This report describes the isolation, molecular characterization, and phylogenetic analysis of the first three complete genomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from three patients involved in the first outbreak of COVID-19 in Lombardy, Italy. Early molecular epidemiological tracing suggests that SARS-CoV-2 was present in Italy weeks before the first reported cases of infection.
To evaluate the incidence and risk factors for liver enzyme elevations (LEE) in patients initiating first-line ART in the ICONA prospective observational cohort, between June 2009 and December 2017.In total, 6575 ART-naive patients were selected, initiating two NRTIs with the third drug being a boosted PI (n=2436; 37.0%), an NNRTI (n=2384; 36.3%) or an integrase strand transfer inhibitor (INSTI) (n=1755; 26.7%). HBV surface antigen and HCV RNA were detected in 3.9% and 5.8% of the study population. Inverse probability weighted Cox regression analysis was used to calculate the HRs, according to first-line regimen, for LEE, defined as ALT or AST increases of ≥2.5× upper limit of normal (ULN) for patients with normal baseline values or ≥2.5× baseline for patients with higher baseline values.One hundred and eighty-three LEE occurred over 20722 patient-years of follow-up. After adjusting for the main confounders, the risk of LEE halved with INSTIs compared with NNRTIs (HR 0.46, 95% CI 0.25-0.86), with a significant reduction in the raltegravir group (HR 0.11, 95% CI 0.02-0.84 using the NNRTI class as reference). HRs for LEE were significantly higher in subjects with HBV or HCV coinfection, in patients with poorly controlled HIV infection and in those who acquired HIV through homosexual transmission.In our study, INSTI use almost halved the risk of LEE compared with other regimens. This finding could be particularly important for choosing ART in patients with risk factors for liver toxicity such as HCV and HBV coinfections.
Warfarin is widely used for the management of clotting disorders and is prone to drug-drug interactions (DDIs) that can result in subtherapeutic anticoagulation or over-anticoagulation [1]. Hence, titration of warfarin dose is usually based on close monitoring of the international normalized ratio (INR). DDIs between warfarin and antiretrovirals are likely, particularly if non-nucleoside reverse transcriptase inhibitors (NNRTIs) or boosted HIV protease inhibitors are included in the antiretroviral regimen [2]. Conversely, no significant DDIs are expected for HIV integrase inhibitors [3]. A 50-year-old woman living with HIV was referred to our outpatient clinic for the management of polypharmacy (Gestione Ambulatoriale Politerapie: GAP) in January 2021 to find a possible explanation for the sudden changes in INR observed in the last 2 months. She was on maintenance warfarin therapy for the treatment of deep venous thrombosis since December 2007. During the last years, the patient was treated with a fixed daily warfarin dose scheme (8.75 mg/daily from Monday to Saturday and 7.5 on Sunday), resulting in a quite stable INR, with values ranging from 1.72 to 2.37 (Fig. 1). On 1 November 2020, the antiretroviral therapy was switched from nevirapine/lamivudine/abacavir to dolutegravir/lamivudine for treatment simplification; a control of INR 20 days later showed a values of 2.29. In early December, the patient had gum bleeding while brushing her teeth. At that time in Italy, we were experiencing the peak of the second COVID-19 pandemic wave. The patient, in fear of SARS-CoV-2 infection, did not go to the hospital for the measurement of the INR and voluntarily reduced the dose of warfarin to 7.5 mg daily. INR, measured on 29 December for the persistence of gingival bleeding, was 4.29. A second assessment, performed 1 week later, resulted 4.43, confirming over-anticoagulation. The antiretroviral therapy was switched back to previous regimen. INR assessments, performed on 15 January and 22 January 2021 were 2.90 and 2.75, respectively.Fig. 1: Time-course of international normalized ratio values before and after the switch from nevirapine to dolutegravir-based antiretroviral therapy. DTG, dolutegravir; NVP, nevirapine. Shaded area represents the therapeutic range for INR.We excluded that the increase of the INR resulting in bleeding was related to a DDI between dolutegravir and warfarin. In fact, the disposition of warfarin is mainly regulated by CYP2C9 and to a lesser extent by CYP4F2, two cytochromial enzymes whose activity is not affected by dolutegravir [4,5]. The only clinically relevant DDIs that involve dolutegravir as perpetrator are those related to the drug-related inhibition of the organic cation transporter 2 (OCT2) [3]. Nevertheless, warfarin is not a substrate of OCT2. We assumed that a DDI is likely to have happened between warfarin and nevirapine. Indeed, Dionisio et al.[6] described three patients who experienced alterations in the INR after taking concomitantly nevirapine and warfarin. The doses of warfarin required to stabilise anticoagulant activity ranged from 5 to 12 mg and, in one case, nevirapine need to be stopped. In our patient, this DDI has been well managed over the years by adjusting warfarin dosing based on INR results. What is likely to have happened in November 2020 is that the switch from nevirapine to dolutegravir has resulted in the removal of the effect of the NNRTI on warfarin activity. This effect was not immediately captured by INR (that resulted apparently in range) because nevirapine has a terminal half-life of 30 h; accordingly, the drug was present in the body for at least 2 weeks after its discontinuation. Subsequently, when dolutegravir completely replaced nevirapine, there was a remodelling of the activity of warfarin that resulted in the episodes of bleeding, confirmed later by the very high INR values. The COVID-19 pandemic has complicated the situation, hampering the proper INR assessment during the switch from nevirapine to dolutegravir. The adjustments in the warfarin dosing, made by the patient independently without consulting with her doctors engaged in COVID-19 clinics, did not reduce INR, leading to suboptimal control of anticoagulant therapy. Noteworthy, a shift from warfarin to a direct oral anticoagulant (edoxaban or dabigatran) was recommended from healthcare providers of GAP outpatient clinic to minimize DDIs and improve the quality of life of the patient. In conclusion, this is a good example on how the lack of a DDI may potentially become clinically relevant if not properly managed. We believe that our case could be instrumental to recall on the importance of a careful assessment of the overall therapies before embarking in antiretroviral treatment switches even with potentially DDIs-free regimens, particularly in situations characterized by a limited patient monitoring, as in the COVID-19 pandemic. Accordingly, the US Guidelines for HIV/AIDS suggest that persons for whom a regimen change is planned should consider delaying the switch until close follow-up and monitoring are possible [7]. Acknowledgements D.C., C.G. planned the study, revised the therapies of the patient and wrote the first draft of the manuscript. D.B. and P.M. took care of the patient and performed outpatient visits. M.B. and C.R. analysed the data. This study was carried out as part of our routine work. Conflicts of interest There are no conflicts of interest.
ABSTRACT This report describes the isolation, the molecular characterization and the phylogenetic analysis of the first three complete genomes of SARS-CoV-2 isolated from three patients involved in the first outbreak of COVID-19 in Lombardy, Italy. Early molecular epidemiological tracing suggests that SARS-CoV-2 was present in Italy weeks before the first reported cases of infection.
Abstract Background Italy was the first European country hit by the COVID-19 pandemic and has the highest number of recorded COVID-19 deaths in Europe. Methods This prospective cohort study of the correlates of the risk of death in COVID-19 patients was conducted at the Infectious Diseases and Intensive Care units of Luigi Sacco Hospital, Milan, Italy. The clinical characteristics of all the COVID-19 patients hospitalised in the early days of the epidemic (21 February -19 March 2020) were recorded upon admission, and the time-dependent probability of death was evaluated using the Kaplan-Meier method (censored as of 20 April 2020). Cox proportional hazard models were used to assess the factors independently associated with the risk of death. Results Forty-eight (20.6%) of the 233 patients followed up for a median of 40 days (interquartile range 33-47) died during the follow-up. Most were males (69.1%) and their median age was 61 years (IQR 50-72). The time-dependent probability of death was 19.7% (95% CI 14.6-24.9%) 30 days after hospital admission. Age (adjusted hazard ratio [aHR] 2.08, 95% CI 1.48-2.92 per ten years more) and obesity (aHR 3.04, 95% CI 1.42-6.49) were independently associated with an increased risk of death, which was also associated with critical disease (aHR 8.26, 95% CI 1.41-48.29), C-reactive protein levels (aHR 1.17, 95% CI 1.02-1.35 per 50 mg/L more) and creatinine kinase levels above 185 U/L (aHR 2.58, 95% CI 1.37-4.87) upon admission. Conclusions Case-fatality rate of patients hospitalized with COVID-19 in the early days of the Italian epidemic was about 20%. Our study adds evidence to the notion that older age, obesity and more advanced illness are factors associated to an increased risk of death among patients hospitalized with COVID-19.
We read with interest the article by C. Dimeglio and colleagues, in which the authors apply a mathematical model to conclude that a SARS-CoV-2 (Severe Acute Respiratory Sindrome Coronavirus 2) seroprevalence of at least 50% is required to avoid an infection rebound after removal of containment measures1Dimeglio C. Loubes J.-.M. Deporte B. et al.The SARS-CoV-2 seroprevalence is the key factor for deconfinement in France.J Infect. 2020; 81: 318-356Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar. In our study we found that SARS-CoV-2 seroprevalence was dramatically lower than this threshold, even in an area of initially unrestricted viral circulation. Italy was the first European country that suffered a wide spread of Coronavirus Disease 2019 (COVID-19), caused by a novel betacoronavirus which was first identified in China and denominated SARS-CoV-2 (Severe Acute Respiratory Sindrome Coronavirus 2), which caused hundreds of thousands of cases2EpiCentro. Coronavirus | Istituto Superiore di Sanità, https://www.epicentro.iss.it/coronavirus/ (accessed 25th of June, 2020).Google Scholar. During the epidemic, testing was restricted to severely symptomatic cases. Consequently, the true extent of the SARS-CoV-2 infection remains unknown. We estimated SARS-CoV-2 seroprevalence in the municipality of Castiglione d'Adda, a rural town of about 4550 inhabitants located South-East of Milan, which has been heavily affected by SARS-CoV-2 infection since the earliest stages of the epidemic. As of June 21, 2020, 184 confirmed cases of COVID-19 were reported, the large majority of which requiring hospitalization, accounting for about 4% of the total population. At the same time, 47 deaths were officially attributed to COVID-19. In this study, the entire population of Castiglione D'Adda was invited to perform a lateral-flow immunocromatographic tests on capillary blood (Prima Lab, Switzerland) from the 18th of May to the 7th of June. News about the mass screening was disseminated by the town municipality. A random sample of 562 subjects (stratified per sex and age) was invited to undergo confirmatory tests by chemiluminescent method on venipuncture drawn blood (CLIA, IgG anti-SARS-CoV-2, Abbott, USA) and SARS-CoV-2 PCR on NPS, regardless of RICT results. More detailed information about the randomization procedure and the study design are available on the complete protocol, published on medrXiv pre-print server3Pagani G. Bernacchia D. Conti F. et al.Dynamics of the SARS-CoV-2 epidemic in the earliest-affected areas in Italy: mass screening for SARS-CoV-2 serological positivity (SARS-2-SCREEN).medRxiv. 2020; (2020.06.06.20124081)Google Scholar. The analysis of IgG prevalence in the different age groups was performed by logistic regression models with response variable equal to 1 for positive IgG results, and 0 for negative IgG results. Age and gender were included as independent variables. Results were reported in terms of estimated probabilities of being positive to IgG test as a function of age, with respective 95% confidence intervals. Results presented in this paper are based on 509 people selected in the random sample who agreed to undergo venipuncture to perform CLIA serologies. Characteristics of the selected population are reported in Table 1.Table 1Characteristics of 509 subjects in the random sample.IgG negative (n = 394)IgG positive (n = 115)Gender (Female)200 (50•8%)49 (42•6%)Age (years; median, SD)46•0, 20•655•4, 19•5Age groups: 0–1956 (91•8%)5 (8•2%) 20–3992 (82•9%)19 (17•1%) 40–59142 (78•0%)40 (22•0%) ≥ 60104 (67•1%)51 (32•9%)Contact with verified case93 (23•6%)61 (53•0%)Smoker92 (23•4%)10 (8•7%)Cardiovascular diseases CAD/MI10 (2•5%)3 (4•3%) Arrhythmias14 (3•6%)5 (4•3%) Hypertension68 (17•3%)32 (27•8%) Other14 (3•6%)14 (12•2%)At least one of the above:84 (21•3%)47 (40•9%)Rheumatic diseases19 (4•8%)11 (9•6%)Diabetes mellitus12 (3•0%)6 (6•2%)Chronic Lung diseases Asthma20 (5•1%)2 (1•7%) COPD1 (0•3%)1 (0•9%) Other9 (2•3%)4 (3•5%)At least one of the above:29 (7•4%)7 (6•1%)Oncological pathologiesSolid Tumors20 (5•1%)6 (5•2%)Oncochematological2 (0•5%)2 (1•7%)At least one of the above:22 (5•6%)8 (7•0%)Symptomatic Fever65 (16•5%)66 (57•4%) Cough57 (14•5%)31 (27•0%) Anosmia23 (5•8%)37 (32•2%) Dysgeusia27 (6•9%)46 (40•0%) Dispnea23 (5•8%)13 (11•3%) Rush:11 (2•8%)4 (3•5%) Arthromyalgia34 (8•6%)36 (31•3%)At least one of the above:124 (31•5%)89 (77•4%)Other symptoms54 (13•7%)23 (20•0%)Numerical variables are presented as means.CAD: Coronary Artery Disease; MI: Miocardial Infarction; COPD: Chronic Obstructive Pulmonary Disease. Open table in a new tab Numerical variables are presented as means. CAD: Coronary Artery Disease; MI: Miocardial Infarction; COPD: Chronic Obstructive Pulmonary Disease. The overall seroprevalence found in the tested sample was 22.6% (95% confidence interval 17.2%- 29.1%). Interestingly, seroprevalence increases with increasing age (as shown in Table 1). In multivariate analyses, a significant effect of age was found (p<0.0001) while no significant association between IgG positivity and gender emerged (p = 0.2560). The possible existence of a non-linear effect of age was tested by including spline polynomials, without significant results (p = 0.9078). Furthermore, an age/gender interaction effect did not result significant (p = 0.5199). Estimates of probabilities of being positive to IgG test, from a model including only age as independent variable, are reported in Fig. 1. Since the early phases of the pandemic, advanced age was identified as an independent predictor for severe disease and worse outcomes4Chen N. Zhou M. Dong X. et al.Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study.Lancet. 2020; 395: 507-513Abstract Full Text Full Text PDF PubMed Scopus (14440) Google Scholar. Beside this, it remains unclear if the limited number of cases reported in children5Lee P.-.I. Hu Y.-.L. Chen P.-.Y. et al.Are children less susceptible to COVID-19?.J Microbiol Immunol Infect. 2020; 53: 371-372Crossref PubMed Scopus (439) Google Scholar is due to a milder course of disease, with a larger percentage of asymptomatic cases, or to a lower susceptibility to infection, as our results seem to suggest. Different ACE2 expression according to age have been postulated to explain clinical expression and susceptibility to the infection. In particular, a higher expression of ACE2 in lung tissues in advanced age groups had been speculated6Li Y. Zhou W. Yang L. et al.Physiological and pathological regulation of ACE2, the SARS-CoV-2 receptor.Pharmacol Res. 2020; 157104833Crossref PubMed Scopus (259) Google Scholar, 7Bourgonje A.R., Abdulle A.E., Timens W., et al. Angiotensin-converting enzyme-2 (ACE2), SARS-CoV-2 and pathophysiology of coronavirus disease 2019 (COVID-19). J Pathol., 2020. DOI: 10.1002/path.5471.Google Scholar. Moreover, a variable susceptibility to other coronavirus such as HCoV-NL63, which also use ACE2 as cell receptor in humans, in different age groups, has been also reported in different age groups8Huang S.-.H. Su M.-.C. Tien N. et al.Epidemiology of human coronavirus NL63 infection among hospitalized patients with pneumonia in Taiwan.J Microbiol Immunol Infect. 2017; 50: 763-770Crossref PubMed Scopus (30) Google Scholar. Another possible explanation may be that an asymptomatic/pauci-symptomatic infection, more common in younger subjects, could elicit a less marked, or transient, antibody response, as already found in the closely related Middle East Respiratory Syndrome Coronavirus (MERS-CoV)9Choe P.G. Perera R.A.P.M. Park W.B. et al.MERS-CoV antibody responses 1 year after symptom onset, South Korea, 2015.Emerg Infect Dis. 2017; 23: 1079-1084Crossref PubMed Scopus (171) Google Scholar. A possible confounding factor in our findings could be related to social distancing measures: schools of any grade were among the first institutions to be closed in Italy, starting from the 5th of March. This could have led to a lower exposure to the infection in children in pre-scholar and scholar age groups. In conclusion, our findings suggest that SARS-CoV-2 IgG seroprevalence increases with increasing age and these data suggest a lower susceptibility to infection in the lower age groups. These findings have important implications in epidemiology and public health, particularly in designing future population screenings, and could be an important contribution in the re-opening process, especially considered that more than three-fourths of the population could be still susceptible to SARS-CoV-2 infection, even in an area of initially unrestricted viral circulation. The authors declare no conflicts of interest. All authors have seen and approved the final manuscript. MG, GP, FC, DB, AG, RR and EB defined the study protocol. RR, AP, FC, DB and GP cooperated in the practical execution of the study and data gathering. GP drafted a first version of the manuscript, which was then revised and integrated by MG, EB, AG, CEG and SC. CO was responsible of serologies and NPSs execution. EB, PB and GM analyzed the data. All authors approved the final version of the manuscript. The study was funded thanks to the non-conditioning economical support from CISOM (Corpo Italiano di Soccorso dell'Ordine di Malta), Banca Mediolanum, Fondazione Rava, Mylan Italia and FC Internazionale Milano.
