HIV-1 RNA monitoring with a dual-target diagnostic assay: A case report.

In a restricted subset of people living with HIV-1 (PLWH) on antiretroviral therapy (ART) with persistent suppressed viral load (i.e., pol-based HIV-RNA repeatedly undetected), a dual-target (pol and LTR) diagnostic assay for HIV-RNA monitoring can measure quantifiable levels of viral loads (VL) above 30 copies/mL exclusively through the amplification of the LTR region, while the pol target results undetected. We report a patient who shows high levels of HIV-RNA detected exclusively through amplification of the LTR region while undetected by the pol region, during a long monitoring period, from 2018 to date. In this follow-up, the ART was modified without reaching LTR-based undetected HIV-RNA values. Immunological and virological parameters remained optimal with a progressive and steady gain of the CD4/CD8 ratio. The clinical history of this patient, shows that LTR-based viremia above 50 copies/mL can be found occasionally or persistently in the plasma of PLWH under suppressive ART, even at high levels. Based on previous studies, VL detected and quantified exclusively through the amplification of the LTR region corresponds to partial or incomplete HIV-RNA transcripts, which cannot trigger new infections. Interestingly, changes in ART do not eliminate repeated findings of these unusual viral elements.

Autochthonous Dengue Fever in 2 Patients, Rome, Italy.

Since August 2023, outbreaks of dengue virus (DENV) infection have occurred in Italy. We report 2 autochthonous case-patients and their extended follow-up. Despite persistent DENV detected in blood by PCR, results for antigenomic DENV RNA were negative after day 5, suggesting that a 5-day isolation period is adequate to avoid secondary cases.

In Vitro and In Vivo Crosstalk between Type I IFN and IL-8 Responses in SARS-CoV-2 Infection.

COVID-19 patients show characteristic over-expression of different cytokines that may interfere with the interferon (IFN) response, delaying its production. Within the overexpressed cytokines, IL-8 plays a key role, and it may impede IFN-I activation. PBMC from eight healthy donors were exposed to 2019-nCoV/Italy-INMI1 isolate and supernatants/cells were collected at different time points; the production of either IFN-alpha or IL-8 was assessed. The same analysis was performed on plasma samples obtained from 87 COVID-19 patients. Antagonism between IFN-alpha and IL-8 was observed, since in those PBMC with medium or high IL-8 levels, IFN-α levels were low. The same scenario was observed in SARS-CoV-2-infected patients that were divided into three groups based on IL-8 low, medium and high levels; the correlation between low levels of IFN-α and high levels of IL-8 was statistically significant in both the IL-8 medium and IL-8 high group. Overall, our results showed a crosstalk/antagonism between IL-8 and IFN-alpha in PBMC from healthy donors challenged with SARS-CoV-2 and inversely proportional IFN-alpha levels to IL-8 concentrations detected in plasma samples from COVID-19 patients, suggesting that the impairment of the innate immune response in COVID-19 patients may be linked to a dysregulated cytokine response, namely through IL-8 production.

Virus-Encoded Circular RNAs: Role and Significance in Viral Infections.

Circular RNAs (circRNAs) have been the focus of intense scientific research to understand their biogenesis, mechanisms of action and regulatory functions. CircRNAs are single stranded, covalently closed RNA molecules lacking the 5'-terminal cap and the 3'-terminal polyadenine chain, characteristics that make them very stable and resistant. Synthesised by both cells and viruses, in the past circRNAs were considered to have no precise function. Today, increasing evidence shows that circRNAs are ubiquitous, some of them are tissue- and cell-specific, and critical in multiple regulatory processes (i.e., infections, inflammation, oncogenesis, gene expression). Moreover, circRNAs are emerging as important biomarkers of viral infection and disease progression. In this review, we provided an updated overview of current understanding of virus-encoded and cellular-encoded circRNAs and their involvement in cellular pathways during viral infection.

Co-Circulation of SARS-CoV-2 and Other Respiratory Pathogens in Upper and Lower Respiratory Tracts during Influenza Season 2022-2023 in Lazio Region.

Lower respiratory tract infections (LRTIs) occur when there is a lower airway tract infection. They are well-known for increasing the susceptibility of patients to bacterial/fungal co-infections and super-infections. In this study, we present the results of our investigation, which involved 381 consecutive patients admitted to our hospital during the Influenza season from October 2022 to April 2023. Among the 381 specimens, 75 were bronchoalveolar (BAL), and 306 were nasopharyngeal swabs (NPSs). Notably, 34.4% of the examined samples tested positive for SARS-CoV-2. Of these, we observed that 7.96% of NPSs showed positivity only for other respiratory viruses, while a substantial percentage (77%) of BAL specimens exhibited positive results only for bacterial co-infections. The results of our study not only confirm the importance of co-infections in COVID-19 but also emphasize the significance of utilizing rapid diagnostic tests (RDTs) for the timely diagnosis of LRTIs. In fact, RDTs allow for the identification of multiple pathogens, providing clinicians with useful and timely information to establish effective therapy.

Virological characterization of HIV-1 RNA elements detected exclusively through the LTR region by the dual-target Aptima HIV-1 Quant Dx assay in a subset of positive patients.

BACKGROUND: In a restricted subset of HIV patients with suppressed viral load (i.e., pol-undetected HIV-RNA), the Aptima HIV-1 Quant Dx Assay (Aptima), a dual-target (pol and LTR) and dual-probe test for viral load (VL) monitoring, can detect HIV-RNA exclusively through amplification of the LTR region. OBJECTIVES: To analyze the virological characteristics of the HIV-RNA elements detected only through LTR amplification (LTR-e). STUDY DESIGN: LTR-e isolated from plasma and peripheral blood mononuclear cells (PBMC) were evaluated for their ability to trigger productive infections. Viral pellets morphology and ultrastructural characteristics of PBMC from LTR-e patients were examined by electron microscopy. Plasma LTR-e underwent Sanger sequencing. Exosomes were examined with Aptima for LTR-e content. RESULTS: In-vitro, LTR-e could not infect PBMC, induce cytopathic effects, or cause syncytia, even at high VL (e.g., >10,000 copies/mL). Under the electron microscope, plasma pellets and PBMC from patients with LTR-e showed atypical vesicles. Sanger sequencing of LTR-e yielded no results. Moreover, in plasma samples, LTR-e were associated with cell debris, never with exosomes. CONCLUSIONS: Differently from other dual-target but single-probe assays, Aptima unveils VL based only on LTR amplification in some HIV patients. Here, we show that LTR-e represent partial/incomplete/non-canonical transcripts unable to trigger productive infection or transmit HIV-1 infection. The recognition of VL based only on LTR-e in infected individuals is crucial as it allows to avoid inappropriate decisions in the clinical management of HIV patients, such as retesting of VL and switching of ART. Physicians and HIV-RNA dual-target assay manufacturers should consider the important implications of not recognizing this singular type of VL.

HIV-1-Host Interaction in Gut-Associated Lymphoid Tissue (GALT): Effects on Local Environment and Comorbidities.

HIV-1 replication in the gastrointestinal (GI) tract causes severe CD4+ T-cell depletion and disruption of the protective epithelial barrier in the intestinal mucosa, causing microbial translocation, the main driver of inflammation and immune activation, even in people living with HIV (PLWH) taking antiretroviral drug therapy. The higher levels of HIV DNA in the gut compared to the blood highlight the importance of the gut as a viral reservoir. CD4+ T-cell subsets in the gut differ in phenotypic characteristics and differentiation status from the ones in other tissues or in peripheral blood, and little is still known about the mechanisms by which the persistence of HIV is maintained at this anatomical site. This review aims to describe the interaction with key subsets of CD4+ T cells in the intestinal mucosa targeted by HIV-1 and the role of gut microbiome and its metabolites in HIV-associated systemic inflammation and immune activation that are crucial in the pathogenesis of HIV infection and related comorbidities.

Comparison of SARS-CoV-2 Detection in Nasopharyngeal Swab and Saliva Samples from Patients Infected with Omicron Variant.

To compare the detection of the SARS-CoV-2 Omicron variant in nasopharyngeal-swab (NPS) and oral saliva samples. 255 samples were obtained from 85 Omicron-infected patients. SARS-CoV-2 load was measured in the NPS and saliva samples by using Simplexa™ COVID-19 direct and Alinity m SARS-CoV-2 AMP assays. Results obtained with the two diagnostic platforms showed very good inter-assay concordance (91.4 and 82.4% for saliva and NPS samples, respectively) and a significant correlation among cycle threshold (Ct) values. Both platforms revealed a highly significant correlation among Ct obtained in the two matrices. Although the median Ct value was lower in NPS than in saliva samples, the Ct drop was comparable in size for both types of samples after 7 days of antiviral treatment of the Omicron-infected patients. Our result demonstrates that the detection of the SARS-CoV-2 Omicron variant is not influenced by the type of sample used for PCR analysis, and that saliva can be used as an alternative specimen for detection and follow-up of Omicron-infected patients.

Intact provirus and integration sites analysis in acute HIV-1 infection and changes after one year of early antiviral therapy.

Background and objectives: HIV-1 provirus integration in host genomes provides a lifelong reservoir of virally infected cells. Although not able to generate viral progeny, the expression of defective proviruses has been associated with activation. Provirus integration may influence host gene transcription and shifts may occur during disease progression or antiretroviral therapy (ART). The study aimed to analyze intact/defective provirus and sites of provirus integration in acute infections: changes after 48 weeks of early therapy were also evaluated. Methods: DNA from peripheral blood lymphomonocytes of 8 acute HIV-1 infections at serodiagnosis (T0) and after 48 weeks of therapy (T1) was used to quantify intact and defective provirus by digital-droplet PCR and to analyze provirus integration sites, by next-generation sequencing of libraries derived from ligation-mediated PCR. Results: A high variability in the amount of intact proviral DNA was observed at both T0 and T1, in the different subjects. Although the ratio of intact/total proviral HIV-1 DNA did not dramatically change between T0 (8.05%) and T1 (9.34%), after early therapy both intact and total HIV-1 DNA declined significantly, p = 0.047 and p = 0.008, respectively. The median number of different (IQR) integration sites in human chromosomes/subject was 5 (2.25-13.00) at T0 and 4 (3.00-6.75) at T1. Of all the integration sites observed at T1, 64% were already present at T0. Provirus integration was observed in introns of transcriptionally active genes. Some sites of integration, among which the most represented was in the neuregulin 2 gene, were shared by different patients, together with the orientation of the insertion. Provirus integration was also observed in intergenic regions, with median (IQR) % of 15.13 (6.81-21.40) at T0 and 18.46 (8.98-22.18) at T1 of all read matches. Conclusions: In acute HIV-1 infection, the amount of intact proviral DNA in peripheral lymphomonocytes did not exceed 10% of total HIV-1 DNA, a percentage that was not substantially changed by early administrated ART. Provirus displayed a relatively small number of recurrent integration sites in introns of transcriptionally active genes, mainly related to cell-cycle control. Consideration should be given to therapeutic strategies able to target the cells harboring defective proviruses, that are not reached by conventional antiviral drugs, these potentially also impacting on replicative competent integrated provirus.

Evaluating the Dual-Target Aptima HIV-1 Quant Dx Assay: Comparison between Viral Loads Measured with pol and LTR Targets in the Same Samples.

For effective management of HIV-1 patients, accurate measurement of HIV-1-RNA viral load (VL) is fundamental. The latest generation molecular assays for monitoring VL perform simultaneous detection of two regions of the viral genome, but without specifying the target used for VL quantitation. By using the "open" software (research use only [RUO]) of Aptima HIV-1 Quant Dx Assay (Aptima) which provides both results obtained with the pol and LTR targets, we were able to compare n = 500 plasma samples results from chronically HIV-1-infected patients under antiretroviral therapy (ART). Correlation and concordance were analyzed. By stratifying VL into two groups (<30 and ≥30 copies/mL HIV-1-RNA) according to pol-based results and matching them with their respective LTR values, concordance was substantial (κ = 0.635; 95%CI = 0.569 to 0.700) as expected. Considering the specimens (n = 224) with VL exactly quantified (i.e., ≥30 copies/mL) with both targets, an optimal correlation subsisted (r = 0.8882; P < 0.0001) and Bland-Altman plot showed no significant mean difference between them. However, by stratifying all these data in three ranges (30 to 200, 201 to 1,000, and >1,000 copies/mL) according to pol-based results, concordance analysis showed fair agreement (κ = 0.344; 95%CI = 0.257 to 0.432). Indeed, after excluding mutually concordant VL values in each range (n = 134), the remaining discordant samples (n = 90; 40.1%) showed significant (P < 0.05) difference between VL measured with the two targets. With the Aptima "open" software, samples with pol-based VL <1,000 copies (cp)/mL HIV-1-RNA, the corresponding LTR values were on average 0.5 log10 cp/mL higher. Further studies on these discrepancies and the nature of viral RNA elements detected only with the LTR despite efficient ART are in progress. IMPORTANCE The last generation dual-target platforms for quantification of HIV-1 RNA return a single value of viral load (VL) derived from a combined reading of two HIV-1 genome targets. By using a modified version of Aptima software, providing both the VL results obtained from pol and LTR amplification separately, we observed discordant VL results in some samples from HIV-1-infected patients on antiretroviral therapy. In particular, some samples with pol-based quantified <1,000 copies/mL VL showed the LTR-based value on average 0.5 log10 copies/mL higher, and other samples, always by treated patients, showed VL exclusively quantified with LTR target while the corresponding pol-based VL results were completely undetected. Standard software of double-target based diagnostic systems does not allow recognizing discrepant VL values in these particular, but not rare, clinical specimens. This issue could have implications for clinical management by leading physicians to consider changing antiretroviral regimen based on presumed failure of antiretroviral therapy.

Monitoring of SARS-CoV-2 circulation using saliva testing in school children in Rome, Italy.

OBJECTIVES: To describe the trend of SARS-CoV-2 RNA in saliva samples from children attending nine schools in Rome in the local surveillance unit RM3 during the period of September 2021-March 2022, in parallel with the trend of SARS-CoV-2 RNA observed in nasopharyngeal swabs (NPSs) from the population in the same catchment area that was routinely tested at our laboratory in the same period. METHODS: Saliva samples were collected using the Copan LolliSpongeTM device and analyzed by Aptima® SARS-CoV-2 Assay on the Panther® System. NPSs were tested using either Aptima® SARS-CoV-2 Assay or Alinity m SARS-CoV-2 Assay. RESULTS: The percentage of positivity in the two populations was different; of the 2222 saliva samples from students, 0.99% had positive results, whereas the percentage was higher (33.43%) in the 8994 NPSs representing the population from local surveillance unit RM3. Interestingly, the trend of SARS-CoV-2 RNA in saliva samples from students was consistent with that observed in NPSs from the population in same catchment area, although with peaks slightly anticipated. CONCLUSION: Overall, screening of saliva in the schools represents a good system to monitor SARS-CoV-2 circulation in the population, allowing early detection and quick isolation of students who are asymptomatic with positive test results and thus prevention of virus transmission.

Longitudinal dynamics of SARS-CoV-2 anti-receptor binding domain IgG antibodies in a wide population of health care workers after BNT162b2 vaccination.

OBJECTIVES: With the availability of vaccines, commercial assays detecting anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies evolved toward quantitative assays directed to the spike glycoprotein or its receptor-binding domain (RBD). The objective was to perform a large-scale, longitudinal study involving health care workers (HCWs), with the aim of establishing the kinetics of immune response throughout the 9-month period after receipt of the second dose of the BNT162b2 vaccine. METHODS: Quantitative determination of immunoglobulin (Ig) G antibodies against the RBD of the S1 subunit of the spike protein of SARS-CoV-2 on the Alinity systems. RESULTS: The highest levels of anti-RBD IgG were measured after 1 month from full vaccination (median: 1432 binding antibody units/ml [BAU/ml]); subsequently, a steep decrease (7.4-fold decrease) in IgG levels was observed at 6 months (median: 194.3 BAU/ml), with a further 2.5-fold decrease at 9 months (median: 79.3 BAU/ml). Furthermore, the same data, when analyzed for sex, showed significant differences between male and female participants at both 1 and 9 months from vaccination, but not at 6 months. CONCLUSION: Our results confirm the tendency of anti-RBD antibodies to decrease over time, also when extending the analysis up to 9 months, and highlight a better ability of the female sex to produce antibodies 1 month and 9 months after vaccination. Overall, these data, obtained in a wide population of HCWs, support the importance of having increased the vaccine doses.

Rapid and qualitative identification of SARS-CoV-2 mutations associated with variants of concern using a multiplex RT-PCR assay coupled with melting analysis.

OBJECTIVES: Considering the spread of new genetic variants and their impact on public health, it is important to have assays that are able to rapidly detect SARS-CoV-2 variants. METHODS: We retrospectively examined 118 positive nasopharyngeal swabs, first characterized by the Sanger sequencing, using the Simplexa® SARS-CoV-2 Variants Direct assay, with the aim of evaluating the performance of the assay to detect N501Y, G496S, Q498R, Y505H, E484K, E484Q, E484A, and L452R mutations. RESULTS: A total of 111/118 nasopharyngeal swabs were in complete agreement with the Sanger sequencing, whereas the remaining seven samples were not amplified due to the low viral load. The evaluation of the ability of the assay to detect the E484Q mutation was performed using a viral isolate of the SARS-CoV-2 Kappa variant, showing concordance in 15/15 samples. Simplexa® SARS-CoV-2 Variant Direct assay was able to detect mutation pattern of Alpha, Beta, Gamma, Delta, and Omicron variants with 100% specificity and 94% sensitivity, whereas 100% sensitivity and specificity for the Kappa variant was observed. CONCLUSION: The assay can be useful to obtain faster results, contributing to a prompt surveillance of SARS-CoV-2 variants; however, it requires to be confirmed by the Sanger method, especially in the case of pattern of mutations that are different from those expected and also requires updates as new variants emerge.

Changes in the Circulation of Common Respiratory Pathogens among Hospitalized Patients with Influenza-like Illnesses in the Lazio Region (Italy) during Fall Season of the Past Three Years.

A descriptive analysis of common respiratory pathogens (CRPs) detected in nasopharyngeal swabs (NPSs) from hospitalized patients with influenza-like illness during the fall seasons of the past three years, 2019-2021, in the Lazio region, Italy, was conducted to assess whether or not CRP circulation changed because of COVID-19 during the fall season. The results observed in a total of 633 NPSs subjected to molecular diagnosis for CRPs by multiplex PCR assay during the autumn seasons (exactly from week 41 to week 50) were compared with each other. In 2019, in 144 NPSs, the more represented CRPs were rhinovirus/enterovirus (7.6%) and influenza A/B (4.2%). In 2020, 55 (21.6%) out of 255 NPSs resulted positive for SARS-CoV-2 and, except for one case of Legionella pneumophila, the CRPs detected were exclusively rhinovirus/enterovirus (4.7%). In 2021, among 234 NPSs, 25.6% resulted positive for SARS-CoV-2, 14.5% for respiratory syncytial virus (RSV), and 12.8% for rhinovirus/enterovirus. Compared with 2019, in 2020, CRP circulation was severely limited to a few cases; in 2021, instead, infections by RSV (detected also among adults), rhinovirus/enterovirus, and other respiratory pathogens were observed again, while influenza was practically absent. The comparison of the CRPs detected in the NPSs depicts a different circulation in the Lazio region during the last three fall seasons. CRP monitoring has a direct impact on the prevention and control strategies of respiratory infectious diseases, such as the non-pharmacological interventions implemented in response to the COVID-19 pandemic. Future studies should investigate the impact of specific interventions on the spread of respiratory infections.

In vitro Evaluation of Antiviral Efficacy of a Standardized Hydroalcoholic Extract of Poplar Type Propolis Against SARS-CoV-2.

Except for specific vaccines and monoclonal antibodies, effective prophylactic or post-exposure therapeutic treatments are currently limited for COVID-19. Propolis, a honeybee's product, has been suggested as a potential candidate for treatment of COVID-19 for its immunomodulatory properties and for its powerful activity against various types of viruses, including common coronaviruses. However, direct evidence regarding the antiviral activities of this product still remains poorly documented. VERO E6 and CALU3 cell lines were infected with SARS-CoV-2 and cultured in the presence of 12.5 or 25 µg/ml of a standardized Hydroalcoholic Extract acronym (sHEP) of Eurasian poplar type propolis and analyzed for viral RNA transcription, for cell damage by optical and electron microscopy, and for virus infectivity by viral titration at 2, 24, 48, and 72 h post-infection. The three main components of sHEP, caffeic acid phenethyl ester, galangin, and pinocembrin, were tested for the antiviral power, either alone or in combination. On both cell lines, sHEP showed significant effects mainly on CALU3 up to 48 h, i.e., some protection from cytopathic effects and consistent reduction of infected cell number, fewer viral particles inside cellular vesicles, reduction of viral titration in supernatants, dramatic drop of N gene negative sense RNA synthesis, and lower concentration of E gene RNA in cell extracts. Interestingly, pre-treatment of cells with sHEP before virus inoculation induced these same effects described previously and was not able to block virus entry. When used in combination, the three main constituents of sHEP showed antiviral activity at the same levels of sHEP. sHEP has a remarkable ability to hinder the replication of SARS-CoV-2, to limit new cycles of infection, and to protect host cells against the cytopathic effect, albeit with rather variable results. However, sHEP do not block the virus entry into the cells. The antiviral activity observed with the three main components of sHEP used in combination highlights that the mechanism underlying the antiviral activity of sHEP is probably the result of a synergistic effect. These data add further emphasis on the possible therapeutic role of this special honeybee's product as an adjuvant to official treatments of COVID-19 patients for its direct antiviral activity.

Differential Dynamics of SARS-CoV-2 Binding and Functional Antibodies upon BNT162b2 Vaccine: A 6-Month Follow-Up.

To investigate the dynamic association among binding and functional antibodies in health-care-workers receiving two doses of BNT162b2 mRNA COVID-19-vaccine, SARS-CoV-2 anti-RBD IgG, anti-Trimeric-S IgG, and neutralizing antibodies (Nabs) were measured in serum samples collected at 2 weeks, 3 months, and 6 months from full vaccination. Despite the high correlation, results for anti-RBD and anti-Trimeric S IgG were numerically different even after recalculation to BAU/mL following WHO standards indications. Moreover, after a peak response at 2 weeks, anti-RBD IgG levels showed a 4.5 and 13 fold decrease at 3 and 6 months, respectively, while the anti-Trimeric S IgG presented a less pronounced decay of 2.8 and 4.7 fold. Further different dynamics were observed for Nabs titers, resulting comparable at 3 and 6 months from vaccination. We also demonstrated that at NAbs titers ≥40, the area under the receiver operating characteristic curve and the optimal cutoff point decreased with time from vaccination for both anti-RBD and anti-Trimeric S IgG. The mutating relation among the anti-RBD IgG, anti-Trimeric S IgG, and neutralizing antibodies are indicative of antibody maturation upon vaccination. The lack of standardized laboratory procedures is one factor interfering with the definition of a correlate of protection from COVID-19.

Comparison of Allplex™ SARS-CoV-2 Assay, Easy SARS-CoV-2 WE and Lumipulse quantitative SARS-CoV-2 antigen test performance using automated systems for the diagnosis of COVID-19.

Diagnostic methods based on SARS-CoV-2 antigen detection are a promising alternative to SARS-CoV-2 RNA amplification. We evaluated the automated chemiluminescence-based Lumipulse® G SARS-CoV-2 Ag assay as compared to real time assays (combined results from RT-PCR Allplex™ SARS-CoV-2 assay and Easy SARS-CoV-2 WE kit) on 513 nasopharyngeal swabs (NPS). Among these, 53.6% resulted positive to RT-PCR, considered as the reference test. Compared to the reference test, overall sensitivity and specificity of Lumipulse® G SARS-CoV-2 Ag assay were 84.0%, and 89.1%, respectively, and overall agreement between the antigen and molecular assays was substantial (κ = 0.727). When stratifying samples into groups based on ranges of RT-PCR cycle threshold (Ct), the antigen test sensitivity was >95% for samples with Ct <30. Linear regression analysis showed strong and highly significant correlation between the Lumipulse Ag concentrations and the RT-PCR Ct values (RdRp gene), irrespective of whether the Ct values from molecular test were combined in a unique regression analysis or analysed separately. Overall, chemiluminescence-based antigen assay may be reliably applied to NPS samples to identify individuals with high viral loads, more likely to transmit the virus.