Increased reports of measles in the Metropolitan City of Milan, northern Italy, September 2023 to March 2024.

Since late 2023, the Metropolitan City of Milan and surrounding areas (northern Italy) have been experiencing a resurgence of measles, with most cases detected starting from January 2024. During this brief period, we observed measles in travellers from endemic areas, participants in international events, vaccinees and healthcare workers. Indigenous cases have also been identified. Even though we have not yet identified large and disruptive outbreaks, strengthening surveillance and vaccination activities is pivotal to help limit the impact of measles spread.

COVID-19 monitoring of school personnel through molecular salivary test and dried blood spot analysis.

Background: When the coronavirus disease 2019 (COVID-19) pandemic broke out, most countries enforced school closures as a precautionary measure. Although COVID-19 is still present three years later, schools have been reopened. We aimed to test the association of molecular salivary testing (MST) and dried blood spot (DBS) analysis for community surveillance by investigating the immunological profile of a group of school staff during and following COVID-19 vaccination. Methods: We conducted the study in a school in Milan from April 2021, when school staff were administered the first dose of vaccine against SARS-CoV-2, until the school year ended in June 2022. Each participant provided samples for MST and DBS one month (T1, W1) after receiving their first dose of vaccine. Subsequently, they collected weekly MST samples for five weeks (W2-W6), plus a DBS sample in the last week (T2). Both samples were collected one (T3), four (T4), and seven months (T5) after the administration of the second vaccine dose in May 2021. A final DBS sample was collected one year (T6) after T3. Results: Sixty participants provided 327 MSTs and 251 DBSs. None of the MST samples tested positive for SARS-CoV-2 RNA during the study period. A total of 201 DBS samples tested positive for the IgG semiquantitative analysis. Negative samples were found only at T1 (20.45%) and T2 (7.32%). We observed borderline results at T1 (4.55%), T2 (7.32%), and T4 (2.70%). The anti-SARS-CoV-2 average antibody ratio increased after the second dose between T2 and T3, and the trend peaked after the third dose between T4 and T6. We performed an immunoenzymatic assay of antibodies against nucleocapsid protein on samples collected at T1 from five participants who reported having been infected before the study and from four subjects with an abnormal increase in the antibody values at T4. Two samples tested positive in the first group and two in the second one. Conclusions: Our findings show that MST and DBS could be effective tools in the active surveillance of school personnel and that schools could be considered safe settings in view of SARS-CoV-2 infection. Vaccines might have contributed to case and/or symptom reduction.

Differential diagnosis of fever and rash cases negative for measles and rubella to complement surveillance activities.

In the quest to eliminate measles virus (MV) and rubella virus (Ruv), every suspected case must be properly identified and diagnosed. Since 2017, in Milan (Italy), a total of 978 measles and rubella suspected cases (fever and rash) were investigated and 310 were not laboratory confirmed (discarded cases). To improve surveillance activities, we investigated the presence in discarded cases of 8 other viral pathogens commonly associated with rash: human herpesvirus 6 (HHV-6) and 7 (HHV-7), parvovirus B19 (B19V), enterovirus (EV), Epstein-Barr virus (EBV), human adenovirus (HAdV), cytomegalovirus (HCMV), and SARS-CoV-2. Differential diagnosis was carried out on 289 discarded cases by multiplex real-time PCR assays. At least one pathogen was detected in 188 cases (65.1%) with HHV-7 being the most frequently detected virus. No difference in the number of detected infections overtime was observed and infections were identified in all age groups. As expected, most HHV-6, EV, HAdV, and HCMV-positive cases were found in children aged 0-4 years and HHV-7 was most frequent in the 15-39 age group. In light of the World Health Organization measles elimination goal, the introduction of laboratory methods for differential diagnosis is required for the final classification of clinically compatible cases. The used screening panel allowed us to increase the percentage of virus-positive cases to 87.5%, allowing us to clarify viral involvement and epidemiology, improve diagnosis, and strengthen surveillance activities. As all investigated pathogens were detected, this diagnostic panel was a suitable tool to complement MV and RuV surveillance activities.

Preschool-located influenza vaccination and influenza-like illness surveillance: an Italian pilot experience.

BACKGROUND: We describe the first school-located influenza vaccination campaign with quadrivalent live-attenuated influenza vaccine (LAIV) among pre-school children in Italy, coupled with an innovative school-centred influenza-like illnesses (ILIs) surveillance using a self-sampling non-invasive saliva collection method. METHODS: The pilot study was proposed during the 2021/2022 influenza season to fifteen pre-schools in the Milan municipality. LAIV was offered directly in school to all healthy children without contraindications. ILI differential diagnosis was conducted by real-time RT-PCR for influenza A/B and SARS-CoV-2. RESULTS: Five pre-schools were involved in the pilot project and overall, 135 families (31.2%) participated in the study, adhering to both surveillance and vaccination; 59% of families had an immigrant background. No pupil experienced adverse reactions after vaccination. Nineteen saliva samples were collected from sixteen children (11.8%). Six samples (31.6%) tested positive for SARS-CoV-2; none was positive for influenza A/B. CONCLUSIONS: The participation in the immunisation campaign was good, considering possible absences due to COVID-19 pandemic, and the intranasal administration was well tolerated and helped to overcome parental hesitancy. Saliva sampling represented a useful tool to reduce children's stress and increase parents' compliance. The high participation of families with an immigrant background suggests that school-based interventions can represent an effective strategy to overcome socioeconomic and cultural barriers.

Serological investigation of SARS-CoV-2 infection in patients with suspect measles, 2017-2022.

BACKGROUND: Several studies suggested that SARS-CoV-2 was already spreading worldwide during the last months of 2019 before the first outbreak was detected in Wuhan, China. Lombardy (Northern Italy) was the first European region with sustained SARS-CoV-2 transmission and recent investigations detected SARS-CoV-2-RNA-positive patients in Lombardy since late 2019. METHODS: We tested for anti-SARS-CoV-2 IgG all serum samples available in our laboratory (N = 235, collected between March 2017 and March 2022) that we received within the framework of measles/rubella surveillance from measles and rubella virus-negative patients. RESULTS: Thirteen of 235 samples (5.5%) were IgG-positive. The positivity rate increased starting in 2019 and was significantly different from the expected false positive rate from 2019 onwards. Additionally, in 2019 the percentage of IgG-positive patients was significantly lower among SARS-CoV-2 RNA-negative patients (3/92) compared to SARS-CoV-2 RNA-positive patients (2/7, p = 0.04). The highest percentage of IgG positivity in the pre-pandemic period was recorded during the second half of 2019. This coincided with an increase in negativity for measles and a widening of the peak of the number of measles discarded cases per 100,000 inhabitants, indicating a higher-than-normal number of measles-negative patients experiencing fever and rash. This also coincided with the first patient positive for SARS-CoV-2 RNA (September 12th, 2019); this patient was also positive for anti-SARS-CoV-2 IgG and IgM. CONCLUSIONS: Although the number of samples was low and one cannot conclusively establish that the virus started circulating in Lombardy around September 2019, our findings should stimulate similar research investigating the possibility of undetected SARS-CoV-2 pre-pandemic circulation.

Detection of human papillomavirus in fresh and dried urine through an automated system for cervical cancer screening in low- and middle-income countries.

The majority of cervical cancer cases and associated deaths occur in low- and middle-income countries (LMICs), where sociocultural barriers, poor access to prevention and care, and technical and practical difficulties hinder screening coverage improvement. Using urine specimens for human papillomaviruses (HPV) molecular screening through automated testing platforms can help to overcome these problems. We evaluated the high-risk (HR) HPV detection performance of the Xpert® HPV test on GeneXpert® System (Cepheid), on fresh and dried urine (Dried Urine Spot [DUS]) samples as compared to an in-house polymerase chain reaction (PCR) genotyping assay. Forty-five concentrated urine samples collected from women with known cytological and HPV infection status, determined through in-house PCR and genotyping assays, were tested "as is" and as DUS with the Xpert® HPV test. This system detected HR-HPV in 86.4% of fresh and in 77.3% of dried urine samples collected from HPV+ women, correctly identifying HR-HPV infection in 100% of women with low- and high-grade lesions. High concordance (91.4%, k = 0.82) was found between PCR test and Xpert® HPV Test from urine. Urine-based Xpert® HPV test seems to be a suitable screening test for detection of HR-HPV infections associated with low- and high-grade lesions requiring follow-up monitoring or treatment. This methodology, relying on noninvasively collected samples and on available rapid testing platforms, could facilitate large, at-scale screening programs, particularly in LMICs and rural areas, thus reducing adverse outcomes of HPV infection and facilitating achievement of the WHO cervical cancer elimination goal.

A large A(H3N2) influenza outbreak with a high attack rate in a drug user community in Italy, April 2022.

Despite the COVID-19 pandemic, influenza remains an important issue. Especially in community settings, influenza outbreaks can be difficult to control and can result in high attack rates. In April 2022, a large A(H3N2) influenza outbreak spread in the largest Italian drug-rehabilitation community. One hundred eighty-four individuals presented influenza-like symptoms (attack rate of 26.2%); 56% previously received the influenza vaccine. Sequence analyses highlighted a genetic drift from the vaccine strain, which may have caused the observed lack of protection.

Breakthrough Infections: A Challenge towards Measles Elimination?

Measles is one of the most contagious diseases known to man. Despite the existence of a safe and effective live attenuated vaccine, measles can appear in vaccinated individuals. Paradoxically, breakthrough cases increase as vaccination coverage in the general population rises. In measles endemic areas, breakthrough cases represent less than 10% of total infections, while in areas with high vaccination coverage these are over 10% of the total. Two different vaccination failures have been described: primary vaccination failure, which consists in the complete absence of humoral response and occurs in around 5% of vaccinated individuals; and secondary vaccination failure is due to waning immunity or incomplete immunity and occurs in 2-10% of vaccinees. Vaccination failures are generally associated with lower viral loads and milder disease (modified measles) since vaccination limits the risk of complicated disease. Vaccination failure seems to occur between six and twenty-six years after the last vaccine dose administration. This review summarizes the literature about clinical, serological, epidemiological, and molecular characteristics of measles breakthrough cases and their contribution to virus transmission. In view of the measles elimination goal, the assessment of the potential decline in antibody protection and the targeted implementation of catch-up vaccination are essential.

A Methodological Approach to Water Concentration to Investigate the Presence of SARS-CoV-2 RNA in Surface Freshwaters.

During the COVID-19 public health emergency, an increasing number of studies reported the occurrence of SARS-CoV-2 in wastewaters worldwide, but little is known about the presence of the virus in surface freshwaters. The aim of the current study was to develop and validate an appropriate and scalable methodological approach for the concentration and detection of SARS-CoV-2 from surface freshwater samples, collected within the Milan rural network subjected to flood spillways activity. Overall, both surface water and distilled water samples spiked with inactivated SARS-CoV-2 were used to validate the concentration method for pathogens determination. Two pre-filtration systems, filter paper and Sartolab® P20 (Sartorius, Germany) and two concentration methods, two-phase (PEG-dextran method) separation and tangential flow ultrafiltration (UF), were compared. The effects of pre-filtration and concentration on viral nucleic acid recovery were assessed through real time RT-PCR targeting SARS-CoV-2 and the internal viral control PMMoV (Pepper Mild Mottle Virus). Our results showed that UF is more sensitive than the PEG-dextran method in viral acid nucleic recovery from surface water samples. Better results were obtained pre-filtering samples with Sartolab® P20 and extracting the nucleic acids with undiluted silica, rather than diluted as required by the standard protocol. The proposed method will be used for the monitoring of surface waters in the Milan area.

Molecular evidence for SARS-CoV-2 in samples collected from patients with morbilliform eruptions since late 2019 in Lombardy, northern Italy.

As a reference laboratory for measles and rubella surveillance in Lombardy, we evaluated the association between SARS-CoV-2 infection and measles-like syndromes, providing preliminary evidence for undetected early circulation of SARS-CoV-2. Overall, 435 samples from 156 cases were investigated. RNA from oropharyngeal swabs (N = 148) and urine (N = 141) was screened with four hemi-nested PCRs and molecular evidence for SARS-CoV-2 infection was found in 13 subjects. Two of the positive patients were from the pandemic period (2/12, 16.7%, March 2020-March 2021) and 11 were from the pre-pandemic period (11/44, 25%, August 2019-February 2020). Sera (N = 146) were tested for anti-SARS-CoV-2 IgG, IgM, and IgA antibodies. Five of the RNA-positive individuals also had detectable anti-SARS-CoV-2 antibodies. No strong evidence of infection was found in samples collected between August 2018 and July 2019 from 100 patients. The earliest sample with evidence of SARS-CoV-2 RNA was from September 12, 2019, and the positive patient was also positive for anti-SARS-CoV-2 antibodies (IgG and IgM). Mutations typical of B.1 strains previously reported to have emerged in January 2020 (C3037T, C14408T, and A23403G), were identified in samples collected as early as October 2019 in Lombardy. One of these mutations (C14408T) was also identified among sequences downloaded from public databases that were obtained by others from samples collected in Brazil in November 2019. We conclude that a SARS-CoV-2 progenitor capable of producing a measles-like syndrome may have emerged in late June-late July 2019 and that viruses with mutations characterizing B.1 strain may have been spreading globally before the first Wuhan outbreak. Our findings should be complemented by high-throughput sequencing to obtain additional sequence information. We highlight the importance of retrospective surveillance studies in understanding the early dynamics of COVID-19 spread and we encourage other groups to perform retrospective investigations to seek confirmatory proofs of early SARS-CoV-2 circulation.

Characterization of Vaccine Breakthrough Cases during Measles Outbreaks in Milan and Surrounding Areas, Italy, 2017-2021.

Despite the existence of an effective live-attenuated vaccine, measles can appear in vaccinated individuals. We investigated breakthrough measles cases identified during our surveillance activities within the measles/rubella surveillance network (MoRoNet) in Milan and surrounding areas (Northern Italy). Between 2017 and 2021, we confirmed measles virus (genotypes B3 or D8) infections in 653 patients and 51 of these (7.8%) were vaccinees. Among vaccinated individuals whose serum was available, a secondary failure was evidenced in 69.4% (25/36) of cases while 11 patients (30.6%) were non-responders. Non-responders were more frequently hospitalized and had significantly lower Ct values in both respiratory and urine samples. Median age and time since the last immunization were similar in the two groups. Importantly, we identified onward transmissions from vaccine failure cases. Vaccinees were involved in 20 outbreaks, in 10 of them they were able to transmit the virus, and in 8 of them, they were the index case. Comparing viral hemagglutinin sequences from vaccinated and non-vaccinated subjects did not show a specific mutation pattern. These results suggest that vaccination failure was likely due to the poor immune response of single individuals and highlights the importance of identifying breakthrough cases and characterizing their clinical and virologic profiles.

Waiting for the truth: is reluctance in accepting an early origin hypothesis for SARS-CoV-2 delaying our understanding of viral emergence?

Two years after the start of the COVID-19 pandemic, key questions about the emergence of its aetiological agent (SARS-CoV-2) remain a matter of considerable debate. Identifying when SARS-CoV-2 began spreading among people is one of those questions. Although the current canonically accepted timeline hypothesises viral emergence in Wuhan, China, in November or December 2019, a growing body of diverse studies provides evidence that the virus may have been spreading worldwide weeks, or even months, prior to that time. However, the hypothesis of earlier SARS-CoV-2 circulation is often dismissed with prejudicial scepticism and experimental studies pointing to early origins are frequently and speculatively attributed to false-positive tests. In this paper, we critically review current evidence that SARS-CoV-2 had been circulating prior to December of 2019, and emphasise how, despite some scientific limitations, this hypothesis should no longer be ignored and considered sufficient to warrant further larger-scale studies to determine its veracity.

HPV Type-Specific Prevalence a Decade after the Implementation of the Vaccination Program: Results from a Pilot Study.

Human papillomavirus infection is a cause of the development of invasive cervical cancer. Three types of vaccine are currently available to prevent precancerous/cancerous lesions due to persistent infection, which is supported mainly by 7 different high-risk genotypes. The aim of this pilot study was to acquire preliminary data on type-specific prevalence 10 years after the implementation of the HPV vaccination program in Italy, in order to subsequently plan appropriate observational studies in the Italian population. First-voided urine samples were collected from 393 consenting subjects, both females and males, aged 18-40 years, and HPV DNA was detected by PCR amplification of a 450 bp L1 fragment. All amplified products were genotyped by means of the Restriction Fragment Length Polymorphism (RFLP) method. The female population was divided into three cohorts ("vaccine-eligible", "pre-screening" and "screening" cohorts) according to the preventive intervention scheduled by age; males were included in the same three cohorts according to their year of birth. The overall prevalence of HPV infection was 19%, being higher in females than in males (22.1% vs. 13.6%, p = 0.03729). In the female population, 10 years after the start of the national immunization program, we observed a reduction in the prevalence of vaccine types and the number of circulating genotypes, especially in the "vaccine-eligible" cohort. The frequency of HPV vaccine types increased with age, particularly in males in the "pre-screening" and "screening" cohorts. Our study highlights the importance of monitoring HPV infection in both genders, to validate the effect of the HPV vaccination program.

Evidence of SARS-CoV-2 RNA in an Oropharyngeal Swab Specimen, Milan, Italy, Early December 2019.

We identified severe acute respiratory syndrome coronavirus 2 RNA in an oropharyngeal swab specimen collected from a child with suspected measles in early December 2019, ≈3 months before the first identified coronavirus disease case in Italy. This finding expands our knowledge on timing and mapping of novel coronavirus transmission pathways.

Molecular Epidemiology of B3 and D8 Measles Viruses through Hemagglutinin Phylogenetic History.

Of the 24 known measles genotypes, only D8 and B3 are responsible for outbreaks in the last years in Europe, Asia, and America. In this study the H gene of 92 strains circulating between 2015 and 2019 in Lombardy, Northern Italy, and 1273 H sequences available in GenBank were analyzed in order to evaluate the genetic variability and to assess the conservation of the immunodominant sites. Overall, in Lombardy we observed the presence of four different B3 and three different D8 clusters, each one of them including sequences derived from viruses found in both vaccinated and unvaccinated subjects. Worldwide, the residue 400 within the H protein, a position located within the main immune epitope, is mutated in all circulating strains that belong to the two globally endemic genotypes, B3 and D8. Our data demonstrate the usefulness of measles virus (MV) H gene sequencing. Indeed, the monitoring the H protein epitopes of circulating strains could be included in the measles laboratory surveillance activities in order to improve and optimize strategies for measles control, as countries go towards elimination phase.

Genetic characterization of variants of HPV­16, HPV­18 and HPV­52 circulating in Italy among general and high­risk populations.

Viral factors, such as high­risk human papillomavirus variants, can increase the risk of viral persistence and influence the progression to cancer. In the present study, the long control region (LCR) of human papillomavirus (HPV)­16 and HPV­52, and the L1 region of HPV­16 and HPV­18, identified from subjects belonging to both general and high­risk populations (migrants, HIV+ subjects and adolescent/young people) residing in Italy, were characterized using molecular and phylogenetic techniques. To the best of our knowledge, this is the first Italian study to analyze a large number of sequences (n=458) and report phylogenetic data on the HPV­52 variants. The phylogenetic analysis showed that 90% of the LCR variants of HPV­16 and HPV­52 clustered within lineage A (European lineage) and only sequences identified from subjects belonging to high­risk populations fell into the non­European lineages. Analysis of the LCRs revealed a high genomic diversity with a large number of changes. Several mutations in the binding sites for viral and cellular transcription factors characterized the HPV­16 LCR variants belonging to the African lineages B and C, were observed in subjects with cytological abnormalities (high squamous intraepithelial lesions). The HPV­16 and HPV­18 L1 molecular characterization identified 30% of changes in the immune­dominant epitope loops. These data give a clear picture of the situation in Italy, and a starting point for understanding the molecular pathogenesis and developing molecular diagnostics for HPV, vaccines and other therapeutic approaches in order to control and/or eliminate virus­induced diseases.

Ongoing large measles outbreak with nosocomial transmission in Milan, northern Italy, March-August 2017.

A large measles outbreak has been ongoing in Milan and surrounding areas. From 1 March to 30 June 2017, 203 measles cases were laboratory-confirmed (108 sporadic cases and 95 related to 47 clusters). Phylogenetic analysis revealed the co-circulation of two different genotypes, D8 and B3. Both genotypes caused nosocomial clusters in two hospitals. The rapid analysis of epidemiological and phylogenetic data allowed effective surveillance and tracking of transmission pathways.