Detection of influenza virus in urban wastewater during the season 2022/2023 in Sicily, Italy.

Introduction: Seasonal influenza generally represents an underestimated public health problem with significant socioeconomic implications. Monitoring and detecting influenza epidemics are important tasks that require integrated strategies. Wastewater-based epidemiology (WBE) is an emerging field that uses wastewater data to monitor the spread of disease and assess the health of a community. It can represent an integrative surveillance tool for better understanding the epidemiology of influenza and prevention strategies in public health. Methods: We conducted a study that detected the presence of Influenza virus RNA using a wastewater-based approach. Samples were collected from five wastewater treatment plants in five different municipalities, serving a cumulative population of 555,673 Sicilian inhabitants in Italy. We used the RT-qPCR test to compare the combined weekly average of Influenza A and B viral RNA in wastewater samples with the average weekly incidence of Influenza-like illness (ILI) obtained from the Italian national Influenza surveillance system. We also compared the number of positive Influenza swabs with the viral RNA loads detected from wastewater. Our study investigated 189 wastewater samples. Results: Cumulative ILI cases substantially overlapped with the Influenza RNA load from wastewater samples. Influenza viral RNA trends in wastewater samples were similar to the rise of ILI cases in the population. Therefore, wastewater surveillance confirmed the co-circulation of Influenza A and B viruses during the season 2022/2023, with a similar trend to that reported for the weekly clinically confirmed cases. Conclusion: Wastewater-based epidemiology does not replace traditional epidemiological surveillance methods, such as laboratory testing of samples from infected individuals. However, it can be a valuable complement to obtaining additional information on the incidence of influenza in the population and preventing its spread.

Whole-Genome Sequencing and Genetic Diversity of Human Respiratory Syncytial Virus in Patients with Influenza-like Illness in Sicily (Italy) from 2017 to 2023.

Monitoring the genetic variability of human respiratory syncytial virus (hRSV) is of paramount importance, especially for the potential implication of key antigenic mutations on the emergence of immune escape variants. Thus, to describe the genetic diversity and evolutionary dynamics of hRSV circulating in Sicily (Italy), a total of 153 hRSV whole-genome sequences collected from 770 hRSV-positive subjects between 2017 and 2023, before the introduction of expanded immunization programs into the population, were investigated. The phylogenetic analyses indicated that the genotypes GA.2.3.5 (ON1) for hRSV-A and GB.5.0.5a (BA9) for hRSV-B co-circulated in our region. Amino acid (AA) substitutions in the surface and internal proteins were evaluated, including the F protein antigenic sites, as the major targets of immunoprophylactic monoclonal antibodies and vaccines. Overall, the proportion of AA changes ranged between 1.5% and 22.6% among hRSV-A, whereas hRSV-B varied in the range 0.8-16.9%; the latter was more polymorphic than hRSV-A within the key antigenic sites. No AA substitutions were found at site III of both subgroups. Although several non-synonymous mutations were found, none of the polymorphisms known to potentially affect the efficacy of current preventive measures were documented. These findings provide new insights into the global hRSV molecular epidemiology and highlight the importance of defining a baseline genomic picture to monitor for future changes that might be induced by the selective pressures of immunological preventive measures, which will soon become widely available.

Induction of significant neutralizing antibodies against SARS-CoV-2 by a highly attenuated pangolin coronavirus variant with a 104nt deletion at the 3'-UTR.

SARS-CoV-2 related coronaviruses (SARS-CoV-2r) from Guangdong and Guangxi pangolins have been implicated in the emergence of SARS-CoV-2 and future pandemics. We previously reported the culture of a SARS-CoV-2r GX_P2V from Guangxi pangolins. Here we report the GX_P2V isolate rapidly adapted to Vero cells by acquiring two genomic mutations: an alanine to valine substitution in the nucleoprotein and a 104-nucleotide deletion in the hypervariable region (HVR) of the 3'-terminus untranslated region (3'-UTR). We further report the characterization of the GX_P2V variant (renamed GX_P2V(short_3UTR)) in in vitro and in vivo infection models. In cultured Vero, BGM and Calu-3 cells, GX_P2V(short_3UTR) had similar robust replication kinetics, and consistently produced minimum cell damage. GX_P2V(short_3UTR) infected golden hamsters and BALB/c mice but was highly attenuated. Golden hamsters infected intranasally had a short duration of productive infection in pulmonary, not extrapulmonary, tissues. These productive infections induced neutralizing antibodies against pseudoviruses of GX_P2V and SARS-CoV-2. Collectively, our data show that the GX_P2V(short_3UTR) is highly attenuated in in vitro and in vivo infection models. Attenuation of the variant is likely partially due to the 104-nt deletion in the HVR in the 3'-UTR. This study furthers our understanding of pangolin coronaviruses pathogenesis and provides novel insights for the design of live attenuated vaccines against SARS-CoV-2.

Treatment of Metastatic Primary Extramammary Paget Disease With Combination Anlotinib and Tislelizumab: A Case Report and Review of the Literature.

Extramammary Paget's disease (EMPD) is a rare cutaneous neoplasm with distant metastases and a poor prognosis. We report the case of a 63-year-old male patient exhibiting stage IV primary EMPD with neuroendocrine differentiation, and harboring a somatic mutation in AMER1. After four cycles of Anlotinib combined with Tislelizumab, the patient achieved partial response for the metastatic lesions according to mRECIST1.1 criteria. Total positron emission tomography and computed tomography (PET-CT) scans revealed a significant reduction in SUV from 18.9 to 5.3, and the serum CEA decreased to normal levels after the treatment regimen. However, the patient developed fractures of the fourth and fifth thoracic vertebrae during the treatment. Therefore, percutaneous vertebroplasty was performed, and the patient experienced severe postoperative pneumonia and died from pulmonary encephalopathy and respiratory failure in June 2021. The overall and progression-free survival of the patient after diagnosis were 9 and 8 months, respectively. During the systemic treatment, the patient suffered grade 1 rash in the back and thigh and grade 1 hypertension. Nevertheless, the combination treatment of anlotinib and tislelizumab had a favorable clinical outcome and provided a survival advantage, and should be considered a therapeutic option for patients with AMER1-mutant metastatic EMPD.

Can We Clinically Distinguish Anejaculation From Retrograde Ejaculation in Patients on α1A-Blockers Therapy for Lower Urinary Tract Symptoms?

OBJECTIVE: To investigate the physiopathology of ejaculatory disorders (EjD) and discriminate between retrograde ejaculation (REj) and anejaculation (AEj) induced by α1A-blockers, through the association between the mean postorgasm seminal vesicle volume and the presence of sperm in midstream urine, in patients with moderate-to-severe lower urinary tract symptoms (LUTS) secondary to benign prostatic enlargement. MATERIALS AND METHODS: Therapy-naïve male patients with LUTS and without previous EjD were treated with α1A-blockers. Pre- and post-treatment EjD were investigated through question 4 of the 4-item Male Sexual Function questionnaire and the Male Sexual Health Questionnaire for Ejaculatory Dysfunction Short Form (MSHQ-EjD-SF). After 12 weeks, postorgasm urine was collected for sperm count and seminal vesicle volume was calculated through transrectal ultrasound. RESULTS: All 42 patients reported with EjD after treatment with α1A-blockers: 4-item Male Sexual Function questionnaire and MSHQ-EjD-SF Q4 scores were significantly higher (P <.001) and MSHQ-EjD-SF Q1-3 score was significantly lower (P <.001) than before. Postorgasm seminal vesicle volume was significantly higher in patients with postorgasm sperm-negative urine (AEj), and lower in patients with postorgasm sperm-positive urine (REj; P <.001). CONCLUSION: We clearly demonstrated an association between the presence of sperm in the midstream urine and seminal vesicle volume after orgasm, strongly confirming and differentiating the hypothesis of a dual etiology for EjD (REj vs AEj) secondary to α1A-blockers therapy for LUTS.

Performance evaluation of gastrointestinal viral ELIte panel multiplex RT-PCR assay for the diagnosis of rotavirus, adenovirus and astrovirus infection.

Rotavirus, adenovirus, norovirus and astrovirus are considered to be among the major causes of sporadic cases and outbreaks of acute gastroenteritis globally. Rapid and accurate identification of enteric viruses is still a challenge for the clinical laboratory. Recently, several molecular platforms for the detection of viral enteric pathogens have become available. In this study, the diagnostic accuracy of InGenius Gastrointestinal Viral (GV) Elite Panel, a newly developed one-step multiplex real-time RT-PCR assay simultaneously detecting rotavirus, adenovirus and astrovirus, was evaluated retrospectively analyzing an archival collection of 128 stool samples of children hospitalized with acute gastroenteritis. The overall sensitivity and specificity for the GV assay was 100% and 96.2% for rotavirus, 96.9% and 100% for astrovirus, 100% and 100% for adenovirus, respectively. The InGenius GV assay showed a high concordance with the reference methods and was able to detect all tested genotypes of rotavirus (including G1, G3, G4, G9 and G12P[8] and G2P[4]), adenovirus and astrovirus (AstV-1 and 2). Studies of considerable sample size are required to determine robust Cycle threshold cut-off values to effectively correlate infection to disease. These preliminary results suggest that InGenius GV assay can be recommended as a valuable method for accurate diagnosis of epidemic and sporadic gastroenteritis.

The reemergence of human rabies and emergence of an Indian subcontinent lineage in Tibet, China.

Coordinated surveillance, vaccination and public information efforts have brought the Chinese rabies epizootic under control, but significant numbers of fatalities are still reported annually with some cases occurring in previously rabies free regions. Tibet has remained virtually rabies free for 16 years, but since 2015 one human rabies case has been reported each year. To better understand the origins of these cases, we sequenced three human samples and an additional sample isolated from a dog in 2012. Three genomes were sequenced from brain samples: human case 1 (reported in 2015), human case 3 (2017), and the 2012 dog case. For human case 2 (2016), the rabies N gene was sequenced from a limited saliva sample. Phylogenetic analysis shows that Case 1 (CXZ1501H) and the dog case (CXZ1201D) belong to China IV lineage (equivalent to Arctic-like-2 in global rabies), suggesting an association with a wildlife spillover event. However, Case 2 (CXZ1601H) is placed within the dominant lineage China I, and was most similar with recent strains from neighboring Yunnan province, indicating the current epizootic has finally reached Tibet. Most surprisingly however, was the finding that Case 3 (CXZ1704H) is distinct from other Chinese isolates. This isolate is placed in the Indian Subcontinent clade, similar to recent Nepal strains, indicating that cross-border transmission is a new source for rabies infections. Thus, the complex mixture of the rabies epizootic in Tibet represents a major new challenge for Tibet and national rabies control.

Inner Mongolia: A Potential Portal for the Spread of Rabies to Western China.

In recent years, the number of human rabies cases in China has decreased annually. However, some western provinces with no human cases for more than 10 years have begun to report rabies cases, and all of the rabies lineages that circulated in western China were found in Inner Mongolia as well. In this study, we generated a phylogenetic tree with all the Inner Mongolia rabies strains available in GenBank and our laboratory, as well as strains from western China and representative viruses from neighboring countries, based on the N gene sequence. Furthermore, the possible relationships underlying the spread of the virus within Inner Mongolia and neighboring regions were analyzed. Three of six rabies lineages of China (China I-VI) were shown to exist in Inner Mongolia, and a spatial cluster analysis supported that the China I lineage, the dominant cluster of China, likely spread to Ningxia from Inner Mongolia. Wild raccoon dog rabies (China IV/Arctic-like-2) may have spread to Inner Mongolia from Russia and likely continued to spread to Qinghai and Tibet. The red fox lineage (China III/Cosmopolitan), which likely spread from Russia and Mongolia, has been shown to circulate in Inner Mongolia and was a serious threat to Xinjiang, which is adjacent to Inner Mongolia. Thus, Inner Mongolia likely became a location where national and international rabies viruses collected and developed into a potential portal for the spread of rabies to western China. To effectively control the spread of rabies in China, both prevention and control of dog and wild animal rabies in Inner Mongolia should be a top priority.

Performance evaluation of a newly developed molecular assay for the accurate diagnosis of gastroenteritis associated with norovirus of genogroup II.

The performance of a newly proposed fully automated cassette-based sample-to-results solution for norovirus (NoV) detection, InGenius Norovirus ELITe MGB®, was evaluated. A total of 120 selected archival stool samples from children hospitalized for acute gastroenteritis were used to compare the results to a reference real-time RT-PCR. The InGenius NoV assay showed optimal diagnostic accuracy (sensitivity, 100%; specificity, 95.7%) and was able to correctly detect the entire wide panel of epidemiologically relevant genotypes tested. These preliminary results suggest that the InGenius NoV assay can be recommended as a valuable method for accurate diagnosis of NoV GII infection in epidemic and sporadic gastroenteritis.

Is Italian population protected from Poliovirus? Results of a seroprevalence survey in Florence, Italy.

OBJECTIVES: Periodical assessments of population susceptibility to polioviruses (PV) is essential for evaluating population protection and planning appropriate vaccination strategies. The aim of the current work was to assess serological protective titers against all three polioviruses in the general population of Florence. METHODS: A convenience sample of 328 sera, collected in 2009 in Florence (Central Italy) was analyzed. Samples were considered protective if neutralizing antibodies were detected at dilutions ≥1:8, according to the WHO protocols. RESULTS: The immune coverage was 75.3%, 69.2% and 46% for PV1, PV2 and PV3, respectively. The protective titers of neutralizing antibodies were generally higher in children up to 14 years of age, with 74.4% (PV1), 75.6% (PV2) and 56.7% (PV3) of seroprevalence. From the age of 11 years, most of the study subjects were seronegative for PV3. CONCLUSIONS: In a polio-free country with strong migration pressures, such as Italy, our results bring clear support to the recent recommendation of Italian health authorities to introduce a fifth dose of IPV vaccine in adolescence all over the country.

Waterborne Norovirus outbreak at a seaside resort likely originating from municipal water distribution system failure.

In May 2016 a Norovirus (NoV) gastroenteritis outbreak involved a high school class visiting a seaside resort near Taormina (Mascali, Sicily). Twenty-four students and a teacher were affected and 17 of them showed symptoms on the second day of the journey, while the others got ill within the following 2 days. Symptoms included vomiting, diarrhoea and fever, and 12 students required hospitalisation. Stool samples tested positive for NoV genome by Real-Time polymerase chain reaction assay in all 25 symptomatic subjects. The GII.P2/GII.2 NoV genotype was linked to the outbreak by ORF1/ORF2 sequence analysis. The epidemiological features of the outbreak were consistent with food/waterborne followed by person-to-person and/or vomit transmission. Food consumed at a shared lunch on the first day of the trip was associated to illness and drinking un-bottled tap water was also considered as a risk factor. The analysis of water samples revealed the presence of bacterial indicators of faecal contamination in the water used in the resort as well as in other areas of the municipal water network, linking the NoV gastroenteritis outbreak to tap water pollution from sewage leakage. From a single water sample, an amplicon whose sequence corresponded to the capsid genotype recovered from patients could be obtained.

Molecular evolutionary analysis of type-1 human astroviruses identifies putative sites under selection pressure on the capsid protein.

Human astroviruses (HAstV) are important enteric pathogens that can be classified into eight sero/genotypes (HAstV-1 to -8). Although the various HAstV types show global spread, type-1 strains tend to be predominant. Molecular analysis of the genomic region encoding the capsid protein (ORF2) has revealed discrete sequence variation, with different lineages within each HAstV type and at least three major lineages have been identified within HAstV-1. Longitudinal epidemiological surveillance has revealed temporal shift of the various HAstV-1 lineages. Metadata analysis of HAstV-1 sequences available in the databases also revealed temporal shifts of the circulation of HAstV-1 lineages, suggesting possible antigenic-related mechanisms of selection at the sub-genotype level. By comparison of HAstV-1 capsid sequences, lineage-defining residues under positive selection were identified. Structural analysis of HAstV-1 capsid allowed identifying at least six residues exposed on the virion surface. Two residues were located in the VP34 (shell region) whilst four residues were mapped in the VP25/27 (protruding region) of HAstV capsid protein, in proximity of the putative receptor binding S site. These findings suggest that mechanisms similar to those observed and/or hypothesized for other enteric viruses are also shaping the evolution of HAstVs, with intra-typic diversification being a possible mechanism to decrease the antigenic pressure to which these viruses are exposed.

Performance analysis of two immunochromatographic assays for the diagnosis of rotavirus infection.

Group A rotaviruses (RVAs) are the primary cause of acute gastroenteritis (AGE) in young children worldwide. Several commercial tests including latex agglutination, enzyme-linked assays (ELISA) and immunochromatographic tests (ICT) have been developed for the diagnosis of RVA infection. In the present study, the performance of two commercially available one-step chromatographic immunoassays, CerTest Rotavirus+Adenovirus (Biotec S.L, Zaragoza, Spain) and Vikia Rota-Adeno (bioMerieux SA, Lyon, France) were retrospectively evaluated using Real-time PCR as reference test. Re-testing by Real-time PCR of 2096 stool samples of children hospitalized with AGE previously screened by ICTs (1467 by CerTest and 629 by Vikia) allowed to calculate higher sensitivity for Vikia (94% vs 85% of CerTest) and higher specificity for CerTest (93% vs 89% of Vikia). Accordingly, higher Positive Predictive Values (87% vs 78%) and Positive Likelihood Ratios (12.32 vs 8.8) were found for CerTest and lower Negative Predictive Values (91% vs 97%) and Negative Likelihood Ratios (0.16 vs 0.06) for Vikia. However, both CerTest and Vikia showed a substantial agreement (κ=0.79) with the Real-time PCR. A correlation between false negative results by ICTs and high Cycle Threshold values of Real-time PCR, indicative of low viral load, was observed. False positive results by the two ICT assays were not related to Norovirus, Adenovirus or Astrovirus infections, therefore the risk of cross-reactions was excluded. Both CerTest and VIKIA were able to detect the wide range of RVA genotypes circulating over the study period (including G1P[8], G2P[4], G3, G4, G9 and G12P[8]). The results of the present study showed a satisfactory efficacy of the two diagnostic tests analyzed.

Significance of borderline hemoglobin A2 values in an Italian population with a high prevalence of beta-thalassemia.

We report a retrospective analysis carried out on 23,485 subjects submitted to a screening program from 2000 to 2006. Of these subjects, 3,934 had borderline HbA(2) values from 3.1 to 3.9%; 410 samples, analyzed previously using PCR methods and sequencing because all of these were partners of a carrier of classical beta-thalassemia, were selected for statistical analysis. Of 410 subjects, 94 (22.9%) were positive for a molecular defect in the beta-, delta- or alpha-globin genes. The most prevalent molecular defects were beta IVS1 nt 6 (HBB c.92+6T C), co-inheritance of severe beta thalassemia and delta mutations, beta-promoter mutations and triplication of alpha genes were detected; alpha-thalassemia and Hb-variants were also evident. Borderline HbA(2) is not a rare event in a population with a high prevalence of beta-thalassemia carriers. These data support the necessity to investigate these cases at a molecular level, particularly if the partner is a carrier of beta-thalassemia.

Hb Marineo [beta70(E14)Ala-->Val]: a silent hemoglobin variant with a mutation within the heme pocket.

We report a new hemoglobin (Hb) variant, Hb Marineo [beta70(E14)Ala --> Val], found in three generations of a family from West Sicily. The mutation is due to a GCC --> GTC substitution at codon 70 of the beta-globin gene. To date, three mutations at codon 70 of the beta-globin gene have been described, presenting with hemolytic anemia. In our case, no anemia or other alteration of hematological indices were found. Cation exchange high performance liquid chromatography (HPLC) showed a peak in the P2 window (VARIANT I), while a peak was detected by VARIANT II HPLC in the P3 window. Reversed phase HPLC analysis showed an abnormal chain amounting to about 40% of the total beta chains.