Terbinafine Resistance in Trichophyton Strains Isolated from Humans and Animals: A Retrospective Cohort Study in Italy, 2016 to May 2024.

Background: In recent decades, globalization and international migration have increased the spread of infectious agents, including dermatophytes. Although considered minor infections, dermatophytoses are highly contagious, and they significantly reduce the quality of life, inducing itching, burning, sleep disturbances, and even depressive states. Moreover, the increasing resistance to antifungals threats the public health and burdens the costs for the healthcare system. Methods: DermaGenius® Resistance Multiplex real-time PCR assay allowed to analyze the terbinafine susceptibility/resistance of 172 Trichophyton strains, which were isolated from human and animal samples collected from 2016 to May 2024 and previously identified by Sanger sequencing. Results: All the 11 animal strains belonged to the T. interdigitale/T. mentagrophytes complex and tested terbinafine sensitive. Out of 161 human strains, 9 (5.6%) showed terbinafine resistance and 7 (4.3%) were identified as T. indotineae. Conclusions: This study provides preliminary data about behavior toward antifungals in animals and finalizes the scientific information currently available about human strains, highlighting the importance of the One Health concept. Moreover, it supports the relevant role of T. indotineae as an emerging dermatophyte with high proportion of terbinafine resistance.

Proof of concept of a new plasma complement Factor H from waste plasma fraction.

Introduction: Complement factor H (FH) is a major regulator of the complement alternative pathway, its mutations predispose to an uncontrolled activation in the kidney and on blood cells and to secondary C3 deficiency. Plasma exchange has been used to correct for FH deficiency and although the therapeutic potential of purified FH has been suggested by in vivo experiments in animal models, a clinical approved FH concentrate is not yet available. We aimed to develop a purification process of FH from a waste fraction rather than whole plasma allowing a more efficient and ethical use of blood and plasma donations. Methods: Waste fractions from industrial plasma fractionation (pooled human plasma) were analyzed for FH content by ELISA. FH was purified from unused fraction III and its decay acceleration, cofactor, and C3 binding capacity were characterized in vitro. Biodistribution was assessed by high-resolution dynamic PET imaging. Finally, the efficacy of the purified FH preparation was tested in the mouse model of C3 glomerulopathy (Cfh-/- mice). Results: Our purification method resulted in a high yield of highly purified (92,07%), pathogen-safe FH. FH concentrate is intact and fully functional as demonstrated by in vitro functional assays. The biodistribution revealed lower renal and liver clearance of human FH in Cfh-/- mice than in wt mice. Treatment of Cfh-/- mice documented its efficacy in limiting C3 activation and promoting the clearance of C3 glomerular deposits. Conclusion: We developed an efficient and economical system for purifying intact and functional FH, starting from waste material of industrial plasma fractionation. The FH concentrate could therefore constitute possible treatments options of patients with C3 glomerulopathy, particularly for those with FH deficiency, but also for patients with other diseases associated with alternative pathway activation.

Forensic Microbiology: When, Where and How.

Forensic microbiology is a relatively new discipline, born in part thanks to the development of advanced methodologies for the detection, identification and characterization of microorganisms, and also in relation to the growing impact of infectious diseases of iatrogenic origin. Indeed, the increased application of medical practices, such as transplants, which require immunosuppressive treatments, and the growing demand for prosthetic installations, associated with an increasing threat of antimicrobial resistance, have led to a rise in the number of infections of iatrogenic origin, which entails important medico-legal issues. On the other hand, the possibility of detecting minimal amounts of microorganisms, even in the form of residual traces (e.g., their nucleic acids), and of obtaining gene and genomic sequences at contained costs, has made it possible to ask new questions of whether cases of death or illness might have a microbiological origin, with the possibility of also tracing the origin of the microorganisms involved and reconstructing the chain of contagion. In addition to the more obvious applications, such as those mentioned above related to the origin of iatrogenic infections, or to possible cases of infections not properly diagnosed and treated, a less obvious application of forensic microbiology concerns its use in cases of violence or violent death, where the characterization of the microorganisms can contribute to the reconstruction of the case. Finally, paleomicrobiology, e.g., the reconstruction and characterization of microorganisms in historical or even archaeological remnants, can be considered as a sister discipline of forensic microbiology. In this article, we will review these different aspects and applications of forensic microbiology.

Genetic barriers more than environmental associations explain Serratia marcescens population structure.

Bacterial species often comprise well-separated lineages, likely emerged and maintained by genetic isolation and/or ecological divergence. How these two evolutionary actors interact in the shaping of bacterial population structure is currently not fully understood. In this study, we investigate the genetic and ecological drivers underlying the evolution of Serratia marcescens, an opportunistic pathogen with high genomic flexibility and able to colonise diverse environments. Comparative genomic analyses reveal a population structure composed of five deeply-demarcated genetic clusters with open pan-genome but limited inter-cluster gene flow, partially explained by Restriction-Modification (R-M) systems incompatibility. Furthermore, a large-scale research on hundred-thousands metagenomic datasets reveals only a partial habitat separation of the clusters. Globally, two clusters only show a separate gene composition coherent with ecological adaptations. These results suggest that genetic isolation has preceded ecological adaptations in the shaping of the species diversity, an evolutionary scenario coherent with the Evolutionary Extended Synthesis.

Cultivation and sequencing-free protocol for Serratia marcescens detection and typing.

Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here "Infectious clone"). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting typing (HLMT) protocol for the fast detection and typing of S. marcescens, with 100% detection capability on mixed samples and a limit of detection that can reach the 10 genome copies. The protocol was able to identify the S. marcescens infectious clone with 97% specificity and 96% sensitivity when compared to WGS, yielding typing results portable among laboratories. The protocol is a cost and time saving method for S. marcescens detection and typing for large environmental/clinical surveillance screenings, also in low-middle income countries.

Increased circulation of echovirus 11 in the general population and hospital patients as elicited by the non-polio enterovirus laboratory-based sentinel surveillance in northern Italy, 2023.

OBJECTIVES: Following the alert of echovirus 11 (E-11) infection in neonates in EU/EEA Member States, we conducted an investigation of E-11 circulation by gathering data from community and hospital surveillance of enterovirus (EV) in northern Italy from 01 August 2021 to 30 June 2023. METHODS: Virological results of EVs were obtained from the regional sentinel surveillance database for influenza-like illness (ILI) in outpatients, and from the laboratory database of ten hospitals for inpatients with either respiratory or neurological symptoms. Molecular characterization of EVs was performed by sequence analysis of the VP1 gene. RESULTS: In our ILI series, the rate of EV-positive specimens showed an upward trend from the end of May 2023, culminating at the end of June, coinciding with an increase in EV-positive hospital cases. The E-11 identified belonged to the D5 genogroup and the majority (83%) were closely associated with the novel E-11 variant, first identified in severe neonatal infections in France since 2022. E-11 was identified sporadically in community cases until February 2023, when it was also found in hospitalized cases with a range of clinical manifestations. All E-11 cases were children, with 14 out of 24 cases identified through hospital surveillance. Of these cases, 60% were neonates, and 71% had severe clinical manifestations. CONCLUSION: Baseline epidemiological data collected since 2021 through EV laboratory-based surveillance have rapidly tracked the E-11 variant since November 2022, alongside its transmission during the late spring of 2023.

New plasma protein C and protein S concentrate: A synergy for therapeutic purposes.

BACKGROUND AND OBJECTIVES: Deficiencies of protein C (PC) or protein S (PS) are rare diseases, characterized by mutations in the PC or PS genes, which encode plasma serine proteases with anti-coagulant activity. Severe PC or PS deficiencies manifest in early life as neonatal purpura fulminans, a life-threatening heamorrhagic condition requiring immediate treatment. First-line treatment involves replacement therapy, followed by maintenance with anti-coagulants. Replacement therapy with specific protein concentrates is currently only limited to PC, and therefore, a PC + PS concentrate represents a useful addition to therapeutic options, particularly for severe PS deficiency. Further, the production of a PC + PS concentrate from unused plasma fractionation intermediates would impact favourably on manufacturing costs, and consequently therapy prices for patients and health systems. MATERIALS AND METHODS: Several chromatographic runs were performed on the same unused plasma fractionation intermediates using different supports to obtain a PC/PS concentrate. The best chromatographic mediums were chosen, in terms of specific activity and recovery. A full process of purification including virus inactivation/removal and lyophilization steps was set up. RESULTS: The final freeze-dried product had a mean PC concentration of 47.75 IU/mL with 11% of PS, and a mean specific activity of 202.5 IU/mg protein, corresponding to over 12,000-fold purification from plasma. CONCLUSION: The development of a novel concentrated PC/PS mixture obtained from a waste fraction of other commercial products could be used for its potential therapeutic role in the management of neonatal purpura fulminans pathology.

Safety of spinal anesthesia and analysis of cerebrospinal fluid in SARS-CoV-2 pregnant women undergoing cesarean section: an observational prospective study.

BACKGROUND: Systemic infection has always been considered a relative contraindication to neuraxial anesthesia, despite the fact that infectious complications are relatively uncommon. Pregnancy-related physiological changes and coronavirus disease (COVID-19) neurotropic features may facilitate the virus' entry into the central nervous system. The principal aim of this study was to test the safety of spinal anesthesia in "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2)-positive pregnant women and to examine cerebrospinal fluid (CSF) characteristics. METHODS: We conducted a prospective observational single-center study in asymptomatic or paucisymptomatic consecutive pregnant SARS-CoV-2 patients who underwent spinal anesthesia for cesarean section. Women with severe infection were excluded because they underwent general anesthesia. At the time of spinal anesthesia, we collected CSF samples, and then we performed a chemical-physical analysis to look for signs of inflammation and for SARS-CoV-2 genome. RESULTS: We included 26 women. No spinal anesthesia complications were reported in the perioperative period and after 2 months. All CSF samples were crystal clear, and all physical-chemical values were within physiological ranges: the median concentration of CSF/plasma glucose ratio was 0.66, IQR 0.5500 (0.6000-0.7100), and the average CSF protein concentration value was 23.2 mg/dl (SD 4.87). In all samples, genomes of SARS-CoV-2 and other neurotropic viruses were not detected. CONCLUSIONS: Spinal anesthesia was safe in SARS-CoV-2 pregnant women with mild disease; no clinical maternal complications were detected, and no CSF changes indicative of inflammatory or infectious diseases that would compromise the safety of the procedure were found.

A territory-wide opportunistic, hospital-based HCV screening in the general population from northern Italy: The 1969-1989 birth-cohort.

BACKGROUND AND AIM: The World Health Organization (WHO) goal of Hepatitis C Virus (HCV) elimination by 2030 rose awareness about the need of screening plans, worldwide. In Italy, graduated screening starting from people born in 1969-1989 might be the most-effective strategy. We performed an opportunistic HCV screening study in the general population attending health facilities in Lombardy region, Northern Italy. METHODS: This is a prospective, multicenter, territory-wide, opportunistic study supported by the Regional Government of Lombardy, Italy. Between June 2022 and December 2022, all subjects born in 1969-1989, hospitalized or accessing blood collection centres were offered anti-HCV and HCV-RNA tests. Patients with known anti-HCV positivity and/or previous anti-HCV treatment were excluded. Demographic features were uploaded into a regional web-based platform. RESULTS: In total, 120 193 individuals were screened in 75 centres. Mean age was 44 (±6) years, 65.2% were females, 83.7% were tested at blood collection centres. Anti-HCV tested positive in 604 (0.50%) subjects: mean age 47 (±5), 51.1% females. HCV seroprevalence was higher in males (p < 0.00001), elderly (p < 0.00001) and in- vs. outpatients (p = 0.0009). HCV-RNA was detectable in 125 out of 441 (28.3%) anti-HCV positive subjects. Actively infected patients were 46 (±6) years old, mainly males (56.8%). The overall prevalence of active HCV infection was 0.10%, higher in elderly (p = 0.0003) and in in-patients (p = 0.0007). Among 93 HCV-RNA positive patients, the median age was 48 years, 58% males, 62% Italian born, median HCV-RNA levels were 6,1 log IU/mL, liver stiffness measurement (LSM) values 5.5 (3.1-29.9) kPa and ALT levels 48 U/L. CONCLUSIONS: The prevalence of active HCV infection in the 1969-1989 population attending health facilities in Lombardy was low. Most viremic patients were Italian-born, with mild liver disease but high-HCV-RNA levels. Due to the higher prevalence in the elderly, the extension of such opportunistic screening programs to lower birth cohorts would be warranted.

Water Safety Plan, Monochloramine Disinfection and Extensive Environmental Sampling Effectively Control Legionella and Other Waterborne Pathogens in Nosocomial Settings: The Ten-Year Experience of an Italian Hospital.

Legionella contamination control is crucial in healthcare settings where patients suffer an increased risk of disease and fatal outcome. To ensure an effective management of this health hazard, the accurate application of a hospital-specific Water Safety Plan (WSP), the choice of a suitable water disinfection system and an extensive monitoring program are required. Here, the ten-year experience of an Italian hospital is reported: since its commissioning, Legionellosis risk management has been entrusted to a multi-disciplinary Working Group, applying the principles of the World Health Organization's WSP. The disinfection strategy to prevent Legionella and other waterborne pathogens relies on the treatment of domestic hot water with a system ensuring the in situ production and dosage of monochloramine. An average of 250 samples/year were collected and analyzed to allow an accurate assessment of the microbiological status of water network. With the aim of increasing the monitoring sensitivity, in addition to the standard culture method, an optimized MALDI-ToF MS-based strategy was applied, allowing the identification of Legionella species and other relevant opportunistic pathogens. Data collected so far confirmed the effectiveness of this multidisciplinary approach: the fraction of positive samples never overcame 1% on a yearly basis and Legionnaires' Disease cases never occurred.

In Vitro Activity of Isavuconazole and Amphotericin B in Association against Mucorales.

Mucormycoses can be treated with the combination of Amphotericin B and Isavuconazole. This study evaluates the effects of these drugs in vitro against 59 strains representing 12 Mucorales. In vitro testing of the two drugs together and alone was performed using the MIC Test strip "Epsilon test synergy-method" (ETSM), which is more standard in clinical practice than microbroth dilution testing. Amphotericin B and Isavuconazole have synergistic/additive effects against L. corymbifera, R. arrhizus and M. circinelloides. Different effects have been shown for other Mucorales. ETSM can help the clinical management of mucormycosis from a practical point of view, due to its feasibility in the laboratory.

Prevention of congenital Chagas disease by screening of mothers and monitoring of serological tests of neonates: the seven years' experience.

Approximately 14000 immigrants coming from the Cochabamba area of Bolivia, with an increased risk of congenital Chagas Disease (CD), are currently living in Bergamo, Italy. According to the World Health Organization (WHO) recommendation (2011), prevention of congenital CD involves testing all pregnant women at risk of infection and performing follow-up of their newborns. In our study, all pregnant women of Latin American origin were tested for the presence of Trypanosoma cruzi antibodies and children, born to mothers found to be positive, were followed up after delivery. T. cruzi antibodies were detected using a chemiluminescence immunoassay. The test was also performed on siblings and fathers of children with CD, and women of childbearing age to prevent the congenital infection, as proposed by 2011 WHO recommendation. In the study period 1105 patients were tested for CD, using a serological test: 934 (85%) were females and 171 (15%) were males. Of the 62 newborns, from mothers who tested positive, 28 were females and 34 were males. The number of positive adults and siblings identified was 148 (14%). Among the adults and siblings born between 1991 and 2011 only 3 (2%) of females tested positive to serological test. All neonates, with the exception of one, were classified as non-infected according to the follow-up of index value of CD serology. This study confirms the usefulness of serological tests and of their index value as follow-up. The difference of positivity rate for CD antibodies between people born before and after 1990 should be further investigated to generate information that potentially improve the prevention and control of CD.

Endemic Systemic Mycoses in Italy: A Systematic Review of Literature and a Practical Update.

Endemic systemic mycoses such as blastomycosis, coccidioidomycosis, histoplasmosis, talaromycosis, paracoccidioidomycosis are emerging as an important cause of morbidity and mortality worldwide. We conducted a systematic review on endemic systemic mycoses reported in Italy from 1914 to nowadays. We found out: 105 cases of histoplasmosis, 15 of paracoccidioidomycosis, 10 of coccidioidomycosis, 10 of blastomycosis and 3 of talaromycosis. Most cases have been reported in returning travelers and expatriates or immigrants. Thirtytwo patients did not have a story of traveling to an endemic area. Fortysix subjects had HIV/AIDS. Immunosuppression was the major risk factor for getting these infections and for severe outcomes. We provided an overview on microbiological characteristics and clinical management principles of systemic endemic mycoses with a focus on the cases reported in Italy.

Impact of SARS-CoV-2 Omicron and Delta variants in patients requiring intensive care unit (ICU) admission for COVID-19, Northern Italy, December 2021 to January 2022.

This multicenter observational study included 171 COVID-19 adult patients hospitalized in the ICUs of nine hospitals in Lombardy (Northern Italy) from December, 1st 2021, to February, 9th 2022. During the study period, the Delta/Omicron variant ratio of cases decreased with a delay of two weeks in ICU patients compared to that in the community; a higher proportion of COVID-19 unvaccinated patients was infected by Delta than by Omicron whereas a higher rate of COVID-19 boosted patients was Omicron-infected. A higher number of comorbidities and a higher comorbidity score in ICU critically COVID-19 inpatients was positively associated with the Omicron infection as well in vaccinated individuals. Although people infected by Omicron have a lower risk of severe disease than those infected by Delta variant, the outcome, including the risk of ICU admission and the need for mechanical ventilation due to infection by Omicron versus Delta, remains uncertain. The continuous monitoring of the circulating SARS-CoV-2 variants remains a milestone to counteract this pandemic.

Vascular graft infections caused by Mycobacterium bovis BCG after BCG immunotherapy for non-muscle-invasive bladder cancer: Case report and review of literature.

Bacillus Calmette-Guerin (BCG) immunotherapy (i.e., intravesical instillation of live attenuated strain of Mycobacterium bovis) is a standard of care for non-muscle-invasive bladder cancer (NMIBC). The risk of infective adverse events is generally low as studies have reported an incidence of systemic BCG infections between 3% and 7%. In the majority of cases, BCG infections are disseminated (34.4%), genitourinary (23.4%), osteomuscular (19.9%), or vascular (6.7%). Regarding vascular involvement, mycotic aortic aneurysm, aorto-enteric fistula and vascular bypass graft infections have been described. A 73-year-old man with a prosthetic femoral-popliteal bypass was treated with BCG immunotherapy for a relapsed NMIBC. Two months later, the patient developed fever and hyporexia. PET-CT and CT scans of the abdomen showed an abscess surrounding the superficial femoral artery, while blood cultures yielded M. bovis BCG, and antitubercular therapy (with RMP + EMB + INH) was started. The prosthetic graft was removed and its cultures tested positive for M. bovis as well. A total of 14 cases of vascular prosthesis infections caused by M. bovis BCG following BCG instillation are so far reported. All the cases occurred in adult symptomatic men. Abdominal aorta was involved in the majority of cases. CT scan played a pivotal role in the diagnostic process. Mycobacterium bovis BCG was isolated from several different sources. Treatment required surgery and medical therapy, the latter showing wide variability. Previous BCG immunotherapy must be considered in the differential diagnosis in patients with infected vascular grafts. These infectious complications are rare and, while the infected grafts should be removed, there are no definite recommendations regarding the type of regimen and duration of treatment.

Yeast Bloodstream Infections in the COVID-19 Patient: A Multicenter Italian Study (FiCoV Study).

Fungemia is a co-infection contributing to the worsening of the critically ill COVID-19 patient. The multicenter Italian observational study FiCoV aims to estimate the frequency of yeast bloodstream infections (BSIs), to describe the factors associated with yeast BSIs in COVID-19 patients hospitalized in 10 hospitals, and to analyze the antifungal susceptibility profiles of the yeasts isolated from blood cultures. The study included all hospitalized adult COVID-19 patients with a yeast BSI; anonymous data was collected from each patient and data about antifungal susceptibility was collected. Yeast BSI occurred in 1.06% of patients, from 0.14% to 3.39% among the 10 participating centers. Patients were mainly admitted to intensive or sub-intensive care units (68.6%), over 60 years of age (73%), with a mean and median time from the hospitalization to fungemia of 29 and 22 days, respectively. Regarding risk factors for fungemia, most patients received corticosteroid therapy during hospitalization (61.8%) and had a comorbidity (25.3% diabetes, 11.5% chronic respiratory disorder, 9.5% cancer, 6% haematological malignancies, 1.4% organ transplantation). Antifungal therapy was administered to 75.6% of patients, mostly echinocandins (64.5%). The fatality rate observed in COVID-19 patients with yeast BSI was significantly higher than that of COVID-19 patients without yeast BSI (45.5% versus 30.5%). Candida parapsilosis (49.8%) and C. albicans (35.2%) were the most fungal species isolated; 72% of C. parapsilosis strains were fluconazole-resistant (range 0-93.2% among the centers). The FiCoV study highlights a high prevalence of Candida BSIs in critically ill COVID-19 patients, especially hospitalized in an intensive care unit, a high fatality rate associated with the fungal co-infection, and the worrying spread of azole-resistant C. parapsilosis.

Multicenter Italian Study on "In Vitro Activities" of Isavuconazole, Voriconazole, Amphotericin B, and Caspofungin for Aspergillus Species: Comparison between SensititreTM YeastOneTM and MIC Test Strip.

In this study the activity of Isavuconazole, Voriconazole, Amphotericin B, and Caspofungin against 224 clinical isolates of Aspergillus spp. originating from seven Italian hospitals, was comparatively evaluated with two commercial antifungal susceptibility tests (AST): SensititreTM YeastOneTM (SYO) and MIC Test Strip. More attention was focused on Isavuconazole activity, given the new introduction of the drug in widely distributed antifungal susceptibilities methods in the clinical microbiology lab. The minimum inhibitory concentrations of antifungal drug that can inhibit the growth of pathogen by 90% (MIC90) for Isavuconazole detected by SYO were 0.5, 1, 0.25, and 2 µg/mL for Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, and Aspergillus niger, respectively, whilst they were 0.25, 0.25, 0.5, and 0.75 µg/mL by MIC Test Strip. Essential agreement between the two tested methods for Isavuconazole is 70% for all the species tested, 75.7% for A. fumigatus, 45.2% for A. flavus, 90.6% for A. terreus, and 40% for A. niger. Although the tested strains do not express any phenotypic resistance, MIC results were quite different if tested with microdilution broth or gradient agar method. This is the first Italian multicenter report on Isavuconazole MIC obtained employing the widely used SensititreTM Yeast OneTM (SYO) and MIC Test Strip on clinical isolates of Aspergillus.

Hypervariable-Locus Melting Typing: a Novel Approach for More Effective High-Resolution Melting-Based Typing, Suitable for Large Microbiological Surveillance Programs.

Pathogen typing is pivotal to detecting the emergence of high-risk clones in hospital settings and to limit their spread. Unfortunately, the most commonly used typing methods (i.e., pulsed-field gel electrophoresis [PFGE], multilocus sequence typing [MLST], and whole-genome sequencing [WGS]) are expensive or time-consuming, limiting their application to real-time surveillance. High-resolution melting (HRM) can be applied to perform cost-effective and fast pathogen typing, but developing highly discriminatory protocols is challenging. Here, we present hypervariable-locus melting typing (HLMT), a novel approach to HRM-based typing that enables the development of more effective and portable typing protocols. HLMT types the strains by assigning them to melting types (MTs) on the basis of a reference data set (HLMT-assignment) and/or by clustering them using melting temperatures (HLMT-clustering). We applied the HLMT protocol developed on the capsular gene wzi for Klebsiella pneumoniae on 134 strains collected during surveillance programs in four hospitals. Then, we compared the HLMT results to those obtained using wzi, MLST, WGS, and PFGE typing. HLMT distinguished most of the K. pneumoniae high-risk clones with a sensitivity comparable to that of PFGE and MLST+wzi. It also drew surveillance epidemiological curves comparable to those obtained using MLST+wzi, PFGE, and WGS typing. Furthermore, the results obtained using HLMT-assignment were consistent with those of wzi typing for 95% of the typed strains, with a Jaccard index value of 0.9. HLMT is a fast and scalable approach for pathogen typing, suitable for real-time hospital microbiological surveillance. HLMT is also inexpensive, and thus, it is applicable for infection control programs in low- and middle-income countries. IMPORTANCE In this work, we describe hypervariable-locus melting typing (HLMT), a novel fast approach to pathogen typing using the high-resolution melting (HRM) assay. The method includes a novel approach for gene target selection, primer design, and HRM data analysis. We successfully applied this method to distinguish the high-risk clones of Klebsiella pneumoniae, one of the most important nosocomial pathogens worldwide. We also compared HLMT to typing using WGS, the capsular gene wzi, MLST, and PFGE. Our results show that HLMT is a typing method suitable for real-time epidemiological investigation. The application of HLMT to hospital microbiology surveillance can help to rapidly detect outbreak emergence, improving the effectiveness of infection control strategies.

The changing pattern of bacterial and fungal respiratory isolates in patients with and without COVID-19 admitted to intensive care unit.

OBJECTIVES: Severe acute respiratory syndrome 2 (SARS-CoV-2) pandemic has had a heavy impact on national health system, especially in the first wave. That impact hit principally the intensive care units (ICUs). The large number of patients requiring hospitalization in ICUs lead to a complete upheaval of intensive wards. The increase in bed, the fewer number of nurses per patient, the constant use of personal protective equipment, the new antimicrobial surveillance protocols could have had deeply effects on microbiological flora of these wards. Moreover, the overconsumption of antimicrobial therapy in COVID-19 patients, like several studies report, could have impact of this aspect. Aim of this study is to evaluate the changing pattern of microbiological respiratory isolates during and before COVID-19 pandemic in a tertiary hospital ICUs. METHODS: A retrospective, observational study was conducted in ICUs of "ASST Papa Giovanni XXIII", a large tertiary referral hospital in Northern Italy. We have retrospectively collected the microbiological data from bronchoalveolar lavage (BAL) and tracheal aspirate (TA) of patients with COVID-19, hospitalized in ICUs from 22nd February 2020 to 31st May 2020 (Period 1), and without COVID-19, from 22nd February 2019 to 31st May 2019 (Period 2). We compared the prevalence and the antibiotic profile of bacterial and fungal species in the two time periods. RESULTS: The prevalence of Pseudomonas spp. shows a statistically significant increase from patients without COVID-19 compared to COVID-19 positive as well as the prevalence of Enterococcus spp. On the contrary, the prevalence of Gram negative non fermenting bacteria (GN-NFB), Haemophilus influenzae and Streptococcus pneumoniae showed a significant reduction between two periods. There was a statistically significant increase in resistance of Pseudomonas spp. to carbapenems and piperacillin/tazobactam and Enterobacterales spp. for piperacillin/tazobactam, in COVID-19 positive patients compared to patients without COVID-19. We did not observe significant changing in fungal respiratory isolates. CONCLUSIONS: A changing pattern in prevalence and resistance profiles of bacterial and fungal species was observed during COVID-19 pandemic.