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.

Prevalence of visceral leishmaniasis among people with HIV: a systematic review and meta-analysis.

Leishmaniasis is a parasitic infection expressing different clinical phenotypes. Visceral leishmaniasis (VL) is considered an opportunistic infection among people with human immunodeficiency virus (HIV). The objective of this review was to identify published data on the prevalence of Leishmania spp. infection among PWH and to define particular determinants that affect critically the epidemiological characteristics of VL-HIV coinfection and, potentially, its burden on public health. Two independent reviewers conducted a systematic literature search until June 30, 2022. Meta-analyses were conducted using random-effects models to calculate the summary prevalence and respective 95% confidence intervals (CI) of leishmaniasis among PWH. Meta-regression analysis was performed to investigate the impact of putative effect modifiers, such as the mean CD4 cell count, on the major findings. Thirty-four studies were eligible, yielding a summary prevalence of 6% (95%CI, 4-11%) for leishmaniasis (n = 1583) among PWH (n = 85,076). Higher prevalence rates were noted in Asia (17%, 95%CI, 9-30%) and America (9%, 95%CI, 5-17%) than in Europe (4%, 95%CI, 2-8%). Prevalence rates were significantly mediated by the age, sex, and CD4 cell count of participants. Heterogeneity remained significant in all meta-analyses (p < 0.0001). In the majority of included studies, people were coinfected with HIV and Leishmania species associated with VL, as opposed to those associated with cutaneous leishmaniasis. No sign of publication bias was shown (p = 0.06). Our summary of published studies on leishmaniasis among PWH is important to provide prevalence estimates and define potential underlying factors that could guide researchers to generate and further explore specific etiologic hypotheses.

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.

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.

Filamentous Fungal Keratitis in Greece: A 16-Year Nationwide Multicenter Survey.

In a multicenter, prospective study of filamentous fungal keratitis in Greece, predisposing factors, etiology, treatment practices, and outcome, were determined. Corneal scrapings were collected from patients with clinical suspicion of fungal keratitis, and demographic and clinical data were recorded. Fungal identification was based on morphology, molecular methods, and matrix assisted laser desorption ionization time-of-flight mass-spectrometry. A total of 35 cases were identified in a 16-year study period. Female to male ratio was 1:1.7 and median age 48 years. Corneal injury by plant material, and soft contact lens use were the main risk factors (42.8% and 31.4%, respectively). Trauma was the leading risk factor for men (68.1%), contact lens use (61.5%) for women. Fusarium species were isolated more frequently (n = 21, 61.8%). F. solani was mostly associated with trauma, F. verticillioides and F. proliferatum with soft contact lens use. Other fungi were: Purpureocillium lilacinum (14.7%), Alternaria (11.8%), Aspergillus (8.8%), and Phoma foliaceiphila, Beauveria bassiana and Curvularia spicifera, one case each. Amphotericin B and voriconazole MIC50s against Fusarium were 2 mg/L and 4 mg/L respectively. Antifungal therapy consisted mainly of voriconazole locally or both locally and systemically, alone or in combination with liposomal AmB. Cure/improvement rate with antifungal therapy alone was 52%, keratoplasty was required in 40% of cases, and enucleation in 8%. In conclusion, filamentous fungal keratitis in Greece is rare, but with considerable morbidity. A large proportion of cases resulted in keratoplasty despite appropriate antifungal treatment.

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.

Evaluation of two automated systems for colistin susceptibility testing of carbapenem-resistant Acinetobacter baumannii clinical isolates.

Background: Colistin is commonly needed for the treatment of infections due to carbapenem-resistant Acinetobacter baumannii (CRAB) and the determination of its in vitro activity is obviously important. However, the accurate routine antimicrobial susceptibility testing (AST) of colistin is still challenging. The only acceptable method for colistin AST is broth microdilution (BMD); disc and gradient diffusion assays are inappropriate and the performance of semi-automated systems has not been validated. Objectives: In the present study, two commonly used semi-automated systems were evaluated for colistin AST of contemporary CRAB clinical isolates. Methods: A total of 117 single-patient CRAB isolates collected randomly during 2015 from distinct tertiary hospitals located throughout Greece were tested. Colistin MICs were determined using the semi-automated systems Phoenix100 and Vitek2 and also agar dilution (AD), compared with the reference BMD. Results: Colistin resistance rates for Phoenix100/Vitek2/AD/BMD were 15.4%/16.2%/35.9%/24.8%. The essential/categorical agreement rates were as follows: Phoenix100, 91.5%/88.9%; Vitek2, 88.9%/89.7%; and AD, 93.2%/87.2%. Alarmingly high rates of very major errors (VMEs) were observed for Phoenix100 (41.4%) and Vitek2 (37.9%), while major errors (MEs) were limited (1.1% by both systems); VMEs were much more common for isolates with MICs of 2 mg/L than for isolates with MICs of ≤ 1 mg/L, as determined by automated methods. AD produced considerably higher colistin MICs, yielding MEs of 15.9%. Conclusions: Colistin resistance of A. baumannii is greatly underestimated by Phoenix100/Vitek2, potentially leading to inappropriate colistin administration. Colistin AST results by automated systems within the susceptible range, particularly those at the susceptibility breakpoint (2 mg/L), need to be validated by BMD.

Medical and Social Aspects of Syphilis in the Balkans from the mid-19th Century to the Interwar.

The current study presents some aspects of syphilis in the Balkan Peninsula from the 19th century until the Interwar. Ever since the birth of modern Balkan States (Greece, Bulgaria, Turkey and Serbia), urbanization, poverty and the frequent wars have been considered the major factors conducive to the spread of syphilis. The measures against sex work and sexually transmitted diseases (STDs) were taken in two aspects, one medical and the other legislative. In this period, numerous hospitals for venereal diseases were established in the Balkan countries. In line with the international diagnostic approach and therapeutic standards, laboratory examinations in these Balkan hospitals included spirochete examination, Wassermann reaction, precipitation reaction and cerebrospinal fluid examination. Despite the strict legislation and the adoption of relevant laws against illegal sex work, public health services were unable to curb the spread of syphilis. Medical and social factors such as poverty, citizen's ignorance of STDs, misguided medical perceptions, lack of sanitary control of prostitution and epidemiological studies, are highlighted in this study. These factors were the major causes that helped syphilis spread in the Balkan countries during the 19th and early 20th century. The value of these aspects as a historic paradigm is diachronic. Failure to comply with the laws and the dysfunction of public services during periods of war or socioeconomic crises are both factors facilitating the spread of STDs.

Administration of a triple versus a standard double antimicrobial regimen for human brucellosis more efficiently eliminates bacterial DNA load.

The effects of doxycycline-streptomycin-rifampin versus a standard doxycycline-streptomycin regimen on residual Brucella DNA were compared in 36 acute brucellosis patients. At admission, all patients given triple (n = 22) and double (n = 14) regimens had detectable Brucella DNA with similar mean loads (P = 0.982). At follow-up, 14 to 20 months postpresentation, significantly more patients receiving triple than double regimens had undetectable Brucella DNA (P = 0.026). The doxycycline-streptomycin-rifampin regimen eliminates Brucella DNA more efficiently than doxycycline-streptomycin, which may result in superior long-term clearance of Brucella.

Modified CLSI extended-spectrum ß-lactamase (ESBL) confirmatory test for phenotypic detection of ESBLs among Enterobacteriaceae producing various ß-lactamases.

The worldwide dissemination of Enterobacteriaceae producing AmpC ß-lactamases and carbapenemases makes difficult the phenotypic detection of extended-spectrum ß-lactamases (ESBLs), as they may be masked by these additional enzymes. A modification of the CLSI ESBL confirmatory test was developed and evaluated in a comparative study for its ability to successfully detect ESBLs among Enterobacteriaceae producing various carbapenemases (Klebsiella pneumoniae carbapenemase [KPC], VIM, NDM, and OXA-48) and plasmidic or derepressed AmpCs. The modified CLSI ESBL confirmatory test was performed with cefotaxime and ceftazidime disks with and without clavulanate, on which both boronic acid (BA) and EDTA were dispensed. A total of 162 genotypically confirmed ESBL-positive Enterobacteriaceae isolates (83 carbapenemase/ESBL producers, 25 AmpC/ESBL producers, and 54 ESBL-only producers) were examined. For comparison, 139 genotypically confirmed ESBL-negative Enterobacteriaceae isolates (94 of them possessed carbapenemases and 20 possessed AmpCs) were also tested. The standard CLSI ESBL confirmatory test was positive for 106 of the 162 ESBL producers (sensitivity, 65.4%) and showed false-positive results for 4 of the 139 non-ESBL producers (specificity, 97.1%). The modified CLSI ESBL confirmatory test detected 158 of 162 ESBL producers (sensitivity, 97.5%) and showed no false-positive results for non-ESBL producers (specificity, 100%). The findings of the study demonstrate that the modified CLSI ESBL confirmatory test using antibiotic disks containing both BA and EDTA accurately detects ESBLs in Enterobacteriaceae regardless of the coexistence of additional ß-lactam resistance mechanisms.

The Balkan region: NDM-1-producing Klebsiella pneumoniae ST11 clonal strain causing outbreaks in Greece.

OBJECTIVES: Despite the fact that the NDM-1 carbapenemase has successfully disseminated worldwide, outbreaks remain uncommon in the European region. We describe the characteristics of the first outbreaks caused by NDM-1-producing Klebsiella pneumoniae clonal isolates in Greece. METHODS: Between January 2010 and June 2013, 132 non-repetitive carbapenem-resistant Enterobacteriaceae isolates, which gave a positive modified Hodge test and were phenotypically suspected of metallo-ß-lactamase production, were recovered from patients hospitalized at Ioannina University Hospital. Resistance genes were identified by PCR and sequencing. Plasmid profiling, conjugation experiments, enterobacterial repetitive intergenic consensus PCR, PFGE and multilocus sequence typing (MLST) were performed. Patient records were retrieved to access patterns of acquisition. RESULTS: Molecular testing verified the presence in 78 K. pneumoniae isolates, collected from 71 patients, of the blaNDM-1 gene. The blaCTX-M-15, blaOXA-1 and blaTEM-1 genes were also present in most isolates. The blaNDM-1 gene was located on a narrow host range IncFII-type plasmid, of ∼95 kb, flanked upstream by a non-truncated ISAba125 element and downstream by the bleMBL gene. Genotyping clustered all K. pneumoniae isolates into a single clonal type with one subtype and MLST assigned them to sequence type 11. Two outbreaks were noted, the first between November and December 2011 involving four patients and the second initiated in May 2012 and ongoing, involving the remaining patients. All but two cases were characterized as hospital acquired. No links to immigration or travel history to endemic areas were established. CONCLUSIONS: This survey highlights the successful undetected dissemination of yet another carbapenemase in Greece and strengthens the hypothesis of a latent NDM-1 cluster in the Balkan region.

From Clinical Suspicion to Diagnosis: A Review of Diagnostic Approaches and Challenges in Fungal Keratitis.

Fungal keratitis is a relatively rare yet severe ocular infection that can lead to profound vision impairment and even permanent vision loss. Rapid and accurate diagnosis plays a crucial role in the effective management of the disease. A patient's history establishes the initial clinical suspicion since it can provide valuable clues to potential predisposing factors and sources of fungal exposure. Regarding the evaluation of the observed symptoms, they are not exclusive to fungal keratitis, but their timeline can aid in distinguishing fungal keratitis from other conditions. Thorough clinical examination of the affected eye with a slit-lamp microscope guides diagnosis because some clinical features are valuable predictors of fungal keratitis. Definitive diagnosis is established through appropriate microbiological investigations. Direct microscopic examination of corneal scrapings or biopsy specimens can assist in the presumptive diagnosis of fungal keratitis, but culture remains the gold standard for diagnosing fungal keratitis. Advanced molecular techniques such as PCR and MALDI-ToF MS are explored for their rapid and sensitive diagnostic capabilities. Non-invasive techniques like in vivo confocal microscopy (IVCM) and optical coherence tomography (OCT) are useful for real-time imaging. Every diagnostic technique has both advantages and drawbacks. Also, the selection of a diagnostic approach can depend on various factors, including the specific clinical context, the availability of resources, and the proficiency of healthcare personnel.

Antimicrobial and Diagnostic Stewardship of the Novel ß-Lactam/ß-Lactamase Inhibitors for Infections Due to Carbapenem-Resistant Enterobacterales Species and Pseudomonas aeruginosa.

Carbapenem-resistant Gram-negative bacterial infections are a major public health threat due to the limited therapeutic options available. The introduction of the new ß-lactam/ß-lactamase inhibitors (BL/BLIs) has, however, altered the treatment options for such pathogens. Thus, four new BL/BLI combinations-namely, ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam, and ceftolozane/tazobactam-have been approved for infections attributed to carbapenem-resistant Enterobacterales species and Pseudomonas aeruginosa. Nevertheless, although these antimicrobials are increasingly being used in place of other drugs such as polymyxins, their optimal clinical use is still challenging. Furthermore, there is evidence that resistance to these agents might be increasing, so urgent measures should be taken to ensure their continued effectiveness. Therefore, clinical laboratories play an important role in the judicious use of these new antimicrobial combinations by detecting and characterizing carbapenem resistance, resolving the presence and type of carbapenemase production, and accurately determining the minimum inhibitor concentrations (MICs) for BL/BLIs. These three targets must be met to ensure optimal BL/BLIs use and prevent unnecessary exposure that could lead to the development of resistance. At the same time, laboratories must ensure that results are interpreted in a timely manner to avoid delays in appropriate treatment that might be detrimental to patient safety. Thus, we herein present an overview of the indications and current applications of the new antimicrobial combinations and explore the diagnostic limitations regarding both carbapenem resistance detection and the interpretation of MIC results. Moreover, we suggest the use of alternative narrower-spectrum antibiotics based on susceptibility testing and present data regarding the effect of synergies between BL/BLIs and other antimicrobials. Finally, in order to address the absence of a standardized approach to using the novel BL/BLIs, we propose a diagnostic and therapeutic algorithm, which can be modified based on local epidemiological criteria. This framework could also be expanded to incorporate other new antimicrobials, such as cefiderocol, or currently unavailable BL/BLIs such as aztreonam/avibactam and cefepime/taniborbactam.

M1UKStreptococcus pyogenes causing community-acquired pneumonia, pleural empyema and streptococcal toxic shock syndrome.

OBJECTIVES: Streptococcus pyogenes causes superficial infections but can also cause deep-seated infections and toxin-mediated diseases. In the present study, phylogenetic and in silico prediction analyses were performed on an antimicrobial resistant M1UKS. pyogenes strain causing severe clinical manifestations during the current surge of invasive group A Streptococcus (iGAS) disease. METHODS: A 40-year-old patient was admitted to the hospital with fever, chest pain and fatigue. Based on the clinical and laboratory findings, a diagnosis of sepsis with disseminated intravascular coagulation, community-acquired pneumonia, pleural empyema and streptococcal toxic shock syndrome was made. Microbial identification was performed by multiplex PCR and conventional culturing. Furthermore, antimicrobial susceptibility testing, whole genome sequencing, phylogenomic analysis and in silico prediction analysis of antimicrobial resistance genes and virulence factors were performed. RESULTS: S. pyogenes isolates were detected in pleural fluid and sputum of the patient. Both isolates belonged to the M1UK lineage of the emm1/ST28 clone, being closely related with an M1UK GAS strain from Australia. They exhibited resistance to erythromycin and clindamycin and susceptibility-increased exposure to levofloxacin and carried genes encoding for protein homologues of antibiotic efflux pumps. Moreover, several virulence factors, and a previously described single-nucleotide polymorphism in the 5' transcriptional leader sequence of the ssrA gene, which enhances expression of SpeA, were detected. CONCLUSIONS: The present antimicrobial-resistant M1UKS. pyogenes strain represents the first report of this emerging lineage associated with such manifestations of iGAS disease.

Candida auris in Greek healthcare facilities: Active surveillance results on first cases and outbreaks from eleven hospitals within Attica region.

BACKGROUND: Candida auris was sporadically detected in Greece until 2019. Thereupon, there has been an increase in isolations among inpatients of healthcare facilities. AIM: We aim to report active surveillance data on MALDI-TOF confirmed Candida auris cases and outbreaks, from November 2019 to September 2021. METHODS: A retrospective study on hospital-based Candida auris data, over a 23-month period was conducted, involving 11 hospitals within Attica region. Antifungal susceptibility testing and genotyping were conducted. Case mortality and fatality rates were calculated and p-values less than 0.05 were considered statistically significant. Infection control measures were enforced and enhanced. RESULTS: Twenty cases with invasive infection and 25 colonized were identified (median age: 72 years), all admitted to hospitals for reasons other than fungal infections. Median hospitalisation time until diagnosis was 26 days. Common risk factors among cases were the presence of indwelling devices (91.1 %), concurrent bacterial infections during hospitalisation (60.0 %), multiple antimicrobial drug treatment courses prior to hospitalisation (57.8 %), and admission in the ICU (44.4 %). Overall mortality rate was 53 %, after a median of 41.5 hospitalisation days. Resistance to fluconazole and amphotericin B was identified in 100 % and 3 % of tested clinical isolates, respectively. All isolates belonged to South Asian clade I. Outbreaks were identified in six hospitals, while remaining hospitals detected sporadic C. auris cases. CONCLUSION: Candida auris has proven its ability to rapidly spread and persist among inpatients and environment of healthcare facilities. Surveillance focused on the presence of risk factors and local epidemiology, and implementation of strict infection control measures remain the most useful interventions.

A combined disk test for direct differentiation of carbapenemase-producing enterobacteriaceae in surveillance rectal swabs.

Carbapenemase-producing Enterobacteriaceae (CPE) are rapidly spreading worldwide. Early detection of fecal CPE carriers is essential for effective infection control. Here, we evaluated the performance of a meropenem combined disk test (CDT) for rapidly differentiating CPE isolates directly from rectal swabs. The screening method was applied for 189 rectal swabs from hospitalized patients at high risk for CPE carriage. Swabs were suspended in 1 ml saline and cultured for confluent growth onto a MacConkey agar plate with a meropenem (MER) disk alone, a MER disk plus phenyl boronic acid (PBA), a MER disk plus EDTA, and a MER disk plus PBA and EDTA. An inhibition zone of ≤ 25 mm around the MER disk alone indicated carriage of carbapenem-resistant organisms. Furthermore, ≥ 5-mm differences in the inhibition zone between MER disks without and with the inhibitors (PBA, EDTA, or both) were considered positive results for detecting Klebsiella pneumoniae carbapenemase (KPC), metallo-ß-lactamase (MBL), or both carbapenemases, respectively. For comparison, rectal suspensions were tested using MacConkey plates with ertapenem (MacERT) disks and PCR (PCR-S) for carbapenemase genes. Of the 189 samples, 97 were genotypically confirmed as CPE positive by one of the three protocols tested. The CDT, MacERT disks, and PCR-S assays exhibited sensitivities of 94.8%, 96.9%, and 94.8% and specificities of 100%, 98.9%, and 100%, respectively, for detecting CPE-positive swabs. Moreover, the CDT correctly differentiated the production of KPC, MBL, or both carbapenemases in 78 of the 97 (80.4%) CPE-positive rectal swabs. Our results demonstrate that the CDT may provide a simple and inexpensive method for detecting and differentiating the carbapenemase type within a single day without requiring further testing and additional delay, supporting the timely implementation of infection control measures.

Outbreak caused by an ertapenem-resistant, CTX-M-15-producing Klebsiella pneumoniae sequence type 101 clone carrying an OmpK36 porin variant.

Although numerous studies have documented outbreaks of carbapenem-resistant Klebsiella pneumoniae (CRKP) possessing various carbapenemases, reports on outbreaks due to CRKP possessing extended-spectrum ß-lactamases (ESBLs) and/or AmpCs with porin lesions have been limited. Here, we describe an outbreak caused by an ertapenem-resistant, CTX-M-15-producing clonal K. pneumoniae strain expressing an OmpK36 porin variant. From May 2012 to November 2012, 37 ertapenem-resistant K. pneumoniae isolates phenotypically negative for carbapenemase production were recovered from 19 patients hospitalized in the intensive care unit of a Greek hospital. The isolates were either susceptible or intermediate to other carbapenems and resistant to all remaining ß-lactams but cefotetan. Phenotypic and molecular analysis revealed the presence in all isolates of the blaCTX-M-15 gene on a conjugative 100-kb plasmid, disruption in the expression of the ompK35 gene, and the production of an Ompk36 porin variant. The index case was a patient admitted from another hospital. Active surveillance upon admission and on a weekly basis was immediately initiated; environmental samples were also periodically tested. Molecular typing showed that all clinical isolates as well as two ertapenem-resistant environmental K. pneumoniae isolates belonged to the same clonal type and were assigned to multilocus sequence typing (MLST) sequence type 101 (ST101). As all colonized/infected patients were hospitalized during overlapping periods, cross-infection was considered the main route for the dissemination of the outbreak strain. Despite reinforcement of infection control measures and active surveillance, the outbreak lasted approximately 7 months. Identification of hidden carriers upon admission and by screening on a weekly basis was found valuable for early recognition and subsequent successful management of the outbreak.