When Does the Cytokine Storm Begin in COVID-19 Patients? A Quick Score to Recognize It.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that is responsible for coronavirus disease 2019 (COVID-19), which has rapidly spread across the world, becoming a pandemic. The "cytokine storm" (CS) in COVID-19 leads to the worst stage of illness, and its timely control through immunomodulators, corticosteroids, and cytokine antagonists may be the key to reducing mortality. After reviewing published studies, we proposed a Cytokine Storm Score (CSs) to identify patients who were in this hyperinflammation state, and at risk of progression and poorer outcomes. We retrospectively analyzed 31 patients admitted to Infectious Disease Department in "St. Maria" Hospital in Terni with confirmed SARS-CoV-2 infections, and analyzed the "CS score" (CSs) and the severity of COVID-19. Then we conducted a prospective study of COVID-19 patients admitted after the definition of the CSscore. This is the first study that proposes and applies a new score to quickly identify COVID-19 patients who are in a hyperinflammation stage, to rapidly treat them in order to reduce the risk of intubation. CSs can accurately identify COVID-19 patients in the early stages of a CS, to conduct timely, safe, and effect administration of immunomodulators, corticosteroids, and cytokine antagonists, to prevent progression and reduce mortality.

Antimicrobial Stewardship Program, COVID-19, and Infection Control: Spread of Carbapenem-Resistant Klebsiella Pneumoniae Colonization in ICU COVID-19 Patients. What Did Not Work?

The Italian burden of disease associated with infections due to antibiotic-resistant bacteria has been very high, largely attributed to Carbapenem-Resistant Klebsiella pneumoniae (CR-Kp). The implementation of infection control measures and antimicrobial stewardship programs (ASP) has been shown to reduce healthcare-related infections caused by multidrug resistance (MDR) germs. Since 2016, in our teaching hospital of Terni, an ASP has been implemented in an intensive care unit (ICU) setting, with the "daily-ICU round strategy" and particular attention to infection control measures. We performed active surveillance for search patients colonized by Carbapenem-Resistant Enterobacteriaceae (CRE). In March 2020, coronavirus disease 2019 (COVID-19) arrived and the same ICU was reserved only for COVID-19 patients. In our retrospective observational study, we analyzed the bimonthly incidence of CRE colonization patients and the incidence of CRE acquisition in our ICU during the period of January 2019 to June 2020. In consideration of the great attention and training of all staff on infection control measures in the COVID-19 era, we would have expected a clear reduction in CRE acquisition, but this did not happen. In fact, the incidence of CRE acquisition went from 6.7% in 2019 to 50% in March-April 2020. We noted that 67% of patients that had been changed in posture with prone position were colonized by CRE, while only 37% of patients that had not been changed in posture were colonized by CRE. In our opinion, the high intensity of care, the prone position requiring 4-5 healthcare workers (HCWs), equipped with personal protective equipment (PPE) in a high risk area, with extended and prolonged contact with the patient, and the presence of 32 new HCWs from other departments and without work experience in the ICU setting, contributed to the spread of CR-Kp in our ICU, determining an increase in CRE acquisition colonization.

Impact of Antimicrobial Stewardship Interventions on Appropriateness of Surgical Antibiotic Prophylaxis: How to Improve.

Surgical site infections (SSIs) are the most common healthcare-associated infections. The appropriate use of Surgical Antibiotic Prophylaxis (SAP) is a key component to reduce SSIs, while its inappropriate application is a major cause of some emerging infections and selects for antibiotic resistance. We describe an Antimicrobial Stewardship (AMS) intervention on SAP appropriateness. The prospective study was conducted in an Italian hospital, in 12 main surgical units, and was organized in three subsequent phases, as follows. Phase 0: Definition of hospital evidence-based guidelines and a new workflow to optimize the process of ordering, dispensing, administering and documenting the SAP. Phase 1: We analysed 2059 elective surgical cases from January to June 2018 for three SAP parameters of appropriateness: indication, choice and dose. Phase 2: In July 2018, an audit was performed to analyse the results; we reviewed 1781 elective surgical procedures from July to December 2018 looking for the same three SAP appropriateness parameters. The comparative analysis between phases 1 and 2 demonstrated that the correct indication, the correct dose and the overall compliance significantly improved (p-value 0.00128, p-value < 2.2·1016 and p-value < 5.6·1012 respectively). Our prospective study demonstrates a model of successful antimicrobial stewardship intervention that improves appropriateness on SAP.

Mycobacterium decipiens sp. nov., a new species closely related to the Mycobacterium tuberculosis complex.

Two mycobacterial strains with close similarity to the Mycobacterium tuberculosis complex (MTBC) were isolated from cutaneous lesions of patients in the USA and Italy. At the phenotypic level, similarities to the MTBC included slow growth rate, rough morphotype of the unpigmented colonies and nearly identical high-performance liquid chromatography profiles of mycolic acids. In contrast to the MTBC, the strains were niacin- and nitrate-negative, and catalase-positive both at 68 °C and in semi-quantitative tests. The clinical isolates were more closely related to M. tuberculosis than to any other known mycobacterium and scored positive with commercial DNA probes (Hologic AccuProbe M. tuberculosis). Both average nucleotide identity and genome-to-genome distance suggested the strains are different from the MTBC. Therefore, given the distinguishing phenotypic and genomic-scale differences, we submit that the strains belong to a new species we have named Mycobacteriumdecipiens with type strain TBL 1200985T (=ATCC TSD-117T=DSM 105360T).

Targeted next generation sequencing in Italian patients with Usher syndrome: phenotype-genotype correlations.

We report results of DNA analysis with next generation sequencing (NGS) of 21 consecutive Italian patients from 17 unrelated families with clinical diagnosis of Usher syndrome (4 USH1 and 17 USH2) searching for mutations in 11 genes: MYO7A, CDH23, PCDH15, USH1C, USH1G, USH2A, ADGVR1, DFNB31, CLRN1, PDZD7, HARS. Likely causative mutations were found in all patients: 25 pathogenic variants, 18 previously reported and 7 novel, were identified in three genes (USH2A, MYO7A, ADGRV1). All USH1 presented biallelic MYO7A mutations, one USH2 exhibited ADGRV1 mutations, whereas 16 USH2 displayed USH2A mutations. USH1 patients experienced hearing problems very early in life, followed by visual impairment at 1, 4 and 6 years. Visual symptoms were noticed at age 20 in a patient with homozygous novel MYO7A missense mutation c.849G > A. USH2 patients' auditory symptoms, instead, arose between 11 months and 14 years, while visual impairment occurred later on. A homozygous c.5933_5940del;5950_5960dup in USH2A was detected in one patient with early deafness. One patient with homozygous deletion from exon 23 to 32 in USH2A suffered early visual symptoms. Therefore, the type of mutation in USH2A and MYO7A genes seems to affect the age at which both auditory and visual impairment occur in patients with USH.

Mycobacterium aquaticum sp. nov., a rapidly growing species isolated from haemodialysis water.

The characterization of five Iranian isolates, four from hospital haemodialysis water and one from the sputum of a patient, led to the detection of a novel mycobacterium species. The strains were characterized by mucoid colonies developing in 3-5 days at temperatures ranging from 25 to 37 °C. The biochemical test pattern was unremarkable while the HPLC profile of mycolic acids resembled that of Mycobacterium fortuitum. The sequences of three major housekeeping genes (16S rRNA, hsp65 and rpoB) were unique and differed from those of any other mycobacterium. Mycobacterium brisbanense, which is the species that shared the highest 16S rRNA gene sequence similarity (99.03 %), was distinct, as shown by the average nucleotide identity and by the genome to genome distance values (91.05 and 43.10 %, respectively). The strains are thus considered to represent a novel species of the genus Mycobacterium, for which the name Mycobacterium aquaticum sp. nov. is proposed. The type strain is RW6T (=DSM 104277T=CIP111198T).

Usher's Syndrome Type II: A Comparative Study of Genetic Mutations and Vestibular System Evaluation.

Objective Usher's syndrome type II (USH2) is characterized by moderate to profound congenital hearing loss, later onset of retinitis pigmentosa, and normal vestibular function. Recently, a study investigating the vestibular function of USH2 patients demonstrated a pathologic response to vestibular tests. In this cross-sectional study we performed vestibular tests of a group patients with genetic diagnosis of USH2 syndrome to demonstrate if vestibular damage is present in USH2 patients. Study Design Cross-sectional study. Setting Tertiary referral center. Subjects and Methods Mutated genes of 7 patients with a clinical diagnosis of USH2 were evaluated. Vestibular function was investigated by audiometry, Fitzgerald-Hallpike caloric vestibular testing, cervical vestibular evoked myogenic potentials (C-VEMPs), ocular vestibular evoked myogenic potentials (O-VEMPs), and video head impulse test (v-HIT). Results Genetic tests confirmed the USH2 diagnosis in 5 of 7 patients examined, with 1 patient reporting a unique mutation on genetic tests. Four (80%) of the 5 patients with a genetic diagnosis of USH2 showed pathological O-VEMPs. Two patients (40%) reported bilateral absent or abnormal values of C-VEMPs. The superior semicircular canal presented a significant deficit in 2 (40%) patients. The same 2 cases showed a pathologic response of the v-HIT of the horizontal semicircular canal. Finally, the posterior semicircular canal presented a significant deficit in 4 (40.0%) patients. Conclusion A vestibular evaluation with vestibular evoked myogenic potentials and v-HIT seems to identify latent damage to the vestibular receptors of USH2 patients.

Mycobacterium alsense sp. nov., a scotochromogenic slow grower isolated from clinical respiratory specimens.

The name 'Mycobacterium alsiense', although reported in 2007, has not been validly published. Polyphasic characterization of three available strains of this species led us to the conclusion that they represent a distinct species within the genus Mycobacterium. The proposed novel species grows slowly and presents pale yellow-pigmented colonies. Differentiation from other mycobacteria is not feasible on the basis of biochemical and cultural features alone while genetic analysis, extended to eight housekeeping genes and one spacer region, reveals its clear distinction from all other mycobacteria. Mycobacterium asiaticum is the most closely related species on the basis of 16S rRNA gene sequences (similarity 99.3 %); the average nucleotide identity between the genomes of the two species is 80.72 %, clearly below the suggested cut-off (95-96 %). The name Mycobacterium alsense sp. nov. is proposed here for the novel species and replaces the name 'M. alsiense', ex Richter et al. 2007, given at the time of isolation of the first strain. The type strain is TB 1906T ( = DSM 45230T = CCUG 56586T).

Mycobacterium angelicum sp. nov., a non-chromogenic, slow-growing species isolated from fish and related to Mycobacterium szulgai.

The name 'Mycobacterium angelicum' dates back to 2003 when it was suggested for a slowly growing mycobacterium isolated from freshwater angelfish. This name is revived here and the novel species is proposed on the basis of the polyphasic characterization of four strains including the original one. The four strains presented 100 % 16S rRNA gene sequence similarity with Mycobacterium szulgai but clearly differed from M. szulgai for the milky white aspect of the colonies. The sequence similarity with the type strain of M. szulgai ranged, in eight additionally investigated genetic targets, from 78.9 to 94.3 %, an evident contrast with the close relatedness that emerged at the level of 16S rRNA gene. The average nucleotide identity between the genomes of M. szulgai DSM 44166T and strain 126/5/03T (type strain of the novel species) was 92.92 %, and supported the status of independent species. The confirmation of the name Mycobacterium angelicum sp. nov. is proposed, with strain 126/5/03T ( = CIP 109313T = DSM 45057T) as the type strain.

Mycobacterium celeriflavum sp. nov., a rapidly growing scotochromogenic bacterium isolated from clinical specimens.

Six strains of a rapidly growing scotochromogenic mycobacterium were isolated from pulmonary specimens of independent patients. Biochemical and cultural tests were not suitable for their identification. The mycolic acid pattern analysed by HPLC was different from that of any other mycobacterium. Genotypic characterization, targeting seven housekeeping genes, revealed the presence of microheterogeneity in all of them. Different species were more closely related to the test strains in various regions: the type strain of Mycobacterium moriokaense showed 99.0 % 16S rRNA gene sequence similarity, and 91.5-96.5 % similarity for the remaining six regions. The whole genome sequences of the proposed type strain and that of M. moriokaense presented an average nucleotide identity (ANI) of 82.9 %. Phylogenetic analysis produced poorly robust trees in most genes with the exception of rpoB and sodA where Mycobacterium flavescens and Mycobacterium novocastrense were the closest species. This phylogenetic relatedness was confirmed by the tree inferred from five concatenated genes, which was very robust. The polyphasic characterization of the test strains, supported by the ANI value, demonstrates that they belong to a previously unreported species, for which the name Mycobacterium celeriflavum sp. nov. is proposed. The type strain is AFPC-000207(T) ( = DSM 46765(T) = JCM 18439(T)).

Characterization of 17 strains belonging to the Mycobacterium simiae complex and description of Mycobacterium paraense sp. nov.

Fourteen mycobacterial strains isolated from pulmonary samples of independent patients in the state of Pará (Brazil), and three strains isolated in Italy, were characterized using a polyphasic approach. Thorough genetic investigation, including whole-genome sequencing, demonstrated that the strains belong to the M. simiae complex, being most closely related to Mycobacterium interjectum. For 14 of the strains, evidence emerged supporting their inclusion in a previously unreported species of the genus Mycobacterium, for which the name Mycobacterium paraense sp. nov. is proposed (type strain, IEC26(T) = DSM 46749(T) = CCUG 66121(T)). The novel species is characterized by slow growth, unpigmented or pale yellow scotochromogenic colonies, and a HPLC mycolic acid profile different from other known mycobacteria. In different genetic regions, high sequence microheterogeneity was detected.

MYO7A and USH2A gene sequence variants in Italian patients with Usher syndrome.

PURPOSE: To analyze the spectrum of sequence variants in the MYO7A and USH2A genes in a group of Italian patients affected by Usher syndrome (USH). METHODS: Thirty-six Italian patients with a diagnosis of USH were recruited. They received a standard ophthalmologic examination, visual field testing, optical coherence tomography (OCT) scan, and electrophysiological tests. Fluorescein angiography and fundus autofluorescence imaging were performed in selected cases. All the patients underwent an audiologic examination for the 0.25-8,000 Hz frequencies. Vestibular function was evaluated with specific tests. DNA samples were analyzed for sequence variants of the MYO7A gene (for USH1) and the USH2A gene (for USH2) with direct sequencing techniques. A few patients were analyzed for both genes. RESULTS: In the MYO7A gene, ten missense variants were found; three patients were compound heterozygous, and two were homozygous. Thirty-four USH2A gene variants were detected, including eight missense variants, nine nonsense variants, six splicing variants, and 11 duplications/deletions; 19 patients were compound heterozygous, and three were homozygous. Four MYO7A and 17 USH2A variants have already been described in the literature. Among the novel mutations there are four USH2A large deletions, detected with multiplex ligation dependent probe amplification (MLPA) technology. Two potentially pathogenic variants were found in 27 patients (75%). Affected patients showed variable clinical pictures without a clear genotype-phenotype correlation. CONCLUSIONS: Ten variants in the MYO7A gene and 34 variants in the USH2A gene were detected in Italian patients with USH at a high detection rate. A selective analysis of these genes may be valuable for molecular analysis, combining diagnostic efficiency with little time wastage and less resource consumption.

Survey of 150 strains belonging to the Mycobacterium terrae complex and description of Mycobacterium engbaekii sp. nov., Mycobacterium heraklionense sp. nov. and Mycobacterium longobardum sp. nov.

A thorough phenotypic and genotypic analysis of 150 strains belonging to the Mycobacterium terrae complex resulted in the identification of a number of previously unreported sequevars (sqvs) within the species known to belong to the complex. For the species Mycobacterium arupense, three sqvs were detected in the 16S rRNA gene, six sqvs in the hsp65 gene and 15 sqvs in the rpoB gene; in Mycobacterium senuense two sqvs were present in each of the three genetic regions; in Mycobacterium kumamotonense four, two and nine sqvs were found, respectively, and in M. terrae three, four and six sqvs were found, respectively. The inappropriate inclusion of Mycobacterium triviale within the M. terrae complex was confirmed. The limited utility of biochemical tests and of mycolic acid analyses for the differentiation of the members of M. terrae complex was also confirmed. The survey allowed the recognition of three previously undescribed species that were characterized by unique sequences in the 16S rRNA, hsp65 and rpoB genes. Mycobacterium engbaekii sp. nov. (proposed previously 40 years ago but never validly published) was characterized by pink photochromogenic pigmentation and rapid growth; phylogenetically it was related to Mycobacterium hiberniae. The type strain of this species, of which eight strains were investigated, is ATCC 27353(T) ( = DSM 45694(T)). A cluster of 24 strains was the basis for the description of Mycobacterium heraklionense sp. nov., which has an intermediate growth rate and is unpigmented; nitrate reductase activity is typically strong. Closely related to M. arupense with respect to the 16S rRNA gene, M. heraklionense sp. nov. could be clearly differentiated from the latter species in the other genetic regions investigated. The type strain is NCTC 13432(T) ( = LMG 24735(T) = CECT 7509(T)). Mycobacterium longobardum sp. nov., represented in the study by seven strains, was characterized by a unique phylogenetic location within the M. terrae complex, clearly divergent from any other species. The type strain is DSM 45394(T) ( = CCUG 58460(T)).

Mycobacterium europaeum sp. nov., a scotochromogenic species related to the Mycobacterium simiae complex.

Four strains isolated in the last 15 years were revealed to be identical in their 16S rRNA gene sequences to MCRO19, the sequence of which was deposited in GenBank in 1995. In a polyphasic analysis including phenotypic and genotypic features, the five strains (including MCRO19), which had been isolated in four European countries, turned out to represent a unique taxonomic entity. They are scotochromogenic slow growers and are genetically related to the group that included Mycobacterium simiae and 15 other species. The novel species Mycobacterium europaeum sp. nov. is proposed to accommodate these five strains. Strain FI-95228(T) ( = DSM 45397(T)  = CCUG 58464(T)) was chosen as the type strain. In addition, a thorough revision of the phenotypic and genotypic characters of the species related to M. simiae was conducted which leads us to suggest the denomination of the 'Mycobacterium simiae complex' for this group.

Unusual Methylobacterium fujisawaense Infection in a Patient with Acute Leukaemia Undergoing Hematopoietic Stem Cell Transplantation: First Case Report.

Microorganisms of the genus Methylobacterium are facultative methylotrophic, gram-negative rods that are ubiquitous in nature and rarely cause human disease, mostly in subjects with preexisting causes of immune depression. Methylobacterium fujisawaense, first proposed as a new species in 1988, has never been reported as a bacterial agent of human infections so far. Here we describe a case of M. fujisawaense infection in a relapsed acute leukaemia undergoing unrelated allogeneic hematopoietic stem cell transplantation. Molecular identification of an M. fujisawaense strain was obtained from multiple mycobacterial blood cultures.

Mycobacterium insubricum sp. nov.

Five independent strains, isolated from clinical samples but probably not responsible for disease, revealed phenotypic and genotypic features that appeared to exclude their belonging to any of the recognized Mycobacterium species. The strains, which are non-pigmented rapid growers, presented a cell-wall lipid pattern resembling those of Mycobacterium brumae and Mycobacterium fallax. Sequencing of the 16S rRNA, hsp65 and rpoB genes and the 16S-23S rRNA gene internal transcribed spacer (ITS) revealed that the strains are clearly distinct from every other Mycobacterium species. While the 16S rRNA and rpoB genes were characterized by a single sequevar, two sequevars were detected in hsp65 and three in the ITS. The divergence shown in the latter region was striking, in which only two short regions (less than 150 bp in all) were comparable with other mycobacteria, apart from Mycobacterium monacense and Mycobacterium gilvum. The PCR restriction analysis pattern of the novel strains also differs from any reported to date. The name Mycobacterium insubricum sp. nov. is proposed for the novel species; the type strain is FI-06250(T) (=DSM 45132(T) =CIP 109609(T)).

Pulmonary disease due to Mycobacterium arosiense, an easily misidentified pathogenic novel mycobacterium.

Mycobacterium arosiense is a newly described species. After noticing it was misidentified as Mycobacterium intracellulare by the commercial identification system GenoType CM (Hein, Nehren, Germany), we detected 4 such strains among 33 that were previously misidentified as M. intracellulare. Three more strains were found among unidentified mycobacteria not tested previously with GenoType. The first case of pulmonary disease due to M. arosiense is reported here, and the novel species, of which so far only one strain had been investigated, is further characterized.