Nasopharyngeal Swab Specimens Research Articles

Hybrid de novo Whole-Genome Assembly and Annotation of SARS-CoV-2 Virus from Nosocomial Infection in Pahang, Malaysia

PurposeSARS-CoV-2 virus is highly contagious and spreads easily that it is difficult to detect in elucidating the pattern of infection for contact tracing purposes. The advent of high-throughput sequencing techniques has improved diagnosis in detection the viral lineages especially involving sporadic infection. The combination of short- and long-reads greatly improved the assembly of the SARS-CoV-2 genome and marked as a new approach to correct erroneous frame-shifts from single sequencing effort.Methods & MaterialsThe samples were recovered from nasopharyngeal and oropharyngeal swab specimens of symptomatic health-care worker. The viral RNA was extracted for RT-qPCR and constructed for genomic library according to the ARTIC nCoV-2019 protocol. We performed whole-genome sequencing using hybrid approach combining both short and long-read sequencing approaches, respectively. The raw reads were reconstructed using a combination of bioinformatic tools for trimming, assembly and annotation. Finally, the consensus sequence was mapped with Geneious mapper using default parameters.ResultsThe hybrid approach resulted in a 29,782bp complete whole-genome with GC content of 38%. The product was deposited to GISAID as hCov-19/Malaysia/IIUM316/2020, and was identified to be originated from B.6 lineage of clade O. Besides, we observed several mutational points such as M153I (spike), P13L (N), T1198K (NSP3), L37F (NSP6), and A97V (NSP12), which may representing the major contributor to early pandemic transmission in Malaysia.ConclusionThe present study highlights the utility of whole-genome sequencing as a diagnostic tool of evaluating sporadic pattern of infection that can help to provide information regarding viruses relatedness, mutational rate, geographical spread and host adaptation. High-quality genome data can be used to assist in epidemiological investigation particularly when combined with other types of data.

Viral Infection and Respiratory Exacerbation in Children: Results from a Local German Pediatric Exacerbation Cohort.

Respiratory viruses play an important role in asthma exacerbation, and early exposure can be involved in recurrent bronchitis and the development of asthma. The exact mechanism is not fully clarified, and pathogen-to-host interaction studies are warranted to identify biomarkers of exacerbation in the early phase. Only a limited number of international exacerbation cohorts were studied. Here, we have established a local pediatric exacerbation study in Germany consisting of children with asthma or chronic, recurrent bronchitis and analyzed the viriome within the nasopharyngeal swab specimens derived from the entire cohort (n = 141). Interestingly, 41% of exacerbated children had a positive test result for human rhinovirus (HRV)/human enterovirus (HEV), and 14% were positive for respiratory syncytial virus (RSV). HRV was particularly prevalent in asthmatics (56%), wheezers (50%), and atopic (66%) patients. Lymphocytes were decreased in asthmatics and in HRV-infected subjects, and patients allergic to house dust mites were more susceptible to HRV infection. Our study thus confirms HRV infection as a strong ‘biomarker’ of exacerbated asthma. Further longitudinal studies will show the clinical progress of those children with a history of an RSV or HRV infection. Vaccination strategies and novel treatment guidelines against HRV are urgently needed to protect those high-risk children from a serious course of disease.

BA.1 Omicron Variant of SARS-CoV-2: First Case Reported in Calabria Region, Italy

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), emerged in Wuhan, China. Several variants of concern (VOCs) have been identified so far. Recently, the B.1.1.529 (Omicron) variant of SARS-CoV-2 spread rapidly worldwide. We describe the first case of the Omicron genetic lineage BA.1 in our region. The patient is a physician who traveled to Johannesburg (South Africa) and returned to Reggio Calabria (Italy). He underwent a SARS-CoV-2 screening before leaving—a required procedure where travelers present a negative PCR test one-day prior to departing. Three days after arriving in Italy, he started experiencing cold symptoms. Clinically, he was without fever or severe respiratory symptoms and reported suffering from a cold and sore throat. The nasopharyngeal swab specimen was tested by TaqPath COVID-19 RT-PCR and sequenced by Sanger sequencing, and next-generation sequencing (NGS) data were processed with their relative software. A peculiar drop-off of the S gene was obtained with TaqPath COVID-19 RT-PCR. S gene mutations indicative of the Omicron variant were obtained with both sequencing methods, pointing out 17 mutations in the 29 recognized by Sanger and the 28 recognized by NGS.

Correlation of COVID-19 Genome with Hypoxemia and Co-morbidities: Experimental Study

Background: The Covid-19 pandemic has wreaked havoc throughout the world, with 150 million cases to date and over 3 million lives claimed worldwide. Aim: To explore the hypoxemia in COVID-19 patients in relation with genomic mutation and co-morbidities. Study Design: Experimental Study. Methodology: A total of 16 COVID-19 positive patients admitted to Aziz Bhatti hospital were included in this study. COVID-19 was confirm through nasopharyngeal swab specimen diluted in normal saline subsequent RT-PCR was performed as per the standard operating procedure. Genome sequencing and interpretation of analysis was done through Illumina MiSeq. Results: There was statistically significant difference (P < 0.05) in SaO2 in patients with N (Nucleocapisd) protein mutation compared with NSP13( Helicase) mutation. Conclusion: It was concluded that mutation of N (Nucleocapisd) protein causes more pronounced hypoxia compared with Helicase mutation of COVID-19 genome. Keywords: COVID-19, Hypoxia, Mutation and Genome.

A short plus long-amplicon based sequencing approach improves genomic coverage and variant detection in the SARS-CoV-2 genome.

High viral transmission in the COVID-19 pandemic has enabled SARS-CoV-2 to acquire new mutations that may impact genome sequencing methods. The ARTIC.v3 primer pool that amplifies short amplicons in a multiplex-PCR reaction is one of the most widely used methods for sequencing the SARS-CoV-2 genome. We observed that some genomic intervals are poorly captured with ARTIC primers. To improve the genomic coverage and variant detection across these intervals, we designed long amplicon primers and evaluated the performance of a short (ARTIC) plus long amplicon (MRL) sequencing approach. Sequencing assays were optimized on VR-1986D-ATCC RNA followed by sequencing of nasopharyngeal swab specimens from fifteen COVID-19 positive patients. ARTIC data covered 94.47% of the virus genome fraction in the positive control and patient samples. Variant analysis in the ARTIC data detected 217 mutations, including 209 single nucleotide variants (SNVs) and eight insertions & deletions. On the other hand, long-amplicon data detected 156 mutations, of which 80% were concordant with ARTIC data. Combined analysis of ARTIC + MRL data improved the genomic coverage to 97.03% and identified 214 high confidence mutations. The combined final set of 214 mutations included 203 SNVs, 8 deletions and 3 insertions. Analysis showed 26 SARS-CoV-2 lineage defining mutations including 4 known variants of concern K417N, E484K, N501Y, P618H in spike gene. Hybrid analysis identified 7 nonsynonymous and 5 synonymous mutations across the genome that were either ambiguous or not called in ARTIC data. For example, G172V mutation in the ORF3a protein and A2A mutation in Membrane protein were missed by the ARTIC assay. Thus, we show that while the short amplicon (ARTIC) assay provides good genomic coverage with high throughput, complementation of poorly captured intervals with long amplicon data can significantly improve SARS-CoV-2 genomic coverage and variant detection.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Mutational Pattern in the Fourth Pandemic Phase in Greece.

The aim of this study is to investigate the circulating variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Athens and from rural areas in Greece during July and August 2021. We also present a rapid review of literature regarding significant SARS-CoV-2 mutations and their impact on public health. A total of 2500 nasopharyngeal swab specimens were collected from suspected COVID-19 cases (definition by WHO 2021b). Viral nucleic acid extraction was implemented using an automatic extractor and the RNA recovered underwent qRT-PCR in order to characterize the specimens as positive or negative for SARS-CoV-2. The positive specimens were then used to identify specific Spike gene mutations and characterize the emerging SARS-CoV-2 variants. For this step, various kits were utilized. From the 2500 clinical specimens, 220 were tested positive for SARS-CoV-2 indicating a prevalence of 8.8% among suspected cases. The RT-PCR Ct (Cycle threshold) Value ranged from 19 to 25 which corresponds to medium to high copy numbers of the virus in the positive samples. From the 220 positive specimens 148 (67.3%) were from Athens and 72 (32.7%) from Greek rural areas. As far as the Spike mutations investigated: N501Y appeared in all the samples, D614G mutation appeared in 212 (96.4%) samples with a prevalence of 87.2% in Athens and 98.6% in the countryside, E484K had a prevalence of 10.8% and 12.5% in Athens and the rural areas, respectively. K417N was found in 18 (12.2%) samples from Athens and four (5.6%) from the countryside, P681H was present in 51 (34.5%) Athenian specimens and 14 (19.4%) specimens from rural areas, HV69-70 was carried in 32.4% and 19.4% of the samples from Athens and the countryside, respectively. P681R had a prevalence of 87.2% in Athens and 98.6% in rural areas, and none of the specimens carried the L452R mutation. 62 (28.2%) samples carried the N501Y, P681H, D614G and HV69-70 mutations simultaneously and the corresponding variant was characterized as the Alpha (UK) variant (B 1.1.7). Only six (2.7%) samples from the center of Athens had the N501Y, E484K, K417N and D614G mutations simultaneously and the virus responsible was characterized as the Beta (South African) variant (B 1.351). Our study explored the SARS-CoV-2 variants using RT-PCR in a representative cohort of samples collected from Greece in July and August 2021. The prevalent mutations identified were N501Y (100%), D614G (96.4%), P681R (90.1%) and the variants identified were the Delta (90.1%), Alpha (28.2%) and Beta (2.7%).

Long-term exposure to air pollution and COVID-19 incidence: a prospective study of residents in the city of Varese, Northern Italy

ObjectivesTo investigate the association between long-term exposure to airborne pollutants and the incidence of SARS-CoV-2 up to March 2021 in a prospective study of residents in Varese city.MethodsCitizens of Varese...

Exploratory Study on Application of MALDI-TOF-MS to Detect SARS-CoV-2 Infection in Human Saliva.

SARS-CoV-2 has caused a large outbreak since its emergence in December 2019. COVID-19 diagnosis became a priority so as to isolate and treat infected individuals in order to break the contamination chain. Currently, the reference test for COVID-19 diagnosis is the molecular detection (RT-qPCR) of the virus from nasopharyngeal swab (NPS) samples. Although this sensitive and specific test remains the gold standard, it has several limitations, such as the invasive collection method, the relative high cost and the duration of the test. Moreover, the material shortage to perform tests due to the discrepancy between the high demand for tests and the production capacities puts additional constraints on RT-qPCR. Here, we propose a PCR-free method for diagnosing SARS-CoV-2 based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling and machine learning (ML) models from salivary samples. Kinetic saliva samples were collected at enrollment and ten and thirty days later (D0, D10 and D30), to assess the classification performance of the ML models compared to the molecular tests performed on NPS specimens. Spectra were generated using an optimized protocol of saliva collection and successive quality control steps were developed to ensure the reliability of spectra. A total of 360 averaged spectra were included in the study. At D0, the comparison of MS spectra from SARS-CoV-2 positive patients (n = 105) with healthy healthcare controls (n = 51) revealed nine peaks that significantly distinguished the two groups. Among the five ML models tested, support vector machine with linear kernel (SVM-LK) provided the best performance on the training dataset (accuracy = 85.2%, sensitivity = 85.1%, specificity = 85.3%, F1-Score = 85.1%). The application of the SVM-LK model on independent datasets confirmed its performances with 88.9% and 80.8% of correct classification for samples collected at D0 and D30, respectively. Conversely, at D10, the proportion of correct classification had fallen to 64.3%. The analysis of saliva samples by MALDI-TOF MS and ML appears as an interesting supplementary tool for COVID-19 diagnosis, despite the mitigated results obtained for convalescent patients (D10).

Impact of Pooling Samples on Analytic Sensitivity of a Real-Time Reverse Transcriptase PCR Assay for SARS CoV-2

During the COVID-19 pandemic, laboratories experienced periods of shortages for certain critical materials required to meet the high demand for SARS-CoV-2 testing. The U.S. Food & Drug Administration provided a template for molecular diagnostic testing, including guidance for a specimen pooling process in order to evaluate performance of the SARS-CoV-2 nucleic acid amplification assay. This study aimed to evaluate the testing of pooled specimens consisting of four nasopharyngeal swab specimens using the Luminex ARIES® nucleic acid amplification platform. Results indicated that there was a loss of analytic sensitivity with pooled nasopharyngeal swab samples, demonstrating that this approach should be balanced against material shortages and the clinical utility of a less sensitive assay.

Symptoms COVID 19 Positive Vapers Compared to COVID 19 Positive Non-vapers.

Objectives:The purpose of the present study was to assess and describe the severity of symptoms reported by Covid-19 positive patients who vaped (smoked e-cigarettes) when compared to those who did not vape or smoke at the time of the diagnosis of Covid-19.Methods:Patients from this study are from a well-characterized patient cohort collected at Mayo Clinic between March 1, 2020 and February 28, 2021; with confirmed COVID-19 diagnosis defined as a positive result on reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assays from nasopharyngeal swab specimens. Among the 1734 eligible patients, 289 patients reported current vaping. The cohort of vapers (N = 289) was age and gender matched to 1445 covid-19 positive patients who did not vape. The data analyzed included: date of birth, gender, ethnicity, race, marital status, as well as lifestyle history such as vaping and smoking and reported covid-19 symptoms experienced.Results:A logistic regression analysis was performed separately for each symptom using generalized estimating equations (GEE) with robust variance estimates in order to account for the 1:5 age, sex, and race matched set study design. Patients who vaped and developed Covid-19 infection were more likely to have chest pain or tightness (16% vs 10%, vapers vs non vapers, P = .005), chills (25% vs 19%, vapers vs non vapers, P = .0016), myalgia (39% vs 32%, vapers vs non vapers, P = .004), headaches (49% vs 41% vapers vs non vapers, P = .026), anosmia/dysgeusia (37% vs 30%, vapers vs non vapers, P = .009), nausea/vomiting/abdominal pain (16% vs 10%, vapers vs non vapers, P = .003), diarrhea (16% vs 10%, vapers vs non vapers, P = .004), and non-severe light-headedness (16% vs 9%, vapers vs non vapers, P < .001).Conclusion:Vapers experience higher frequency of covid-19 related symptoms when compared with age and gender matched non-vapers. Further work should examine the impact vaping has on post-covid symptom experience.

Polydopamine decorated MoS2 nanosheet based electrochemical immunosensor for sensitive detection of SARS-CoV-2 nucleocapsid protein in clinical samples.

The outbreak of the highly contagious disease COVID-19, which is triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demands a rapid, low-cost, and highly sensitive immunosensor that can detect and identify the virus efficiently. Here, an electrochemical immunosensor based on a nanocomposite consisting of molybdenum disulfide nanosheets decorated with polydopamine (MoS2-PDA) is developed for highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein). The MoS2-PDA nanocomposite possesses various hydroxyl and amine groups that have excellent chemistry with crosslinkers and act as adhesive agents to bind with the working electrode surface. Furthermore, the optical, functional, structural, vibrational, and morphological properties of the MoS2-PDA nanocomposite are studied using various characterization techniques such as UV-vis, FTIR, and Raman spectroscopies, XRD, and TEM. The electrochemical immunosensor is fabricated by functionalizing the MoS2-PDA nanocomposite with anti-SARS-CoV-2 nucleocapsid IgG antibody (Ab) and has a very high sensitivity against the N protein with a linear range between 10 ag mL-1 and 100 ng mL-1. The electrochemical immunosensor exhibits a lowest limit of detection (LOD) of 2.80 ag mL-1 and a limit of quantification (LOQ) of 8.48 ag mL-1via electrochemical impedance spectroscopy (EIS). Furthermore, the electrochemical immunosensor is successfully employed to detect the N protein in nasopharyngeal swab specimens and displays good consistency with the conventional RT-PCR test results. The results show that the MoS2-PDA nanocomposite-based electrochemical platform can serve as a highly sensitive and selective detector of N protein and will pave the way for the development of a point-of-care (POC) electrochemical immunosensor for rapid detection of other infectious viruses.

Saliva as an Alternative to Nasopharyngeal Swab Specimen for Reverse-transcription Polymerase Chain Reaction (RT-PCR) Diagnosis of SARS-CoV-2 Infection: A Systematic Review and Meta-analysis

Objectives. To assess the accuracy of reverse-transcription polymerase chain reaction (RT-PCR) of saliva specimens compared to RT-PCR of nasopharyngeal swabs (NPS) in the diagnosis of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection. Methods. A database search was conducted in MEDLINE, the Cochrane COVID Study Register, the COVID-19 Living Evidence Database, medRxiv, and bioRxiv for published articles and preprints dated between 1 January and 26 December 2020 of studies on the diagnostic accuracy of saliva RT-PCR compared to nasopharyngeal swab (NPS) RT-PCR. Studies included cross-sectional, cohort, and case-control designs. Risk of bias assessment was done using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. Pooled sensitivity and specificity estimates were determined using a bivariate random-effects model. Subgroup analysis was done based on patients’ diagnostic and symptom status, healthcare setting, and collection method. Certainty of evidence was determined using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach for all pooled analyses. Results. Fifty-one studies with 16,476 paired specimens were included. Overall pooled analysis showed that saliva RT-PCR had 84% (95% confidence interval 80-88%) sensitivity and 96% (95% CI 94-98%) specificity compared to NPS RT-PCR. No significant difference in sensitivity or specificity was found between subgroups. Substantial heterogeneity (&gt;70%) was noted for all analyses. The certainty of evidence for the overall study was moderate for both sensitivity and specificity. Conclusion. Using saliva RT-PCR has comparable sensitivity and specificity to NPS in diagnosing SARS-CoV-2 infection regardless of patient diagnostic or symptom status, healthcare setting, or collection method. It may be used as an alternative specimen for diagnosis.

Detection of SARS-CoV-2 RNA in Urine by RT-LAMP: A Very Rare Finding.

Detection of SARS-CoV-2 is routinely performed in naso/oropharyngeal swabs samples from patients via RT-qPCR. The RT-LAMP technology has also been used for viral RNA detection in respiratory specimens with both high sensitivity and specificity. Recently, we developed a novel RT-LAMP test for SARS-CoV-2 RNA detection in nasopharyngeal swab specimens (named, N15-RT-LAMP) that can be performed as a single-tube colorimetric method, in a real-time platform, and as dry-LAMP. To date, there has been very little success in detecting SARS-CoV-2 RNA in urine by RT-qPCR, and the information regarding urine viral excretion is still scarce and not comprehensive. Here, we tested our N15-RT-LAMP on the urine of 300 patients admitted to the Hospital of Salamanca, Spain with clinical suspicion of COVID-19, who had a nasopharyngeal swab RT-qPCR-positive (n = 100), negative (n = 100), and positive with disease recovery (n = 100) result. The positive group was also tested by RT-qPCR for comparison to N15-RT-LAMP. Only a 4% positivity rate was found in the positive group via colorimetric N15-RT-LAMP and 2% via RT-qPCR. Our results are consistent with those obtained in other studies that the presence of SARS-CoV-2 RNA in urine is a very rare finding. The absence of SARS-CoV-2 RNA in urine in the recovered patients might suggest that the urinary route is very rarely used for viral particle clearance.

Isolation and characterization of SARS-CoV-2 Beta variant from UAE travelers

BackgroundThe emergence of SARS-CoV-2 variants in places where the virus is uncontained poses a global threat from the perspective of public health and vaccine efficacy. Travel has been important factor for the easy spread of SARS-CoV-2 variants worldwide. India has also observed the importation of SARS-CoV-2 variants through international travelers. MethodsIn this study, we have collected the oropharyngeal and nasopharyngeal swab specimens from 58 individuals with travel history from United Arab Emirates (UAE), East, West and South Africa, Qatar, Ukraine and Saudi Arabia arrived in India during February–March 2021. The clinical specimens were initially screened for SARS-CoV-2 using Real time RT-PCR. All the specimens were inoculated on to Vero CCL-81 cells for virus isolation. The viral isolates were further sequenced using Next-Generation Sequencing. ResultsAll 58 cases were tested positive for SARS-CoV-2 using Real time RT-PCR. Four specimens showed progressive infectivity with fusion of the infected cells with neighboring cells leading to large mass of cells. Replication competent virus was confirmed from culture supernatant of the passage 2 using Real time RT-PCR. Two plaque purified SARS-CoV-2 isolates demonstrated high viral RNA load of 3.8–7.5 × 1011 and 1.1–1.6 × 1011 at passage 4 and 5 respectively. Nucleotide variations along with amino acid changes were also observed among these two isolates at passage 2–5. All four cases were male with no symptoms and co-morbidity. The sequence analysis has shown two different clusters, first cluster with nucleotide deletions in the ORF1ab and the spike, while second cluster with deletions in spike region. The viral isolates demonstrated 99.88–99.96% nucleotide identity with the representative sequences of Beta variant (B.1.351). ConclusionThese findings suggest easier transmission of SARS-CoV-2 variants with human mobility through international travel. The isolated Beta variant would be useful to determine the protective efficacy of the currently available and upcoming COVID-19 vaccines in India.

Factors influencing the performance of rapid SARS-CoV-2 antigen tests under field condition.

BackgroundGlobally, real‐time reverse transcription–polymerase chain reaction (rRT‐PCR) is the reference detection technique for SARS‐CoV‐2, which is expensive, time consuming, and requires trained laboratory personnel. Thus, a cost‐effective, rapid antigen test is urgently needed. This study evaluated the performance of the rapid antigen tests (RATs) for SARS‐CoV‐2 compared with rRT‐PCR, considering different influencing factors.MethodsWe enrolled a total of 214 symptomatic individuals with known COVID‐19 status using rRT‐PCR. We collected and tested paired nasopharyngeal (NP) and nasal swab (NS) specimens (collected from same individual) using rRT‐PCR and RATs (InTec and SD Biosensor). We assessed the performance of RATs considering specimen types, viral load, the onset of symptoms, and presenting symptoms.ResultsWe included 214 paired specimens (112 NP and 100 NS SARS‐CoV‐2 rRT‐PCR positive) to the analysis. For NP specimens, the average sensitivity, specificity, and accuracy of the RATs were 87.5%, 98.6%, and 92.8%, respectively, when compared with rRT‐PCR. While for NS, the overall kit performance was slightly lower than that of NP (sensitivity 79.0%, specificity 96.1%, and accuracy 88.3%). We observed a progressive decline in the performance of RATs with increased Ct values (decreased viral load). Moreover, the RAT sensitivity using NP specimens decreased over the time of the onset of symptoms.ConclusionThe RATs showed strong performance under field conditions and fulfilled the minimum performance limit for rapid antigen detection kits recommended by World Health Organization. The best performance of the RATs can be achieved within the first week of the onset of symptoms with high viral load.

Paper-based immunoassay based on 96-well wax-printed paper plate combined with magnetic beads and colorimetric smartphone-assisted measure for reliable detection of SARS-CoV-2 in saliva

Coronavirus disease 2019 (COVID-19) has been recognized as a global pandemic outbreak, opening the most severe socio-economic crisis since World War II. Different scientific activities have been emerged in this global scenario, including the development of innovative analytical tools to measure nucleic acid, antibodies, and antigens in the nasopharyngeal swab, serum, and saliva for prompt identification of COVID-19 patients and to evaluate the immune response to the vaccine. The detection of SARS-CoV-2 in saliva remains a challenge for the lack of sufficient sensitivity. To address this issue, we developed a novel paper-based immunoassay using magnetic beads to support the immunological chain and 96-well wax-printed paper plate as a platform for color visualization by using a smartphone combined with Spotxel free-charge app. To assess the reliability of the measurement of SARS-CoV-2 in saliva, untreated saliva was used as a specimen and the calibration curve demonstrated a dynamic range up to 10 μg/mL, with a detection limit equal to 0.1 μg/mL. The effectiveness of this sustainable analytical tool in saliva was evaluated by comparing the data with the nasopharyngeal swab specimens sampled by the same patients and tested with Real-Time PCR reference method, founding 100% of agreement, even in the case of high Cycle Threshold (CT) numbers (low viral load). Furthermore, the positive saliva samples were characterized by the next-generation sequencing method, demonstrating the capability to detect the Delta variant, which is actually (July 2021) the most relevant variant of concern.

Comparative Evaluation of Allplex Respiratory Panels 1, 2, 3, and BioFire FilmArray Respiratory Panel for the Detection of Respiratory Infections.

Multiplex nucleic acid amplification assays that simultaneously detect multiple respiratory pathogens in a single nasopharyngeal swab (NPS) specimen are widely used for rapid clinical diagnostics. We evaluated Allplex Respiratory Panel (RP) 1, 2, 3, and the BioFire FilmArray RP assay for detecting respiratory pathogens from NPS specimens. In all, 181 NPS specimens obtained from patients suspected of having respiratory infections during the non-influenza season (August–December 2019) were included. The Allplex RP 1, 2, and 3 detected 154 samples positive for respiratory viruses, whereas the BioFire FilmArray detected viruses in 98 samples. Co-infection with two or more viruses was detected in 41 and 17 NPS specimens by Allplex RP and the BioFire FilmArray RP, respectively. For adenoviruses, Allplex RP 1 detected 31 specimens, compared to 34 by the BioFire FilmArray. In all, 64 NPS specimens were positive for human enterovirus (HEV) and human rhinovirus (HRV) on the Allplex RP, in contrast to 39 HEV/HRV on the BioFire FilmArray. The parainfluenza virus (PIV-1–4) detection rate differed between the two systems. Most discrepant results were observed for NPS specimens with high cycle threshold values obtained by Allplex RP. This study showed concordant performance of the Allplex RP 1, 2, 3, and the BioFire FilmArray RP for the simultaneous detection of multiple respiratory viruses.

Histopathological Evaluation of Deceased Persons in Lusaka, Zambia With or Without Coronavirus Disease 2019 (COVID-19) Infection: Results Obtained From Minimally Invasive Tissue Sampling.

BackgroundAlthough much has been learned about the pathophysiology of coronavirus disease 2019 (COVID-19) infections, pathology data from patients who have died of COVID-19 in low- and middle-income country settings remain sparse. We integrated minimally invasive tissue sampling (MITS) into an ongoing postmortem surveillance study of COVID-19 in deceased individuals of all ages in Lusaka, Zambia.MethodsWe enrolled deceased subjects from the University Teaching Hospital Morgue in Lusaka, Zambia within 48 hours of death. We collected clinical and demographic information, a nasopharyngeal swab, and core tissue biopsies from the lung, liver, and kidneys for pathologic analysis. Individuals were considered eligible for MITS if they had a respiratory syndrome prior to death or a COVID-19+ polymerase chain reaction (PCR) nasopharyngeal swab specimen. Samples were retested using quantitative reverse transcriptase PCR.ResultsFrom June to September 2020 we performed MITS on 29 deceased individuals. PCR results were available for 28/29 (96.5%) cases. Three had a COVID-19+ diagnosis antemortem, and 5 more were identified postmortem using the recommended cycle threshold cut-point <40. When expanding the PCR threshold to 40 ≤ cycle threshold (Ct) ≤ 45, we identified 1 additional case. Most cases were male and occurred in the community The median age at death was 47 years (range 40–64). Human immunodeficiency virus (HIV)/AIDS, tuberculosis, and diabetes were more common among the COVID-19+ cases. Diffuse alveolar damage and interstitial pneumonitis were common among COVID-19+ cases; nonspecific findings of hepatic steatosis and acute kidney injury were also prevalent in the COVID-19+ group. Vascular thrombi were rarely detected.ConclusionsLung abnormalities typical of viral pneumonias were common among deceased COVID-19+ individuals, as were nonspecific findings in the liver and kidneys. Pulmonary vascular thrombi were rarely detected, which could be a limitation of the MITS technique. Nonetheless, MITS offers a valuable alternative to open autopsy for understanding pathological changes due to COVID-19.

Diagnostic Accuracy of Nasopharyngeal Swab Cultures in Children Less Than Five Years with Chronic Wet Cough.

Background: It is necessary to find a non-invasive and accurate procedure to predict persistent bacterial bronchitis (PBB) causative organisms and guide antibiotic therapy. The study objective was to compare the diagnostic accuracy of nasopharyngeal swab cultures with bronchoalveolar lavage (BAL) cultures in children with PBB. Methods: Nasopharyngeal swab and BAL fluid specimens were collected and cultured for bacterial pathogens prospectively from less than five-year-old children undergoing flexible bronchoscopy for chronic wet cough. Results: Of the 59 children included in the study, 26 (44.1%) patients had a positive BAL bacterial culture with neutrophilic inflammation. Prevalence of positive cultures for any of the four common respiratory pathogens implicated in PBB (Moraxella catarrhalis, Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae) was significantly higher (p = 0.001) in NP swabs compared to BAL fluids (86.4% and 44.1% of PBB cases, respectively). NP swab cultures for any of the four main bacterial pathogens had 85% (95% CI: 65–96%) and 48% (95% CI: 31–66%) sensitivity and specificity of detecting PBB, respectively. Positive and negative predictive values were 56% (95% CI: 47–65%) and 80% (95% CI: 60–91%), respectively. In conclusion, in children less than 5 years of age with chronic wet cough (PBB-clinical), a negative NP swab result reduces the likelihood of lower airway infection; however, a positive NP swab does not accurately predict the presence of lower airway pathogens. Flexible bronchoscopy should be considered in those with recurrent PBB-clinical or with clinical pointers of central airway anomalies.

340. Outcomes of COVID-19 in Hospitalized SOT Recipients: Experence in Colombia, South America

BackgroundSOTs (SOT) recipients with COVID‐19 are considered to be at high risk of severe clinical outcomes. Several descriptive studies have reported a high frequency of intensive care unit admission and death rates. There is a lack of evidence regarding the best approach for immunosuppressive therapy in SOT recipients with COVID-19.MethodsWe performed a single-centered, retrospective, observational study of all SOT recipients with SARS-CoV-2 confirmed infection RT-PCR from nasopharyngeal swab specimens who were admitted to the emergency department from March 25 to September 1, 2020. Glucocorticoid therapy was administered according to the criteria of the attending physician. We classified glucocorticoid dose as low dose therapy if the patient received dexamethasone 6 mg/day or methylprednisolone 40 mg/day, and a high dose if the patient received methylprednisolone 80–160 mg/day. Specimens collected within the first 48 hourswere defined coinfection, while specimens collected after 48 hours were defined as hospital-acquired superinfection.ResultsOf a total of 43 SOT recipients with COVID-19, 17 (39%) required intensive care unit admission. 32 (74.4%) required glucocorticoid therapy: 13 received low dose and 19 high dose. 15 (34.8%) had secondary infections. A total of 12 (27.9%) presented hospital-acquired bacterial superinfections, mostly caused by P. aeruginosa, most of isolations were from respiratory tract cultures. The median time from hospital admission to superinfection diagnosis was 9 (7-13) days. Community-acquired co-infection at COVID-19 diagnosis was documented only in 3 (6.9%) patients, mostly caused by K. Pneumoniae, all isolations were from urine culture. Glucocorticoid therapy was indicated in 32 (80%) patients, 19 received high dose and 13 low doses. Overall hospital mortality was 17.5%. ICU mortality was 41%. Overall mortality in the high dose steroids group was 37 % vs . 0% in the low dose group.ConclusionOur results showed a higher frequency of superinfection in SOT recipients with COVID-19 compared to previous reports, and higher ICU mortality. Further studies are needed to establish the best approach for glucocorticoid therapy in SOT recipients with COVID-19.Disclosures All Authors: No reported disclosures