Development of the one-step qualitative RT-PCR assay to detect SARS-CoV-2 Omicron (B.1.1.529) variant in respiratory specimens

A new SARS-CoV-2 Omicron (B.1.1.529) Variant of Concern has been emerging worldwide. We are seeing an unprecedented surge in patients due to Omicron in this COVID-19 pandemic. A rapid and accurate molecular test that effectively differentiates Omicron from other SARS-CoV-2 variants would be important for both epidemiologic value and for directing variant-specific therapies such as monoclonal antibody infusions. In this study, we developed a real-time RT-PCR assay for the qualitative detection of Omicron from routine clinical specimens sampling the upper respiratory tract. The limit of detection of the SARS-CoV-2 Omicron variant RT-PCR assay was 2 copies/l. Notably, the assay did not show any cross-reactivity with other SARS-CoV-2 variants including Delta (B.1.617.2). This SARS-CoV-2 Omicron variant RT-PCR laboratory-developed assay is sensitive and specific to detect Omicron in nasopharyngeal and nasal swab specimens.

Evaluation of the Cepheid Xpert Xpress SARS-CoV-2 test for bronchoalveolar lavage

Accurate and rapid laboratory tests are essential for the prompt diagnosis of COVID-19, which is important to patients and infection control. The Xpert Xpress SARS-CoV-2 test is a real-time RT-PCR intended for the qualitative detection of nucleic acid from SARS-CoV-2 in upper respiratory specimens. In this study, we assessed the analytical and clinical performance characteristics of this rapid test for SARS-CoV-2 in 60 bronchoalveolar lavage (BAL) specimens. BAL is a specimen type that is not authorized under EUA for the Xpert Xpress SARS-CoV-2 test. The limit of detection of the Xpert Xpress SARS-CoV-2 test was 500 copies/ml. The overall agreement of the Xpert Xpress SARS-CoV-2 test was 100%. The Xpert Xpress SARS-CoV-2 test is sensitive and specific to aid in diagnosis of COVID-19 using bronchoalveolar lavage.

Evaluation of viral loads in patients with SARS-CoV-2 Delta variant infection: Higher loads do not translate into different testing scenarios

The Delta SARS-CoV-2 variant is very infectious, and it is spreading quickly during this pandemic. In the study, we compared viral loads in surging cases infected with the SARS-CoV-2 Delta variant in the fourth wave of COVID-19 with the three prior waves. The data comprised viral loads from positive cases detected within the UPMC health care system in Allegheny County, Pennsylvania. A total of 2,059 upper airway samples were collected and tested for SARS-CoV-2 positive by RT-PCR during March 2020-September 2021. We did not observe significant difference in viral load difference between the third (December 2020 - January 2021) and fourth (June 2021 - September 2021) waves; however, they had the higher viral load than the first (March 2020 - June 2020) and second waves (June 2020 - August 2020). We did find an age-related effect with the elderly presenting with lower viral loads, which was also seen in the earlier waves. However, the level of viral load in the fourth wave was not sufficient higher to qualitatively change our expected detected rates using various testing modalities.

Differences in detected viral loads guide use of SARS-CoV-2 antigen-detection assays towards symptomatic college students and children.

Limitations in timely testing for SARS-CoV-2 drive the need for new approaches in suspected COVID-19 disease. We queried whether viral load (VL) in the upper airways at presentation could improve the management and diagnosis of patients. This study was conducted in a 9 hospital system in Allegheny County, Pennsylvania between March 1-August 31 2020. Viral load was determined by PCR assays for patients presenting to the Emergency Departments (ED), community pediatrics practices and college health service. We found that for the ED patients, VL did not vary substantially between those admitted and not. VL was relatively equivalent across ages, except for the under 25 age groups that tended to present with higher loads. To determine if rapid antigen testing (RAT) could aid diagnosis in certain populations, we compared BD Veritor and Quidel Sofia to SOC PCR-based tests. The antigen assay provided a disease-detection sensitivity of >90% in a selection of 32 positive students and was modeled to have an 80% sensitivity in all positive students. In the outpatient pediatric population, the antigen assay detected 70% of PCR-positives. Extrapolating these findings to viral loads in older hospitalized patients, a minority would be detected by RAT (40%). Higher loads did correlate with death, though the prognostic value was marginal (ROC AUC of only 0.66). VL did not distinguish between those needing mechanical ventilation and routine inpatients. We conclude that VL in upper airways, while not prognostic for disease management, may aid in selecting proper testing methodologies for certain patient populations.

Detection, genetic characterization, and quantification of norovirus RNA from sera of children with gastroenteritis.

BACKGROUND: Norovirus (NoV) infection is thought to be confined to the intestines, whereas many reports suggest antigenemia and viremia occur during rotavirus gastroenteritis. OBJECTIVES: To detect NoV RNA in sera and cerebrospinal fluids (CSF) from NoV-infected children, and to quantify and genetically characterize the NoV found in these compartments. STUDY DESIGN: Semi-nested PCR was conducted on stool, serum and CSF samples from 56 patients with acute gastroenteritis. Positive samples for NoV were analyzed further by sequencing and real-time PCR. RESULTS: From 39 patients with NoV RNA in stools, 6 also had NoV RNA in sera and none had NoV RNA in CSF. Genotypes of the NoV in stool and serum from the same patient matched completely. The strains in this study had high homology (98.1-100%) with registered strains in the database. The median viral load in stools of the serum-positive patients was greater than that of the serum-negative patients, but this difference was not statistically significant (9.8 x 10(9)copies/g versus 1.1 x 10(9)copies/g (p=0.117)). CONCLUSIONS: NoV RNA appeared in the blood stream in 15% of the patients of NoV gastroenteritis. Although the viral load in stool was not statistically correlated with NoV appearance in serum, genetic analysis indicated that NoV RNA in sera originated from the NoV gastroenteritis.

Development of a rapid immunochromatographic test for noroviruses genogroups I and II.

Norovirus (NoV) is known to cause acute gastroenteritis in children worldwide. Although reverse transcription-PCR (RT-PCR) method is considered to be the "gold standard" for diagnosis of this viral infection, it requires skillful personnel and well-equipped laboratory. In this study, a rapid and easily performable diagnostic kit was developed using immunochromatographic method with rabbit polyclonal antibodies raised against recombinant virus-like particles (rVLPs) of most prevalent genotypes, genogroup II genotypes 3 and 4. This kit was evaluated for reactivity to rVLPs and detection of natural viruses in stool samples collected from children with diarrhea in comparison to the results obtained by RT-PCR. In the prospective assessment, the kit showed agreement rate of 84.1%, sensitivity of 69.8% and specificity of 93.7%. Genotyping of the RT-PCR positive samples by sequence analysis revealed that some heterogeneous genotypes were also detected while some in homogeneous genotypes occasionally showed false negative records resulting in lower sensitivity. No cross-reactivity with other common viral pathogens was observed. Taken together with the result of the detection limit of viral load as small as approximately 10(6-7)copies/g of stool, the current immunochromatography test is justified for screening for NoV infection with simple laboratory support.

Characterizations of adenovirus type 41 isolates from children with acute gastroenteritis in Japan, Vietnam, and Korea.

Genetic and antigenic characterizations of 70 strains of adenovirus type 41 (Ad41), isolated between 1998 and 2001 from children in Japan, Vietnam, and Korea, were done by DNA restriction enzyme (RE) analysis, sequencing analysis, and monoclonal antibody (MAb)-based enzyme-linked immunosorbent assay (ELISA). Eight genome types were observed in the present study, among which D25, D26, D27, and D28 were novel genome types. These eight genome types were divided into two genome-type clusters (GTCs) based on phylogenetic analysis of the hypervariable regions (HVRs) of the hexon. GTC1 includes D1, D25, D26, D27, and D28, and the GTC2 contains D4, D12, and D22. The amino acid homologies among the members within a GTC were 97 to 100%, whereas between the members of different GTCs the homologies were 92 to 94%. The specificity of the GTC classification was confirmed by ELISA with MAb 1F, which was selected by the Ad41 prototype Tak strain. It was found that only the isolates of GTC1 but not of GTC2 reacted with MAb 1F. These results suggest that Ad41 isolates from the three countries should be classified into two subtypes. The accumulation of amino acid mutations located in HVRs of hexon are indicative for the classification of Ad41 subtype.