Comparison of genotypic and phenotypic antimicrobial profile in carbapenemases producing Klebsiella pneumoniae.

BACKGROUND AND AIM: Prompt administration of appropriate antibiotic therapy is crucial in improving outcomes, particularly in cases sustained by multi-drug resistant strains. Although phenotypic antimicrobial susceptibility testing (AST) represents the gold standard to address antibiotics treatment, the long time required to obtained affordable results could negatively affect the prognosis. In contrast, rapid genotypic AST provide essential information for treatment and surveillance program. In order to evaluate the potential adoption of rapid AST in clinical routine, we compared the genotypic and phenotypic antimicrobial profiles of different K.pneumoniae strains, characterized by different expression of carbapenemases-encoding genes. METHODS: A set of 109 strains of Cr-Kp were tested for the antimicrobial drugs by the automatized Vitek II system and, in parallel, to the new combination of ß-lactams/ß-lactamases inhibitors (BL/BLI) by Etest. An antimicrobial resistance index (ARI) was calculated for each strain, assigning each 1 or 0 points based on observed resistance/susceptibility, and dividing the total by the number of antibiotics tested. Kruskal-Wallis test, followed by Dunn's post hoc test (Bonferroni correction), were used to compare quantitative variables among resistance gene subgroups. RESULTS: We observed a higher ARI score in KPC/OXA-48 strains, similar profile in KPC alone and KPC/CTX-M groups and a significant lower resistance in no-carbapenemases-producing group. Same trend was observed in AST for BL/BLI. CONCLUSIONS: These preliminary results showed a close link between genotypic and phenotypic AST, supporting the adoption of rapid AST in cases of severe infections, ensuring to saving time and providing, the surveillance of MDR strains and improving stewardship programs.

A Whole-Genome Sequencing-Based Approach for the Characterization of Klebsiella pneumoniae Co-Producing KPC and OXA-48-like Carbapenemases Circulating in Sardinia, Italy.

BACKGROUND: Whole-genome sequencing (WGS) provides important information for the characterization, surveillance, and monitoring of antimicrobial resistance (AMR) determinants, particularly in cases of multi- and extensively drug-resistant microorganisms. We reported the results of a WGS analysis carried out on carbapenemases-producing Klebsiella pneumoniae, which causes hospital-acquired infections (HAIs) and is characterized by a marked resistance profile. METHODS: Clinical, phenotypic, and genotypic data were collected for the AMR surveillance screening program of the University Hospital of Sassari (Italy) during 2020-2021. Genomic DNA was sequenced using the Illumina Nova Seq 6000 platform. Final assemblies were manually curated and carefully verified for the detection of antimicrobial resistance genes, porin mutations, and virulence factors. A phylogenetic analysis was performed using the maximum likelihood method. RESULTS: All 17 strains analyzed belonged to ST512, and most of them carried the blaKPC-31 variant blaOXA-48-like, an OmpK35 truncation, and an OmpK36 mutation. Phenotypic analysis showed a marked resistance profile to all antibiotic classes, including ß-lactams, carbapenems, aminoglycosides, fluoroquinolone, sulphonamides, and novel ß-lactam/ß-lactamase inhibitors (BL/BLI). CONCLUSION: WGS characterization revealed the presence of several antibiotic resistance determinants and porin mutations in highly resistant K. pneumoniae strains responsible for HAIs. The detection of blaKPC-31 in our hospital wards highlights the importance of genomic surveillance in hospital settings to monitor the emergence of new clones and the need to improve control and preventive strategies to efficiently contrast AMR.

Infection Prevention Control Strategies of New Delhi Metallo-ß-lactamase Producing Klebsiella pneumoniae.

The spread of multi-drug resistant organisms (MDROs) is increasing at an alarming rate worldwide. Among these, Carbapenemase-producing New Delhi Metallo-ß-lactamase (NDM) poses a significant clinical threat, and appropriate measures must be taken to prevent or limit its penetration into still-free territories. The present report describes two independent cases of patients from Ukraine colonized by NDM-producing Klebsiella pneumoniae and admitted to two separate wards of an acute university hospital in a territory not yet affected by Carbapenemase producers of this class. Moreover, this report illustrates the infection prevention control (IPC) strategies promptly implemented by the IPC operational team to verify the possible spread of the microorganism in the ward and avoid any possible further contamination. The identification of genes coding for Carbapenemases, performed using real-time PCR, revealed no other cases within the wards involved. These cases emphasize the importance of early case recognition of multidrug-resistant bacteria, the necessity of effective inter-hospital communication, the need for effective antimicrobial stewardship protocol, and the importance of adequate IPC policies. Additionally, we highlight the need to improve screening procedures in the case of patients from countries with a high prevalence of MDRO, as essential measures to prevent potential nosocomial outbreaks and/or endemization.

Preliminary Results of Feasibility and Acceptability of Self-Collection for Cervical Screening in Italian Women.

BACKGROUND: Given the diagnostic accuracy of HPV-DNA tests in terms of self-collected samples, in order to implement self-sampling in cervical screening programs, the standardization of the pre-analytical phase, including decisions concerning the choice of medium, the volume of elution, and storage conditions, are necessary, in addition to understanding the potential factors involved in acceptability by women. On this basis, we carried out a cross-sectional study to assess (i) the stability of dry vaginal self-collected samples stored at room temperature for up to 4 weeks after elution in 2 mL of eNat® (Copan) medium, and (ii) the acceptability of self-collection in enrolled women. METHODS: 185 women were enrolled in the LILT (Italian League Against Tumors) regional project. A self-sampling kit, including a dry FLOQSwab® (Copan), instructions for use, and a satisfaction questionnaire, were supplied for each woman and sent by mail to the laboratory. The HPV-DNA test was carried out using the Anyplex™ II HPV HR (Seegene) kit. To evaluate the specimen's stability, 185 dry vaginal swabs were eluted in eNat®, a lyses-based molecular medium and tested for HPV detection at two different time points (<6 days and 1 month after elution). The Cohen's Kappa coefficients and McNemar test were used to assess the agreement of HPV-DNA at different times. RESULTS: We found high agreement in terms of HPV-DNA results among the samples tested at two different time points (Cohen K = 0.98; p < 0.0001). Moreover, most of the women found it easy to use self-collection devices and the pictorial instructions clear to understand. Approximately half of the enrolled women declared preferring self-sampling to clinician-collected methods. CONCLUSION: Our results display the high reliability and accuracy of HPV-DNA tests using dry vaginal self-collection FLOQSwabs® devices eluted in 2 mL of molecular medium. The analysis of the questionnaire showed a high acceptability of self-collection among women, although a high percentage preferred standard collection devices. Overall, our preliminary results support the adoption of self-collection in screening programs, even though further analyses should be performed to optimize and standardize protocols for HPV tests on self-samples, and educational campaigns are needed to adequately inform and increase responsiveness in a target population.

Prevalence of Human Papilloma Virus Infection in Bladder Cancer: A Systematic Review.

The etiology of bladder cancer is known to be associated with behavioral and environmental factors. Moreover, several studies suggested a potential role of HPV infection in the pathogenesis with controversial results. A systematic review was conducted to assess the role of HPV. A total of 46 articles that reported the prevalence of HPV infection in squamous (SCC), urothelial (UC), and transitional cell carcinomas (TCC) were selected. A pooled prevalence of 19% was found, with a significant difference in SCC that was mainly driven by HPV-16. Moreover, infection prevalence in case-control studies showed a higher risk of bladder cancer in HPV-positive cases (OR: 7.84; p-value < 0.00001). The results may suggest an etiologic role of HPV in bladder cancer. HPV vaccine administration in both sexes could be key to prevent the infection caused by high-risk genotypes.

High-Risk Clone of Klebsiella pneumoniae Co-Harbouring Class A and D Carbapenemases in Italy.

Background: Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is endemic globally, causing severe infections in hospitalized patients. Surveillance programs help monitor and promptly identify the emergence of new clones. We reported the rapid spread of a novel clone of K. pneumoniae co-harbouring class A and D carbapenemases in colonized patients, and the potential risk factors involved in the development of infections. Methods: Rectal swabs were used for microbiological analyses and detection of the most common carbapenemase encoding genes by real-time PCR (i.e., blaKPC, blaOXA-48, blaNDM, blaVIM, and blaIMP). All strains co-harbouring KPC and OXA-48 genes were evaluated. For each patient, the following variables were collected: age, sex, length and ward of stay, device use, and outcome. Clonality of CR-Kp was assessed by preliminary pulsed field gel electrophoresis (PFGE), followed by multi-locus sequence typing (MLST) analyses. Results: A total of 127 isolates of K. pneumoniae co-harbouring KPC and OXA-48 were collected between September 2019 and December 2020. The median age (IQR) of patients was 70 (61-77). More than 40% of patients were admitted to intensive care unit (ICU). Around 25% of patients developed an invasive infection, the majority of which were respiratory tract infections (17/31; 54.8%). ICU stay and invasive infection increased the risk of mortality (OR: 5.39, 95% CI: 2.42-12.00; OR 6.12, 95% CI: 2.55-14.69, respectively; p-value ≤ 0.001). The antibiotic susceptibility test showed a resistance profile for almost all antibiotics considered. Monoclonal origin was confirmed by PFGE and MLST showing a similar restriction pattern and belonging to ST-512. Conclusions: We report the spread and the marked antibiotic resistance profiles of K. pneumoniae strains co-producing KPC and OXA-48. Further study could clarify the roles of clinical and microbiological variables in the development of invasive infection and increasing risk of mortality, in colonized patients.

Genomic Characterization of KPC-31 and OXA-181 Klebsiella pneumoniae Resistant to New Generation of ß-Lactam/ß-Lactamase Inhibitor Combinations.

BACKGROUND: Carbapenem resistant Klebsiella pneumoniae (cr-Kp) causes serious infections associated with a high mortality rate. The clinical efficacy of ceftazidime/avibactam (CZA), meropenem/vaborbactam (M/V), and imipenem/relebactam (I/R) against cr-Kp is challenged by the emergence of resistant strains, making the investigation and monitoring of the main resistance mechanisms crucial. In this study, we reported the genome characterization of a Klebsiella pneumoniae strain isolated from a critically ill patient and characterized by a multidrug resistant (MDR) profile, including resistance to CZA, M/V, and I/R. METHODS: An antimicrobial susceptibility test (AST) was performed by an automated system and E-test and results were interpreted following the EUCAST guidelines. Genomic DNA was extracted using a genomic DNA extraction kit and it was sequenced using the Illumina Nova Seq 6000 platform. Final assembly was manually curated and carefully verified for detection of antimicrobial resistance genes, porins modifications, and virulence factors. RESULTS: The K. pneumoniae isolate belonged to sequence type ST512 and harbored 23 resistance genes, conferring resistance to all antibiotic classes, including blaKPC-31 and blaOXA-181, leading to carbapenems resistance. The truncation of OmpK35 and mutation OmpK36GD were also observed. CONCLUSIONS: The genomic characterization demonstrated the high resistant profile of new cr-Kp coharboring class A and D carbapenemases. The presence of KPC-31, as well as the detection of OXA-181 and porin modifications, further limit the therapeutic options, including the novel combinations of ß-lactam/ß-lactamase inhibitor antibiotics in patients with severe pneumonia caused by cr-Kp.

Correlations Between Olfactory Psychophysical Scores and SARS-CoV-2 Viral Load in COVID-19 Patients.

OBJECTIVES/HYPOTHESIS: The aim of this study was to evaluate the correlations between the severity and duration of olfactory dysfunctions (OD), assessed with psychophysical tests, and the viral load on the rhino-pharyngeal swab determined with a direct method, in patients affected by coronavirus disease 2019 (COVID-19). STUDY DESIGN: Prospective cohort study. METHODS: Patients underwent psychophysical olfactory assessment with Connecticut Chemosensory Clinical Research Center test and determination of the normalized viral load on nasopharyngeal swab within 10 days of the clinical onset of COVID-19. RESULTS: Sixty COVID-19 patients were included in this study. On psychophysical testing, 12 patients (20% of the cohort) presented with anosmia, 11 (18.3%) severe hyposmia, 13 (18.3%) moderate hyposmia, and 10 (16.7%) mild hyposmia with an overall prevalence of OD of 76.7%. The overall median olfactory score was 50 (interquartile range [IQR] 30-72.5) with no significant differences between clinical severity subgroups. The median normalized viral load detected in the series was 2.56E+06 viral copies/106 copies of human beta-2microglobulin mRNA present in the sample (IQR 3.17E+04-1.58E+07) without any significant correlations with COVID-19 severity. The correlation between viral load and olfactory scores at baseline (R2  = 0.0007; P = .844) and 60-day follow-up (R2  = 0.0077; P = .519) was weak and not significant. CONCLUSIONS: The presence of OD does not seem to be useful in identifying subjects at risk for being super-spreaders or who is at risk of developing long-term OD. Similarly, the pathogenesis of OD is probably related to individual factors rather than to viral load and activity. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:2312-2318, 2021.