Are the Conventional Commercial Yeast Identification Methods Still Helpful in the Era of New Clinical Microbiology Diagnostics? A Meta-Analysis of Their Accuracy.

Accurate identification of pathogenic species is important for early appropriate patient management, but growing diversity of infectious species/strains makes the identification of clinical yeasts increasingly difficult. Among conventional methods that are commercially available, the API ID32C, AuxaColor, and Vitek 2 systems are currently the most used systems in routine clinical microbiology. We performed a systematic review and meta-analysis to estimate and to compare the accuracy of the three systems, in order to assess whether they are still of value for the species-level identification of medically relevant yeasts. After adopting rigorous selection criteria, we included 26 published studies involving Candida and non-Candida yeasts that were tested with the API ID32C (674 isolates), AuxaColor (1,740 isolates), and Vitek 2 (2,853 isolates) systems. The random-effects pooled identification ratios at the species level were 0.89 (95% confidence interval [CI], 0.80 to 0.95) for the API ID32C system, 0.89 (95% CI, 0.83 to 0.93) for the AuxaColor system, and 0.93 (95% CI, 0.89 to 0.96) for the Vitek 2 system (P for heterogeneity, 0.255). Overall, the accuracy of studies using phenotypic analysis-based comparison methods was comparable to that of studies using molecular analysis-based comparison methods. Subanalysis of studies conducted on Candida yeasts showed that the Vitek 2 system was significantly more accurate (pooled ratio, 0.94 [95% CI, 0.85 to 0.99]) than the API ID32C system (pooled ratio, 0.84 [95% CI, 0.61 to 0.99]) and the AuxaColor system (pooled ratio, 0.76 [95% CI, 0.67 to 0.84]) with respect to uncommon species (P for heterogeneity, <0.05). Subanalysis of studies conducted on non-Candida yeasts (i.e., Cryptococcus, Rhodotorula, Saccharomyces, and Trichosporon) revealed pooled identification accuracies of ≥98% for the Vitek 2, API ID32C (excluding Cryptococcus), and AuxaColor (only Rhodotorula) systems, with significant low or null levels of heterogeneity (P > 0.05). Nonetheless, clinical microbiologists should reconsider the usefulness of these systems, particularly in light of new diagnostic tools such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, which allow for considerably shortened turnaround times and/or avoid the requirement for additional tests for species identity confirmation.

Development and validation of an in-house database for matrix-assisted laser desorption ionization-time of flight mass spectrometry-based yeast identification using a fast protein extraction procedure.

In recent studies evaluating the usefulness of the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based identification of yeasts for the routine diagnosis of fungal infections, preanalytical sample processing has emerged as a critical step for reliable MALDI-TOF MS outcomes, especially when the Bruker Daltonics Biotyper software was used. In addition, inadequate results often occurred due to discrepancies between the methods used for clinical testing and database construction. Therefore, we created an in-house MALDI-TOF MS library using the spectra from 156 reference and clinical yeast isolates (48 species in 11 genera), which were generated with a fast sample preparation procedure. After a retrospective validation study, our database was evaluated on 4,232 yeasts routinely isolated during a 6-month period and fast prepared for MALDI-TOF MS analysis. Thus, 4,209 (99.5%) of the isolates were successfully identified to the species level (with scores of ≥2.0), with 1,676 (39.6%) having scores of >2.3. For the remaining 23 (0.5%) isolates, no reliable identification (with scores of <1.7) was obtained. Interestingly, these isolates were almost always from species uniquely represented or not included in the database. As the MALDI-TOF MS results were, except for 23 isolates, validated without additional phenotypic or molecular tests, our proposed strategy can enhance the rapidity and accuracy of MALDI-TOF MS in identifying medically important yeast species. However, while continuous updating of our database will be necessary to enrich it with more strains/species of new and emerging yeasts, the present in-house MALDI-TOF MS library can be made publicly available for future multicenter studies.

Forecasting ESKAPE infections through a time-varying auto-adaptive algorithm using laboratory-based surveillance data.

BACKGROUND: Mathematical or statistical tools are capable to provide a valid help to improve surveillance systems for healthcare and non-healthcare-associated bacterial infections. The aim of this work is to evaluate the time-varying auto-adaptive (TVA) algorithm-based use of clinical microbiology laboratory database to forecast medically important drug-resistant bacterial infections. METHODS: Using TVA algorithm, six distinct time series were modelled, each one representing the number of episodes per single 'ESKAPE' (E nterococcus faecium, S taphylococcus aureus, K lebsiella pneumoniae, A cinetobacter baumannii, P seudomonas aeruginosa and E nterobacter species) infecting pathogen, that had occurred monthly between 2002 and 2011 calendar years at the Università Cattolica del Sacro Cuore general hospital. RESULTS: Monthly moving averaged numbers of observed and forecasted ESKAPE infectious episodes were found to show a complete overlapping of their respective smoothed time series curves. Overall good forecast accuracy was observed, with percentages ranging from 82.14% for E. faecium infections to 90.36% for S. aureus infections. CONCLUSIONS: Our approach may regularly provide physicians with forecasted bacterial infection rates to alert them about the spread of antibiotic-resistant bacterial species, especially when clinical microbiological results of patients' specimens are delayed.

In-depth characterization of multidrug-resistant NDM-1 and KPC-3 co-producing Klebsiella pneumoniae bloodstream isolates from Italian hospital patients.

Bloodstream infection (BSI) caused by carbapenem-resistant Klebsiella pneumoniae (KP) poses significant challenges, particularly when the infecting isolate carries multiple antimicrobial resistance (AMR) genes/determinants. This study, employing short- and long-read whole-genome sequencing, characterizes six New Delhi metallo-ß-lactamase (NDM) 1 and KP carbapenemase (KPC) 3 co-producing KP isolates, the largest cohort investigated in Europe to date. Five [sequence type (ST) 512] and one (ST11) isolates were recovered from patients who developed BSI from February to August 2022 or February 2023 at two different hospitals in Rome, Italy. Phylogenetic analysis revealed two distinct clusters among ST512 isolates and a separate cluster for the ST11 isolate. Beyond blaNDM-1 and blaKPC-3, various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a blaNDM-1 plasmid (IncC) and a blaKPC-3 plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a blaNDM-1 plasmid [IncFII(pKPX1)] and a blaKPC-3 plasmid [IncFIB(K)/IncFII(K)]. The blaNDM-1 plasmids carried genes conferring resistance to clinically relevant antimicrobial agents, and the aminoglycoside resistance gene aac(6')-Ib was found on different plasmids. Colistin resistance-associated mgrB/pmrB gene mutations were present in all isolates, and the yersiniabactin-encoding ybt gene was unique to the ST11 isolate. In conclusion, our findings provide insights into the genomic context of blaNDM-1/blaKPC-3 carbapenemase-producing KP isolates.IMPORTANCEThis study underscores the critical role of genomic surveillance as a proactive measure to restrict the spread of carbapenemase-producing KP isolates, especially when key antimicrobial resistance genes, such as blaNDM-1/blaKPC-3, are plasmid borne. In-depth characterization of these isolates may help identify plasmid similarities contributing to their intra-hospital/inter-hospital adaptation and transmission. Despite the lack of data on patient movements, it is possible that carbapenem-resistant isolates were selected to co-produce KP carbapenemase and New Delhi metallo-ß-lactamase via plasmid acquisition. Studies employing long-read whole-genome sequencing should be encouraged to address the emergence of KP clones with converging phenotypes of virulence and resistance to last-resort antimicrobial agents.

In vitro interaction between alginate lyase and amphotericin B against Aspergillus fumigatus biofilm determined by different methods.

Aspergillus fumigatus biofilms represent a problematic clinical entity, especially because of their recalcitrance to antifungal drugs, which poses a number of therapeutic implications for invasive aspergillosis, the most difficult-to-treat Aspergillus-related disease. While the antibiofilm activities of amphotericin B (AMB) deoxycholate and its lipid formulations (e.g., liposomal AMB [LAMB]) are well documented, the effectiveness of these drugs in combination with nonantifungal agents is poorly understood. In the present study, in vitro interactions between polyene antifungals (AMB and LAMB) and alginate lyase (AlgL), an enzyme degrading the polysaccharides produced as extracellular polymeric substances (EPSs) within the biofilm matrix, against A. fumigatus biofilms were evaluated by using the checkerboard microdilution and the time-kill assays. Furthermore, atomic force microscopy (AFM) was used to image and quantify the effects of AlgL-antifungal combinations on biofilm-growing hyphal cells. On the basis of fractional inhibitory concentration index values, synergy was found between both AMB formulations and AlgL, and this finding was also confirmed by the time-kill test. Finally, AFM analysis showed that when A. fumigatus biofilms were treated with AlgL or polyene alone, as well as with their combination, both a reduction of hyphal thicknesses and an increase of adhesive forces were observed compared to the findings for untreated controls, probably owing to the different action by the enzyme or the antifungal compounds. Interestingly, marked physical changes were noticed in A. fumigatus biofilms exposed to the AlgL-antifungal combinations compared with the physical characteristics detected after exposure to the antifungals alone, indicating that AlgL may enhance the antibiofilm activity of both AMB and LAMB, perhaps by disrupting the hypha-embedding EPSs and thus facilitating the drugs to reach biofilm cells. Taken together, our results suggest that a combination of AlgL and a polyene antifungal may prove to be a new therapeutic strategy for invasive aspergillosis, while reinforcing the EPS as a valuable antibiofilm drug target.

Rapid antifungal susceptibility testing by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis.

The widespread use of antifungal agents, which is likely to expand with their enhanced availability, has promoted the emergence of drug-resistant strains. Antifungal susceptibility testing (AFST) is now an essential procedure for guiding appropriate antifungal therapy. Recently, we developed a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based method that enables the detection of fungal isolates with reduced echinocandin susceptibility, relying on the proteome changes that are detectable after a 15-h exposure of fungal cells to serial drug concentrations. Here, we describe a simplified version of this approach that facilitates discrimination of the susceptible and resistant isolates of Candida albicans after a 3-h incubation in the presence of "breakpoint" level drug concentrations of the echinocandin caspofungin (CSF). Spectra at concentrations of 0 (null), 0.03 (intermediate), and 32 (maximal) µg/ml of CSF were used to create individual composite correlation index (CCI) matrices for 65 C. albicans isolates, including 13 fks1 mutants. Isolates are then classified as susceptible or resistant to CSF if the CCI values of spectra at 0.03 and 32 µg/ml are higher or lower, respectively, than the CCI values of spectra at 0.03 and 0 µg/ml. In this way, the drug resistance of C. albicans isolates to echinocandin antifungals can be quickly assessed. Furthermore, the isolate categorizations determined using MALDI-TOF MS-based AFST (ms-AFST) were consistent with the wild-type and mutant FKS1 genotypes and the AFST reference methodology. The ms-AFST approach may provide a rapid and reliable means of detecting emerging antifungal resistance and accelerating the initiation of appropriate antifungal treatment.

Comparative evaluation of BD Phoenix and vitek 2 systems for species identification of common and uncommon pathogenic yeasts.

The BD Phoenix system was evaluated for species-level identification of yeasts (250 clinical isolates) and compared with the Vitek 2 system, using ribosomal internal transcribed spacer (ITS) sequence analysis as the gold standard. Considering only the species included in each system's database, 96.3% (236/245) and 91.4% (224/245) of the isolates were correctly identified by BD Phoenix and Vitek 2, respectively.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method for discrimination between molecular types of Cryptococcus neoformans and Cryptococcus gattii.

We evaluated the usefulness of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for Cryptococcus identification at the species and subspecies levels by using an in-house database of 25 reference cryptococcal spectra. Eighty-one out of the 82 Cryptococcus isolates (72 Cryptococcus neoformans and 10 Cryptococcus gattii) tested were correctly identified with respect to their molecular type designations. We showed that MALDI-TOF MS is a practicable alternative to conventional mycology or DNA-based methods.

Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry for caspofungin susceptibility testing of Candida and Aspergillus species.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for testing susceptibility to caspofungin of wild-type and fks mutant isolates of Candida and Aspergillus. Complete essential agreement was observed with the CLSI reference method, with categorical agreement for 94.1% of the Candida isolates tested. Thus, MALDI-TOF MS is a reliable and accurate method to detect fungal isolates with reduced caspofungin susceptibility.

Uncommon Neosartorya udagawae fungus as a causative agent of severe corneal infection.

We report the first documented case of a posttraumatic fungal keratitis caused by Neosartorya udagawae. The patient was empirically treated with fluconazole until a corneal scraping grew an Aspergillus fumigatus-like fungus, and itraconazole therapy was then established. A sequence-based approach assigned the isolate to the species. Five months after completion of antifungal therapy, endophthalmitis occurred and orbital exenteration was necessary.

Echinocandin antifungal drug resistance in Candida species: a cause for concern?

The echinocandins, the newest generation of antifungal agents, are inhibitors of ß-1,3-D-glucan synthesis, an action that damages fungal cell walls. Despite a relatively broad spectrum of activity, these drugs are rapidly fungicidal against most Candida spp and have established noninferiority over existing antifungal drugs in the treatment of invasive candidiasis. Clinical resistance to this class of agent is rare, although point mutations in the target Fksp have been shown to confer in vitro resistance to echinocandins in a range of Candida spp, which can result in clinical failures. In addition, the echinocandin treatment can induce a salvage mechanism involving the compensatory upregulation of chitin synthesis in the cell wall. Further elucidation of the echinocandin resistance mechanisms could be exploited to envisage new therapeutic strategies for the treatment of life-threatening Candida infections.

Different detection capabilities by mycological media for Candida isolates from mono- or dual-species cultures.

The aim of this study was to compare the Candida bromcresol green (BCG) medium with the chromogenic (CHROM) Brilliance Candida agar and Sabouraud dextrose agar (SDA) media in regard to their capability of detecting Candida isolates from mono- or dual-species cultures. We prepared Candida isolates' suspensions to obtain mono-species (n = 18) or dual-species (n = 153) culture plates per each medium, and three readers independently observed 513 plates at 24-h, 48-h and 72-h incubation time. We scored reading results as correct, over or under detection compared to the expected species number(s). BCG showed significantly higher correct-detection and lower under-detection rates for all Candida species when observed by at least one reader. At 24-h reading, 12 mono-species cultures had correct (or over) detections in all media, whereas 106, 60 and 78 dual-species cultures had correct (or over) detections in BCG, CHROM or SDA, respectively. BCG provides the basis for an accurate laboratory diagnosis of Candida infections.

First bloodstream infection caused by Prevotella copri in a heart failure elderly patient with Prevotella-dominated gut microbiota: a case report.

BACKGROUND: Bloodstream infection (BSI) is a constant threat for hospitalized patients, and elderly patients are particularly susceptible to BSI caused by anaerobic bacteria. Changes in the gut microbiota composition may lead to pathogen overgrowth and translocation into the bloodstream. Consequently, domination of specific taxa in the intestinal bacterial community seems to be associated with a higher risk of bacteremia in some patient populations. CASE PRESENTATION: Here, we report the case of a 90-year-old heart failure (HF) patient who was admitted to the hospital for an acute state of cardiac decompensation. Twenty days after admission, he was febrile to 38.2 °C whereas his white blood count and C-reactive protein increased to 6190 cells/µL and 31.2 mg/L, respectively. Of the patient's blood culture (BC) bottle pairs collected under the suspicion of infection, the anaerobic bottle yielded an organism that was later identified as Prevotella copri. Concomitantly, the patient's fecal sample was obtained for the intestinal microbiota characterization by sequencing the V3/V4/V6 regions of the bacterial 16S rRNA gene. The analysis revealed highest relative abundances of Bacteroidales (34.1%), Prevotellaceae (19.0%), Prevotella (15.2%), and P. copri (6.1%) taxa, indicating that the patient's gut microbiota was dominated by Prevotella organisms. The patient was successfully treated with metronidazole, and was discharged to a long-term care facility at 35 days of admission. CONCLUSIONS: We provide the first evidence for a clinically significant BSI caused by P. copri and its relationship to a Prevotella-rich gut microbiota in the HF patient setting. When strengthening the pathogenicity of P. copri, the present case suggests that the gut may be a source of BSI caused by the rare anaerobic organism. Future studies are necessary to assess the role of the gut microbiota profiling for precise identification and targeted treatment of patients at high risk of BSI.

Actoxumab + bezlotoxumab combination: what promise for Clostridium difficile treatment?

INTRODUCTION: Clostridium difficile infection (CDI) is the most common healthcare-associated infection worldwide. As standard CDI antibiotic therapies can result in unacceptably high recurrence rates, novel therapeutic strategies for CDI are necessary. A recently emerged immunological therapy is a monoclonal antibody against C. difficile toxin B. Areas covered: In this review, the authors summarize the available pharmacological, preclinical, and clinical data for the CDI treatment based on anti-toxin A (actoxumab) and anti-toxin B (bezlotoxumab) human monoclonal antibodies (HuMabs), and discuss about the potentiality of a therapy that includes HuMab combined administration for CDI. Expert opinion: Although only bezlotoxumab is indicated to reduce recurrence of CDI, experimental studies using a combination of HuMabs actoxumab and bezlotoxumab have shown that bolstering the host immune response against both the C. difficile toxins may be effective in primary and secondary CDI prevention. Besides neutralizing both the key virulence factors, combination of two HuMabs could potentially offer an advantage for a yet to emerge C. difficile strain, which is a steady threat for patients at high risk of CDI. However, as actoxumab development was halted, passive immunotherapy with actoxumab/bezlotoxumab is actually impracticable. Future research will be needed to assess HuMab combination as a therapeutic strategy in clinical and microbiological cure of CDI.

Fatal fulminant cryptococcemia complicating sarcoidosis: Is it to be expected?

Cryptococcosis may be a life-threatening complication of sarcoidosis. We describe a case of cryptococcemia that rapidly progressed toward fatality without apparent other sites of infection. We discuss on the importance of serum cryptococcal polysaccharide antigen testing for identifying at-risk patients who might benefit from timely diagnosis and treatment of cryptococcosis.

Epidermolysis bullosa simplex with mottled pigmentation due to de novo P25L mutation in keratin 5 in an Italian patient.

Epidermolysis bullosa simplex with mottled pigmentation (EBS-MP) is an autosomal dominant inherited disorder of the skin, which manifests as recurrent blistering, punctate palmo-plantar hyperkeratoses, and mottled pigmentation of the trunk and extremities. Previous reports have identified the P25L mutation within the non-helical V1 domain of keratin 5 as the unique cause of the disease. We found this mutation in the first Italian case of EBS-MP. The proband was heterozygous for the P25L mutation, which occurred de novo as this change was not detected in the peripheral blood DNA of the healthy parents. Our report extends the limited number of EBS-MP cases and gives further evidence that mutation P25L is responsible for this unusual phenotype.

Laminin-5 mutational analysis in an Italian cohort of patients with junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a rare genodermatosis characterized by dermal-epidermal separation that is caused by mutations in the genes encoding hemidesmosomal components and laminin-5, the major epithelial adhesion ligand. Here, we report on the mutational analysis of LAMA3, LAMB3, and LAMC2 genes encoding laminin-5 chains in 19 Italian patients, 11 affected with the severe Herlitz (H JEB) and eight with the mild non-Herlitz variant of JEB (non-H JEB). Eighteen mutations, seven of which were novel, were identified and their consequences analyzed at the mRNA and protein level. Premature termination codon mutations in both alleles of LAMB3 or LAMC2 genes were found in nine of the 11 H JEB patients, with a prevalence of mutations in LAMC2. In one case, a homozygous frameshift mutation in LAMB3 was associated to illegitimate splicing leading to non-H JEB. One H JEB patient showed a large intragenic duplication within LAMC2, a genetic defect so far uncovered in laminin-5 genes. Splicing or missense mutations, were prevalent in non-H JEB patients. Collectively, five mutations appeared to be frequent in laminin-5 JEB patients: R635X, 29insC, E210K, W143X in LAMB3 and R95X in LAMC2. These recurrent mutations account for approximately 44% of laminin-5 JEB alleles in Italian patients.

Genotype-phenotype correlation in italian patients with dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a rare skin disorder that is clinically heterogeneous and is transmitted either in dominant (DDEB) or recessive (RDEB) mode. Nevertheless, all variants of DEB are caused by mutations in type VII collagen gene (COL7A1). We report an analysis of COL7A1 mutations in 51 Italian DEB patients, 27 affected with Hallopeau-Siemens RDEB, 19 with non Hallopeau-Siemens RDEB, two with DDEB, two with pretibial RDEB, and one with inversa RDEB. Forty-one mutations were identified, 18 of which are novel. Mutation consequences were analyzed at the mRNA and protein level and genotype-phenotype correlation was determined. Recessive inheritance of a new case of pretibial RDEB was also established. In RDEB patients, six recurrent mutations were identified: 7344G-->A, 425A-->G, 8441-14del21, 4783-1G-->A, 497insA, and G1664A, the last three being found only in Italian patients. Indeed, haplotype analysis supported propagation of ancestral mutated alleles within the Italian population for these particular mutations. Altogether recurrent mutations account for approximately 43% of RDEB alleles in Italian patients and therefore new DEB patients should first be screened for the presence of these mutations.