Culturing of SARS-CoV-2 from patient samples: Protocol for optimal virus recovery and assessment of infectious viral load.

Optimal sampling, preservation, and culturing of SARS-CoV-2 from COVID-19 patients are critical for successful recovery of virus isolates and to accurately estimate contagiousness of the patient. In this study, we investigated the influence of the type of sampling media, storage time, freezing conditions, sterile filtration, and combinations of these to determine the optimal pre-analytic conditions for virus recovery and estimation of infectious viral load in COVID-19 patients. Further, we investigated the viral shedding kinetics and mucosal antibody response in 38 COVID-19 hospitalized patients. We found Universal Transport Medium (Copan) to be the most optimal medium for preservation of SARS-CoV-2 infectivity. Our data showed that the probability of a positive viral culture was strongly correlated to Ct values, however some samples did not follow the general trend. We found a significant correlation between plaque forming units and levels of mucosal antibodies and found that high levels of mucosal antibodies correlated with reduced chance of isolating the virus. Our data reveals essential parameters to consider from specimen collection over storage to culturing technique for optimal chance of isolating SARS-CoV-2 and accurately estimating patient contagiousness.

Humoral antibody response following mRNA vaccines against SARS-CoV-2 in solid organ transplant recipients; a status after a fifth and bivalent vaccine dose.

Background: In solid organ transplant (SOT) recipients, the humoral response following COVID-19 vaccination is reduced, as a result of their immunosuppressed treatment. In this study, we investigated antibody concentrations after booster vaccinations until the fifth dose, the latter by monovalent or bivalent BA1 or BA4/5 vaccines. In addition, we evaluated the efficacy of vaccination by recording breakthrough infections, hospitalizations, and deaths. Method: This prospective cohort study included 438 SOT recipients (>18 years) vaccinated with mRNA vaccines against COVID-19 from January 2021 until March 2023. Blood samples were drawn before and after each vaccination and tested for SARS-CoV-2 spike RBD IgG antibodies with the lowest and highest cut-off at 7.1 and 5,680 BAU/mL, respectively. Vaccine information, breakthrough infections, and hospitalizations were collected from the medical records. Results: Most participants received BNT162b2 and 61.4% received five vaccine doses. The response proportion in SOT recipients increased from 86.7% after the fourth dose to 93.0% following the fifth dose. Antibody concentration decreased with 142.7 BAU/mL between the third and fourth dose (median 132 days, Quartile 1: 123, Quartile 3: 148) and 234.3 BAU/mL between the fourth and fifth (median 250 days, Quartile 1: 241, Quartile 3: 262) dose among those without breakthrough infection (p=0.34). When comparing the Omicron BA.1 or Omicron BA.4/BA.5 adapted vaccines, no significant differences in antibody concentration were found, but 20.0% of SOT recipients receiving a monovalent fifth vaccine dose had a breakthrough infection compared to 4.0% and 7.9% among those who received BA.1 and BA.4/BA.5 adapted vaccines, respectively (p=0.04). Since January 2021, 240 (54.8%) participants had a breakthrough infection, and 22 were hospitalized, but no deaths were observed. Conclusions: The fifth COVID-19 vaccine dose raised antibody response to 93.0% of the study population. Additional booster doses, as well as bivalent vaccines, led to higher levels of antibody concentration in SOT recipients. We found a lower incidence of breakthrough infections among SOT recipients after receiving a bivalent vaccine as a fifth dose compared to those receiving a monovalent dose. Antibody concentrations did not wane when the time between doses was prolonged from four to eight months.

Viral dynamics of SARS-CoV-2 in immunocompromised patients.

OBJECTIVES: Immunocompromised patients infected with SARS-CoV-2 have been shown to shed replicable virus for a prolonged period of time, and the duration of isolation can therefore be difficult to estimate. The objective of this study was to evaluate the viral load dynamic in non-hospitalized immunocompromised patients infected with SARS-CoV-2 and treated with monoclonal antibodies (mAbs) or antivirals. METHODS: Oropharyngeal swabs for RT-PCR and viral culture were collected from 29 immunocompromised patients before treatment with mAbs or antivirals and at days 5 and 15 after treatment. Overall, 12 patients were infected with the subvariant Omicron BA.1, 12 with Omicron BA.2, two with the Delta variant and for three patients determination of the variant were inconclusive. RESULTS: Before treatment with mAbs or antivirals, 22 of 29 patients (76% [95% CI, 56-90]) shed replicative SARS-CoV-2. At day 5, 21 patients (72% [95% CI, 53-87]) still tested RT-PCR-positive, but for 14 patients (48% [95% CI, 29-67]) there were no replicative virus in culture. At day 15, 16 patients (55% [95% CI, 36-74%]) tested positive but only two patients (7% [95%CI, 1-23]) had replicative virus. DISCUSSION: Half of the patients in this cohort had no viable virus after 5 days and only two patients had replicative virus after 15 days. This could indicate that the current CDC recommendations of an isolation period of 20 days for immunocompromised patients infected with SARS-CoV-2 could be reduced, but larger studies are needed to estimate the isolation duration for immunocompromised patients.

BQ.1.1, XBB.1, and XBB.1.5 neutralization after bivalent mRNA COVID-19 booster in patients with cancer.

Ehmsen et al. evaluate the neutralizing capacity to current SARS-CoV-2 variants in patients with cancer before and after receiving the BNT162b2 bivalent mRNA vaccine booster. Bivalent vaccine provides some protection against BQ.1.1 but fails to protect against XBB.1 and XBB.1.5 in patients with cancer.

Omicron BA.5 Neutralization among Vaccine-Boosted Persons with Prior Omicron BA.1/BA.2 Infections.

Worldwide, millions of persons have received multiple COVID-19 vaccinations and subsequently recovered from SARS-CoV-2 Omicron breakthrough infections. In 2 small, matched cohorts (n = 12, n = 24) in Denmark, we found Omicron BA.1/BA.2 breakthrough infection after 3-dose BNT162b2 vaccination provided improved Omicron BA.5 neutralization over 3-dose vaccination alone.

Serum Neutralization of SARS-CoV-2 Omicron BA.1 and BA.2 after BNT162b2 Booster Vaccination.

The SARS-CoV-2 Omicron variant BA.2 sublineage is rapidly replacing earlier Omicron lineages, suggesting BA.2 has increased vaccine evasion properties. We measured neutralization titers of authentic BA.1 and BA.2 isolates in serum samples from persons who received the BNT162b2 booster vaccine. All samples neutralized BA.1 and BA.2 at equal median values.

A Method for Quantification of Epithelium Colonization Capacity by Pathogenic Bacteria.

Most bacterial infections initiate at the mucosal epithelium lining the gastrointestinal, respiratory, and urogenital tracts. At these sites, bacterial pathogens must adhere and increase in numbers to effectively breach the outer barrier and invade the host. If the bacterium succeeds in reaching the bloodstream, effective dissemination again requires that bacteria in the blood, reestablish contact to distant endothelium sites and form secondary site foci. The infectious potential of bacteria is therefore closely linked to their ability to adhere to, colonize, and invade epithelial and endothelial surfaces. Measurement of bacterial adhesion to epithelial cells is therefore standard procedure in studies of bacterial virulence. Traditionally, such measurements have been conducted with microtiter plate cell cultures to which bacteria are added, followed by washing procedures and final quantification of retained bacteria by agar plating. This approach is fast and straightforward, but yields only a rough estimate of the adhesive properties of the bacteria upon contact, and little information on the ability of the bacterium to colonize these surfaces under relevant physiological conditions. Here, we present a method in which epithelia/endothelia are simulated by flow chamber-grown human cell layers, and infection is induced by seeding of pathogenic bacteria on these surfaces under conditions that simulate the physiological microenvironment. Quantification of bacterial adhesion and colonization of the cell layers is then performed by in situ time-lapse fluorescence microscopy and automatic detection of bacterial surface coverage. The method is demonstrated in three different infection models, simulating Staphylococcus aureus endothelial infection and Escherichia coli intestinal- and uroepithelial infection. The approach yields valuable information on the fitness of the bacterium to successfully adhere to and colonize epithelial surfaces and can be used to evaluate the influence of specific virulence genes, growth conditions, and antimicrobial treatment on this process.

First clinical description of Eggerthia catenaformis bacteremia in a patient with dental abscess.

We present a case of Eggerthia catenaformis bacteremia originating from a dental abscess and imitating necrotizing fasciitis in a previously healthy adult. The isolates were easily identified by MALDI-TOF MS. The clinical course, surgical and antibiotic treatment as well as the successful outcome are reported.

Species differentiation of Bacteroides dorei from Bacteroides vulgatus and Bacteroides ovatus from Bacteroides xylanisolvens - back to basics.

We present the results from 16S sequencing and phenotypic tests for differentiation of Bacteroides dorei from Bacteroides vulgatus and Bacteroides ovatus from Bacteroides xylanisolvens, which was not possible with MALDI-TOF MS. Testing with ß-glucosidase could differentiate B. dorei from B. vulgatus and a negative catalase reaction could identify B. xylanisolvens.