Evaluation of Two-Assay Serological Testing Strategies for Anti-HCV Screening in Italian Populations: A Dual Screening Approach.

(1) Background: Hepatitis C virus (HCV) screening mostly uses a one-assay anti-HCV testing approach, which has a higher probability of false-positive results in populations with low HCV prevalence. (2) Methods: In this investigation, 17,926 participants were screened for HCV, and the reactives were tested using a two-assay anti-HCV approach: Elecsys ElectroChemiLuminescence (ECL) and a ChemiLuminescence ImmunoAssay (CLIA), respectively. A recombinant immunoblot assay (RIBA) was performed to confirm anti-HCV positivity. Statistical analysis was performed. (3) Results: A total of 350 specimens were reactive in the ECL screening, of which CLIA retesting showed that 292 (83.4%) were anti-HCV reactive (283 positives, 9 indeterminate, none negative by RIBA), but 58 (16.6%) were not anti-HCV reactive (15 positive, 12 indeterminate, 31 negatives by RIBA). The two-assay strategy significantly improved the positive predictive value (PPV: 95.00%) with χ2: 7.59 (p < 0.01) compared to the PPV assessed by one assay (PPV: 90.6%) with χ2: 34.51 (p < 0.001). The ROC curve defined a sensibility and specificity for the dual approach of 99.66% and 100.00%. (4) Conclusions: Compared with a one-assay testing strategy, the two-assay testing strategy may significantly reduce false positives in anti-HCV testing and identify inactive HCV infection in low seroprevalence populations.

The Role of Erythrocyte Sedimentation Rate (ESR) in Myeloproliferative and Lymphoproliferative Diseases: Comparison between DIESSE CUBE 30 TOUCH and Alifax Test 1.

(1) Background: The erythrocyte sedimentation rate (ESR) is widely diffused in hematology laboratories to monitor inflammatory statuses, response to therapies (such as antibiotics), and oncologic diseases. However, ESR is not a specific diagnostic marker but needs to be contextualized and compared with clinical and other laboratory findings. This study aimed to investigate the performance of two automated instruments, namely the DIESSE CUBE 30 TOUCH (DIESSE, Siena, Italy) and the Alifax Test 1 (Alifax Srl, Polverara, Italy), in comparison with the gold standard, the Westergren method, in lymphoproliferative and myeloproliferative patients. (2) Methods: 97 EDTA samples were selected from the hematology department of Roma Tor Vergata Hospital and analyzed. Statistical analysis was applied. (3) A good correlation between CUBE 30 TOUCH and the gold standard was observed in the overall sample (R2 = 0.90), as well as in patients with lymphoproliferative diseases (R2 = 0.90) and myeloproliferative diseases (R2 = 0.90). The correlation between Test 1 and the gold standard was observed in the overall sample (R2 = 0.68), as well as in patients with lymphoproliferative diseases (R2 = 0.79) and myeloproliferative diseases (R2 = 0.53). (4) Conclusions: The CUBE 30 TOUCH appears to be a more trustworthy tool for evaluating ESR in these pathologies.

Validation of the New MINI-CUBE for Clinic Determination of Erythrocyte Sedimentation Rate.

Background: Erythrocyte sedimentation rate (ESR) indirectly measures blood fibrinogen, and it is altered by all those pathological conditions that modify the aggregation of red blood cells. The international guidelines by the International Council for Standardization in Hematology (ICSH) define the Westergren method as the gold standard for ESR, although it is completely operator-dependent, time-consuming, and requires a patient's blood consumption. Therefore, the validation of new ESR analyzers is needed. The aim of this study is the validation of a new automated ESR analyzer, MINI-CUBE (DIESSE, Diagnostica Senese, Italy). Methods: The samples (n = 270) were collected at the University Hospital of the University of Rome Tor Vergata. A comparison between the automated instrument and the gold standard was performed. Statistical analyses were processed by MedCalc software. Results: The comparison analysis performed on the overall samples reported a good agreement, showing a Spearman rank correlation coefficient of 0.94 (P < 0.001), compared to the Westergren test. The Bland-Altman analysis showed a mean bias of 1.5 (maximum (max.):19.6; minimum (min.): -16.6). Inter-run (level 1 coefficient of variation (CV): 4.9%; level 2 CV: 0.8%), intra-run (level 1 CV: 21.1%; level 2 CV: 3.2%), and inter-instrument (level 1 CV: 27.1%; level 2 CV: 5.6%) precision were also assessed. The hematocrit value did not interfere with the analysis: Spearman rank correlation coefficient of 0.929 (P < 0.001); mean bias of 1.3 (max.:18.3; min.: -15.6). Conclusions: Overall results from MINI-CUBE asserted a good correlation rate with the gold standard, and it could be considered an accurate, and objective alternative for the Westergren test.

The Antibodies' Response to SARS-CoV-2 Vaccination: 1-Year Follow Up.

The use of vaccines has allowed the containment of coronavirus disease 2019 (COVID-19) at a global level. The present work aims to add data on vaccination by evaluating the level of neutralizing antibodies in individuals who have received a three-vaccination series. For this purpose, we ran a surveillance program directed at measuring the level of IgG Abs against the Receptor Binding Domain (RBD) and surrogate virus neutralizing Ab (sVNT) anti-SARS-CoV-2 in the serum of individuals undergoing vaccination. This study was performed on employees from the University of Rome Tor Vergata and healthcare workers from the University Hospital who received the Vaxzevria vaccine (n = 56) and Comirnaty vaccine (n = 113), respectively. After the second dose, an increase in both RBD and sVNT Ab values was registered. In individuals who received the Comirnaty vaccine, the antibody titer was about one order of magnitude higher after 6 months from the first dose. All participants in this study received the Comirnaty vaccine as the third dose, which boosted the antibody response. Five months after the third dose, nearly one year from the first injection, the antibody level was >1000 BAU/mL (binding antibody units/mL). According to the values reported in the literature conferring protection against SARS-CoV-2 infection, our data indicate that individuals undergoing three vaccine doses present a low risk of infection.

Saliva as Biomarker for Oral and Chronic Degenerative Non-Communicable Diseases.

Saliva is a very complex fluid and it is essential to maintain several physiological processes and functions, including oral health, taste, digestion and immunological defenses. Saliva composition and the oral microbiome can be influenced by several factors, like diet and smoking habits, and their alteration can represent an important access point for pathogens and, thus, for systemic illness onset. In this review, we explore the potentiality of saliva as a new tool for the early detection of some pathological conditions, such as oral diseases, chronic degenerative non-communicable diseases, among these chronic kidney disease (CKD). We also examined the possible correlation between oral and systemic diseases and oral and gut microbiota dysbiosis. In particular, we deeply analyzed the relationship between oral diseases and CKD. In this context, some salivary parameters can represent a new device to detect either oral or systemic pathologies. Moreover, the positive modulation of oral and gut microbiota induced by prebiotics, postbiotics, or symbiotics could represent a new possible adjuvant therapy in the clinical management of oral diseases and CKD.

Trends in Antibiotic Resistance of Nosocomial and Community-Acquired Infections in Italy.

The World Health Organization has recently identified three categories of pathogens, namely: critical, high, and medium priority, according to the need for new antibiotics. Critical priority pathogens include carbapenem-resistant microorganism (CPO) such as A. baumannii and P. aeruginosa, K. pneumoniae, and Enterobacter spp., whereas vancomycin-resistant E. faecium (VRE), methicillin and vancomycin-resistant S. aureus (MRSA) are in the high priority list. We compared the trend of antimicrobial resistants (AMRs) in clinical isolates, divided by year and bacteria spp., of samples obtained from nosocomial and community patients. Patient records were collected, including age, sex, site of infection, isolated organisms, and drug susceptibility patterns. From 2019 to 2022, a total of 113,635 bacterial isolates were tested, of which 11,901 resulted in antimicrobial resistants. An increase in the prevalence of several antibiotics resistant bacteria was observed. Specifically, the percentage of CPO cases increased from 2.62% to 4.56%, the percentage of MRSA increased from 1.84% to 2.81%, and the percentage of VRE increased from 0.58% to 2.21%. AMRs trend resulted in increases in CPO and MRSA for both community and nosocomial. Our work aims to highlight the necessity of preventive and control measures to be adopted in order to reduce the spread of multidrug-resistant pathogens.

Characterization of Gut Microbiota Composition in Type 2 Diabetes Patients: A Population-Based Study.

(1) Background: A clinical laboratory index to assess gut dysbiosis is the F/B ratio < 0.8. In fact, an elevated proportion of Firmicutes and a reduced population of Bacteroides in diabetes type 2 (T2D) subjects has been observed. This study aimed to detail the dysbiosis status in the Italian population, focusing on some pathogenic spectra (T2D) or metabolic disorders. (2) Material and methods: A quantity of 334 fecal samples was analyzed in order to perform genetic testing and sequencing. (3) Results: A trend in over imbalance was observed in the percentage of Proteobacteria (median value: 6.75%; interquartile range (IQR): 3.57−17.29%). A statistically significant association (χ2p = 0.033) was observed between type of dysbiosis and T2D, corresponding to an Odds Ratio (OR) of 1.86. It was noted that females with cystitis/candidiasis are significantly prevalent in T2D patients (p < 0.01; OR: 3.59; 95% CI: 1.43−8.99). Although, in non-diabetic males, a sugar craving is significantly associated with the rate of dysbiosis in non-diabetic males (p < 0.05; OR 1.07; 95% CI 1.00−1.16). (4) Conclusion: In T2D patients, the Bacteroidetes/Firmicutes ratio was biased in favor of Proteobacteria, to be expected due to the nutritional habits of the patients. Thus, T2D females had altered gut permeability favoring the development of infections in the vaginal tract.

T-Cell Assay after COVID-19 Vaccination Could Be a Useful Tool? A Pilot Study on Interferon-Gamma Release Assay in Healthcare Workers.

Background: SARS-CoV-2 T-cells are crucial for long-term protection against reinfection. The aim was to demonstrate the Interferon-gamma Release Assay (IGRA) test could be useful for vaccination monitoring. Methods: In a prospective cohort of 98 vaccinated healthcare workers for SARS-CoV-2, we selected 23 people in low-antibodies (Group 1, N = 8), high-antibodies (Group 2, N = 9), and negative control groups (Group 3, N = 6). SARS-CoV-2-specific humoral and cellular responses were analyzed at 8 months after two doses of Pfizer BioNTech, evaluating anti-RBD (Receptor Binding Domain) and RBD-ACE2 (Angiotensin Converting Enzyme-2) blocking antibodies in sera through a Chemiluminescence Immunoassay (CLIA) and T-cells through the IGRA test in heparinized plasma. Moreover, lymphocyte subtyping was executed by a flow cytometer. Statistical analysis was performed. Results: The data confirmed that RBD and RBD-ACE2 blocking ACE2 antibody levels of Group 1 were significantly lower than Group 2; p < 0.001. However, T-cells showed no significant difference between Group 1 and Group 2. Conclusions: This work suggests the need for new strategies for booster doses administration.

Evaluation of Natural and Vaccine-Induced Anti-SARS-CoV-2 Immunity: A Comparative Study between Different Groups of Volunteers.

(1) Background: The production of anti-SARS-CoV-2 antibodies should help minimize the severity of COVID-19 disease. Our focus was to investigate and compare different vaccination schedules, monitoring circulating S-RBD Ab (antibodies anti-Spike protein-Receptor Binding Domain) levels after administering two doses in naïve patients. Likewise, vaccine-stimulated immunity in naïve and previously infected patients was compared. (2) Methods: We included 392 patients. Sera were evaluated by Elecsys anti-SARS-CoV-2 S. Statistical analyses were conducted by MedCalc and JASP. (3) Results: In COVID-19 patients, the median value of Ab levels was 154 BAU/mL, stable up to 9 months after the infection. From the data observed in vaccinated patients, higher median values were recorded in COVID-19/Pfizer BioNTech (18913 BAU/mL) than in other groups (Pfizer BioNTech: 1841; ChadOx1 961; heterologous vaccination: 2687) BAU/mL. (4) Conclusions: In conclusion, a single booster dose given to previously infected patients raised an antibody response much higher than two doses given to naïve individuals and heterologous vaccination generated a robust persistent antibody response at high levels, steady up to three months after administration.

Validation of a quantitative lateral flow immunoassay (LFIA)-based point-of-care (POC) rapid test for SARS-CoV-2 neutralizing antibodies.

With the widespread use of coronavirus disease 2019 (COVID-19) vaccines, a rapid and reliable method to detect SARS-CoV-2 neutralizing antibodies (NAbs) is extremely important for monitoring vaccine effectiveness and immunity in the population. The purpose of this study was to evaluate the performance of the RapiRead™ reader and the TestNOW™ COVID-19 NAb rapid point-of-care (POC) test for quantitative measurement of antibodies against the spike protein receptor-binding domain of severe respiratory syndrome coronavirus 2 (SARS-CoV-2) in different biological matrices compared to chemiluminescence immunoassay (CLIA) methods. Ninety-four samples were collected and analyzed using a RapiRead™ reader and TestNOW™ COVID-19 NAb kits for detecting neutralizing antibodies, and then using two CLIAs. The data were compared statistically using the Kruskal-Wallis test for more than two groups or the Mann-Whitney test for two groups. Specificity and sensitivity were evaluated using a receiver operating characteristic (ROC) curve. Good correlation was observed between the rapid lateral flow immunoassay (LFIA) test system and both CLIA methods. RapiRead™ reader/TestNOW™ COVID-19 NAb vs. Maglumi: correlation coefficient (r) = 0.728 for all patients; r = 0.841 for vaccinated patients. RapiRead™ reader/TestNOW™ COVID-19 NAb vs. Mindray: r = 0.6394 in all patients; r = 0.8724 in vaccinated patients. The time stability of the POC serological test was also assessed considering two times of reading, 12 and 14 minutes. The data revealed no significant differences. The use of a RapiRead™ reader and TestNOW™ COVID-19 NAb assay is a quantitative, rapid, and valid method for detecting SARS-CoV-2 neutralizing antibodies and could be a useful tool for screening studies of SARS-CoV-2 infection and assessing the efficacy of vaccines in a non-laboratory context.

Performance evaluation of four surrogate Virus Neutralization Tests (sVNTs) in comparison to the in vivo gold standard test.

BACKGROUND: Several commercial surrogate Virus Neutralization Tests (sVNTs) have been developed in the last year. Neutralizing anti-SARS-CoV-2 antibodies through interaction with Spike protein Receptor Binding Domain (S-RBD) can block the virus from entering and infecting host cells. However, there is a lack of information about the functional activity of SARS-CoV-2 antibodies that may be associated with protective responses. For these reasons, to counteract viral infection, the conventional virus neutralization test (VNT) is still considered the gold standard. The aim of this study was to contribute more and detailed information about sVNTs' performance, by determining in vitro the anti-SARS-CoV-2 neutralizing antibody concentration using four different commercial assays and then comparing the obtained data to VNT. METHODS: Eighty-eight samples were tested using two chemiluminescence assays (Snibe and Mindray) and two ELISA assays (Euroimmun and Diesse). The antibody titers were subsequently detected and quantified by VNT. RESULTS: The overall agreement between each sVNT and VNT was 95.45% for Euroimmun and 98.86% for Diesse, Mindray and Snibe. Additionally, we investigated whether the sVNTs were closer to the gold standard than traditional anti-SARS-CoV-2 antibody assays S-RBD or S1 based, finding a higher agreement mean value for sVNTs (98.01 ± 1.705% vs 95.45 ± 1.921%; p < 0.05). Furthermore, Spearman's statistical analysis for the correlation of sVNT versus VNT showed r = 0.666 for Mindray; r = 0.696 for Diesse; r = 0.779 for Mindray and r = 0.810 for Euroimmun. CONCLUSIONS: Our data revealed a good agreement between VNT and sVNTs. Despite the VNT still remains the gold standard, the sVNT might be a valuable tool for screening wider populations.

Evaluation of serological anti-SARS-CoV-2 chemiluminescent immunoassays correlated to live virus neutralization test, for the detection of anti-RBD antibodies as a relevant alternative in COVID-19 large-scale neutralizing activity monitoring.

The Spike-Receptor Binding Domain (S-RBD) is considered the most antigenic protein in SARS-CoV-2 and probably the key player in SARS-CoV-2 immune response. Quantitative immunoassays may help establish an anti-RBD Abs threshold as an indication of protective immunity. Since different immunoassays are commercial, the standard reference method for the neutralizing activity is the live Virus Neutralization Test (VNT). In this study, anti-RBD IgG levels were detected with two chemiluminescent immunoassays in paucisymptomatic, symptomatic and vaccinated subjects, and their neutralizing activity was correlated to VNT titer, using SARS-CoV-2 original and British variant strains. Both immunoassays confirmed higher anti-RBD Abs levels in vaccinated subjects. Furthermore, despite different anti-RBD Abs median concentrations between the immunoassays, a strong positive correlation with VNT was observed. In conclusion, although the SARS-CoV-2 immune response heterogeneity, the use of immunoassays can help in large-scale monitoring of COVID-19 samples, becoming a valid alternative to VNT test for diagnostic routine laboratories.

Evaluation of ECLIA antigen detection tests as screening methods for COVID-19 in comparison with molecular analysis.

BACKGROUND: Data from literature shows that antigen tests are rapid and helpful tools for diagnosis of COVID-19. AIM: This work aimed to evaluate the performances of the Elecsys SARS-CoV-2 Antigen test, in comparison to RT-qPCR, the gold standard. METHODS: A total of 110 swabs were tested; according to rRT-PCR, 76 were positive, and 34 were negative. The swabs were processed by Elecsys SARS CoV 2 Antigen assay (Roche Diagnostics GmbH, Mannheim, Germany), an electrochemiluminescence immunoassay (ECLIA). RESULTS: In a first evaluation, the overall sensitivity and specificity were 85% and 100%, respectively. It was noted that most of the discordant cases had cycle threshold (Ct) values > 28. Therefore, it was assumed a new measure to evaluate sensitivity and specificity, then samples with Ct values < 28 were selected. In this way, it was achieved a Ct < 28 sensitivity of 94%. The level of agreement between the two tests was 89. 1% with κ value of 0.77 for total data and 95.9% with κ value of 0.95 for samples with < 28 Ct. The antigen test performs well in the presence of high viral loads, whereas lower levels are missed. CONCLUSIONS: The comparison data obtained in this study support that this method seems a proper approach for rapid screening of patients with high SARS-CoV-2 viral load; however, the rate of sensitivity is highly Ct-dependent.

Serological anti-SARS-CoV-2 neutralizing antibodies association to live virus neutralizing test titers in COVID-19 paucisymptomatic/symptomatic patients and vaccinated subjects.

A large number of immunoassays have been developed to detect specific anti-SARS-CoV-2 antibodies; however, not always they are functional to neutralize the virus. The reference test for the anti-spike neutralizing antibodies (nAbs) ability to counteract the viral infection is the virus neutralization test (VNT). Great interest is developing on reliable serological assays allowing antibodies concentration and antibody protective titer correlation. The aim of our study was to detect nAbs serum levels in paucisymptomatic, symptomatic and vaccinated subjects, to find a cut-off value able to protect from virus infection. nAbs serum levels were detected by a competitive automated immunoassay, in association to VNT with the SARS-CoV-2 original and British variant strains. The median nAbs concentrations were: 281.3 BAU/ml for paucisymptomatics; 769.4 BAU/ml for symptomatics; 351.65 BAU/ml for the vaccinated cohort; 983 BAU/ml considering only the second dose vaccinated individuals. The original strain VNT analysis showed 1:80 median neutralization titers in paucisymptomatic and vaccinated subjects; 1:160 in symptomatic patients; 1:160 in the second dose groups. The British variant VNT analysis showed lower neutralization titers in paucisymptomatic and vaccinated groups (1:40); the same titer in symptomatic patients (1:160); the second dose group confirmed the original strain titer (1:160). In conclusion, our data showed optimal correlations with a proportional increase between neutralizing activity and antibody concentration, making nAbs detection a good alternative to virus neutralization assays, difficult to carry out in routine laboratories. Finally, ROC curve analysis established a cut-off of 408.6 BAU/ml to identify subjects with a low risk of infection.

The WHO International Standard for COVID-19 serological tests: towards harmonization of anti-spike assays.

BACKGROUND AND AIMS: SARS-CoV-2 antibody assays are relevant in managing the COVID-19 pandemic, providing valuable data on the immunization status of the population. However, current serology tests are highly variable, due to their different characteristics and to the lack of reference materials. The aim of the World Health Organization (WHO) first International Standard (IS) for anti-SARS-CoV-2 immunoglobulin is to harmonize humoral immune response assessment after natural infection or vaccination, and recommend reporting the results for binding activity in Binding Antibody Units (BAU). MATERIALS AND METHODS: This study analyzed six commercial quantitative anti-SARS-CoV-2 S-protein assays in a head-to-head comparison, using the manufacturers' conversion factors for the WHO IS to obtain BAU/mL values. RESULTS: Our data showed good alignment up to 1000 BAU/mL, then began to disperse, exhibiting some discrepancies. Moreover, correlations among methods varied with Cohen's Kappa ranging from 0.580 to 1.00, with the lowest agreement values for kits using different target antigens or different antibody isotypes, making it clear that the laboratory report should include this information. Values expressed as BAU/ml showed a reduced between-assays variability compared to AU/ml (median coefficients of variation 0.38 and 0.68, respectively; p < 0.001). CONCLUSION: On the basis of these data at present anti-SARS CoV-2 serological assays' results are not interchangeable, and, more importantly, individual immune monitoring should be performed with the same method.

Evaluation of S-RBD and high specificity ACE-2-binding antibodies on SARS-CoV-2 patients after six months from infection.

The antibody response to SARS-CoV-2 has not yet fully defined, but the availability of sensitive and specific serological assays is crucial to observe the presence of specific antibodies against the human receptor binding domain (S-RBD) and high specificity ACE-2-binding antibodies or neutralizing antibodies (NT) in response to vaccines. Indeed, these peculiar antibodies should prevent viral interaction between RBD and Angiotensin-Converting Enzyme 2 (ACE2) receptor, located on surface of host cells. In this study, 72 samples from 37 hospitalized COVID-19 patients and 35 not-hospitalized patients were analyzed longitudinally. The detection of S-RBD and NT antibodies was carried out using CLIA tests. Hospitalized patients showed elevated serum levels of S-RBD (97.22%) and NT (77.78%) antibodies, differently, not-hospitalized, who were paucisymptomatic or asymptomatic patients, showed lower serum levels of S-RBD (65.71%) and NT (38.14%) antibodies. The results suggest that the NT serum level is strongly related to disease severity (p < 0.001) and to the serum level of S-RBD antibodies (p < 0.0001).