High-throughput peptide array analysis and computational techniques for serological profiling of flavivirus infections: Implications for diagnostics and vaccine development.

Arthropod-borne viruses, such as dengue virus (DENV), pose significant global health threats, with DENV alone infecting around 400 million people annually and causing outbreaks beyond endemic regions. This study aimed to enhance serological diagnosis and discover new drugs by identifying immunogenic protein regions of DENV. Utilizing a comprehensive approach, the study focused on peptides capable of distinguishing DENV from other flavivirus infections through serological analyses. Over 200 patients with confirmed arbovirus infection were profiled using high-density pan flavivirus peptide arrays comprising 6253 peptides and the computational method matrix of local coupling energy (MLCE). Twenty-four peptides from nonstructural and structural viral proteins were identified as specifically recognized by individuals with DENV infection. Six peptides were confirmed to distinguish DENV from Zika virus (ZIKV), West Nile virus (WNV), Yellow Fever virus (YFV), Usutu virus (USUV), and Chikungunya virus (CHIKV) infections, as well as healthy controls. Moreover, the combination of two immunogenic peptides emerged as a potential serum biomarker for DENV infection. These peptides, mapping to highly accessible regions on protein structures, show promise for diagnostic and prophylactic strategies against flavivirus infections. The described methodology holds broader applicability in the serodiagnosis of infectious diseases.

Concomitant diagnosis of sexually transmitted infections and human monkeypox in patients attending a sexual health clinic in Milan, Italy.

In 2022, many monkeypox (MPX) outbreaks have been documented in countries where MPX was not endemic. It spread all around the world, especially in European Union and United States. While MPX is classically considered to be transmitted through close contact with lesions, the hypothesis of sexual transmission has been proposed. This study considered a total of 49 patients suspected for MPX that were also tested for other sexually transmitted infections (STIs), including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and Trichomonas vaginalis. The data from coinfected patients suggested that MPXV and STIs might share the same route of inoculum, corroborating the hypothesis of possible sexual transmission for the emerging poxvirus. And like any other STI, MPX should be considered without stigmatization.

Mpox DNA clearance in semen over 6-month follow-up.

Sexual intercourse is a well-established way of transmission of mpox infection. However, it is still uncertain whether semen may represent a viral reservoir. The aim of the study was to evaluate the clearance of viral DNA in semen samples from individuals diagnosed with mpox infection over 6-month follow-up. This prospective, observational, single-center study was conducted at IRCCS San Raffaele Scientific Institute, Milan, Italy, between May and October 2022 in 140 individuals who attended Sexual Health Clinic and diagnosed with mpox infection. Semen samples were collected and analyzed by real-time polymerase chain reaction assays. The baseline collection was performed in 64 (46%) of 140 men diagnosed with mpox infection. The viral DNA was detected in 43 (67%) with median cycle threshold (Ct) 34 (interquartile range [IQR] 31-36). The research was repeated in 32 (74%) and viral DNA clearance was observed in all within 6 months in a median time of 10.5 days (IQR 7-33). Viral clearance occurred in all tested individuals, mostly within 2 weeks since the first positive test. These findings suggest a transient presence of viral DNA in semen and do not support the hypothesis of reservoir. More studies on mpox DNA detection in semen with viral culture and extended follow-up are needed.

First Identification of the New Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant (B.1.1.529) in Italy.

We identified the first case in Italy of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.529 variant, using whole-genome sequencing in an Italian subject traveling from Mozambique. Specific mutation profiles deserve further investigations to clarify potential effects on vaccination efficacy. This case highlights the crucial role of rapid and continuous surveillance of SARS-CoV-2 variant circulation.

Isolation of viable monkeypox virus from anal and urethral swabs, Italy, May to July 2022.

Anal and urethral samples from confirmed cases of monkeypox were screened for monkeypox virus (MPXV) by real-time PCR. Isolation of the virus was subsequently attempted in cell culture. Actively-replicating virus was demonstrated in 13 of 18 and 11 of 15 PCR-positive anal and urethral swabs, respectively, collected within 7 days from symptoms onset. Two asymptomatic secondary cases had detectable MPXV genetic material in urethral secretion and for one, MPXV was successfully isolated, supporting a potential MPXV sexual transmission hypothesis.

Geographical reconstruction of the SARS-CoV-2 outbreak in Lombardy (Italy) during the early phase.

The first identification of autochthonous transmission of SARS-CoV-2 in Italy was documented by the Laboratory of Clinical Microbiology, Virology and Bioemergencies of L. Sacco Hospital (Milano, Italy) on 20th February 2020 in a 38 years old male patient, who was found positive for pneumonia at the Codogno Hospital. Thereafter Lombardy has reported the highest prevalence of COVID-19 cases in the country, especially in Milano, Brescia and Bergamo provinces. The aim of this study was to assess the potential presence of different viral clusters belonging to the six main provinces involved in Lombardy COVID-19 cases in order to highlight peculiar province-dependent viral characteristics. A phylogenetic analysis was conducted on 20 full length genomes obtained from patients addressing to several Lombard hospitals from February 20th to April 4th, 2020, aligned with 41 Italian viral genome assemblies available on GISAID database as of 30th March, 2020: two main monophyletic clades, containing 8 and 53 isolates, respectively, were identified. Noteworthy, Bergamo isolates mapped inside the small clade harbouring M gene D3G mutation. The molecular clock analysis estimated a cluster divergence approximately one month before the first patient identification, supporting the hypothesis that different SARS-CoV-2 strains had spread worldwide at different times, but their presence became evident only in late February along with Italian epidemic emergence. Therefore, this epidemiological reconstruction suggests that virus initial circulation in Lombardy was ascribable to multiple introduction. The phylogenetic reconstruction robustness, however, will be improved when more genomic sequences are available, in order to guarantee a complete epidemiological surveillance.

Compassionate remdesivir treatment of severe Covid-19 pneumonia in intensive care unit (ICU) and Non-ICU patients: Clinical outcome and differences in post-treatment hospitalisation status.

SARS-CoV-2 is causing an increasing number of deaths worldwide because no effective treatment is currently available. Remdesivir has shown in vitro activity against coronaviruses and is a possible antiviral treatment for SARS-CoV-2 infection. This prospective (compassionate), open-label study of remdesivir, which was conducted at Luigi Sacco Hospital, Milan, Italy, between February 23 and March 20, 2020, involved patients with SARS-CoV-2 pneumonia aged ≥18 years undergoing mechanical ventilation or with an oxygen saturation level of ≤94 % in air or a National Early Warning Score 2 of ≥4. The primary outcome was the change in clinical status based on a 7-category ordinal scale (1 = not hospitalised, resuming normal daily activities; 7 = deceased). The 35 patients enrolled from February 23 to March 20, 2020, included 18 in intensive care unit (ICU), and 17 in our infectious diseases ward (IDW). The 10-day course of remdesivir was completed by 22 patients (63 %) and discontinued by 13, of whom eight (22.8 %) discontinued because of adverse events. The median follow-up was 39 days (IQR 25-44). At day 28, 14 (82.3 %) patients from IDW were discharged, two were still hospitalized and one died (5.9 %), whereas in ICU 6 (33.3 %) were discharged, 8 (44.4 %) patients died, three (16.7 %) were still mechanically ventilated and one (5.6 %) was improved but still hospitalized. Hypertransaminasemia and acute kidney injury were the most frequent severe adverse events observed (42.8 % and 22.8 % of the cases, respectively). Our data suggest that remdesivir can benefit patients with SARS-CoV-2 pneumonia hospitalised outside ICU where clinical outcome was better and adverse events are less frequently observed. Ongoing randomised controlled trials will clarify its real efficacy and safety, who to treat, and when.

European Multicenter Study on Analytical Performance of Veris HIV-1 Assay.

The analytical performance of the Veris HIV-1 assay for use on the new, fully automated Beckman Coulter DxN Veris molecular diagnostics system was evaluated at 10 European virology laboratories. The precision, analytical sensitivity, performance with negative samples, linearity, and performance with HIV-1 groups/subtypes were evaluated. The precision for the 1-ml assay showed a standard deviation (SD) of 0.14 log10 copies/ml or less and a coefficient of variation (CV) of ≤6.1% for each level tested. The 0.175-ml assay showed an SD of 0.17 log10 copies/ml or less and a CV of ≤5.2% for each level tested. The analytical sensitivities determined by probit analysis were 19.3 copies/ml for the 1-ml assay and 126 copies/ml for the 0.175-ml assay. The performance with 1,357 negative samples demonstrated 99.2% with not detected results. Linearity using patient samples was shown from 1.54 to 6.93 log10 copies/ml. The assay performed well, detecting and showing linearity with all HIV-1 genotypes tested. The Veris HIV-1 assay demonstrated analytical performance comparable to that of currently marketed HIV-1 assays. (DxN Veris products are Conformité Européenne [CE]-marked in vitro diagnostic products. The DxN Veris product line has not been submitted to the U.S. FDA and is not available in the U.S. market. The DxN Veris molecular diagnostics system is also known as the Veris MDx molecular diagnostics system and the Veris MDx system.).

Monkeypox Virus Neutralizing Antibodies at Six Months from Mpox Infection: Virologic Factors Associated with Poor Immunologic Response.

A natural monkeypox virus infection may not induce sufficient neutralizing antibody responses in a subset of healthy individuals. The aim of this study was to evaluate monkeypox virus-neutralizing antibodies six months after infection and to assess the virological factors predictive of a poor immunological response. Antibodies were assessed using a plaque reduction neutralization test at six months from mpox infection; mpox cutaneous, oropharyngeal, and anal swabs, semen, and plasma samples were tested during infection. Overall, 95 people were included in the study; all developed detectable antibodies. People who were positive for the monkeypox virus for more days had higher levels of antibodies when considering all tested samples (p = 0.029) and all swabs (p = 0.005). Mpox cycle threshold values were not predictive of antibody titers. This study found that the overall days of monkeypox virus detection in the body, irrespective of the viral loads, were directly correlated with monkeypox virus neutralizing antibodies at six months after infection.