Francesco Cerutti

Background: Monitoring HIV-1 subtype circulation and transmitted drug resistance (TDR) remains a key priority, particularly since the rollout of high-sensitivity next-generation sequencing (NGS). Methods: Routine plasma HIV-1 RNA NGS genotyping data were collected from newly diagnosed individuals in Italy over 2022–24. HIV-1 TDR and genotypic susceptibility were evaluated through HIVdb with NGS set at 10% and 20%. Subtype and transmission clusters (TC) were determined through the maximum likelihood phylogeny based on the GTR + F + R9 model. Results: Seven hundred and forty-two individuals were included, 51.9% harbouring non-B strains [CRF02_AG (18.1%); CRF BF (6.1%); A1/A3/A6 (7.1%); others (20.5%)]. TDR prevalence to any class was 11.7% at Sanger-like NGS-setting (>20%), slightly increased (15.0%) at 10% NGS-setting, and significantly varied across subtypes, with the highest prevalence observed in B subtype. Most antiretrovirals showed full genotypic activity in nearly 99% of individuals, except for efavirenz and rilpivirine (proportion of individuals with full activity 2 sequences). Thirteen TC (22.8%, 8 pairs, 5 clusters) involved individuals harbouring TDR. TDR was detected as minority mutations in five TC. Conclusions: A high proportion of HIV-1 non-B subtypes circulate in Italy. TDR prevalence is around 12% using NGS at Sanger-like threshold and moderately increases to 15% when NGS is set at 10%. However, the impact of the detected TDR on the susceptibility to currently used antiretrovirals in clinical practice is negligible.

Background: Human metapneumovirus (hMPV) infections have a significant impact on public health. However, the extent of this burden in Italy remains poorly defined due to a lack of comprehensive data. The aim of this cross-sectional multicenter study was to understand the epidemiology of hMPV in Italy, particularly in older adults. Methods: We analyzed laboratory data from molecular respiratory viral diagnostic tests conducted at 17 centers across Italy from September 2022 to August 2024. Respiratory viruses were tested from outpatients for epidemiologic surveillance and from patients presenting to tertiary hospitals for diagnostic purpose. G gene sequencing was performed on a limited number of circulating strains. Results: Data from 96 460 tests yielded an overall hMPV positivity rate of 3.4%; the hMPV positivity rate was 2.6% in adults aged 50 years and older, a third of whom were aged >80 years. In north-west Italy, hMPV was detected more frequently in outpatients than in hospitalized patients. The temporal distribution of cases showed seasonal peaks in February 2023 and April 2024, which exhibited some geographic variation but overlapped in the general population and in the elderly. Phylogenetic analysis suggested an even distribution of hMPV-A and -B, with a predominance of clades A2c with a 111-nucleotide duplication and B2b, and the possible extinction of previously circulating clades A2c with a 180-nucleotide duplication and B2a. Conclusions: hMPV was shown to be a relevant respiratory pathogen in older adults, who could be more likely to have severe outcomes. These findings may inform hMPV surveillance and the development of prevention strategies.

Background: Next-generation sequencing (NGS) is gradually replacing Sanger sequencing for HIV genotypic drug resistance testing (GRT). This work evaluated the concordance among different NGS-GRT interpretation tools in a real-life setting. Methods: Routine NGS-GRT data were generated from viral RNA at 11 Italian laboratories with the AD4SEQ HIV-1 Solution v2 commercial kit. NGS results were interpreted by the SmartVir system provided by the kit and by two online tools (HyDRA Web and Stanford HIVdb). NGS-GRT was considered valid when the coverage was >100 reads (100×) at each PR/RT/IN resistance-associated position listed in the HIVdb 9.5.1 algorithm. Results: Among 629 NGS-GRT, 75.2%, 74.2%, and 70.9% were valid according to SmartVir, HyDRA Web, and HIVdb. Considering at least two interpretation tools, 463 (73.6%) NGS-GRT had a valid coverage for resistance analyses. The proportion of valid samples was affected by viremia 10% showed fair concordance among different interpretation tools. Conclusion: This Italian survey on NGS resistance testing suggests that viremia levels and HIV subtype affect NGS-GRT coverage. Within the current routine method for NGS-GRT, only mutations with frequency >10% seem reliably detected across different interpretation tools.

S ummary: We propose a new method to estimate the ratio between the basic reproduction numbers of a newly emerged variant and the one currently dominating. We use public data of two kinds: the proportions of the daily infected from each strain obtained from a random sample that has been sequenced, and the epidemic curves of total infections and recoveries. Our method is based on a new discrete-time SIR model with two strains, considered both in the deterministic and stochastic versions. In the deterministic case we use maximum likelihood, while in the stochastic setting, since we need to reconstruct the missing incidence and prevalence information of the new variant, we decided to use a hierarchical Bayesian hidden Markov model. This new methodology is applied to data from the Piedmont Italian region in December-January 2022, when the Omicron variant started to be observed and quickly became prevalent. With both approaches, we obtain an estimate of the contagiousness ratio that is consistent with other studies specifically designed for the aim.

While mass-scale vaccination campaigns are ongoing worldwide, genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to monitor the emergence and global spread of viral variants of concern (VOC). Here, we present a streamlined workflow—COVseq—which can be used to generate highly multiplexed sequencing libraries compatible with Illumina platforms from hundreds of SARS-CoV-2 samples in parallel, in a rapid and cost-effective manner. We benchmark COVseq against a standard library preparation method (NEBNext) on 29 SARS-CoV-2 positive samples, reaching 95.4% of concordance between single-nucleotide variants detected by both methods. Application of COVseq to 245 additional SARS-CoV-2 positive samples demonstrates the ability of the method to reliably detect emergent VOC as well as its compatibility with downstream phylogenetic analyses. A cost analysis shows that COVseq could be used to sequence thousands of samples at less than 15 USD per sample, including library preparation and sequencing costs. We conclude that COVseq is a versatile and scalable method that is immediately applicable for SARS-CoV-2 genomic surveillance and easily adaptable to other pathogens such as influenza viruses.

To date, there is no severe acute respiratory syndrome coronavirus 2-(SARS-CoV-2)-specific prognostic biomarker available. We assessed whether SARS-CoV-2 cycle threshold (Ct) value at diagnosis could predict novel CoronaVirus Disease 2019 (COVID-19) severity, clinical manifestations, and six-month sequelae. Hospitalized and outpatient cases were randomly sampled from the diagnoses of March 2020 and data collected at 6 months by interview and from the regional database for COVID-19 emergency. Patients were stratified according to their RNA-dependent-RNA-polymerase Ct in the nasopharyngeal swab at diagnosis as follows: Group A ≤ 20.0, 20.0 < group B ≤ 28.0, and Group C > 28.0. Disease severity was classified according to a composite scale evaluating hospital admission, worst oxygen support required, and survival. Two hundred patients were included, 27.5% in Groups A and B both, 45.0% in Group C; 90% of patients were symptomatic and 63.7% were hospitalized. The median time from COVID-19 onset to swab collection was five days. Lethality, disease severity, type, and number of signs and symptoms, as well as six-month sequelae distributed inversely among the groups with respect to SARS-CoV-2 Ct. After controlling for confounding, SARS-CoV-2 Ct at diagnosis was still associated with COVID-19-related death (p = 0.023), disease severity (p = 0.023), number of signs and symptoms (p < 0.01), and presence of six-month sequelae (p < 0.01). Early quantification of SARS-CoV-2 may be a useful predictive marker to inform differential strategies of clinical management and resource allocation.

The fan mussel, Pinna nobilis, represents the largest bivalve endemic to the Mediterranean Sea. Since 2016, dramatic mass mortality of this species has been observed in several areas. The first surveys suggested that Haplosporidium pinnae (currently considered species-specific) was the main etiological agent, but recent studies have indicated that a multifactorial disease may be responsible for this phenomenon. In this study, we performed molecular diagnostic analyses on P. nobilis, P. rudis, and bivalve heterologous host species from the island of Sardinia to shed further light on the pathogens involved in the mass mortality. The results support the occurrence of a multifactorial disease and that Mycobacterium spp. and H. pinnae are not necessarily associated with the illness. Indeed, our analyses revealed that H. pinnae is not species-specific for P. nobilis, as it was present in other bivalves at least three years before the mass mortality began, and species of Mycobacterium were also found in healthy individuals of P. nobilis and P. rudis. We also detected the species Rhodococcus erythropolis, representing the first report in fan mussels of a bacterium other than Mycobacterium spp. and Vibrio spp. These results depict a complicated scenario, further demonstrating how the P. nobilis mass mortality event is far from being fully understood.