Search

Due to the global spread of pan resistant organisms, colistin is actually considered as one of the last resort antibiotics against MDR and XDR bacterial infections. The emergence of colistin resistant strains has been observed worldwide in Gram-negative bacteria, such as Enterobacteriaceae and especially in K. pneumoniae, in association with increased morbidity and mortality. This landscape implies the exploration of novel assays able to target colistin resistant strains rapidly. In this study, we developed and evaluated a new MALDI-TOF MS assay in positive-ion mode that allows quantitative or qualitative discrimination between colistin susceptible (18) or resistant (32) K. pneumoniae strains in 3 h by using the “Autof MS 1000” mass spectrometer. The proposed assay, if integrated in the diagnostic workflow, may be of help for the antimicrobial stewardship and the control of the spread of K. pneumoniae colistin resistant isolates in hospital settings.

Direct use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) on positive blood culture (PBC) bottles may accelerate the identification of bloodstream infection (BSI) causing organisms (1), particularly anaerobic bacteria for which BSI-associated mortality rates are high (2)..

Introduction: Mucormycoses represent a rare but very aggressive class of mold infections occurring in patients with hematological malignancies (HMs). In the past, patients at high risk of invasive mucomycosis (IM) were those affected by acute myeloid leukemia but over the last ten years the prophylaxis with a very effective mold-active drug, such as posaconazole, has completely modified the epidemiology. In fact, IM is now observed more frequently in patients with lymphoproliferative disorders who do not receive antifungal prophylaxis. Areas covered: The attention was focused on the epidemiology, diagnosis, prophylaxis and treatment of IM in HMs. Authors excluded pediatric patients considering the different epidemiology and differences in treatment given the limitation of the use of azoles in the pediatric field. A systematic literature review was performed using PubMed database listings between February 2014 and February 2020 using the following MeSH terms: leukemia, hematological malignancies, stem cell transplantation, mucormycosis, molds, prophilaxis, treatment. Expert opinion: The epidemiology of mucormycosis in HMs is changing in the last years. The availability of drugs more effective than in the past against this infection has reduced the mortality; however, a timely diagnosis remains a relevant problem potentially influencing the outcome of hematologic patients with IM.

Sporothrix schenckii is a worldwide-distributed thermally dimorphic fungus, which usually causes a subacute to chronic infection through traumatic implantation or inoculation of its infectious propagules. The fungus encompasses a group of phylogenetically closely related species, thus named the S. schenckii complex, of which S. schenckii sensu stricto and S. brasiliensis are main causative species of sporotrichosis. Owing to a multifaceted molecular dynamic, the S. schenckii complex can switch between the mycelium and the yeast form. This characteristic along with a varying cell wall composition account for significant species-specific differences in the host range, virulence, and susceptibility to antifungal drugs. While culture remains the gold standard to diagnose sporotrichosis, polymerase chain reaction (PCR) or matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry-based methods have become an essential for accurate species identification in many clinical laboratories. If directly applied on tissue samples, molecular methods are helpful to improve both sensitivity of and time to the etiological diagnosis of sporotrichosis. This mini-review aims to put together the old and new knowledge on the S. schenckii complex biology and identification, with particular emphasis on the laboratory diagnosis-related aspects of dis-ease.

Gastrointestinal basidiobolomycosis (GIB), an unusual fungal infection caused by the fungus Basidiobolus ranarum, is rarely reported in the medical literature. GIB is difficult to diagnose because its clinical presentation is non-specific and has no identifiable risk factors. We report here the first case of GIB diagnosed in Italy in a patient suffering from a duodenal ulcer with perforation. The patient was successfully treated with itraconazole. The absence of non-specific signs and symptoms of GIB may delay a definitive diagnosis and treatment. A microbiological investigation should always be requested in order to reach a rapid and definitive diagnosis and to rule out other intestinal diseases.

Diagnosis of invasive aspergillosis (IA) is challenging, particularly in high-risk patients with lung lesions other than typical according to 2008-EORTC/MSG criteria. Even if microbiology is positive, they still remain unclassified according to 2008-EORTC/MSG. Quantitative polymerase chain reaction (qPCR) provides new mycological documentation of IA. This retrospective study assessed Aspergillus fumigatus real time qPCR (MycoGENIE®) in BAL to diagnose IA and identify azole-resistant strains. Clinical, radiological, and microbiological data from 114 hematology patients (69% HSCT recipients; 29% on mould active agents) from years 2012-2017 were collected; and 123 BAL samples were tested with qPCR (cutoff: Ct < 40) and galactomannan (GM, Platelia®, cutoff: 0.5 ODI). Patients were classified as proven/probable, possible, and no-IA. "Atypical-IA" referred to patients with lesions other than typical according to 2008-EORTC/MSG and positive mycology. Proven IA was diagnosed in two cases (1.6%), probable in 28 (22.8%), possible in 27 (22%), atypical in 14 (11.4%). qPCR was positive in 39 samples (31.7%). Sensitivity and specificity of qPCR for proven/probable IA (vs no-IA; atypical-IA excluded) were 40% (95% confidence interval [CI]: 23-59) and 69% (95%CI: 55-81), respectively. Sensitivity of qPCR was higher when combined with GM (83%, 95%CI: 65-94) and in those receiving mould-active agents at BAL (61%, 95%CI: 32-86). One sample had TR34/L98H mutation. In conclusion, in high-risk hematology patients with various lung lesions, A. fumigatus qPCR in BAL contributes to diagnosing IA, particularly if combined with GM and in patients receiving mould-active agents might allow detecting azole-resistant mutations in culture negative samples.

Invasive fungal infection (IFI) remains the major complication in patients with either acute leukemia, allogeneic stem cell transplantation setting, or both, especially regarding pulmonary lo-calization. We report an experience of a 74-year-old Caucasian male with a Philadelphia-positive (BCR-ABL p190) Common B-acute lymphoblastic leukemia (ALL) who developed a pulmonary infection due to Geosmithia argillacea. Furthermore, we describe the management of this complication and the results of microbiological tests useful to guide the treatment. All cases reported show failure of voriconazole treatment. In the majority of cases a good susceptibility to posaconazole has been reported, which seems to have a good clinical impact; however, only L-AmB shows a clinical effect to produce quick clinical improvement and so it should be a drug of choice. A literature revision shows that only a few papers have thus far described this infection, at present only one case was reported in a hematological setting like a gastrointestinal graft versus host disease in an allogeneic HSCT recipient. The severity of clinical conditions in hematological malignancy settings requires improving the management of this emerging invasive fungal infection. Indeed, a molecular diag-nostic approach with a tight laboratory collaboration and targeted therapy should become the gold standard.

To support the clinical laboratory diagnosis of Pneumocystis jirovecii (PJ) pneumonia (PCP), an invasive fungal infection mainly occurring in HIV‐negative patients, in‐house or commercial PJ‐specific real‐time quantitative PCR (qPCR) assays are todays’ reliable options. The performance of these assays depends on the type of PJ gene (multi‐copy mitochondrial versus single‐copy nuclear) targeted by the assay. We described the development of a PJ‐PCR assay targeting the dihydrofolate reductase (DHFR)‐encoding gene. After delineating its analytical performance, the PJ‐PCR assay was used to test bronchoalveolar lavage (BAL) fluid samples from 200 patients (only seven were HIV positive) with suspected PCP. Of 211 BAL fluid samples, 18 (8.5%) were positive and 193 (91.5%) were negative by PJ‐PCR. Of 18 PJ‐PCR‐positive samples, 11 (61.1%) tested positive and seven (38.9%) tested negative with the immunofluorescence assay (IFA). All (100%) of the 193 PJ‐PCR‐negative samples were IFA negative. Based on IFA/PCR results, patients were, respectively, classified as having (n = 18) and not having (n = 182) proven (PJ‐ PCR+/IFA+) or probable (PJ‐PCR+/IFA−) PCP. For 182 patients without PCP, alternative infectious or non‐infectious etiologies were identified. Our PJ‐PCR assay was at least equivalent to IFA, fostering studies aimed at defining a qPCR‐based standard for PCP diagnosis in the future.

The occurrence of multidrug-resistant Candida auris isolates and the increased mortality associated with invasive infections or outbreaks due to this Candida species have been reported in many healthcare settings. Therefore, accurate and rapid identification at the species level of clinical C. auris isolates as well as their timely differentiation as susceptible or resistant to antifungal drugs is mandatory. Aims of the present study were to implement the MALDI-TOF mass spectrometry (MS) Bruker Daltonics Biotyper® database with C. auris spectrum profiles and to develop a fast and reproducible MS assay for detecting anidulafungin (AFG) resistance in C. auris isolates. After creation of main C. auris spectra, a score-oriented dendrogram was generated from hierarchical cluster analysis, including spectra of isolates from C. auris and other Candida (C. glabrata, C. guilliermondii, C. haemulonii, C. lusitaniae, and C. parapsilosis) or non-Candida (Rhodotorula glutinis) species. Cluster analysis allowed to group and classify the isolates according to their species designation. Then, a three-hour incubation antifungal susceptibility testing (AFST) assay was developed. Spectra obtained at null, intermediate, or maximum AFG concentrations were used to create composite correlation index matrices for eighteen C. auris isolates included in the study. All six resistant C. auris isolates were detected as resistant whereas 11 of 12 susceptible C. auris isolates were detected as susceptible by the MS-AFST assay. In conclusion, our MS-based assay offers the possibility of rapidly diagnosing and appropriately treating patients with C. auris infection.

The therapeutic armamentarium for the treatment of patients with lymphoproliferative diseases has grown considerably over the most recent years, including a large use of new immunotherapeutic agents. As a consequence, the epidemiology of infectious complications in this group of patients is poorly documented, and even more importantly, the potential benefit of antimicrobial prophylaxis remains a matter of debate when considering the harmful effect from the emergence of multidrug resistant pathogens. The present position paper is addressed to all hematologists treating patients affected by lymphoproliferative malignancies with the aim to provide clinicians with a useful tool for the prevention of bacterial, fungal and viral infections.

No abstract

An autochthonous case of lymphocutaneous sporotrichosis caused by Sporothrix schenckii is reported. The patient developed skin lesions localized along the lymphatics that appeared after he suffered an injury while collecting wicker canes in marshy water. The fungus was identified as Sporothrix schenckii by MALDI-TOF and sequencing. Phylogenetic analysis was also performed. Low MIC values were detected for all tested echinocandins and azoles except for fluconazole. The patient was treated with itraconazole without significant improvement. A regression of lesions was observed after 3 months of therapy with voriconazole. Few cases of sporotrichosis have been reported in Europe. However, several cases of sporotrichosis have been described in Italy. The incidence of sporotrichosis in Italy may be underestimated and microbiologists, and clinicians must be aware of this fungal infection.

Coinfections with bacteria or fungi may be a frequent complication of COVID-19, but coinfections with Candida species in COVID-19 patients remain rare. We report the 53-day clinical course of a complicated type-2 diabetes patient diagnosed with COVID-19, who developed bloodstream infections initially due to methicillin-resistant Staphylococcus aureus, secondly due to multidrug-resistant Gram-negative bacteria, and lastly due to a possibly fatal Candida glabrata. The development of FKS-associated pan-echinocandin resistance in the C. glabrata isolated from the patient after 13 days of caspofungin treatment aggravated the situation. The patient died of septic shock shortly before the prospect of receiving potentially effective antifungal therapy. This case emphasizes the importance of early diagnosis and monitoring for antimicrobial drug-resistant coinfections to reduce their unfavorable outcomes in COVID-19 patients.

The most prevalent cause of nosocomial bloodstream infection (BSI) among non-C. albicans Candida species, Candida parapsilosis, may not only be resistant to azole antifungal agents but also disseminate to vulnerable patients. In this survey of BSIs occurring at a large Italian hospital between May 2014 and May 2019, C. parapsilosis accounted for 28.5% (241/844) of all Candida isolates causing BSI episodes. The majority of episodes (151/844) occurred in medical wards. Across the 5 yearly periods, the rates of azole non-susceptibility were 11.8% (4/34), 17.8% (8/45), 28.6% (12/42), 32.8% (19/58), and 17.7% (11/62), respectively, using the Sensititre YeastOne® method. Among azole non-susceptible isolates (54/241; 22.4%), 49 were available for further investigation. Using the CLSI reference method, all 49 isolates were resistant to fluconazole and, except one (susceptible dose-dependent), to voriconazole. Forty (81.6%) isolates harbored the Erg11p Y132F substitution and nine (18.4%) isolates the Y132F in combination with the Erg11p R398I substitution. According to their genotypes, as defined using a microsatellite analysis based on six short tandem repeat markers, 87.7% of isolates (43/49) grouped in two major clusters (II and III), whereas 4.1% of isolates (2/49) belonged to a separate cluster (I). Interestingly, all the isolates from cluster II harbored the Y132F substitution, and those from cluster III harbored both Y132F and R398I substitutions. Of 56 non-Italian isolates included as controls, two Indian isolates with the Y132F substitution had a genotype clearly differing from that of the isolates from clusters II and I. In conclusion, these findings show the dominance of clonal Y132F isolates in our hospital and suggest detection of the Y132F substitution as helpful tool to prevent transmission among hospitalized patients at risk of BSI.

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.

Background: Nowadays, the global spread of resistance to oxyimino-cephalosporins in Enterobacteriaceae implies the need for novel diagnostics that can rapidly target resistant organisms from these bacterial species. Methods: In this study, we developed and evaluated a Direct Mass Spectrometry assay for Beta-Lactamase (D-MSBL) that allows direct identification of (oxyimino)cephalosporin-resistant Escherichia coli or Klebsiella pneumoniae from positive blood cultures (BCs), by using the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) technology. Results: The D-MSBL assay was performed on 93 E. coli or K. pneumoniae growing BC samples that were shortly co-incubated with cefotaxime (CTX) as the indicator cephalosporin. Susceptibility and resistance defining peaks from the samples’ mass spectra were analyzed by a novel algorithm for bacterial organism classification. The D-MSBL assay allowed discrimination between E. coli and K. pneumoniae that were resistant or susceptible to CTX with a sensitivity of 86.8% and a specificity of 98.2%. Conclusion: The proposed algorithm-based D-MSBL assay, if integrated in the routine laboratory diagnostic workflow, may be useful to enhance the establishment of appropriate antibiotic therapy and to control the threat of oxyimino-cephalosporin resistance in hospital.

The echinocandins are relatively new antifungal drugs that represent, together with the older azoles, the recommended and/or preferred agents to treat candidaemia and other forms of invasive candidiasis in human patients. If "time is of the essence" to reduce the mortality for these infections, the administration of appropriate antifungal therapy could be accelerated by the timely reporting of laboratory test results. In this study, we attempted to validate a MALDI-ToF mass spectrometry-based assay for the antifungal susceptibility testing (AFST) of the potentially multidrug-resistant pathogen Candida glabrata against anidulafungin and fluconazole. The practical applicability of the assay, reported here as MS-AFST, was assessed with a panel of clinical isolates that were selected to represent phenotypically and genotypically/molecularly susceptible or resistant strains. The data show the potential of our assay for rapid detection of antifungal resistance, although the MS-AFST assay performed at 3 h of the in vitro antifungal exposure failed to detect C. glabrata isolates with echinocandin resistance-associated FKS2 mutations. However, cell growth kinetics in the presence of anidulafungin revealed important cues about the in vitro fitness of C. glabrata isolates, which may lead to genotypic or phenotypic antifungal testing in clinical practice.

not reported

We tested 59 common and 27 uncommon Aspergillus species isolates for the susceptibility to the mold-active azole antifungal agents, itraconazole, voriconazole, and posaconazole, using the Sensititre method. The overall essential agreement with the CLSI reference method was 96.5% for itraconazole and posaconazole, and 100% for voriconazole. By Sensititre, as well as CLSI, all of 10 A. fumigatus isolates with a cyp51 mutant genotype were classified as being non-wild-type (MIC > ECV) to triazoles.

Fungal infections have increased significantly in the last few years, and their outcomes are in part complicated by the emergence of antifungal drug-resistant pathogens. Together with Candida species, the mould Aspergillus fumigatus is one of the most prevalent organisms to cause invasive fungal disease. The molecular detection of (tri)azole resistance in both Candida and Aspergillus species may represent a useful means of monitoring the incidence of clinical isolates with antifungal resistance-associated gene alterations. Here, we describe molecular methods that have been developed to allow for accurate detection of azole-resistant isolates among C. glabrata and A. fumigatus fungal species.

In the last years, life-threatening fungal diseases have increased signifi cantly, due to the rising number of human individuals susceptible to fungal infections, which are in part complicated by the emergence of antifungal drug-resistant pathogens. Among yeasts, Candida albicans and Candida glabrata are the most common organisms responsible for invasive fungal diseases. The molecular detection of echinocandin resistance in Candida species may represent a useful means of monitoring the incidence of clinical isolates with antifungal resistance-associated gene alterations. Here, we describe the current methods that enable researchers and/or clinical microbiologists to accurately detect echinocandin-resistant isolates of C. albicans and C. glabrata .

Candida parapsilosis is the most frequent cause of catheter-related candidemia among non-Candida albicans species. This may be related to intrinsic capabilities as adhering and forming a biofilm on abiotic surfaces such as on medical devices. As previously demonstrated, patients infected with high biofilm-producing C. parapsilosis isolates had a greater mortality risk compared to patients infected with low biofilm-producing C. parapsilosis isolates. We developed the BIOF–HILO assay, a MALDI–TOF mass spectrometry (MS)-based assay, which compares mass spectra obtained from attached and suspended isolate cells during the early (i.e., 3-h) adhesion phase of in vitro biofilm formation. The composite correlation index (CCI) analysis was used to discriminate between mass spectra differences of the two cell types, classifying all 50 C. parapsilosis clinical isolates, included in the study, after only 3-h of testing, in high or low biofilm producers. All high (n = 25) or low (n = 25) biofilm producers had, according to CCI mass spectra comparison values, higher or lower than one CCI ratios, which were obtained by dividing the CCIsuspendedcells by the CCIattachedcells . In conclusion, the BIOF–HILO assay allows a rapid categorization of C. parapsilosis clinical isolates in high or low biofilm producers. This information, if timely provided to physicians, may improve treatment outcomes in patients with C. parapsilosis candidemia.

N/A

Aspergillus species are the cause of invasive mold infections in immunocompromised patients: Aspergillus fumigatus, A. flavus and A. terreus account for most cases of invasive aspergillosis (IA). As certain species are associated with higher mortality and vary in their resistance to antifungal therapy, diagnosis requires increasingly rapid molecular methods that enable sensitive detection and species discrimination. We have developed PCR and Multiplex PCR assays for the detection of six medically important Aspergillus spp. species DNA in bronchoalveolar lavage (BAL) specimens from hematology and intensive care unit (ICU) patients at risk of IA, using different species and genus-specific PCR primers, selected within the SCW4 gene, encoding a cell wall glucanase of A. fumigatus, similar to mannoprotein Mp65 of Candida albicans. The genus-specific PCR primers were able to amplify only Aspergillus DNAs but not that belonging to other fungal genera tested. The species-specific PCR primers allowed differentiation of each Aspergillus species by the amplicon length produced. The methods described in this study are rapid (less than 4 h), reproducible, simple and specific and demonstrate potential application in the clinical laboratory.

Infections by Nocardia spp. are generally regarded as opportunistic diseases in immunocompromised patients, but can also affect immunocompetent subjects. Such infections represent an important diagnostic challenge for clinicians and microbiologists, and diagnosis is frequently delayed or even conducted post mortem. A 54-year-old man was admitted to our hospital because of ventriculitis and relapsing brain abscess. Five months prior, this patient had undergone external ventricular drain and surgery for a cerebellar abscess. Histopathology demonstrated pyogenic inflammatory reaction, microbiologic investigations proved negative and empiric antimicrobial therapy was administered for a total of eight weeks. Six weeks later, the patient developed relapsing neurologic manifestations. On reviewing the patient's clinical history it emerged that the patient had suffered pneumonia two months prior to neurosurgery, treated with amoxicillin/clavulanate 3g a day and levofloxacin 500mg a day for three weeks. On the CNS relapsing manifestations, nocardiosis was suspected and DNA sequencing from the formalin-fixed paraffin-embedded cerebellar tissue collected during neurosurgery allowed diagnosis of Nocardia paucivorans infection. The patient received medical therapy for 11 months. At follow-up, eight months after treatment was discontinued, the patient was aymptomatic. Nocardia spp. infections need to be suspected not only in immunocompromised, but also in immunocompetent patients. Proper samples need to be collected for proper microbiologic investigations. Paraffin-embedded tissue genomic sequencing can be a useful tool for diagnosis of nocardiosis.