Evaluation of the Vitek 2 system for antifungal susceptibility testing of Candida auris using a representative international panel of clinical isolates: overestimation of amphotericin B resistance and underestimation of fluconazole resistance.

Although the Vitek 2 system is broadly used for antifungal susceptibility testing of Candida spp., its performance against Candida auris has been assessed using limited number of isolates recovered from restricted geographic areas. We therefore compared Vitek 2 system with the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method using an international collection of 100 C. auris isolates belonging to different clades. The agreement ±1 twofold dilution between the two methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention's (CDC's) tentative resistance breakpoints and Vitek 2-specific wild-type upper limit values (WT-ULVs) were determined. The CLSI-Vitek 2 agreement was poor for 5-flucytosine (0%), fluconazole (16%), and amphotericin B (29%), and moderate for voriconazole (61%), micafungin (67%), and caspofungin (81%). Significant interpretation errors were recorded using the CDC breakpoints for amphotericin B (31% CA, 69% major errors; MaEs) and fluconazole (69% CA, 31% very major errors; VmEs), but not for echinocandins (99% CA, 1% MaEs for both micafungin and caspofungin) for which the Vitek 2 allowed correct categorization of echinocandin-resistant FKS1 mutant isolates. Discrepancies were reduced when the Vitek 2 WT-ULV of 16 mg/L for amphotericin B (98% CA, 2% MaEs) and of 4 mg/L for fluconazole (96% CA, 1% MaEs, 3% VmEs) were used. In conclusion, the Vitek 2 system performed well for echinocandin susceptibility testing of C .auris. Resistance to fluconazole was underestimated whereas resistance to amphotericin B was overestimated using the CDC breakpoints of ≥32 and ≥2 mg/L, respectively. Vitek 2 minimun inhibitory concentrations (MICs) >4 mg/L indicated resistance to fluconazole and Vitek 2 MICs ≤16 mg/L indicated non-resistance to amphotericin B.

Evaluation of the mannan antigen assay in neonates with or without Candida albicans colonization.

Mannan antigen (MA) in neonates as a marker of invasive candidemia is not well studied, although 4% of all neonatal intensive care unit admissions are attributed to Candida spp. infections. The aim of this case-control study was to evaluate the performance of MA (Platelia™ Candida AgPluskit, Bio-Rad) in neonates who had rectal Candida colonization or in non-colonized controls. We cultured 340 rectal swabs of neonates and MA was negative in 24/25 C. albicans colonized (96% specificity) and in 30/30 non-colonized neonates (100% specificity). The results indicate a high specificity of the assay, which could be useful in neonates with possible candidemia.

The present study aimed to evaluate the use of mannan antigen (MA) assay in a neonatal unit and compared between C. albicans colonized and non-colonized infants. According to our results, MA found to have high specificity in both groups.

SARS-CoV-2 Reinfections and Long COVID in the Post-Omicron Phase of the Pandemic.

We are reviewing the current state of knowledge on the virological and immunological correlates of long COVID, focusing on recent evidence for the possible association between the increasing number of SARS-CoV-2 reinfections and the parallel pandemic of long COVID. The severity of reinfections largely depends on the severity of the initial episode; in turn, this is determined both by a combination of genetic factors, particularly related to the innate immune response, and by the pathogenicity of the specific variant, especially its ability to infect and induce syncytia formation at the lower respiratory tract. The cumulative risk of long COVID as well as of various cardiac, pulmonary, or neurological complications increases proportionally to the number of SARS-CoV-2 infections, primarily in the elderly. Therefore, the number of long COVID cases is expected to remain high in the future. Reinfections apparently increase the likelihood of long COVID, but less so if they are mild or asymptomatic as in children and adolescents. Strategies to prevent SARS-CoV-2 reinfections are urgently needed, primarily among older adults who have a higher burden of comorbidities. Follow-up studies using an established case definition and precise diagnostic criteria of long COVID in people with or without reinfection may further elucidate the contribution of SARS-CoV-2 reinfections to the long COVID burden. Although accumulating evidence supports vaccination, both before and after the SARS-CoV-2 infection, as a preventive strategy to reduce the risk of long COVID, more robust comparative observational studies, including randomized trials, are needed to provide conclusive evidence of the effectiveness of vaccination in preventing or mitigating long COVID in all age groups. Thankfully, answers not only on the prevention, but also on treatment options and rates of recovery from long COVID are gradually starting to emerge.

Adherence of Candida albicans to Five Long-Term Silicone-Based Denture Lining Materials Bonded to CAD-CAM Denture Base.

PURPOSE: Knowledge about quantifying the number as well as the retention and adhesion of Candida albicans blastoconidia to silicone denture liners is limited. Thus, the aim of this in vitro study was to explore the adherence of C. albicans to the surface of five long-term silicone-based soft denture lining materials, using artificial saliva. MATERIALS AND METHODS: A total of 50 specimens (10 × 10 × 3 mm) of five long-term resilient liners (Molloplast-B; GC Reline Soft; Elite Soft Relining; Tokuyama Sofreliner S; Ufigel SC), bonded to a computer-aided design and computer-aided manufacturing denture base, were prepared. The specimens were inoculated and incubated in artificial saliva for 1 and 24 hours with a standardized (2.8 × 106 cfu/ml) C. albicans suspension. At the end of the incubation period, the specimens were stained with acridine orange and observed using fluorescence microscopy. RESULTS: After 1 hour and in 24 hours, Molloplast B demonstrated significantly earlier adherence of C. albicans cells compared to the other chairside materials (p < 0.001 and p < 0.001, respectively), where the mean number of cells also increased in the frontal parts. Regarding the rate of C. albicans proliferation from 1 to 24 hours within the materials, there was an increase in all materials (Molloplast B: p < 0.001; GC Reline Soft: p = 0.220; Elite Soft Relining: p = 0.032; Tokuyama Sofreliner S: p = 0.001; Ufigel Sc: p = 0.001). The Ufigel Sc showed a significant 2.5-fold increase at 24 hours. CONCLUSIONS: Long-term silicone denture liners accumulate a significant amount of C. albicans blastoconidia and their coverage by them increases progressively over time.

A study of the evolution of the third COVID-19 pandemic wave in the Athens metropolitan area, Greece, through two cross-sectional seroepidemiological surveys: March, June 2021.

We studied the third coronavirus disease 2019 (COVID-19) pandemic wave in Athens metropolitan area (3 738 901 inhabitants) through two seroepidemiological surveys. Persons presenting in 12 healthcare facilities across Athens in March and June 2021 were studied (764 and 901, respectively). Immunoglobulin G antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein were measured by a chemiluminescent microparticle immunoassay. In March the seroprevalence rate was 11.6%, meaning that 435 208 residents of Athens had evidence of immunity. The respective values in June were 55.7% and 2 082 568 residents. The highest seroprevalence rates attributed to SARS-CoV-2 infection were recorded in persons <18 years (16.3% in March and 31.6% in June), while immunity was mainly vaccine-induced in persons 18-64 years and >65 years. Infection-attributed immunity also increased in older-age groups. Wide ranges in seroprevalence rates were noted across areas in March and June. The highest seroprevalence rates were recorded in Piraeus (47.2%) and West Attica (37.5%). However, the highest increase (>5 times) occurred in Piraeus and the South Section of Athens, which are among the most densely populated areas in Athens. In both study periods, history of COVID-19 or febrile episode, and having a cohabitant with COVID-19 were associated with increased risk for seropositivity among unvaccinated persons (p values <0.001 for all). Residing in Piraeus, the South Section or West Attica was associated with increased risk for seropositivity in June (p values <0.001). Wide heterogeneity in seroprevalence rates was found across areas in Athens, which is mainly attributed to population density. The impact of population mobility and socioeconomic status should be explored.

Impact of a 4-year antimicrobial stewardship program implemented in a Greek tertiary hospital.

This study aimed to investigate the effects of a 4-year antibiotic stewardship program (ASP) in a tertiary hospital. We monitored data for 2015 (pre-intervention) and 2016-2019 (post-intervention) about antibiotic consumption (DDD/100 bed days), Clostridioides difficile infections (CDIs), resistance rates, length of stay (LOS), and annual antibiotic costs. Significant reductions were observed for total antibiotics/colistin/carbapenems/quinolones/tigecycline consumption and resistance rates of Pseudomonas aeruginosa, Klebsiella pneumoniae, and vancomycin-resistant enterococci. Considerable reductions occurred for LOS (4.18 [2015]/3.0 [2019] days), CDIs (1.47 [2015]/0.86 [2019] per 1000 patients), antibiotic cost/patient (39.45€ [2015]/23.69€ [2019]). The ASP was successful in reducing antibiotic consumption, antibiotic costs, length of hospital stay, and CDIs.

Aspergillus spp. osteoarticular infections: an updated systematic review on the diagnosis, treatment and outcomes of 186 confirmed cases.

Aspergillus spp. osteoarticular infections are destructive opportunistic infections, while there is no clear consensus on their management. The purpose of this review is to investigate the current literature regarding Aspergillus spp. osteoarticular infections. An electronic search of the PubMed and Scopus databases was conducted considering studies that assessed osteoarticular infections from Aspergillus spp. We included only studies with biopsy proven documentation of positive cultures or histological findings for Aspergillus spp., and those with essential information for each case such as the anatomical location of the infection, the type of treatment (conservative, surgical, combination), the antifungal therapy, and the outcome. Overall, 148 studies from 1965 to 2021 including 186 patients were included in the review. One hundred and seven (57.5%) patients underwent surgical debridement in addition to antifungal therapy, while 79 (42.7%) patients were treated only conservatively. Complete infection resolution was reported in 107 (57.5%) patients, while partial resolution in 29 (15.5%) patients. Surgical debridement resulted in higher complete infection resolution rate compared to only antifungal therapy (70.0% vs. 40.5%, P < 0.001), while complete resolution rate was similar for antifungal monotherapy and combination/sequential therapy (58.3% vs. 54.5%; P = 0.76). Last, complete resolution rate was also similar for monotherapy with amphotericin B (58.1%) and voriconazole (58.6%; P = 0.95). The results of this study indicate that antifungal monotherapy has similar efficacy with combination/sequential therapy, while voriconazole has similar efficacy with amphotericin B. Moreover, surgical debridement of the infected focus results in better outcomes in terms of infection eradication compared to conservative treatment. LAY SUMMARY: Antifungal monotherapy has similar efficacy with combination/sequential therapy, and voriconazole has similar efficacy with amphotericin B for the treatment of Aspergillus spp. osteoarticular infections, while surgical debridement of the infected focus improves the infection eradication rate.

Microbiological findings in prepubertal and pubertal girls with vulvovaginitis.

Vulvovaginitis is a common and challenging gynaecological problem in prepubertal and pubertal girls. Such an infection, owing to a wide range of aetiologies, if not responding to hygienic measures, needs further investigation through vaginal cultures, since treatment should be tailored accordingly. This study aimed to investigate the pathogens isolated in prepubertal and pubertal girls with signs and symptoms of vulvovaginitis. A total of 2314 symptomatic girls, 1094 prepubertal and 1220 pubertal, aged 2 to 16 years, were included. Vaginal samples were inoculated on specific culture plates followed by incubation in aerobic, anaerobic or CO2 atmosphere at 37 °C for 24 or 48 h, as appropriate. The identification of the isolated pathogens was carried out using Gram stain, conventional methods and the automated system VITEK 2 (BioMerieux, Marcy l'Etoile, France). Positive cultures were obtained from 587 (53.7%) of prepubertal girls and 926 (75.9%) of pubertal girls. A total of 613 and 984 pathogens were detected in prepubertal and pubertal subjects, respectively. Isolated bacteria included 40.1% and 22.8% Gram-positive cocci, 35.6% and 24.8% Gram-negative rods in the prepubertal and pubertal groups, respectively, with faecal pathogens being the most prevalent. Bacterial vaginosis was diagnosed in 22.8% of prepubertal and 37.9% of pubertal girls. Candida species were isolated mostly in the pubertal girls (14.5%). CONCLUSION: Culture results should be evaluated with caution in children with vulvovaginitis. In the prepubertal girls, the most common isolated pathogens were opportunistic bacteria of faecal origin while girls in late puberty were more susceptible to bacterial vaginosis and vulvovaginal candidiasis. WHAT IS KNOWN: • Vulvovaginitis is the most frequent and challenging reason for referral to paediatric and adolescent gynaecology services. • Microbiological examination can prove to be a significant tool to help diagnosis although results should be evaluated with caution in children. WHAT IS NEW: • Significantly more positive vaginal cultures and pathogens were recorded in symptomatic pubertal girls compared to prepubertal children. • The prevalence of bacterial vaginosis was increased in both prepubertal and pubertal girls with vulvovaginitis although significantly more in girls at puberty.

Association Between Upper Respiratory Tract Viral Load, Comorbidities, Disease Severity, and Outcome of Patients With SARS-CoV-2 Infection.

BACKGROUND: There is limited information on the association between upper respiratory tract (URT) viral loads, host factors, and disease severity in SARS-CoV-2-infected patients. METHODS: We studied 1122 patients (mean age, 46 years) diagnosed by polymerase chain reaction (PCR). URT viral load, measured by PCR cycle threshold, was categorized as high, moderate, or low. RESULTS: There were 336 (29.9%) patients with comorbidities; 309 patients (27.5%) had high, 316 (28.2%) moderate, and 497 (44.3%) low viral load. In univariate analyses, compared to patients with moderate or low viral load, patients with high viral load were older, more often had comorbidities, developed Symptomatic disease (COVID-19), were intubated, and died. Patients with high viral load had longer stay in intensive care unit and longer intubation compared to patients with low viral load (P values < .05 for all comparisons). Patients with chronic cardiovascular disease, hypertension, chronic pulmonary disease, immunosuppression, obesity, and chronic neurological disease more often had high viral load (P value < .05 for all comparisons). In multivariate analysis high viral load was associated with COVID-19. Level of viral load was not associated with any other outcome. CONCLUSIONS: URT viral load could be used to identify patients at higher risk for morbidity or severe outcome.

A main event and multiple introductions of SARS-CoV-2 initiated the COVID-19 epidemic in Greece.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Chains of infections starting from various countries worldwide seeded the outbreak of COVID-19 in Athens, capital city of Greece. A full-genome analysis of isolates from Athens' hospitals and other healthcare providers revealed the variety of SARS-CoV-2 that initiated the pandemic before lockdown and passenger flight restrictions. A dominant variant, encompassing the G614D amino acid substitution, spread through a major virus dispersal event, and sporadic introductions of rare variants characterized the local initiation of the epidemic. Mutations within the genome highlighted the genetic drift of the virus as rare variants emerged. An important variant contained a premature stop codon in orf7a leading to the truncation of a possibly important for viral pathogenesis domain. This study may serve as a reference for resolving future lines of infection in the area, especially after resumption of passenger flight connections to Athens and Greece during summer of 2020.

Transmission dynamics of SARS-CoV-2 within families with children in Greece: A study of 23 clusters.

There is limited information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection clustering within families with children. We aimed to study the transmission dynamics of SARS-CoV-2 within families with children in Greece. We studied 23 family clusters of coronavirus disease 2019 (COVID-19). Infection was diagnosed by reverse-transcriptase polymerase chain reaction in respiratory specimens. The level of viral load was categorized as high, moderate, or low based on the cycle threshold values. There were 109 household members (66 adults and 43 children). The median attack rate per cluster was 60% (range: 33.4%-100%). An adult member with COVID-19 was the first case in 21 (91.3%) clusters. Transmission of infection occurred from an adult to a child in 19 clusters and/or from an adult to another adult in 12 clusters. There was no evidence of child-to-adult or child-to-child transmission. In total 68 household members (62.4%) tested positive. Children were more likely to have an asymptomatic SARS-CoV-2 infection compared to adults (40% vs 10.5%; P = .021). In contrast, adults were more likely to develop a severe clinical course compared with children (8.8% vs 0%; P = .021). In addition, infected children were significantly more likely to have a low viral load while adults were more likely to have a moderate viral load (40.7% and 18.6% vs 13.8% and 51.7%, respectively; P = .016). In conclusion, while children become infected by SARS-CoV-2, they do not appear to transmit infection to others. Furthermore, children more frequently have an asymptomatic or mild course compared to adults. Further studies are needed to elucidate the role of viral load on these findings.

Human genetic factors associated with susceptibility to SARS-CoV-2 infection and COVID-19 disease severity.

BACKGROUND: The emergence of the novel coronavirus in Wuhan, Hubei Province, China, in December 2019 marked the synchronization of the world to a peculiar clock that is counting infected cases and deaths instead of hours and minutes. The pandemic, highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has indeed caused considerable morbidity and mortality and drastically changed our everyday lives. As we continue to become acquainted with the seventh coronavirus known to infect our species, a number of its characteristics keep surprising us. Among those is the wide spectrum of clinical manifestations of the resulting coronavirus disease 2019 (COVID-19), which ranges from asymptomatic or mildly symptomatic infections to severe pneumonia, respiratory failure, and death. MAIN BODY: Data, now from patient populations, are beginning to accumulate on human genetic factors that may contribute to the observed diversified disease severity. Therefore, we deemed it prudent to review the associations between specific human genetic variants and clinical disease severity or susceptibility to infection that have been reported in the literature to date (at the time of writing this article in early August 2020 with updates in mid-September). With this work, we hope (i) to assist the fast-paced biomedical research efforts to combat the virus by critically summarizing current knowledge on the potential role of host genetics, and (ii) to help guide current genetics and genomics research towards candidate gene variants that warrant further investigation in larger studies. We found that determinants of differing severity of COVID-19 predominantly include components of the immune response to the virus, while determinants of differing susceptibility to SARS-CoV-2 mostly entail genes related to the initial stages of infection (i.e., binding of the cell surface receptor and entry). CONCLUSION: Elucidating the genetic determinants of COVID-19 severity and susceptibility to SARS-CoV-2 infection would allow for the stratification of individuals according to risk so that those at high risk would be prioritized for immunization, for example, if or when safe and effective vaccines are developed. Our enhanced understanding of the underlying biological mechanisms could also guide personalized therapeutics. Such knowledge is already beginning to provide clues that help explain, at least in part, current epidemiologic observations regarding the typically more severe or benign disease course in older males and children, respectively.

Major vault protein/lung resistance related protein: a novel biomarker for rheumatoid arthritis.

OBJECTIVES: Rheumatoid arthritis (RA) can lead to joint destruction and early institution of effective treatment can preserve joint function. Biomarkers can establish early diagnosis and predict effect of treatment. Vault particles, large cytoplasmic ribonucleoprotein particles that participate in inflammation, might serve as biomarkers. The aim of this study was to assess the diagnostic and the prognostic value of major vault protein (MVP) and their antibodies in RA. METHODS: Serum samples from 159 RA patients, 26 early RA (ERA) patients, 21 patients with osteoarthritis (OA) and 30 healthy individuals were tested for MVP, anti-cyclic citrullinated peptide (anti-CCP) and C-reactive protein (CRP) using enzyme-linked immunosorbent assays (ELISA). Rheumatoid factor (RF) was tested by nephelometry, and anti-MVP antibodies were detected by anti-MVP peptide ELISA using an in-house protocol. RESULTS: MVP levels were higher in RA and ERA, compared to OA and healthy controls (p<0.00001). A combination of MVP with RF or anti-CCP showed an improved diagnostic accuracy compared to RF or anti-CCP alone in RA and ERA. MVP exhibited similar AUC levels to anti-CCP and RF in RA whereas in ERA, MVP exhibited the same or slightly higher AUC levels, compared to anti-CCP and RF, respectively. High MVP levels were associated with lack of response to treatment. Levels of anti-MVP peptide 2 antibodies were significantly higher in RA compared to healthy controls (t= 2.73, p=0.007). CONCLUSIONS: MVP and autoantibodies against MVP may have the potential to serve as diagnostic and prognostic biomarkers in RA.

Staphylococcus aureus Carriage Status in Patients with Hidradenitis Suppurativa: An Observational Cohort Study in a Tertiary Referral Hospital in Athens, Greece.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic, recurrent, auto-inflammatory disease that affects the pilosebaceous unit in apocrine gland-bearing areas. Bacteria are thought to play a role in the development and progression of disease. In addition, antibiotics are frequently used as first-line management for HS. We sought to determine the carriage status of Staphylococcus aureus and its resistance to antibiotics among patients with HS in a tertiary referral hospital in Athens, Greece. METHODS: In this observational cohort study, 68 consecutive patients attending the HS clinic of "Attikon" General University Hospital in Athens, Greece, during a 9-month period were enrolled. All patients had not received any antibiotic therapy for any reason during the previous 3 months before enrollment. Nasal and oropharyngeal samplingwere obtained, and specimens were tested for the presence of S. aureus.Antibiotic susceptibility testing was performed using the VITEK 2 system. Standard statistical tests, descriptive statistics tests, and χ2 and Pearson correlation tests were performed, using IBM SPSS Statistics 25.The level of significance was set at a pvalue <0.05. RESULTS: Sixty-eight patients with HS were studied. There were 44 females (64.7%) and 24 males (35.3%). The mean age was 36.63 ± 13.0 (IQR = 21), and the mean age at onset of disease was 23.90 ± 11.53 (IQR = 14). The mean duration of disease was 12.74 ± 10.20 years (IQR = 15). Fifteen (22.1%) of the patients were Hurley stage I, 22 (32.4%) were Hurley stage II, and 31 (45.6%) were Hurley stage III. S. aureus carriage was detected in 17 patients (25%). Six of them (35.3%) had MRSA strains. There was an increased prevalence of S. aureus colonization (p = 0.058) and MRSA (p = 0.101) in Hurley stage III patients, but this result was not statistically significant. CONCLUSIONS: We found a 25% prevalence of S. aureus colonization (17/68 patients) and a 35.3% prevalence of MRSA (6/17) among our HS patients. There was an increased prevalence of S. aureusand MRSA positivity in HS patients with Hurley stage III. Further studies are needed to clarify the possible clinical significance of S. aureus carriage in the disease development and progression as well as in the treatment outcome.

Phylogenetic Analysis of Bird-Virulent West Nile Virus Strain, Greece.

We report the full polyprotein genomic sequence of a West Nile virus strain isolated from Eurasian magpies dying with neurologic signs in Greece. Our findings demonstrate the local genetic evolution of the West Nile virus strain responsible for a human disease outbreak in the country that began in 2010.

Minocycline susceptibility breakpoints for Acinetobacter baumannii: do we need to re-evaluate them?

Minocycline is an old broad-spectrum tetracycline indicated for the treatment of various infections, including those due to minocycline-susceptible Acinetobacter spp. Susceptibility data worldwide are showing increasing rates of resistance of Acinetobacter baumannii to almost all antimicrobial classes, whereas minocycline seems to remain relatively potent against this significant pathogen. Since no new effective drugs have been released against MDR A. baumannii, minocycline is an attractive choice. Tracing back minocycline CLSI susceptibility breakpoints, it is evident that they have been based on old pharmacokinetic approaches. In an attempt to integrate the scarce new pharmacodynamic data, a Monte Carlo simulation was performed. It seems that the currently used breakpoints are, 8-fold elevated according to the approved dosage regimen, giving erroneously higher rates of minocycline susceptibility of A. baumannii. Therefore, current minocycline breakpoints merit re-evaluation in order to deliver reliable susceptibility profiles for selecting the appropriate therapy.

Emergence of NDM-1-producing Klebsiella pneumoniae in Greece: evidence of a widespread clonal outbreak.

OBJECTIVES: NDM-producing Enterobacteriaceae clinical isolates remain uncommon in the European region. We describe the emergence and broad dissemination of one successful NDM-1-producing Klebsiella pneumoniae clone in Greek hospitals. METHODS: During a 4 year survey (January 2013-December 2016), 480 single-patient carbapenem non-susceptible K. pneumoniae isolates, phenotypically MBL positive, were consecutively recovered in eight Greek hospitals from different locations and subjected to further investigation. Antimicrobial susceptibility testing, combined-disc test, identification of resistance genes by PCR and sequencing, molecular fingerprinting by PFGE, plasmid profiling, replicon typing, conjugation experiments and MLST were performed. RESULTS: Molecular analysis confirmed the presence of the blaNDM-1 gene in 341 (71%) K. pneumoniae isolates. A substantially increasing trend of NDM-1-producing K. pneumoniae was noticed during the survey (R2 = 0.9724). Most blaNDM-1-carrying isolates contained blaCTX-M-15, blaOXA-1, blaOXA-2 and blaTEM-1 genes. PFGE analysis clustered NDM-1 producers into five distinct clonal types, with five distinct STs related to each PFGE clone. The predominant ST11 PFGE clonal type was detected in all eight participating hospitals, despite adherence to the national infection control programme; it was identical to that observed in the original NDM-1 outbreak in Greece in 2011, as well as in a less-extensive NDM-1 outbreak in Bulgaria in 2015. The remaining four ST clonal types (ST15, ST70, ST258 and ST1883) were sporadically detected. blaNDM-1 was located in IncFII-type plasmids in all five clonal types. CONCLUSIONS: This study gives evidence of possibly the largest NDM-1-producing K. pneumoniae outbreak in Europe; it may also reinforce the hypothesis of an NDM-1 clone circulating in the Balkans.

Exploring colistin pharmacodynamics against Klebsiella pneumoniae: a need to revise current susceptibility breakpoints.

Objectives: Because the pharmacokinetic/pharmacodynamic (PK/PD) characteristics of colistin against Enterobacteriaceae are not well explored, we studied the activity of colistin against K. pneumoniae in an in vitro PK/PD model simulating different dosing regimens. Methods: Three clinical isolates of K. pneumoniae with MICs of 0.5, 1 and 4 mg/L were tested in an in vitro PK/PD model following a dose-fractionation design over a period of 24 h. A high and low inoculum of 107 and 104 cfu/mL with and without a heteroresistant subpopulation, respectively, were used. PK/PD indices associated with colistin activity were explored and Monte Carlo analysis was performed in order to determine the PTA for achieving a bactericidal effect (2 log kill). Results: The fAUC/MIC (R2 = 0.64-0.68) followed by fCmax/MIC (R2 = 0.55-0.63) best described colistin's 24 h log10 cfu/mL reduction for both low and high inocula. Dosing regimens with fCmax/MIC ≥6 were always associated with a bactericidal effect (P = 0.0025). However, at clinically achievable concentrations, usually below fCmax/MIC = 6, an fAUC/MIC ≥25 was more predictive of a bactericidal effect. Using a dosing regimen of 9 MU/day, the PTA for this pharmacodynamic target was 100%, 5%-70% and 0%, for isolates with MICs of ≤0.5, 1 and ≥2 mg/L, respectively. Dosing regimens that aim for a trough level of 1 mg/L achieve coverage of strains up to 0.5 mg/L (target trough/MIC = 2 mg/L). Conclusions: Characterization of the pharmacodynamics of colistin against Enterobacteriaceae in an in vitro model of infection indicates that a revision of current susceptibility breakpoints is needed. Therapeutic drug monitoring of colistin to achieve pharmacodynamic targets in individual patients is highly recommended.