Combination regimen of nirmatrelvir/ritonavir and molnupiravir for the treatment of persistent SARS-CoV-2 infection: A case report and a scoping review of the literature.

Immunocompromised patients still experience unpredictable courses of COVID-19, despite that effective vaccines and drugs against SARS-CoV-2 are now available. Antiviral combination regimens may have a role in SARS-CoV-2 infection in immunocompromised hosts, but current knowledge is still limited. We describe the case of a 73-year-old Italian man affected by follicular lymphoma with persistent SARS-CoV-2 infection who was successfully treated with co-administration of oral antivirals (10-day molnupiravir and nirmatrelvir/ritonavir). The therapy was well tolerated both from a clinical and biochemical standpoint, with no signs of toxicity. We also performed a scoping review, to sum up available knowledge on combined antiviral regimens including remdesivir, molnupiravir, or nirmatrelvir/ritonavir. Pending further studies on larger cohorts of patients, our report is consistent with available pre-clinical and clinical data, supporting the possible use of combination therapy in selected difficult-to-treat COVID-19 cases.

Fourth Dose of mRNA COVID-19 Vaccine Transiently Reactivates Spike-Specific Immunological Memory in People Living with HIV (PLWH).

Background: People Living With HIV (PLWH), with advanced disease, lower CD4+ T cell counts or an unsuppressed HIV viral load can have a suboptimal vaccine response. For this reason, in the current COVID-19 pandemic, they represent a prioritized population for the SARS-CoV-2 fourth (or second booster) vaccine dose. This work aims to investigate the effects of a second booster on the reactivation of the spike-specific humoral and cell-mediated immune responses in PLWH. Methods: A total of eight PLWH, who received a fourth dose of the original mRNA vaccines were enrolled. They were evaluated before and then 7 days, 1 month and 2 months after the injection. The humoral response was assessed via a chemiluminescent immunoassay. Immunophenotyping and the functional evaluation of the SARS-CoV-2-specific cellular immune responses were performed via flow cytometry. Results: Anti-spike IgG levels were above the cut-off value for all subjects at all timepoints. The spike-specific CD4+ T cell response was reactivated one week after the fourth vaccine dose, and on average declined at two months post-vaccination. A similar trend was observed for the spike-specific B cells. A low percentage of spike-specific CD4+ T cells was activated by the B.1.1.529 BA.1 Omicron-spike mutated peptides, and the majority of these cells were reactive to the conserved portions of the spike protein. Similarly, the majority of the spike-specific memory B cells were able to bind both Wuhan and Omicron-spike entire protein. Conclusions: Spike-specific adaptive immune responses are transiently reactivated in PLWH following the fourth mRNA vaccine dose. The breadth of the immune responses to the mutated spike protein provides insight on the possible cross-reactivity for the SARS-CoV-2 variants of concern (VOCs).

Antiviral Activity of Oligonucleotides Targeting the SARS-CoV-2 Genomic RNA Stem-Loop Sequences within the 3'-End of the ORF1b.

Increased evidence shows vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibited no long-term efficacy and limited worldwide availability, while existing antivirals and treatment options have only limited efficacy. In this study, the main objective was the development of antiviral strategies using nucleic acid-based molecules. To this purpose, partially overlapped 6-19-mer phosphorothioate deoxyoligonucleotides (S-ONs) designed on the SARS-CoV-2 genomic RNA stem-loop packaging sequences within the 3' end of the ORF1b were synthetized using the direct and complementary sequence. Among the S-ONs tested, several oligonucleotides exhibited a fifty percent inhibitory concentration antiviral activity ranging from 0.27 to 34 µM, in the absence of cytotoxicity. The S-ON with a scrambled sequence used in the same conditions was not active. Moreover, selected 10-mer S-ONs were tested using different infectious doses and against different SARS-CoV-2 variants, showing comparable antiviral activity that was abrogated when the central sequence was mutated. Experiments to evaluate the intracellular functional target localization of the S-ON inhibitory activity were also performed. Collectively the data indicate that the SARS-CoV-2 packaging region in the 3' end of the ORF1b may be a promising target candidate for further investigation to develop innovative nucleic-acid-based antiviral therapy.

Rapid diagnostic tests in the management of pneumonia.

INTRODUCTION: Pneumonia is one of the main causes of mortality associated with infectious diseases worldwide. Several challenges have been identified in the management of patients with pneumonia, ranging from accurate and cost-effective microbiological investigations, prompt and adequate therapeutic management, and optimal treatment duration. AREAS COVERED: In this review, an updated summary on the current management of pneumonia patients is provided and the epidemiological issues of infectious respiratory diseases, which in the current pandemic situation are of particular concern, are addressed. The clinical and microbiological approaches to pneumonia diagnosis are reviewed, including discussion about the new molecular assays pointing out both their strengths and limitations. Finally, the current recommendations about antibiotic treatment are examined and discussed depending on the epidemiological contexts, including those with high prevalence of multidrug-resistant bacteria. EXPERT OPINION: We claim that rapid diagnostic tests, if well-positioned in the diagnostic workflow and reserved for the subset of patients who could most benefit from these technologies, may represent an interesting and feasible tool to optimize timing of targeted treatments especially in terms of early de-escalation or discontinuation of antibiotic therapy.

Clinical and Immunological Features of SARS-CoV-2 Breakthrough Infections in Vaccinated Individuals Requiring Hospitalization.

BACKGROUND AND PURPOSE: Waning immunity and the surge of SARS-CoV-2 variants are responsible for breakthrough infections, i.e., infections in fully vaccinated individuals. Although the majority of vaccinated infected subjects report mild or no symptoms, some others require hospitalization. The clinical and immunological features of vaccinated hospitalized COVID-19 patients are currently unknown. METHODS: Twenty-nine unvaccinated and 36 vaccinated hospitalized COVID-19 patients were prospectively enrolled and clinical and laboratory data were gathered. Immunophenotyping of leukocytes' subsets, T and B cell SARS-CoV-2-specific responses were evaluated via flow cytometry. Anti-IFN-α autoantibodies were measured via ELISA. RESULTS: Despite vaccinated patients were older and with more comorbidities, unvaccinated subjects showed higher levels of pro-inflammatory markers, more severe disease, and increased mortality rate. Accordingly, they presented significant alterations in the circulating leukocyte composition, typical of severe COVID-19. Vaccinated patients displayed higher levels of anti-Spike IgGs and Spike-specific B cells. Of all participants, survivors showed higher levels of anti-Spike IgGs and Spike-specific CD4+ T cells than non-survivors. At hospital admission, 6 out of 65 patients (9.2%) displayed high serum concentrations of autoantibodies targeting IFN-α. Remarkably, 3 were unvaccinated and eventually died, while the other 3 were vaccinated and survived. CONCLUSION: Despite more severe pre-existing clinical conditions, vaccinated patients have good outcome. A rapid activation of anti-SARS-CoV-2-specific immunity is fundamental for the resolution of the infection. Therefore, prior immunization through vaccination provides a significant contribution to prevention of disease worsening and can even overcome the presence of high-risk factors (i.e., older age, comorbidities, anti-IFN-α autoantibodies).

Long-term SARS-CoV-2 Asymptomatic Carriage in an Immunocompromised Host: Clinical, Immunological, and Virological Implications.

PURPOSE: SARS-CoV-2 infection in immunocompromised hosts is challenging, and prolonged viral shedding can be a common complication in these patients. We describe the clinical, immunological, and virological course of a patient with eosinophilic granulomatosis with polyangiitis, who developed the status of long-term asymptomatic SARS-CoV-2 carrier for more than 7 months. METHODS: Over the study period, the patient underwent 20 RT-PCR tests for SARS-CoV-2 detection on nasopharyngeal swabs. In addition, viral cultures and genetic investigation of SARS-CoV-2 were performed. As for immunological assessment, serological and specific T-cell testing was provided at different time points. RESULTS: Despite the patient showing a deep drug-induced B and T adaptive immunity impairment, he did not experience COVID-19 progression to severe complications, and the infection remained asymptomatic during the follow-up period, but he was not able to achieve viral clearance for more than 7 months. The infection was finally cleared by SARS-CoV-2-specific monoclonal antibody treatment, after that remdesivir and convalescent plasma failed in this scope. The genetic investigations evidenced that the infection was sustained by multiple viral subpopulations that had apparently evolved intra-host during the infection. CONCLUSION: Our case suggests that people with highly impaired B- and T-cell adaptive immunity can prevent COVID-19 progression to severe complications, but they may not be able to clear SARS-CoV-2 infection. Immunocompromised hosts with a long-term infection may play a role in the emergence of viral variants.

A rapid and cost-effective diagnostic algorithm for the detection of SARS-CoV-2 infection in the emergency area by combining highly sensitive antigenic test and RT-PCR.

A diagnostic algorithm for SARS-CoV-2 infection in patients admitted to the emergency area, based on a combination of rapid antigen and molecular testing, has been evaluated with 3070 nasopharyngeal swabs. Compared to molecular test alone, the proposed algorithm allowed to significantly reduce costs and average time to results.

Implementing Early Phase Treatments for COVID-19 in Outpatient Settings: Challenges at a Tertiary Care Center in Italy and Future Outlooks.

We present a brief commentary illustrating the current COVID-19 outpatient treatment options in Italy. We also report our experience setting up a service dedicated to these patients in the wake of the rise in COVID-19 cases observed in January 2022. We also gathered data on the daily costs faced by our outpatient service, based at a tertiary care center located in Florence, Italy. We present them with some considerations on future outlooks on the use of outpatient treatment in COVID-19.

First-dose mRNA vaccination is sufficient to reactivate immunological memory to SARS-CoV-2 in subjects who have recovered from COVID-19.

The characterization of the adaptive immune response to COVID-19 vaccination in individuals who recovered from SARS-CoV-2 infection may define current and future clinical practice. To determine the effect of the 2-dose BNT162b2 mRNA COVID-19 vaccination schedule in individuals who recovered from COVID-19 (COVID-19-recovered subjects) compared with naive subjects, we evaluated SARS-CoV-2 Spike-specific T and B cell responses, as well as specific IgA, IgG, IgM, and neutralizing antibodies titers in 22 individuals who received the BNT162b2 mRNA COVID-19 vaccine, 11 of whom had a previous history of SARS-CoV-2 infection. Evaluations were performed before vaccination and then weekly until 7 days after second injection. Data obtained clearly showed that one vaccine dose is sufficient to increase both cellular and humoral immune response in COVID-19-recovered subjects without any additional improvement after the second dose. On the contrary, the second dose proved mandatory in naive subjects to further enhance the immune response. These findings were further confirmed at the serological level in a larger cohort of naive (n = 68) and COVID-19-recovered (n = 29) subjects, tested up to 50 days after vaccination. These results question whether a second vaccine injection in COVID-19-recovered subjects is required, and indicate that millions of vaccine doses may be redirected to naive individuals, thus shortening the time to reach herd immunity.

Assessment of the feasibility of pool testing for SARS-CoV-2 infection screening.

BACKGROUND: SARS-CoV-2 pandemic represented a huge challenge for national health systems worldwide. Pooling nasopharyngeal (NP) swabs seems to be a promising strategy, saving time and resources, but it could reduce the sensitivity of the RT-PCR and exacerbate samples management in terms of automation and tracing. In this study, taking advantage of the routine implementation of a screening plan on health workers, we evaluated the feasibility of pool testing for SARS-CoV-2 infection diagnosis in the presence of low viral load samples. METHOD: Pools were prepared with an automated instrument, mixing 4, 6 or 20 NP specimens, including one, two or none positive samples. Ct values of positive samples were on average about 35 for the four genes analyzed. RESULTS: The overall sensitivity of 4-samples and 6-samples pools was 93.1 and 90.0%, respectively. Focussing on pools including one sample with Ct value ≥35 for all analyzed genes, sensitivity decreased to 77.8 and 75.0% for 4- and 6-samples, respectively; pools including two positive samples, resulted positive in any size as well as pools including positive samples with Ct values <35. CONCLUSION: Pool testing strategy should account the balance between cost-effectiveness, dilution effect and prevalence of the infection. Our study demonstrated the good performances in terms of sensitivity and saving resources of pool testing mixing 4 or 6 samples, even including low viral load specimens, in a real screening context possibly affected by prevalence fluctuation. In conclusion, pool testing strategy represents an efficient and resources saving surveillance and tracing tool, especially in specific context like schools, even for monitoring changes in prevalence associated to vaccination campaign.

SARS-CoV-2 infection and vaccination trigger long-lived B and CD4+ T lymphocytes with implications for booster strategies.

BACKGROUNDImmunization against SARS-CoV-2, the causative agent of COVID-19, occurs via natural infection or vaccination. However, it is currently unknown how long infection- or vaccination-induced immunological memory will last.METHODSWe performed a longitudinal evaluation of immunological memory to SARS-CoV-2 up to 1 year after infection and following mRNA vaccination in naive individuals and individuals recovered from COVID-19 infection.RESULTSWe found that memory cells are still detectable 8 months after vaccination, while antibody levels decline significantly, especially in naive individuals. We also found that a booster injection is efficacious in reactivating immunological memory to spike protein in naive individuals, whereas it was ineffective in previously SARS-CoV-2-infected individuals. Finally, we observed a similar kinetics of decay of humoral and cellular immunity to SARS-CoV-2 up to 1 year following natural infection in a cohort of unvaccinated individuals.CONCLUSIONShort-term persistence of humoral immunity, together with the reduced neutralization capacity versus the currently prevailing SARS-CoV-2 variants, may account for reinfections and breakthrough infections. Long-lived memory B and CD4+ T cells may protect from severe disease development. In naive individuals, a booster dose restored optimal anti-spike immunity, whereas the needs for vaccinated individuals who have recovered from COVID-19 have yet to be defined.FUNDINGThis study was supported by funds to the Department of Experimental and Clinical Medicine, University of Florence (Project Excellence Departments 2018-2022), the University of Florence (project RICTD2122), the Italian Ministry of Health (COVID-2020-12371849), and the region of Tuscany (TagSARS CoV 2).

SARS-CoV-2 Spike-Specific CD4+ T Cell Response Is Conserved Against Variants of Concern, Including Omicron

Although accumulating data have investigated the effect of SARS-CoV-2 mutations on antibody neutralizing activity, less is known about T cell immunity. In this work, we found that the ancestral (Wuhan strain) Spike protein can efficaciously reactivate CD4+ T cell memory in subjects with previous Alpha variant infection. This finding has practical implications, as in many countries only one vaccine dose is currently administered to individuals with previous COVID-19, independently of which SARS-CoV-2 variant was responsible of the infection. We also found that only a minority of Spike-specific CD4+ T cells targets regions mutated in Alpha, Beta and Delta variants, both after natural infection and vaccination. Finally, we found that the vast majority of Spike-specific CD4+ T cell memory response induced by natural infection or mRNA vaccination is conserved also against Omicron variant. This is of importance, as this newly emerged strain is responsible for a sudden rise in COVID-19 cases worldwide due to its increased transmissibility and ability to evade antibody neutralization. Collectively, these observations suggest that most of the memory CD4+ T cell response is conserved against SARS-CoV-2 variants of concern, providing an efficacious line of defense that can protect from the development of severe forms of COVID-19.

Evaluation of extraction-free RT-PCR methods for faster and cheaper detection of SARS-CoV-2 using two commercial systems.

OBJECTIVE: When using high-throughput batched diagnostic platforms based on RT-PCR for SARS-CoV-2 detection, avoidance of the conventional nucleic acid extraction step can help to reduce the turnaround time and increase processivity. This approach can also spare reagents and plasticware, which have experienced a shortage during the initial waves of the pandemic, reducing the overall testing costs. METHODS: This study evaluated the performance of extraction-free protocols based on simple dilution of the specimen in sterile RNAse free water (with or without a heating step) in comparison to standard RNA extraction protocols, using two commercial kits for molecular detection of SARS-CoV-2 (Allplex™ SARS-CoV-2 assay and Allplex™ SARS-CoV-2/FluA/FluB/RSV assay) in nasopharyngeal swabs (NPS). RESULTS: Compared with conventional protocols, extraction-free protocols based on sample dilution without a heating step exhibited a lower analytical sensitivity: 74.0% and 82.1% with the Allplex™ SARS-CoV-2 assay (tested with 139 NPS samples) and the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay (tested with 69 NPS samples), with a mean increase of Ct values of +2.04 and +1.32, respectively. Most false negative results were observed with sampled low viral load. Including a step of heat exposure did not improve but actually decreased the analytical sensitivity of the assay. CONCLUSIONS: Results confirmed that extraction-free protocols could be a faster and cheaper approach to SARS-CoV-2 detection in NPS samples, which could improve processivity of diagnostic platforms.

Validation of Two Commercial Multiplex Real-Time PCR Assays for Detection of SARS-CoV-2 in Stool Donors for Fecal Microbiota Transplantation.

Recurrent infection by Clostridioides difficile has recently been treated by fecal microbiota transplantation (FMT). As viable SARS-CoV-2 was recovered from stool of asymptomatic individuals, the FMT procedure could be a potential risk of SARS-CoV-2 transmission, thus underlying the need to reliably detect SARS-CoV-2 in stool. Here, we performed a multicentric study to explore performances of two commercially available assays for detection of SARS-CoV-2 RNA in stool of potential FMT donors. In three hospitals, 180 stool samples were spiked with serial 10-fold dilutions of a SARS-CoV-2 inactivated lysate to evaluate the Seegene Allplex™ SARS-CoV-2 (SC2) and SARS-CoV-2/FluA/FluB/RSV (SC2FABR) Assays for the detection of viral RNA in stool of FMT donors. The results revealed that both assays detected down to 2 TCID50/mL with comparable limit of detection values, SC2 showing more consistent target positivity rate than SC2FABR. Beyond high amplification efficiency, correlation between CT values and log concentrations of inactivated viral lysates showed R2 values ranging from 0.88 to 0.90 and from 0.87 to 0.91 for the SC2 and SC2FABR assay, respectively. The present results demonstrate that both methods are highly reproducible, sensitive, and accurate for SARS-CoV-2 RNA detection in stool, suggesting a potential use in FMT-donor screening.

Male reproductive system inflammation after healing from coronavirus disease 2019.

BACKGROUND: There is evidence that, after severe acute respiratory syndrome coronavirus 2 infection, male reproductive function and semen quality may be damaged OBJECTIVES: To evaluate a panel of inflammatory mediators in semen in patients recovered from coronavirus disease 2019. MATERIAL AND METHODS: Sexually active men with previous severe acute respiratory syndrome coronavirus 2 infection and proven recovery from coronavirus disease 2019 were enrolled in a prospective cohort study. Clinical, uro-andrological data and semen specimens were prospectively collected. For previously hospitalized coronavirus disease 2019 patients, data on serum inflammatory markers were retrospectively collected. RESULTS: A total of 43 men were enrolled in the study. Of these, 32 men were normozoospermic, three were oligozoospermic, and eight were crypto-azoospermic. Serum inflammatory markers (procalcitonin and C-reactive protein) were analyzed in previously hospitalized patients both at admission and at peak of infection. Levels at admission were statistically significantly higher in patients resulting in crypto-azoospermic with respect to those resulting in normozoospermic (p = 0.05; p = 0.03 and p = 0.02, respectively) after healing. Seminal cytokine levels were similar among all groups. Interleukin-1ß and tumor necrosis factor-α levels were significantly negatively related to sperm total number and concentration, whereas interleukin-4 was correlated with sperm motility. DISCUSSION AND CONCLUSION: Negative correlations between interleukin-1ß and tumor necrosis factor-α and sperm number and the overall high levels of semen cytokines indicate a potential detrimental role of severe acute respiratory syndrome coronavirus 2 driven inflammation on spermatogenesis. Overall, our results indicate that male patients recovering from coronavirus disease 2019 deserve accurate follow-up for their fertility status.

AIDS patient with severe T cell depletion achieved control but not clearance of SARS-CoV-2 infection.

A late presenter AIDS patient with severe T cell depletion presented non-severe COVID-19 symptoms, with prolonged viral shedding. Our case report supports the hypothesis that an effective T cell response may be dispensable for the control of COVID-19 progression to severe forms, while it may be necessary for SARS-CoV-2 clearance.

Rapid screening for SARS-CoV-2 VOC-Alpha (202012/01, B.1.1.7) using the Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay.

BACKGROUND: The emergence of SARS-CoV-2 variants of concern (VOCs) for increased transmissibility and being potentially capable of immune-escape mandates for epidemiological surveillance. Genomic alterations present in VOCs can affect the results of RT-qPCR assays for routine diagnostic purposes, leading to peculiar profiles that can be used for rapid screening of variants. This study reports a peculiar profile observed with the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay and VOC-Alpha (202012/01, lineage B.1.1.7, also named VOC-UK), which was the first identified SARS-CoV-2 VOC. METHODS: Samples were analyzed by two RT-qPCR assays: the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay (ASFR, Seegene Technologies Inc; Seoul, South Korea) and the TaqPath COVID-19 RT-PCR (Thermo Fisher Scientific, USA). Definition of the SARS-CoV-2 variant was carried out by Sanger sequencing of the relevant S-gene regions and, in some cases, by whole genome sequencing (WGS) using the ARTIC-nCoV workflow on a MiniION (Oxford Nanopore Technologies, Oxford, UK) or a Illumina MiSeq platform (San Diego, California, USA). RESULTS: Of the 173 SARS-CoV-2-positive specimens, all those of lineage B.1.1.7 (N=71) showed an average Cq difference between the N and S genes of +11±2 (range, +8/+15). None of the other specimens, including several different lineages (Wild-type for the analyzed regions, N=22; Gamma, N=63; Delta, N=9; B.1.258Δ, N=3; B.1.160, N=3; B.1.177.7, N=1; B.1.1.420, N=1), exhibited a similar difference in Cq values. CONCLUSIONS: The peculiar pattern of delayed N gene positivity could constitute a convenient method for VOC-Alpha screening, simultaneous to viral detection, when using the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay.