes"]). CONCLUSION: One in four clinical students reported exposure to BBF. Most students expressed a positive attitude toward PEP, but knowledge and uptake of PEP was sub-optimal. We recommend strengthening training curricula for infection control and prevention and enhancing protocols for timely post-exposure evaluation and follow up for all exposure incidents.

Frequency and Duration of SARS-CoV-2 Shedding in Oral Fluid Samples Assessed by a Modified Commercial Rapid Molecular Assay.

BACKGROUND: RT-PCR on nasopharyngeal (NPS)/oropharyngeal swabs is the gold standard for diagnosis of SARS-CoV-2 infection and viral load monitoring. Oral fluid (OF) is an alternate clinical sample, easy and safer to collect and could be useful for COVID-19 diagnosis, monitoring viral load and shedding. METHODS: Optimal assay conditions and analytical sensitivity were established for the commercial Simplexa™ COVID-19 Direct assay adapted to OF matrix. The assay was used to test 337 OF and NPS specimens collected in parallel from 164 hospitalized patients; 50 bronchoalveolar lavage (BAL) specimens from a subgroup of severe COVID-19 cases were also analysed. RESULTS: Using Simplexa™ COVID-19 Direct on OF matrix, 100% analytical detection down to 1 TCID50/mL (corresponding to 4 × 103 copies (cp)/mL) was observed. No crossreaction with other viruses transmitted through the respiratory toute was observed. Parallel testing of 337 OF and NPS samples showed highly concordant results (κ = 0.831; 95 % CI = 0.771-0.891), and high correlation of Ct values (r = 0.921; p < 0.0001). High concordance and elevated correlation was observed also between OF and BAL. Prolonged viral RNA shedding was observed up to 100 days from symptoms onset (DSO), with 32% and 29% positivity observed in OF and NPS samples, respectively, collected between 60 and 100 DSO. CONCLUSIONS: Simplexa™ COVID-19 Direct assays on OF have high sensitivity and specificity to detect SARS-CoV-2 RNA and provide an alternative to NPS for diagnosis and monitoring SARS-CoV-2 shedding.

Microbiological ascertainment in patients with pneumonia: the experience of a teaching hospital in Rome.

OBJECTIVES: Pneumonia still remains a problem from the clinical and public health viewpoint because of the relevant epidemiological burden. The etiological diagnosis is important in the light of avoiding unnecessary antibiotic treatment and choosing the most appropriate therapeutical approach. This study is aimed at providing evidence on the proportion of microbiological ascertainment in pneumonia-related hospitalizations in one of the most important teaching hospitals in Rome. METHODS: The study relied on the record linkage of two administrative databases of the same hospital: the electronic hospital discharge register and the microbiology laboratory surveillance database. RESULTS: 2819 records were identified, where 46% had a microbiological ascertainment, significantly higher in males than in females (51% vs 40%) and in cases of pneumonia reported in secondary diagnosis instead of primary diagnosis (52% vs 42%). Medical patients had significantly lower proportion of ascertainment compared to surgical patients (43% vs 67%) whereas there were not differences between patients with emergency and elective admission. The overall mortality was 17%. Mortality was significantly higher: in surgical compared to medical patients (27% vs 15%), in ventilated compared to not ventilated patients (41% vs 11%), in cases with secondary diagnosis of pneumonia compared to a primary diagnosis (23% vs 11% ) and in hospitalized in intensive care unit-ICU- rather than in non-ICU (71% vs 12%). CONCLUSION: The proportion of microbiological ascertaiment in pneumonia remains less than 50%. Albeit in line with other evidence, this result should call the attention on the impact of unknown etiological diagnosis on antibiotic treatment and resistance.

Torquetenovirus Titer in Vaginal Secretions from Pregnant and Postpartum Women: Association with Absence of Lactobacillus crispatus and Levels of Lactic Acid and Matrix Metalloproteinase-8.

Vaginal samples from women with term deliveries were tested for torquetenovirus (TTV) by gene amplification, matrix metalloproteinase (MMP)-8 and D- and L-lactic acid by ELISA, and microbiome composition by analysis of the bacterial 16S ribosomal RNA gene. TTV was detected in 43.2%, 31.5%, and 41.4% of first trimester, third trimester, and postpartum samples, respectively. The viral titer was higher in postpartum than in the first (p = 0.0018) or third (p = 0.0013) trimester. The mean gestational age at delivery was lower in women positive for TTV in their first trimester (p = 0.0358). In the first and third trimester, the MMP-8 level was higher if TTV was also present (p < 0.0091). The D-lactic acid level was lower in first trimester samples if TTV was present (p = 0.0334). Lactobacillus crispatus dominance in first and third trimester samples was higher when TTV was absent (p < 0.0033). We conclude that TTV is present in the vagina in many women with normal pregnancy outcomes and that its occurrence is associated with a lack of L. crispatus dominance, an increase in vaginal MMP-8 and a decrease in D-lactic acid.

The kinetics of viral load and antibodies to SARS-CoV-2.

OBJECTIVES: The aim was to understand persistence of the virus in body fluids the and immune response of an infected host to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), an agent of coronavirus disease 2019 (COVID-19). METHODS: We determined the kinetics of viral load in several body fluids through real time reverse transcription polymerase chain reaction, serum antibodies of IgA, IgG and IgM by enzyme-linked immunosorbent assay and neutralizing antibodies by microneutralization assay in 35 COVID-19 cases from two hospitals in Guangdong, China. RESULTS: We found higher viral loads and prolonged shedding of virus RNA in severe cases of COVID-19 in nasopharyngeal (1.3 × 106 vs 6.4 × 104, p < 0.05; 7∼8 weeks) and throat (6.9 × 106 vs 2.9 × 105, p < 0.05; 4∼5 weeks), but similar in sputum samples (5.5 × 106 vs 0.9 × 106, p < 0.05; 4∼5 weeks). Viraemia was rarely detected (2.8%, n = 1/35). We detected early seroconversion of IgA and IgG at the first week after illness onset (day 5, 5.7%, n = 2/35). Neutralizing antibodies were produced in the second week, and observed in all 35 included cases after the third week illness onset. The levels of neutralizing antibodies correlated with IgG (rs = 0.85, p < 0.05; kappa = 0.85) and IgA (rs = 0.64, p < 0.05; kappa = 0.61) in severe, but not mild cases (IgG, rs = 0.42, kappa = 0.33; IgA, rs = 0.32, kappa = 0.22). No correlation with IgM in either severe (rs = 0.17, kappa = 0.06) or mild cases (rs = 0.27, kappa = 0.15) was found. DISCUSSION: We revealed a prolonged shedding of virus RNA in the upper respiratory tract, and evaluated the consistency of production of IgG, IgA, IgM and neutralizing antibodies in COVID-19 cases.

Clinical efficacy of combination therapy of itraconazole and prednisolone for treating effusive feline infectious peritonitis.

A 3-month-old male Scottish Fold kitten with pleural fluid and low ratio of albumin to globulin (A/G ratio) was brought to our small animal hospital. Since RNA from the type I feline coronavirus (FCoV) were detected in drained pleural fluid, the cat was tentatively diagnosed with effusive feline infectious peritonitis (FIP). Following the administration of itraconazole and prednisolone, the A/G ratio increased, and the pleural fluid mostly disappeared. The fecal FCoV levels temporarily decreased. However, the cat showed neurological manifestations and was eventually euthanized due to status epilepticus after 38 days of treatment. In conclusion, itraconazole partly exerted a beneficial effect in a cat with FIP. However, further investigation of a possible role of itraconazole in FIP treatment is warranted.

Reusable respirators as personal protective equipment during ENT surgery.

BACKGROUND: Robust personal protective equipment is essential in preventing the transmission of coronavirus disease 2019 to head and neck surgeons who are routinely involved in aerosol generating procedures. OBJECTIVE: This paper describes the collective experience, across 3 institutes, of using a reusable half-face respirator in 72 head and neck surgery cases. METHOD: Cost analysis was performed to demonstrate the financial implications of using a reusable respirator compared to single-use filtering facepiece code 3 masks. CONCLUSION: The reusable respirator is a cost-effective alternative to disposable filtering facepiece code 3 respirators. Supplying reusable respirators to individual staff members may increase the likelihood of them having appropriate personal protective equipment during their clinical duties.

Development of A4 antibody for detection of neuraminidase I223R/H275Y-associated antiviral multidrug-resistant influenza virus.

The emergence and spread of antiviral drug-resistant viruses have been a worldwide challenge and a great concern for patient care. We report A4 antibody specifically recognizing and binding to the mutant I223R/H275Y neuraminidase and prove the applicability of A4 antibody for direct detection of antiviral multidrug-resistant viruses in various sensing platforms, including naked-eye detection, surface-enhanced Raman scattering-based immunoassay, and lateral flow system. The development of the A4 antibody enables fast, simple, and reliable point-of-care assays of antiviral multidrug-resistant influenza viruses. In addition to current influenza virus infection testing methods that do not provide information on the antiviral drug-resistance of the virus, diagnostic tests for antiviral multidrug-resistant viruses will improve clinical judgment in the treatment of influenza virus infections, avoid the unnecessary prescription of ineffective drugs, and improve current therapies.

Simulation-based training and assessment of mobile pre-hospital SARS-CoV-2 diagnostic teams in Styria, Austria.

The World Health Organization has declared coronavirus disease 2019 (COVID-19) a pandemic. Polymerase chain reaction testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the diagnostic gold standard of COVID-19. We have developed a simulation-based training program for mobile prehospital diagnostic teams in the province of Styria, Austria, and performed a prospective observational study on its applicability and effectivity.The 1-day curriculum uses theoretical instruction, technical skills training, and simulator-based algorithm training to teach and train prehospital patient identification and communication, donning the personal protective equipment, collection of naso-/oropharyngeal swabs for SARS-CoV-2 polymerase chain reaction testing, doffing the personal protective equipment, and sample logistics. Trainings were conducted at the SIM CAMPUS simulation hospital, Eisenerz, using high-fidelity patient simulation. To ensure achievement of predefined learning outcomes, participants had to undergo a final simulator-based objective structured clinical examination.In March 2020, 45 emergency medical assistants and 1 physician of the Austrian Red Cross participated on a voluntary basis. Forty-five of the 46 participants (97.8%) completed the curriculum successfully, with mean objective structured clinical examination ratings of 98.6%.Using several proven educational concepts, we have successfully drafted and implemented a training program for mobile prehospital SARS-CoV-2 diagnostic teams. Based on simulation-based objective structured examinations, it has prepared participants effectively for preclinical duties.

Restructuring ENT out-patient services during the coronavirus disease 2019 pandemic - an iterative approach.

BACKGROUND: Coronavirus disease 2019 has demanded enormous adjustments to National Health Service provisions. Non-urgent out-patient work was initially postponed or performed virtually, but is now being re-established. In ENT surgery, aerosol-generating procedures pose a particular challenge in out-patient settings. OBJECTIVE: A rapid restructuring of ENT out-patient services is required, to safely accommodate aerosol-generating procedures and increase in-person attendances, whilst coronavirus disease 2019 persists. METHODS: Data were collected prospectively over four consecutive cycles. Two surveys were conducted. Results were analysed and disseminated, with recommendations for service restructuring implemented at cycle end-points. RESULTS: Out-patient activity increased four-fold, associated with a significant rise in aerosol-generating procedures during the study period. Mean aerosol-generating procedure duration dropped weekly, implying a learning curve. Service restructuring occurred at cycle end-points. CONCLUSION: Iterative data gathering, results analysis and outcome dissemination enabled a swift, data-driven approach to the restructuring of ENT out-patient services. Patient and staff safety was ensured, whilst out-patient capacity was optimised.