A case of extremely prolonged viral shedding: Could cell cultures be a diagnostic tool to drive COVID-19 patient discharge?

This study addressed the case of a patient with prolonged COVID-19 viral shedding, reported by Real-Time PCR, until 71 days from symptom onset. However, viral culture received negative results after 30 days from symptom onset. Therefore, viral culture may be a worthwhile test for patients requiring discharge, in particular for those presenting prolonged viral shedding.

Geographical reconstruction of the SARS-CoV-2 outbreak in Lombardy (Italy) during the early phase.

The first identification of autochthonous transmission of SARS-CoV-2 in Italy was documented by the Laboratory of Clinical Microbiology, Virology and Bioemergencies of L. Sacco Hospital (Milano, Italy) on 20th February 2020 in a 38 years old male patient, who was found positive for pneumonia at the Codogno Hospital. Thereafter Lombardy has reported the highest prevalence of COVID-19 cases in the country, especially in Milano, Brescia and Bergamo provinces. The aim of this study was to assess the potential presence of different viral clusters belonging to the six main provinces involved in Lombardy COVID-19 cases in order to highlight peculiar province-dependent viral characteristics. A phylogenetic analysis was conducted on 20 full length genomes obtained from patients addressing to several Lombard hospitals from February 20th to April 4th, 2020, aligned with 41 Italian viral genome assemblies available on GISAID database as of 30th March, 2020: two main monophyletic clades, containing 8 and 53 isolates, respectively, were identified. Noteworthy, Bergamo isolates mapped inside the small clade harbouring M gene D3G mutation. The molecular clock analysis estimated a cluster divergence approximately one month before the first patient identification, supporting the hypothesis that different SARS-CoV-2 strains had spread worldwide at different times, but their presence became evident only in late February along with Italian epidemic emergence. Therefore, this epidemiological reconstruction suggests that virus initial circulation in Lombardy was ascribable to multiple introduction. The phylogenetic reconstruction robustness, however, will be improved when more genomic sequences are available, in order to guarantee a complete epidemiological surveillance.

Presence and infectivity of SARS-CoV-2 virus in wastewaters and rivers.

The presence of SARS-CoV-2 in raw wastewaters has been demonstrated in many countries affected by this pandemic. Nevertheless, virus presence and infectivity in treated wastewaters, but also in the receiving water bodies are still poorly investigated. In this study, raw and treated samples from three wastewater treatment plants, and three river samples within the Milano Metropolitan Area, Italy, were surveyed for SARS-CoV-2 RNA detection by means of real time RT-PCR and infectivity test on culture cells. SARS-CoV-2 RNA was detected in raw, but not in treated wastewaters (four and two samples, respectively, sampled in two dates). The isolated virus genome was sequenced, and belonged to the strain most spread in Europe and similar to another found in the same region. RNA presence in raw wastewater samples decreased after eight days, probably following the epidemiological trend estimated for the area. Virus infectivity was always null, indicating the natural decay of viral pathogenicity in time from emission. Samples from receiving rivers (three sites, sampled in the same dates as wastewaters) showed in some cases a positivity to real time RT-PCR, probably due to non-treated, or inefficiently treated discharges, or to the combined sewage overflows. Nevertheless, also for rivers infectivity was null. Risks for public health should be limited, although a precautionary approach to risk assessment is here advocated, giving the preliminary nature of the presented data.

The Role of Staphylococcus aureus in Mastitis : A Multidisciplinary Working Group Experience.

BACKGROUND: Breastfeeding women are at risk of developing mastitis during the lactation period. Staphylococcus aureus has emerged as the community-acquired pathogen responsible for virulence (methicillin resistance and Panton-Valentine leukocidin toxin producing). RESEARCH AIM: The aim was to compare the microorganisms responsible for mastitis and breast abscesses during breastfeeding. METHODS: This observational study was conducted with a sample of women (N = 60) admitted to our hospital between 2016 and 2018. Participants affected by mastitis and breast abscess were studied and cared for by a multidisciplinary working group. A diagnostic breast ultrasound identified the pathology. RESULTS: Twenty-six participants (43.3%) were affected by mastitis and 34 (56.7%) by breast abscess. The most common microorganism identified was Staphylococcus aureus (S. aureus; mastitis, n = 13; abscesses, n = 24). Methicillin resistance was identified in 21 (44.7%) S. aureus strains: 17 (80.9%) cases of abscess and four (19.1%) cases of mastitis. The median number of months of breastfeeding was smaller in the methicillin-resistant S. aureus (MRSA) cases (median = 3, range = 1-20 months) than in the methicillin-sensitive S. aureus (MSSA) cases (median = 6.5, range = 3-21 months). The Panton-Valentine leukocidin toxin gene was detected in 12 (25.5%) cases (MRSA, n = 8, 66.7%; MSSA, n = 4, 33.3%). Hospitalization was required more frequently in MRSA (n = 8, 38%; five Panton-Valentine leukocidin positive) than in MSSA cases (n = 5, 19%; one Panton-Valentine leukocidin positive). Four women out of the eight MRSA cases (50%) that were Panton-Valentine leukocidin positive stopped breastfeeding during mammary pathologies, three (37.5%) participants continued breastfeeding until the follow-up recall, and one case was lost at follow-up. CONCLUSION: Clinical severity was probably complicated by the presence of the Panton-Valentine leukocidin toxin, which required hospitalization more frequently.

Microbial biofilm correlates with an increased antibiotic tolerance and poor therapeutic outcome in infective endocarditis.

BACKGROUND: Infective endocarditis (IE) is associated with high rates of mortality. Prolonged treatments with high-dose intravenous antibiotics often fail to eradicate the infection, frequently leading to high-risk surgical intervention. By providing a mechanism of antibiotic tolerance, which escapes conventional antibiotic susceptibility profiling, microbial biofilm represents a key diagnostic and therapeutic challenge for clinicians. This study aims at assessing a rapid biofilm identification assay and a targeted antimicrobial susceptibility profile of biofilm-growing bacteria in patients with IE, which were unresponsive to antibiotic therapy. RESULTS: Staphylococcus aureus was the most common isolate (50%), followed by Enterococcus faecalis (25%) and Streptococcus gallolyticus (25%). All microbial isolates were found to be capable of producing large, structured biofilms in vitro. As expected, antibiotic treatment either administered on the basis of antibiogram or chosen empirically among those considered first-line antibiotics for IE, including ceftriaxone, daptomycin, tigecycline and vancomycin, was not effective at eradicating biofilm-growing bacteria. Conversely, antimicrobial susceptibility profile of biofilm-growing bacteria indicated that teicoplanin, oxacillin and fusidic acid were most effective against S. aureus biofilm, while ampicillin was the most active against S. gallolyticus and E. faecalis biofilm, respectively. CONCLUSIONS: This study indicates that biofilm-producing bacteria, from surgically treated IE, display a high tolerance to antibiotics, which is undetected by conventional antibiograms. The rapid identification and antimicrobial tolerance profiling of biofilm-growing bacteria in IE can provide key information for both antimicrobial therapy and prevention strategies.

Genomic Characterization of an ST1153 PVL-producing Methicillin Resistant Staphylococcus aureus Clinical Isolate in Italy.

Methicillin-resistant Staphylococcus aureus (MRSA) clones are rapidly increasing beyond the hospital into the community, livestock farming and environmental settings. An Italian man, a professional diver working in Egypt, was admitted to Infectious Diseases Clinic-ASST Fatebenefratelli Sacco for ulcerative skin lesions. An MRSA strain was isolated from the lesions' purulent exudate and the nasal colonization was also ascertained. The strain, characterized by whole genome sequencing, resulted to be Panton-Valentine Leukocidin (PVL) positive, SCCmecI - spa-type t504, and belonging to the sequence type 1153, sporadically described worldwide.

Identification of blaVIM-1 Gene in ST307 and ST661 Klebsiella pneumoniae Clones in Italy: Old Acquaintances for New Combinations.

In the past years, the Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae sequence type 258 (ST258) became an important worldwide spread nosocomial pathogen. Recent evidence shows that the global epidemiology is changing, with the rise of new lineages. In this study we report the microbiological and genomic features of two VIM-1-producing K. pneumoniae isolates belonging to the emerging ST307. Two extensively drug-resistant K. pneumoniae strains, collected between May and June 2017, were confirmed as blaVIM positive by GeneXpert system. The whole-genome sequencing revealed that both KpV_S_1 and KpV_S_2 isolates harbored blaVIM-1 and blaCTX-M-15 genes, besides qnrS1 and qnrB1, strB, mphA, tetR, and tetA determinants. KpV_S_1 and KpV_S_2 isolates belonged to ST661 and ST307, respectively. Both STs have been recently reported as responsible of outbreaks in several European countries. The detection of blaVIM-1 gene in nonpredominant K. pneumoniae clones in a hospital setting should alert on the changing of the epidemiological situation in Italy, usually endemic reservoir of KPC enzyme.

Detection of ST1702 Escherichia coli blaNDM-5 and blaCMY-42 genes positive isolates from a Northern Italian hospital.

We describe two multi drug-resistant (MDR) carbapenemase-producing Escherichia coli clinical isolates from an acute hospital in Milan. Both strains, isolated from a surgical wound sample and a surveillance rectal swab respectively, were positive for a blaNDM-type gene by Xpert Carba-R test. The whole-genome sequence characterization disclosed several resistance determinants: blaNDM-5, blaCMY-42, blaTEM-198, rmtB, mphA. The two isolates belonged to phylogenetic group A, sequence type (ST) 1702 and serotype O89:H9. PCR-based replicon typing and conjugation assay demonstrated an IncI1 plasmid localization for both blaNDM-5 and blaCMY-42 genes. This is the first report of a ST1702 NDM-5 and CMY-42- producing E. coli clone in Italy.

First Report of an ST410 OXA-181 and CTX-M-15 Coproducing Escherichia coli Clone in Italy: A Whole-Genome Sequence Characterization.

We investigated an Italian OXA-181-producing Escherichia coli clinical isolate (ECS1_14) by whole-genome sequencing. The strain coharbored blaCTX-M-15, blaCMY-2, and qnrS1 genes; it belonged to ST410(Achtman)/ST692(Pasteur) and phylogroup A. The blaOXA-181 gene was harbored on a plasmid highly similar (99% identity) to the pOXA181_EC14828 plasmid, recently reported in China.