Rapid Detection of the Omicron (B.1.1.529) SARS-CoV-2 Variant Using a COVID-19 Diagnostic PCR Assay.

The Omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the last variant of concern (VOC) identified to date. Compared to whole-genome or gene-specific sequencing methods, reverse-transcription PCR assays may be a simpler approach to study VOCs. We used a point-of-care COVID-19 diagnostic PCR assay to detect the Omicron SARS-CoV-2 variant in the respiratory tract samples of COVID-19 patients who had tested positive for SARS-CoV-2 RNA between April 2021 and January 2022. Sequencing analyses had shown that 87 samples were positive for the Omicron variant and 43 samples were positive for a non-Omicron variant (Delta, 18 samples; Alpha, 13 samples; Gamma, 10 samples; Beta, 1 sample; or Epsilon, 1 sample). According to results by the PCR assay, whose primers anneal a nucleocapsid (N) gene region that comprises the E31/R32/S33 deletion (also termed the del31/33 mutation), we found that N gene target failure/dropout (i.e., a negative/low result) occurred in 86 (98.8%) of 87 Omicron variant-positive samples tested. These results were assessed in relation to those of the spike (S) gene, which expectedly, was detected in all (100%) 130 samples. A total of 43 (100%) of 43 Delta, Alpha, Gamma, Beta, or Epsilon variant-positive samples had a positive result with the N gene. Importantly, in 86 of 87 Omicron variant-positive samples, the del31/33 mutation was detected together with a P13L mutation, which was, instead, detected alone in the Omicron variant-positive sample that had a positive N-gene result. IMPORTANCE Rapid detection of the Omicron SARS-CoV-2 variant in patients' respiratory tract samples may influence therapeutic choices, because this variant is known to escape from certain monoclonal antibodies. Our findings strengthen the importance of manufacturers' efforts to improve the existing COVID-19 diagnostic PCR assays and/or to develop novel variant-specific PCR assays. Furthermore, our findings show that only a small fraction of SARS-CoV-2-positive samples may require whole-genome sequencing analysis, which is still crucial to validate PCR assay results. We acknowledge that the emergence of novel variants containing mutations outside the PCR assay target region could, however, allow an assay to work as per specifications without being able to identify a SARS-CoV-2-positive sample as a variant. Future work and more experience in this topic will help to reduce the risk of misidentification of SARS-CoV-2 variants that is unavoidable when using the current PCR assays.

SARS-CoV-2 Antigen Test Results to Infer Active or Non-Active Virus Replication Status in COVID-19 Patients.

We used nasopharyngeal swab samples of patients with a symptomatic (n = 82) or asymptomatic (n = 20) coronavirus disease 2019 (COVID-19) diagnosis to assess the ability of antigen detection tests to infer active (potentially transmissible) or inactive (potentially non-transmissible) infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the subgenomic RNA (sgRNA) as an active replication marker of SARS-CoV-2, 48 (76.2%), 56 (88.9%), and 63 (100%) of 63 samples with sgRNA positive results tested positive with the SD BIOSENSOR STANDARD Q COVID-19 Ag (Standard Q), the SD BIOSENSOR STANDARD F COVID-19 Ag FIA (Standard F), or the Fujirebio LUMIPULSE G SARS-CoV-2 Ag (Lumipulse) assay, respectively. Conversely, 37 (94.9%), 29 (74.4%), and 7 (17.9%) of 39 samples with sgRNA negative results tested negative with Standard Q, Standard F, or Lumipulse, respectively. Stratifying results by the number of days of symptoms before testing revealed that most antigen positive/sgRNA positive results were among samples tested at 2-7 days regardless of the assay used. Conversely, most antigen negative/sgRNA negative results were among samples tested at 16-30 days only when Standard Q or Standard F were used. In conclusion, based on our findings, a negative antigen test, especially with the Lumipulse assay, or a positive antigen test, especially with the Standard F assay, may suggest, respectively, the absence or presence of replication-competent SARS-CoV-2.

Pan-Echinocandin-Resistant Candida glabrata Bloodstream Infection Complicating COVID-19: A Fatal Case Report.

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.

A case of Trichosporon asahii urinary tract infection in a frail elderly patient.

Trichosporon urinary tract infection (UTI) is an unusual emerging infection, caused mostly by Trichosporon asahii, described especially in hospitalized patients. To date the interpretation and management of Trichosporon positive urinary culture remains a diagnostic and therapeutic dilemma for which there are no precise indications, and the challenge can be even more complicated in comorbid frail elderly patients. Triazoles are known to be the most effective antifungal drugs but can raise concerns about pharmacological interaction. We report a case of Trichosporon asahii nosocomial UTI in an elderly patient.

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.

A rapid diagnostic workflow for cefotaxime-resistant Escherichia coli and Klebsiella pneumoniae detection from blood cultures by MALDI-TOF mass spectrometry.

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.

Potential Use of MALDI-ToF Mass Spectrometry for Rapid Detection of Antifungal Resistance in the Human Pathogen Candida glabrata.

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.

Susceptibility Testing of Common and Uncommon Aspergillus Species against Posaconazole and Other Mold-Active Antifungal Azoles Using the Sensititre Method.

We tested 59 common and 27 uncommon Aspergillus species isolates for 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 was 100% for voriconazole. By the Sensititre method as well as the CLSI reference method, all of 10 A. fumigatus isolates with a cyp51 mutant genotype were classified as being non-wild-type isolates (MIC > epidemiological cutoff value [ECV]) with respect to triazole susceptibility.

Molecular Detection of Resistance to Echinocandins.

In the last years, life-threatening fungal diseases have increased significantly, 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.

Molecular Detection of Resistance to Azole Components.

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.

Use of SCW4 gene primers in PCR methods for the identification of six medically important Aspergillus species.

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.

Targeted gene disruption in Candida parapsilosis demonstrates a role for CPAR2_404800 in adhesion to a biotic surface and in a murine model of ascending urinary tract infection.

Candida parapsilosis is an emerging opportunistic pathogen, second in frequency only to C. albicans and commonly associated with both mucosal and systemic infections. Adhesion to biotic surfaces is a key step for the development of mycoses. The C. parapsilosis genome encodes 5 predicted agglutinin-like sequence proteins and their precise role in the adhesion process still remains to be elucidated. In this study, we focused on the putative adhesin Cpar2_404800, in view of its high homology to the most important adhesion molecule in C. albicans. Two independent lineages of C. parapsilosis CPAR2_404800 heterozygous and null mutants were obtained by site-specific deletion. CPAR2_404800 mutants did not differ from wild-type strain in terms of in vitro growth or in their ability to undergo morphogenesis. However, when compared for adhesion to a biotic surface, CPAR2_404800 null mutants exhibited a marked reduction in their adhesion to buccal epithelial cells (>60% reduction of adhesion index). Reintroduction of one copy of CPAR2_404800 gene in the null background restored wild type phenotype. A murine model of urinary tract infection was used to elucidate the in vivo contribution of CPAR2_404800. A 0.5 and 1 log10 reduction in colony forming unit numbers (per gram) was observed respectively in bladder and kidneys obtained from mice infected with null mutant compared to wild-type infected ones. Taken together, these findings provide the first evidence for a direct role of CPAR2_404800 in C. parapsilosis adhesion to host surfaces and demonstrate its contribution to the pathogenesis of murine urinary candidiasis.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for identification and clustering of Neisseria gonorrhoeae.

BACKGROUND: The sexually transmitted infection gonorrhea remains a public health concern for becoming resistant to drug treatments available. The purpose of this study was to evaluate the usefulness of the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify and cluster Neisseria gonorrhoeae. From a current monitoring in Italy, as part of the European Gonococcal Antimicrobial Surveillance Programme (EURO-GASP), 93 gonococci collected from 2007 to 2012 susceptible (44 isolates) and resistant (49 isolates) to cefixime were selected. Minimum Inhibitory Concentration (MIC) values for cefixime was assessed by Etest carried out in agreement with the manufacturer's instructions and interpreted referring to European Committee on Antimicrobial Susceptibility testing (EUCAST) clinical breakpoints criteria. Data obtained by N. gonorrhoeae multiantigen sequence typing (NG-MAST) and the dendrogram based on the concatenation of porB and tbpB genes were evaluated. MALDI-TOF MS, to reconfirm gonorrhea identification, analyzed single colonies from freshly grown isolates and applied directly on a ground-steel MALDI target plate. For the MALDI-TOF dendrogram cluster analysis, MSPs (Main Spectrum Profile) from each isolate were created acquiring 5000 shots from 10 technical replicates obtained from bacteria extraction. RESULTS: Molecular typing by NG-MAST showed 28 sequence types (STs); G1407 was the predominant accounting for 75 gonococci. All the 93 gonococci, except one, were correctly identified at species level by MALDI-TOF MS and G1407 isolates were divided into two clusters. CONCLUSION: MALDI-TOF MS for a real-time detection and cluster analysis of gonorrhea is a promising tool for surveillance purposes. Moreover, additional studies are required to collect more data on the performance of MALDI-TOF MS for gonococci.

Geotrichum capitatum septicaemia in a haematological patient after acute myeloid leukaemia relapse: identification using MALDI-TOF mass spectrometry and review of the literature.

Emerging fungal pathogens, such as Geotrichum capitatum, are often associated with poor prognosis and represent a new challenge in modern medicine. Invasive Geotrichum capitatum infection is rare and has been reported exclusively in patients who showed signs of severe immunodeficiency, particularly those affected by haematological malignancies. The optimal therapy against systemic geotricosis has not yet been identified due to limited data about its antifungal susceptibility. The use of several therapeutic strategies and the low number of cases treated does not allow identification of specific therapeutic protocols. Furthermore, in spite of antifungal therapy, mortality rates reach very high levels. We report a case of systemic Geotrichum capitatum infection in a 78-year-old male treated with salvage therapy after acute myeloid leukaemia (AML) relapse. Geotrichum capitatum was isolated from his blood culture and identified by using Vitek 2 and Maldi time-of-flight system (MALDI-TOF). The infection was unsuccessfully treated, despite in vitro susceptibility, with micafungin and liposomal amphotericin B.

Antifungal susceptibility profiles of bloodstream yeast isolates by Sensititre YeastOne over nine years at a large Italian teaching hospital.

Sensititre YeastOne (SYO) is an affordable alternative to the Clinical and Laboratory Standards Institute (CLSI) reference method for antifungal susceptibility testing. In this study, the MICs of yeast isolates from 1,214 bloodstream infection episodes, generated by SYO during hospital laboratory activity (January 2005 to December 2013), were reanalyzed using current CLSI clinical breakpoints/epidemiological cutoff values to assign susceptibility (or the wild-type [WT] phenotype) to systemic antifungal agents. Excluding Candida albicans (57.4% of all isolates [n = 1,250]), the most predominant species were Candida parapsilosis complex (20.9%), Candida tropicalis (8.2%), Candida glabrata (6.4%), Candida guilliermondii (1.6%), and Candida krusei (1.3%). Among the non-Candida species (1.9%), 7 were Cryptococcus neoformans and 17 were other species, mainly Rhodotorula species. Over 97% of Candida isolates were susceptible (WT phenotype) to amphotericin B and flucytosine. Rates of susceptibility (WT phenotype) to fluconazole, itraconazole, and voriconazole were 98.7% in C. albicans, 92.3% in the C. parapsilosis complex, 96.1% in C. tropicalis, 92.5% in C. glabrata, 100% in C. guilliermondii, and 100% (excluding fluconazole) in C. krusei. The fluconazole-resistant isolates consisted of 6 C. parapsilosis complex isolates, 3 C. glabrata isolates, 2 C. albicans isolates, 2 C. tropicalis isolates, and 1 Candida lusitaniae isolate. Of the non-Candida isolates, 2 C. neoformans isolates had the non-WT phenotype for susceptibility to fluconazole, whereas Rhodotorula isolates had elevated azole MICs. Overall, 99.7% to 99.8% of Candida isolates were susceptible (WT phenotype) to echinocandins, but 3 isolates were nonsusceptible (either intermediate or resistant) to caspofungin (C. albicans, C. guilliermondii, and C. krusei), anidulafungin (C. albicans and C. guilliermondii), and micafungin (C. albicans). However, when the intrinsically resistant non-Candida isolates were included, the rate of echinocandin nonsusceptibility reached 1.8%. In summary, the SYO method proved to be able to detect yeast species showing antifungal resistance or reduced susceptibility.

Candida utilis catheter-related bloodstream infection.

Central venous catheter-related fungemia are increasing in the last years, also due to rare fungi. We report the case of a Candida utilis catheter-related bloodstream infection in a patient with metastatic carcinoma of the bladder and a long term totally implanted venous catheter. The diagnosis was done by paired blood cultures and differential time to positivity. The Candida species was rapidly identified by MALDI-TOF mass spectrometry. The patient was successfully treated with anidulafungine.

Application of MALDI-TOF mass spectrometry in clinical diagnostic microbiology.

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a powerful technique for identification of microorganisms, changing the workflow of well-established laboratories so that its impact on microbiological diagnostics has been unparalleled. In comparison with conventional identification methods that rely on biochemical tests and require long incubation procedures, MALDI-TOF MS has the advantage of identifying bacteria and fungi directly from colonies grown on culture plates in a few minutes and with simple procedures. Numerous studies on different systems available demonstrate the reliability and accuracy of the method, and new frontiers have been explored besides microbial species level identification, such as direct identification of pathogens from positive blood cultures, subtyping, and drug susceptibility detection.

Identification and typing of the Candida parapsilosis complex: MALDI-TOF MS vs. AFLP.

In this study we compare the capability of amplification fragment-length polymorphism (AFLP) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify and subtype isolates of members of the Candida parapsilosis complex (C. parapsilosis, C. orthopsilosis, C. metapsilosis) and Lodderomyces elongisporus, which cannot be differentiated with biochemical methods. Both techniques correctly identified all isolates included in this study and clustered isolates within the different species. DNA-based and mass spectrum-based dendrograms yielded similar outcomes with regard to phylogenetic distance within C. orthopsilosis and C. parapsilosis species. However, a different clustering was obtained for C. metapsilosis for which AFLP was highly effective in differentiating. While MALDI-TOF MS was found to be a reliable method for species-level identification, further studies are required to assess its value as a fungal typing tool.

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.