The mitigation measures adopted to reduce the spread of COVID-19 have not only impacted the transmission of respiratory infections but also other infectious diseases. In this study, we explored the effect of the pandemic on the epidemiology of Central Nervous System (CNS) infections by conducting a retrospective analysis of diagnostic data obtained from cerebrospinal fluids (CSFs) collected before, during, and after the pandemic from patients with acute-suspected neuro-infectious syndrome. The samples were analyzed using a meningitis/encephalitis molecular syndromic panel. A total of 4203 CSFs were split into three groups: pre-pandemic (August 2018–February 2020), pandemic (March 2020–March 2022) and post-pandemic (April 2022–March 2024). Each pathogen was statistically assessed individually, comparing one group vs. another. Results showed that viral pathogens were the most frequently detected across all groups; however, Streptococcus pneumoniae was the single most identified pathogen. Comparison of the pre-pandemic and pandemic groups by statistical analysis showed a significant reduction in neuro-infections caused by enterovirus, S. pneumoniae and N. meningitidis, while the other pathogens were not affected. In conclusion, a reduction in CNS infections caused by respiratory and close-contact-transmitted pathogens, including both viral and bacterial agents, was observed during the period when COVID-19 containment measures were in effect.

BACKGROUND:Chagas disease and strongyloidiasis are endemic in Latin America, but both infections have become diseases of global concern due to migration flows. During the last fifteen years, these infections have become emerging infections in Italy as a consequence of the huge immigration from Latin American countries. The aim of this study is to assess the prevalence of Chagas disease, strongyloidiasis, and their co-infection in a cohort of Latin American migrants living in Rome. Additionally, it seeks to evaluate whether informational outreach campaigns-offered directly within communities and supported by local leaders-could represent a possible approach to reveal the hidden public health burden of these neglected infections among migrant populations. METHODS:Six community-based information campaigns on Chagas disease and strongyloidiasis were performed in Rome (Italy) in public events or in homes occupied by migrants from Latin America, inviting people to carry out screening tests at the National Institute for Health, Migration and Poverty clinic. RESULTS:344 adults were tested for Chagas disease and strongyloidiasis. The overall prevalence of Trypanosoma cruzi infection was 7.8% (27/344). Of the positive results, 77.8% (21/27) were observed in persons originating from Bolivia. The prevalence of strongyloidiasis was 10.5% (36/344). Of the positive results, 69.4% (25/36)were among persons originating from Bolivia, out 27(22.2%) individuals tested positive for both Trypanosoma cruzi and Strongyloides stercoralis. CONCLUSIONS:Our findings indicate that targeted informational outreach campaigns-particularly those embedded within cultural, recreational, and sporting events-can be an effective strategy for promoting systematic and combined screening for Chagas disease and strongyloidiasis. Such initiatives not only raise public health awareness among Latin American migrants in non-endemic settings but also help to uncover a largely overlooked public health issue.

Long COVID (LC) is characterized by a wide range of symptoms, the causes of which remain unclear. We investigated associations between inflammatory and coagulation factors, adaptive immune response to SARS-CoV-2, and LC. We enrolled 196 unvaccinated individuals with SARS-CoV-2 (March–June 2020). Blood samples were collected at three (T3M) and twelve (T12M) months post infection. Plasma concentrations of coagulation factors (D-Dimer, E-Selectin, ICAM-1, VCAM-1) and inflammatory markers (IL-6, IL-8, TNF-α, IL-1β) were measured by ELISA, and SARS-CoV-2-specific T-cell response was assessed by Elispot. LC occurred in 66/196 patients (34%); 77.8% had been hospitalized. Respiratory symptoms were present in 54%, fatigue in 30%, and neuropsychological symptoms in 14%. At T3M, hospitalized patients exhibited higher levels of ICAM-1, VCAM-1, and IL-6, along with increased immunoreactivity. LC patients exhibited elevated IL-8 and TNF-α and enhanced immunoreactivity at T3M, though these differences were not observed at T12M. Inflammatory and coagulation markers were altered at three months after acute infection, with some changes persisting at one year, suggesting a long-term immunological impact of SARS-CoV-2 on the inflammatory response. A SARS-CoV-2-specific T-cell response was still detectable at T12M, albeit at a lower level than at T3M, suggesting the persistence of protective memory T-cells beyond the acute phase.

The impact of anti-Spike monoclonal antibody (mAbs) treatment on the immune response of COVID19-patients is poorly explored. In particular, a comparison of the immunological influence of different therapeutic regimens has not yet been performed. Aim of the study was to compare the kinetic of innate and adaptive immune response as well as the SARS-CoV-2 specific humoral and T cell response in two groups of SARS-CoV-2-infected patients treated with two different mAbs regimens: Bamlanivimab/Etesevimab (BAM/ETE) or Casirivimab/Imdevimab (CAS/IMD). SARS-CoV-2-infected patients (n = 39) with mild/moderate disease were enrolled before (T0) and after 7 days (T7) and 30 day (T30) from mAbs infusion. Patients were divided in two groups on the basis of the mAb regimen: BAM/ETE (n = 15) and CAS/IMD (n = 24). The phenotype/function of immune cell subsets was evaluated by flow-cytometry and by ELISA. The Spike-specific T cell response (IFN-γ) and anti-Nucleocapside IgG were evaluated by chemiluminescence assay. SARS CoV-2 RNA in nasal swabs was evaluated by RT-PCR. Eleven out of the thirty-nine enrolled patients tested negative at T7, among which nine (81.8 %) had been treated with CAS/IMD regimen. A comparable increase in CD4 and CD8 T cells was observed in both treatment groups. Moreover, a reduction of CD38 expression on T (CD4, CD8 and Vδ2) and on NK cells was observed in both groups, as well as a reduction overtime of the perforin expression in T (CD8, Vδ2) and in NK cells reaching significance only in CAS/IMD-treated patients. The SARS-CoV-2-specific T cells response increased at T7 in BAM/ETE-treated patients and at T30 in CAS/IND group. Of note, at T30 SARS-CoV2-specific T cells was higher in CAS/IMD than in BAM/ETE group. Furthermore, the titre of anti-N IgG increased overtime in both groups with a faster kinetic in CAS/IMD group. The spontaneous production of inflammatory cytokines by monocytes and neutrophils was similar the two mAb regimens, as well as the level of plasmatic IL-6. Finally, patients were also analysed according to sex. The male group showed a higher frequency of activated CD4 T cells, NKG2A-expressing CD8 T cells and perforin-expressing Vδ2 T cells compared to female group. Moreover, a higher specific T cell response at T30 was observed in the male compared to female group. In conclusion, these results show similar effects of both mAb regimens in restoring T and NK cell homeostasis and in reducing inflammation. In contrast, CAS/IMD allows a better humoral and cellular SARS-CoV2 specific immunization.

BACKGROUND:Chagas disease, caused by the parasite Trypanosoma cruzi (T. cruzi), affects around 6-7 million people in Latin America and hundreds of thousands in the United States and Europe. A main complication of chronic Chagas disease is cardiomyopathy, possibly manifesting as arrhythmias, heart failure, or sudden cardiac death. Understanding the link between T. cruzi infection and cardiomyopathy is essential for early diagnosis and adequate treatment. METHODS:We sequenced small RNAs in serum samples from 228 Chagas patients recruited in Chile, Bolivia and Italy. After bioinformatic processing of sequencing data to quantify serum miRNA expression, robust logistic regression was applied to identify miRNAs differentially expressed in Chagas patients with abnormalities in electrocardiography (ECG), bundle-branch block on ECG, and high Kuschnir scores. We also investigated the association between genotype-based miRNA expression and the risk of abnormal ECG findings. FINDINGS:As reported, the risk of abnormal ECG findings was higher in male patients and increased with age. Three miRNAs showed lower serum expression levels in patients with abnormal ECG: miRNA-101-3p, miRNA-576-3p and miRNA-629-5p (p < 0.0002), especially in patients with high Kuschnir scores. The expression of miRNA-629-5p was negatively correlated with the CCL5 expression (p = 3.7×10-8), a chemokine frequently reported in Chagas disease. Gene enrichment analyses indicated involvement of cytokine signalling in Chagas cardiomyopathy. INTERPRETATION:The findings demonstrate the potential of circulating miRNAs as diagnostic biomarkers for Chagas cardiomyopathy. The associations found with disease severity and immune response may help to improve our knowledge of complications' development in Chagas disease.

BACKGROUND:Although evidence exists on the potential involvement of circular RNAs (circRNAs) in the pathogenesis of several viral infections, the expression levels, and the exact role that hsa_circ_0006459 and hsa_circ_0015962 could play during the Dengue virus (DENV) infection are still unclear. These two circRNAs were identified as possible biomarkers for diagnosis and prognosis of DENV disease in peripheral blood mononuclear cells (PBMC) of Dengue-positive patients. This study aimed to evaluate the expression levels of hsa_circ_0006459 and hsa_circ_0015962 in DENV-infected patients and compare them with healthy donors (HD) to provide new insights into the biological significance of these two circRNAs' expression. METHODS:We examined the presence and expression levels of hsa_circ_0006459 and hsa_circ_0015962 in PBMC of DENV-patients throughout a period of 28 days after the DENV diagnosis. HD was used as a control group. RESULTS:Our results show different expression levels and patterns between hsa_circ_0006459 and hsa_circ_0015962, both in DENV patients and HD. CONCLUSION:Possible change in the hsa_circ_0006459 expression during DENV infection was observed, mainly at the time of diagnosis, but without a consistent pattern among patients during follow-up. Further studies are needed to clarify their expression levels and function both in Dengue-positive patients and HD.

Introduction: Coronavirus disease 2019 (COVID-19) is a significant and novel cause of acute respiratory distress syndrome (ARDS). During the COVID-19 pandemic, there has been an increase in the incidence of cases involving pneumothorax and pneumomediastinum. However, the risk factors associated with poor outcomes in these patients remain unclear. Methods: This observational study collected clinical and imaging data from COVID-19 patients with PTX and/or PNM across five tertiary hospitals in central Italy between 1 March 2020 and 1 March 2022. This study also calculated the incidence of PTX and PNM and utilized multivariable regression analysis and Kaplan–Meier curve analysis to identify predictor factors for 28-day mortality and 3-day orotracheal intubation after PTX/PNM. This study also considered the impact of the three main variants of concern (VoCs) (alfa, delta, and omicron) circulating during the study period. Results: During the study period, a total of 11,938 patients with COVID-19 were admitted. This study found several factors independently associated with a higher risk of death in COVID-19 patients within 28 days of pulmonary barotrauma. These factors included a SOFA score ≥ 4 (OR 3.22, p = 0.013), vasopressor/inotropic therapy (OR 11.8, p < 0.001), hypercapnia (OR 2.72, p = 0.021), PaO2/FiO2 ratio < 150 mmHg (OR 10.9, p < 0.001), and cardiovascular diseases (OR 7.9, p < 0.001). This study also found that a SOFA score ≥ 4 (OR 3.10, p = 0.015), PCO2 > 45 mmHg (OR 6.0, p = 0.003), and P/F ratio < 150 mmHg (OR 2.9, p < 0.042) were factors independently associated with a higher risk of orotracheal intubation (OTI) within 3 days from PTX/PNM in patients with non-invasive mechanical ventilation. SARS-CoV-2 VoCs were not associated with 28-day mortality or the risk of OTI. The estimated cumulative probability of OTI in patients after pneumothorax was 44.0% on the first day, 67.8% on the second day, and 68.9% on the third day, according to univariable survival analysis. In patients who had pneumomediastinum only, the estimated cumulative probability of OTI was 37.5%, 46.7%, and 57.7% on the first, second, and third days, respectively. The overall incidence of PTX/PNM among hospitalized COVID-19 patients was 1.42%, which increased up to 4.1% in patients receiving invasive mechanical ventilation. Conclusions: This study suggests that a high SOFA score (≥4), the need for vasopressor/inotropic therapy, hypercapnia, and PaO2/FiO2 ratio < 150 mmHg in COVID-19 patients with pulmonary barotrauma are associated with higher rates of intubation, ICU admission, and mortality. Identifying these risk factors early on can help healthcare providers anticipate and manage these patients more effectively and provide timely interventions with appropriate intensive care, ultimately improving their outcomes.

BACKGROUND:Since August to November 2023, 82 cases of autochthonous or non-travel related Dengue virus (DENV) infection have been reported in Italy, highlighting a concerning trend of local transmission. We describe the clinical and laboratory findings of 10 autochthonous DENV in the metropolitan area of Rome admitted to the Lazzaro Spallanzani National Institute for Infectious Diseases. METHOD AND RESULTS:Ten patients (3 males, 7 females; median age: 51) with classic dengue fever symptoms were admitted between August and November 2023. Laboratory tests confirmed dengue infection through DENV non-structural protein 1 and/or immunoglobulins (IgM/IgG) positive tests, moreover leukopenia, thrombocytopenia, elevated transaminases were detected. A subset of patients underwent extensive biological sampling, including real-time RT-PCR and immunofluorescence, to monitor DENV-RNA and antibody levels over 30 days. DENV-1 was detected in 8 patients and DENV-3 in 2. Upon admission specific IgM antibodies were found in 7 patients while IgG antibodies in 4 patients. DENV RNA was consistently detected in blood within the first 8 days but was less common in saliva and urine. No DENV RNA was detected after day 24. CONCLUSION:These findings contribute to the understanding of the clinical course of DENV infection in a non-endemic setting as integrated epidemiological and clinical model to increase syndromic surveillance and timely diagnosis of DENV infections.

Chagas disease (CD) is a parasitic infection endemic in Latin America and also affects patients in Western countries due to migration flows. This has a significant impact on health services worldwide due to its high morbidity and mortality burden. This paper aims to share our experience at the National Institute for Infectious Diseases “Lazzaro Spallanzani”, IRCCS, in Rome, Italy, where to date, a total of 47 patients—mainly Bolivian women—diagnosed with CD have received treatment with benznidazole, with all but one presenting with chronic disease. Most of the patients were recruited through the first extensive screening program held in 2014 at our Institute. About a quarter of our patients showed adverse effects to benznidazole, including a case of severe drug-induced liver injury, but 83% completed a full course of treatment. In addition to the description of our cohort, the paper reports a brief overview of the disease compiled through a review of the existing literature on CD in non-endemic countries. The growing prevalence of CD in Western countries highlights the importance of screening at-risk populations and urges public concern and medical awareness about this neglected tropical disease. There are still many unanswered questions that need to be addressed to develop a personalized approach in treating patients.

Since spring 2022, the global epidemiology of the monkeypox virus (MPXV) has changed. The unprecedented increase of human clade II MPXV cases worldwide heightened concerns about this emerging zoonotic disease. We analysed the positivity rates, viral loads, infectiousness, and persistence of MPXV DNA for up to 4 months in several biological samples from 89 MPXV-confirmed cases. Our data showed that viral loads and positivity rates were higher during the first two weeks of symptoms for all sample types. Amongst no-skin-samples, respiratory specimens showed higher MPXV DNA levels and median time until viral clearance, suggesting their usefulness in supporting MPXV diagnosis, investigating asymptomatic patients, and monitoring viral shedding. Infectious virus was cultured from respiratory samples, semen, and stools, with high viral loads and collected within the first 10 days. Notably, only one saliva and one semen were found positive for viral DNA after 71 and 31 days from symptoms, respectively. The focus on bloodstream samples showed the best testing sensitivity in plasma, reporting the overall highest MPXV DNA detection rate and viral loads during the 3-week follow-up as compared to serum and whole-blood. The data here presented can be useful for MPXV diagnostics and a better understanding of the potential alternative routes of its onward transmission.

Background:We aim to investigate the proportion of patients (pts) with long-term cognitive outcomes (CO) of PACC and identify associated features. Methods:We assessed participants through a neuropsychological assessment. The chi-square test was used for comparisons according with time of NPA (within or beyond 6 months since COVID19) and with previously hospitalization status (hospitalized patients, PH; not hospitalized patients, nPH). Results:520 participants: mean age 54 years (SD 12), 53 % female, 14 years of education (SD 3.4), 35 % with >1 comorbidity, 48 % previously hospitalized. Overall, we found CO in 89 % of pts, in particular 88 % evaluated in w6M and 89 % in b6M (p = 0.801) while 90 % and 87 % in nPH and PH, respectively (p = 0.239). By fitting multivariable analysis, PH for COVID19 and female gender were associated with an increased risk of an altered PSQI [Odd Ratio, OR 2.48, 95 % CI 1.54 to 3.99, p < 0.001 and OR 2.59, 95 % CI 1.60 to 4.17, p < 0.001, respectively) and BAI [F vs M: OR 1.67, 95 % CI 1.16 to 2.40, p = 0.005). Conclusions:We show a substantial proportion of PACC-CO; hospitalization leads to impaired memory, anxiety and sleep disorders. Women seem to be at higher risk for anxious-depressive symptoms and worse sleep quality than men.

Neurocysticercosis (NCC) is caused by the larval stage of Taenia solium. This parasitic disease is endemic in many areas of the world and is emerging in Europe. NCC can affect different brain regions, but simultaneous involvement of the parenchymal, subarachnoid, and ventricular regions is rare. We report the case of a 39-year-old woman from Honduras, resident in Rome for 10 years, who presented to the Emergency Department complaining of headaches, transient hemianopsia, and bilateral papilledema. MRI showed a concomitant parenchymal, subarachnoid, and ventricular involvement in the brain. T. solium IgG antibodies were detected in the blood. The etiological diagnosis of NCC was obtained by identifying T. solium in cerebrospinal fluid using Next Generation Sequencing. Endoscopic neurosurgery with the placement of a ventricular shunt and medical long-term anti-parasitic treatment with a cumulative number of 463 days of albendazole and 80 days of praziquantel were performed. A successful 4-year follow-up is reported. NCC is one of the most common parasitic infections of the human CNS, but it is still a neglected tropical disease and is considered to be an emerging disease in Europe. Its diagnosis and clinical management remain a challenge, especially for European clinicians.

BACKGROUND:The aim of this study is to design ad hoc malaria learning (ML) approaches to predict clinical outcome in all patients with imported malaria and, therefore, to identify the best clinical setting.METHODS:This is a single-centre cross-sectional study, patients with confirmed malaria, consecutively hospitalized to the Lazzaro Spallanzani National Institute for Infectious Diseases, Rome, Italy from January 2007 to December 2020, were recruited. Different ML approaches were used to perform the analysis of this dataset: support vector machines, random forests, feature selection approaches and clustering analysis.RESULTS:A total of 259 patients with malaria were enrolled, 89.5% patients were male with a median age of 39 y/o. In 78.3% cases, Plasmodium falciparum was found. The patients were classified as severe malaria in 111 cases. From ML analyses, four parameters, AST, platelet count, total bilirubin and parasitaemia, are associated to a negative outcome. Interestingly, two of them, aminotransferase and platelet are not included in the current list of World Health Organization (WHO) criteria for defining severe malaria.CONCLUSION:In conclusion, the application of ML algorithms as a decision support tool could enable the clinicians to predict the clinical outcome of patients with malaria and consequently to optimize and personalize clinical allocation and treatment.

Background and Aims: Several studies reported the effect of COVID-19 on inducing gut dysbiosis, which is also correlated with disease severity. This study aims to investigate the effect of a nutraceutical formula on the shift of microbiota profiles and, secondly, on the clinical–pathological parameters of acute and post-acute COVID-19 patients. Methods: In this randomised, double-blind, placebo-controlled trial conducted at National Institute for Infectious diseases (INMI) Lazzaro Spallanzani (Italy), 52 patients were randomly assigned (1:1) to receive a multistrain synbiotic formula (Kebirah®) or placebo orally for 35 days at COVID-19 diagnosis. Health professionals, investigators, and patients were masked to group assignments. The V3–V4 hypervariable region of 16S rRNA gene sequencing was employed to study the gut microbiota composition in the two groups. Results: Supplementation with Kebirah® prevented the decrease in the Shannon diversity index of gut microbiota, which was instead observed in patients receiving the placebo. In addition, decreases in lymphocyte count and haemoglobin levels were observed only in the placebo group and not in the treated group, which was also characterised by an amelioration of the gut microbial profile, with an enrichment in beneficial bacteria and a preservation of species diversity. Conclusions: Our data suggest that modulating the gut microbiota in acute disease through administration of a specific symbiotic formula could be a useful strategy in the frame of SARS-CoV-2 infections.

Background After the acute phase, symptoms or sequelae related to post-COVID-19 syndrome may persist for months. In a population of patients, previously hospitalized and not, followed up to 12 months after the acute infection, we aim to assess whether and to what extent post-COVID-19 syndrome may have an impact on health-related quality of life (HRQoL) and to investigate influencing factors. Methods We present the cross-sectional analysis of a prospective study, including patients referred to the post-COVID-19 service. Questionnaires and scales administered at 3, 6, 12 months were: Short-Form 36-item questionnaire (SF-36); Visual Analogue Scale of the EQ5D (EQ-VAS); in a subgroup, Beck Anxiety Inventory (BAI), Beck Depression Inventory (BDI-II) and Pittsburgh Sleep Quality Index (PSQI). Linear regression models were fitted to identify factors associated with HRQoL. Results We considered the first assessment of each participant ( n = 572). The mean scores in SF-36 and in EQ-VAS were significantly lower than the Italian normative values and remained stable over time, except the mental components score (MCS) of the SF-36 and EQ-VAS which resulted in lower ratings at the last observations. Female gender, presence of comorbidities, and corticosteroids treatment during acute COVID-19, were associated with lower scores in SF-36 and EQ-VAS; patients previously hospitalized (54%) reported higher MCS. Alterations in BAI, BDI-II, and PSQI ( n = 265)were associated with lower ratings in SF-36 and EQ-VAS. Conclusions This study provides evidence of a significantly bad perception of health status among persons with post-COVID-19 syndrome, associated with female gender and, indirectly, with disease severity. In case of anxious-depressive symptoms and sleep disorders, a worse HRQoL was also reported. A systematic monitoring of these aspects is recommended to properly manage the post-COVID-19 period.

Despite the number of cholera outbreaks reported worldwide, only a few cases are recorded among returning European travellers. We describe the case of a 41-year-old male, returning to Italy after a stay in Bangladesh, his origin country, who presented with watery diarrhoea. Vibrio cholerae and norovirus were detected in the patient's stools via multiplex PCR methods. Direct microscopy, Gram staining, culture and antibiotic susceptibility tests were performed. The isolates were tested using end-point PCR for the detection of potentially enteropathogenic V. cholera. Serotype and cholera toxins identification were carried out. Whole genome sequencing and bioinformatics analysis were performed, and antimicrobial resistance genes identified. A phylogenetic tree with the most similar genomes of databases previously described was built. Sample of the food brought back by the patient were also collected and analysed. The patient was diagnosed with V. cholerae O1, serotype Inaba, norovirus and SARS-CoV-2 concomitant infection. The isolated V. cholerae strain was found to belong to ST69, encoding for cholera toxin, ctxB7 type and was phylogenetically related to the 2018 outbreak in Dhaka, Bangladesh. Adopting a multidisciplinary approach in a cholera non-endemic country ensured rapid and accurate diagnosis, timely clinical management, and epidemiological investigation at national and international level.

Dear Editor, We read with interest the manuscript by Li D. and colleagues, recently published in this Journal, in which the authors revealed the potential binding mode for tecovirimat with a poxvirus phospholipase from monkeypox (MPX) virus [1Li D Liu Y Li K Zhang L. Targeting F13 from monkeypox virus and variola virus by tecovirimat: Molecular simulation analysis.J Infect. 2022; 85: e99-e101Google Scholar]. Tecovirimat and cidofovir are potential options for severe cases of MPX, but limited data on their efficacy and safety are available [2Adler H Gould S Hine P et al.Clinical features and management of human monkeypox : a retrospective observational study in the UK.Lancet Infect Dis. 2022; 3099: 1-10Google Scholar, 3Desai A George T Neumeister S Arutyunova A Stuart C. Compassionate Use of Tecovirimat for the Treatment of Monkeypox Infection.JAMA. 2022; : 1-3Google Scholar, 4Matias WR Koshy JM Nagami EH et al.Tecovirimat for the Treatment of Human Monkeypox: An Initial Series From Massachusetts, United States.Open forum Infect Dis. 2022; 9: ofac377Google Scholar, 5Tarín-Vicente EJ Alemany A Agud-Dios M et al.Clinical presentation and virological assessment of confirmed human monkeypox virus cases in Spain: a prospective observational cohort study.Lancet. 2022; 400: 661-669Google Scholar, 6Mailhe M Beaumont A-L Thy M et al.Clinical characteristics of ambulatory and hospitalised patients with monkeypox virus infection: an observational cohort study.Clin Microbiol Infect. 2022; (Available at)https://doi.org/10.1016/j.cmi.2022.08.012Google Scholar]. Here we retrospectively describe clinical presentation, evolution, management and viral kinetics of the first 19 MPX cases treated with antivirals at the INMI Lazzaro Spallanzani IRCCS in Rome, Italy. The decision regarding treatments was based on international medical consensus and availability of drugs. Viral DNA was extracted by the automatic extractor QIAsymphony (Qiagen, Hilden, Germany), and amplified using the real-time PCR method targeting the tumor necrosis factor receptor gene, G2R. Monkeypox virus (MPXV) DNA concentration was measured using threshold cycles (Ct) values of the MPXV-specific PCR. To obtain an absolute quantification of MPXV DNA in the clinical samples, the PCR assay was adapted to run in digital droplet PCR (ddPCR). The nucleic acid extracted from each sample was loaded into specific nanoplate and distributed, amplified and read in each one of the 26,000 partitions of each well, with a detection limit of the assay of 5 copies/µL. The study was conducted as a part of biological studies on emerging infections approved by the Ethical Committee of the Lazzaro Spallanzani Institute (approval number 14/2015 and amendments). Patients provided written informed consent. As of September 19, 2022, 19/128 (15%) diagnosed cases of MPXV infection at INMI L. Spallanzani received antiviral treatment. All patients were males aged between 27 and 50 years, all but one patients self-identified as men who have sex with men or bisexual and seven patients (37%) were HIV-positive. Systemic symptoms were reported in all but one patient. Muco-cutaneous lesions were observed in all patients (skin lesions in 89% and mucosal lesions in 95%) and in half of them preceded systemic symptoms. The majority (79%) of patients complained of a painful lymphadenopathy. Patients were admitted to hospital within a median of 8 days (IQR 5-10) from date of symptoms onset (OD), mainly for mucosal inflammation caused by MPXV and/or superinfection of the lesions and/or management of severe pain due to the lesions. Specifically, proctitis was diagnosed in four patients (21%) and severe pharyngo-tonsillitis in six patients (32%). One patient presented ocular localization complicated by periorbital edema and conjunctival hyperemia. Nine patients (47%) presented with superinfection of the soft tissues, one of which was complicated by abscess of a finger. Finally, one patient was admitted and treated for worsening of genital lesions. Antiviral treatment was started with a median time of 11 days (IQR 8-12) from OD with oral tecovirimat in 15 (79%) patients and intravenous (IV) cidofovir in 4 (21%) patients All patients treated with oral tecovirimat completed a 14-day course of therapy. Similarly, IV cidofovir was well tolerated. Symptoms improvement and no new lesion appearance were observed 72 hours after the start of treatment in all but one patient treated with cidofovir. No significative alterations of blood tests were observed, apart from a transient increase of alanine aminotransferase after cidofovir. Complete recovery was observed in all patients with a median of 15 days (IQR 11-19) from treatment start. Three patients had still persistence of signs of MPX-mucosal involvement after the resolution of lesions (Table 1).Table 1Patients’ characteristics and clinical course.PT1PT2PT3PT4PT5PT6PT7PT8PT9PT10PT 11PT 12PT 13PT 14PT 15PT 16PT 17PT 18PT 19Gender/Age/ EthnicityM/28 y/ CaucasianM/33 y/CaucasianM/35 y/CaucasianM/46 y/ CaucasianM/33 y/ CaucasianM/33 y/HispanicM/42y/AsianM/40y/ CaucasianM/47y/ CaucasianM/27y/CaucasianM/36 y/ CaucasianM/38y/HispanicM/48y/ CaucasianM/45y/CaucasianM/35y/ CaucasianM/50y/AfricanM/36y/HispanicM/38y/ CaucasianM/47y/ CaucasianSexual orientationBisexualMSMMSMMSMMSMMSMMSMMSMMSMMSMMSMMSMMSMMSMMSMHetero-sexualMSMMSMMSMHIV status(ART; last CD4 (cell/mm3)/VL)NegNegNegPos(BIC/TAF/FTC;1622/ND)Pos(3TC/DT;872 /ND)Pos*(TDF/FTC+DTG;526/26 cp/mL)NegNegPos(3TC/DTG;828 /ND)Pos**(BIC/TAF/FTC;140/<30cp/mL)NegNegNegNegNegNegPos(TAF/FTC/DRV/c+DTG;253/22 cp/mL)***Pos(TDF/FTC/EFV;1323; ND)NegHbsAg/HCVAbNeg/NegNeg/NegNeg/NegNeg/NegNeg/NegNA/NegNeg/NegNeg/NegNeg/NegNeg/NegNeg/NegNeg/NegNeg/NAPos/NegNA/NANeg/NegNeg/NegNA/NegNeg/NegPREPNoYesYesNoNoNoNoYesNoNoNoYesNoNoNoNoNoNoNoSmallpox vaccinationNoNoNoNoNoNoNoNoNoYesNoNoYesNoNoNoNoNoNoSystemic symptomsFever, headacheFever, sore throat, myalgias, diarrhoeaFeverFever, myalgias, rectal pain with discharg, bleedingFever, headache, sore throatFeverFever, sore throat, odynophagiaFever, sore throat, odynophagia, myalgias, headacheFever, sore throat, odynophagia, diarrhoeaNoFever, headacheFever, rectal pain, discharge and bleedingFever, sore throat; odynophagiaFeverFeverFever, myalgiasfever, sore throat myalgias, headache, rectal pain, diarrhoeafever, headache,rectal painfever, sore throat, headacheCutaneous lesionHead, trunk, right leg, suprapubic and perinealTrunk, limbs including handsHead, trunk, limbsTrunk, legsHead, trunk, limbsHead, trunk, limbsHead, trunk, limbs, including palms and solesHead, trunk, limbsNoHead including eyelids, trunk, limbs including palms and solesHead, trunk, limbsHead, trunk, limbsNoTrunkUpper lipHead including scalp, trunk, arms including handsHead, trunk, legsFeet, trunkHead including upper lip and scalp, limbs, trunkMucosal lesionEyelidsPenisPenis, scrotum, perianalPerianal and oropharyngealOropha-ryngealPenis, oropharyngealOropha-ryngeal penis and perianalOrophar-yngealOropha-ryngealPenis, scrotum, perianalPenisPenis, scrotum, perianalPenis; oropharyngealPenisNoPenisPerianal, scrotumPerianalPenis, oropharyngealNumber of lesions11-2011-2011-20<5<511-20≥2011-20<5≥205-10≥20<55-10<5≥20≥20<55-10Systemic symptoms onset after lesionsYesYesYesNoNoYesYesYesNo-NoYesYesYesNoNoYesNoYesLymphad-enopathyInguinalInguinalInguinalNoNeckInguinalInguinalNeckNeckInguinalInguinalNoNeckAxillaryNoYesInguinal, neckInguinalNeckLocalized diseaseOcularNoNoProctitisPhary-ngoton-sillitisNoPhary-ngoton-sillitisPhary-ngoton-sillitisPhary-ngoton-sillitisNoNoProctitisPhary-ngoton-sillitisNoNoNoProctitisProctitisPhary-ngoton-sillitisType of treatmentCidofovirTeco-virimatTeco-virimatTeco-virimatTeco-virimatTeco-virimatCidofovirCidofovirTeco-virimatCidofovirTeco-virimatTeco-virimatTeco-virimatTeco-virimatTeco-virimatTeco-virimatTeco-virimatTeco-virimatTeco-virimatReason for treatmentOcular involvementSuper-infection of cutaneous lesionSoft-tissue superinfectionProctitisPhary-ngotons-illitisSoft-tissue superinfectionPhary-ngotons-illitis, pain managementCompli-cated pharyn-goton-sillitis (right peritonsillar abscess)Phary-ngoton-sillitisSuper-infection of cutaneous lesions, pain managementPain managementCompli-cated proctitisPhary-ngoton-sillitisSuper-infection of cut-aneous lesionsSoft-tissue superinfection(upper lip)Super-infection of cut-aneous lesionsSuper-infection of cut-aneous lesions/ProctitisProctitisSoft-tissue superinfection(upper lip)Days from OD to admission/treatment5/1210/187/109/1111/136/73/69/124/118/1111/1210/119/107/95/69/910/127/85/7Days from treatment to recovery131021912111447186211420718172715* HIV diagnosis 2 months before MPX; ** AIDS presenters with HIV/AIDS diagnosis six months before MPX (multidrug resistant disseminated tuberculosis on treatment); *** recent virological failure. Abbreviations: M, male; y, years, MSM men who have sex with men; Neg, negative; Pos, positive; Unk, unknown; ART, antiretroviral therapy; VL, viral load; ND, not detectable, PREP, pre-exposure prophylaxis; BIC, bictegravir; TAF, tenofovir alafenamide fumarate; FTC, emtricitabine; 3TC, lamivudine; DTF dolutegravir, TDF, tenofovir disoproxil fumarate; DRV/c, darunavir/cobicistat; EFV, efavirenz; NA, not available; STD, sexual transmitted disease; CT computed tomography; OD, onset date. Open table in a new tab * HIV diagnosis 2 months before MPX; ** AIDS presenters with HIV/AIDS diagnosis six months before MPX (multidrug resistant disseminated tuberculosis on treatment); *** recent virological failure. Abbreviations: M, male; y, years, MSM men who have sex with men; Neg, negative; Pos, positive; Unk, unknown; ART, antiretroviral therapy; VL, viral load; ND, not detectable, PREP, pre-exposure prophylaxis; BIC, bictegravir; TAF, tenofovir alafenamide fumarate; FTC, emtricitabine; 3TC, lamivudine; DTF dolutegravir, TDF, tenofovir disoproxil fumarate; DRV/c, darunavir/cobicistat; EFV, efavirenz; NA, not available; STD, sexual transmitted disease; CT computed tomography; OD, onset date. Finally, viral kinetics have been evaluated in 12 patients (Fig. 1). In all of them, MPXV-DNA was detected in at least one sample from at least one compartment. Particularly, during the follow-up, MPXV-DNA was detected by real-time PCR in: 10/12 patients on oropharyngeal swab (OPS), including 9 at the start of antiviral treatment, with a median Ct of 36 (IQR 33-41); 8/9 patients on blood samples with a median Ct of 41 (IQR 37-41); 6/6 patients on feces with a median Ct of 41 (IQR 36-41); 3/3 patients on saliva with a median Ct of 38 (IQR 32-40); 3/3 patients on seminal fluids with a median Ct of 39 (IQR 37-41). In almost all patients, a progressive decline in viral load was observed over the course of treatment. Most biological samples were negative at the last available observation. DdPCR results approximately mirrored the viral shedding expressed with real-time PCR. It is worth noting that, given the low threshold used for the ddPCR, several samples with high Ct values in real-timePCR, resulted negative in ddPCR. Limited data on clinical effectiveness of tecovirimat are available, however, several recent reports on its use has shown good tolerability and no evolution versus severe disease in treated subjects [2Adler H Gould S Hine P et al.Clinical features and management of human monkeypox : a retrospective observational study in the UK.Lancet Infect Dis. 2022; 3099: 1-10Google Scholar, 3Desai A George T Neumeister S Arutyunova A Stuart C. Compassionate Use of Tecovirimat for the Treatment of Monkeypox Infection.JAMA. 2022; : 1-3Google Scholar, 4Matias WR Koshy JM Nagami EH et al.Tecovirimat for the Treatment of Human Monkeypox: An Initial Series From Massachusetts, United States.Open forum Infect Dis. 2022; 9: ofac377Google Scholar,7O'Laughlin K Tobolowsky FA Elmor R et al.Clinical Use of Tecovirimat (Tpoxx) for Treatment of Monkeypox Under an Investigational New Drug Protocol — United States.MMWR Morb Mortal Wkly Rep. 2022; (ePub: 9 September 2022. DOI:)http://dx.doiGoogle Scholar]. Additionally, preliminary results from the first 549 MPX-positive patients treated with tecovirimat in United States (US), showed median time to subjective improvement of 3 days [7O'Laughlin K Tobolowsky FA Elmor R et al.Clinical Use of Tecovirimat (Tpoxx) for Treatment of Monkeypox Under an Investigational New Drug Protocol — United States.MMWR Morb Mortal Wkly Rep. 2022; (ePub: 9 September 2022. DOI:)http://dx.doiGoogle Scholar]. To the best of our knowledge, this is the first report of the use of antivirals for MPX with both clinical and virological results in this current outbreak. One case series of patients treated in 2018-2021 reported viral decay in one patient during tecovirimat treatment showing a shorter duration of viral shedding compared to the other patients [2Adler H Gould S Hine P et al.Clinical features and management of human monkeypox : a retrospective observational study in the UK.Lancet Infect Dis. 2022; 3099: 1-10Google Scholar]. Additionally, in a pre-print publication, outcomes, including viral kinetics, of 14 patients treated before February 2022 with tecovirimat were reported [8Festus Mbrenga, Emmanuel Nakouné, Christian Malaka, Josephine Bourner, J ake Dunning, Guy Vernet, Peter Horby PO. Monkeypox treatment with tecovirimat in the Central African Republic under an Expanded Access Programme Author. medRxiv Prepr doi:10.1101/2022.08.24.22279177.Google Scholar]. In contrast to that report, where rate of appearance of lesions decreased during treatment with a median of 5 days from treatment start [8Festus Mbrenga, Emmanuel Nakouné, Christian Malaka, Josephine Bourner, J ake Dunning, Guy Vernet, Peter Horby PO. Monkeypox treatment with tecovirimat in the Central African Republic under an Expanded Access Programme Author. medRxiv Prepr doi:10.1101/2022.08.24.22279177.Google Scholar], in our patients clinical improvement and no new lesions were reported in almost all patients 72 hours after tecovirimat initiation. The longer time elapsed from symptoms onset to treatment start (21 days) compared to our study (12 days) might partially explain this different result. Of note, in our case series, 15% of MPX cases diagnosed received antiviral treatment, consistently with US data [9Https://www.cdc.gov/poxvirus/monkeypox/response/2022/demographics-TPOXX.html accessed 26 September 2022.Google Scholar]. Concerning viral kinetics, it should be noted that low viral loads were observed. Additionally, some patients had all available samples negative in ddPCR since antiviral starting, in line with previous evidence showing that viral shedding occurs mainly during the first two weeks of the disease [10Peiro-Mestres A Fuertes I Camprubi-Ferrer D et al.Frequent detection of monkeypox virus DNA in saliva, semen, and other clinical samples from 12 patients, Barcelona, Spain, May to June 2022.Eurosurveillance. 2022; 27 (Available at)http://dx.doi.org/10.2807/1560-7917.ES.2022.27.28.2200503Google Scholar]. Due to the median time of 12 days from symptoms onset to starting treatment in this series, we cannot exclude a reduced impact of antiviral therapy on viral shedding or clinical resolution. The main limitations of this study was the lack of control group, so that any conclusions on the effectiveness of antiviral therapy cannot be drawn, the small number of patients included, the heterogeneity of samples and the impossibility to collect samples for all the patients at each timepoint. Data collected on observational studies such as this can help improve our knowledge of the use of antivirals for MPXV, waiting more robust results from the placebo-controlled randomized trial of tecovirimat for MPX. This study was supported by Ricerca Corrente Linea 1 and 2, funded by the Italian Ministry of Health.