Phylogenetic analysis revealed that all sequenced samples belonged to the D8 genotype. Notably, the four mutational patterns identified corresponded to distinct groups on the phylogenetic tree, providing substantial evidence for the likely multiple introductions of MeV in Lazio. By combining molecular and epidemiological investigations, we were able to identify four clusters, associating 12 of the 28 measles cases reported in the study period, and 9 of the 18 sporadic cases. By examining the three clusters previously unidentified during epidemiological investigations, we found that cases in each cluster were spatiotemporally related, and we hypothesize that the cases came into contact randomly in previously unidentified locations, such as public places or other neighbourhood settings frequented by cases. One cluster originated from an imported case from the Czech Republic. Two additional sequenced cases were imported, but no associated sequences were identified among endemic cases; however, since 14 cases could not be sequenced, we cannot exclude that these were involved in other transmission chains.

In July 2017 the Italian government made 10 vaccines, including measles/mumps/rubella (MMR), mandatory for access to schools, following a decline in vaccination coverage between 2013 and 2015 reaching a level as low as 85% for the MMR vaccine, and in the midst of a large measles outbreak [9, 10]. Of the 28 cases, two fell within the age group requiring mandatory vaccination, but only one was fully vaccinated; the remaining cases were adults. In Lazio, by the end of 2024, the highest incidence of measles was observed in the 15–39 age group (80.7 per million inhabitants, more than double the national average of 38.2), followed by the 40–64 age group (24 per million inhabitants, against the national average of 12) [11]. An ad hoc analysis of regional vaccination coverage performed in March 2024 to estimate the susceptible population of Lazio, following the age distribution of cases, found that 77.8% of the population in the 20–50 age group was not vaccinated. Supplementary immunization activities have therefore been implemented at the regional level, targeting this age group.

Concerns about the increase in measles cases in Europe [2] and the contemporary alarm of a new MeV variant impacting molecular diagnostics [7, 8], highlight the need to characterize circulating strains and evaluate the potential of this variant to hinder laboratory diagnostics.

WGS analysis revealed that samples sequenced at RRL-INMI were characterized by four different mutational patterns at the reverse primer annealing site, one of which had previously been associated with a slight impact on molecular diagnostics [7, 8]. The appropriateness of the molecular diagnostic test used at RRL-INMI to detect the currently circulating MeV strain was assessed, and we found no reduction in sensitivity between the mutated and WT strains: both triple-mutated and WT samples were detected up to in 2/2 (100%) replicates down to a 10− 4 dilution equal to 36 Ct. More recently, Beck et al. designed and tested a new mixed reverse primer strategy able to rescue the sensitivity of CDC assay [12]. Nonetheless, monitoring of variants remains important to continuously update molecular diagnostics.

The present work has some limitations, firstly that the diagnostic procedures and the sensitivity tests presented in this study are based on a commercial assay, so primers and probes sequences are not available, and cannot be checked for primer mismatch. However, the RealCycler assay is widely used by many Subnational Reference Laboratories of the measles and rubella surveillance network MoRoNet (https://doi.org/10.1136/sextrans-ICAR-2024.177). Moreover, the relatively small number of whole genome sequences obtained in this study limits the strength of phylogenetic conclusions. Anyway, the measles virus genotype of all positive samples was confirmed based on phylogenetic analyses of N-450. A comparison between the WGS tree and the N450 tree confirmed the genomic clusters identified in the WGS tree; however, the added value of WGS may be that of elucidating specific chains of transmission previously undetected, to support both the accuracy of epidemiological investigation and the implementation of additional measures to control the spread of measles. Moreover, through the integration of WGS and molecular characterization, we were able to rule out the existence of other circulating variants with mutations that could potentially affect the efficacy of existing assays.