Rare multi-fungal sepsis: a case of triple-impact immunoparalysis.

Patients with burn injury and inhalation injury are highly susceptible to infectious complications, including opportunistic pathogens, due to the loss of skin cover and mucosal damage of respiratory tract as well as the disruption of homeostasis. This case report, a 34-year-old man suffered critical burns, provides the first literature description of triple-impact immunoparalysis (critical burns, inhalation injury, and SARS-CoV-2 infection), leading to a lethal multifocal infection caused by several fungi including very rare environmental representatives Metschnikowia pulcherrima and Wickerhamomyces anomalus. The co-infection by these common environmental yeasts in a human is unique and has not yet been described in the literature. Importantly, our patient developed refractory septic shock and died despite targeted antifungal therapy including the most potent current antifungal agent-isavuconazole. It can be assumed that besides immunoparalysis, effectiveness of therapy by isavuconazole was impaired by the large distribution volume in this case. As this is a common situation in intensive care patients, routine monitoring of plasmatic concentration of isavuconazole can be helpful in personalization of the treatment and dose optimization. Whatmore, many fungal species often remain underdiagnosed during infectious complications, which could be prevented by implementation of new methods, such as next-generation sequencing, into clinical practice.

New Multilocus Sequence Typing Scheme for Enterococcus faecium Based on Whole Genome Sequencing Data.

The MLST scheme currently used for Enterococcus faecium typing was designed in 2002 and is based on putative gene functions and Enterococcus faecalis gene sequences available at that time. As a result, the original MLST scheme does not correspond to the real genetic relatedness of E. faecium strains and often clusters genetically distant strains to the same sequence types (ST). Nevertheless, typing has a significant impact on the subsequent epidemiological conclusions and introduction of appropriate epidemiological measures, thus it is crucial to use a more accurate MLST scheme. Based on the genome analysis of 1,843 E. faecium isolates, a new scheme, consisting of 8 highly discriminative loci, was created in this study. These strains were divided into 421 STs using the new MLST scheme, as opposed to 223 STs assigned by the original MLST scheme. The proposed MLST has a discriminatory power of D = 0.983 (CI95% 0.981 to 0.984), compared to the original scheme's D = 0.919 (CI95% 0.911 to 0.927). Moreover, we identified new clonal complexes with our newly designed MLST scheme. The scheme proposed here is available within the PubMLST database. Although whole genome sequencing availability has increased rapidly, MLST remains an integral part of clinical epidemiology, mainly due to its high standardization and excellent robustness. In this study, we proposed and validated a new MLST scheme for E. faecium, which is based on genome-wide data and thus reflects the tested isolates' more accurate genetic similarity. IMPORTANCE Enterococcus faecium is one of the most important pathogens causing health care associated infections. One of the main reasons for its clinical importance is a rapidly spreading resistance to vancomycin and linezolid, which significantly complicates antibiotic treatment of infections caused by such resistant strains. Monitoring the spread and relationships between resistant strains causing severe conditions represents an important tool for implementing appropriate preventive measures. Therefore, there is an urgent need to establish a robust method enabling strain monitoring and comparison at the local, national, and global level. Unfortunately, the current, extensively used MLST scheme does not reflect the real genetic relatedness between individual strains and thus does not provide sufficient discriminatory power. This can lead directly to incorrect epidemiological measures due to insufficient accuracy and biased results.

Rapid Identification of Pseudomonas aeruginosa International High-Risk Clones Based on High-Resolution Melting Analysis.

The Pseudomonas aeruginosa population has a nonclonal epidemic structure. It is generally composed of a limited number of widespread clones selected from a background of many rare and unrelated genotypes recombining at high frequency. Due to the increasing prevalence of nosocomial infections caused by multidrug-resistant/extensively drug-resistant (MDR/XDR) strains, it is advisable to implement infection control measures. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) are considered the gold standard methods in bacterial typing, despite being limited by cost, staff, and instrumental demands. Here, we present a novel mini-MLST scheme for P. aeruginosa rapid genotyping based on high-resolution melting analysis. Using the proposed mini-MLST scheme, 3,955 existing sequence types (STs) were converted into 701 melting types (MelTs), resulting in a discriminatory power of D = 0.993 (95% confidence interval [CI], 0.992 to 0.994). Whole-genome sequencing of 18 clinical isolates was performed to support the newly designed mini-MLST scheme. The clonal analysis of STs belonging to MelTs associated with international high-risk clones (HRCs) performed by goeBURST software revealed that a high proportion of the included STs are highly related to HRCs and have also been witnessed as responsible for serious infections. Therefore, mini-MLST provides a clear warning for the potential spread of P. aeruginosa clones recognized as MDR/XDR strains with possible serious outcomes. IMPORTANCE In this study, we designed a novel mini-MLST typing scheme for Pseudomonas aeruginosa. Its great discriminatory power, together with ease of performance and short processing time, makes this approach attractive for prospective typing of large isolate sets. Integrating the novel P. aeruginosa molecular typing scheme enables the development and spread of MDR/XDR high-risk clones to be investigated.

Sequencing Independent Molecular Typing of Staphylococcus aureus Isolates: Approach for Infection Control and Clonal Characterization.

Staphylococcus aureus is a major bacterial human pathogen that causes a wide variety of clinical manifestations. The main aim of the presented study was to determine and optimize a novel sequencing independent approach that enables molecular typing of S. aureus isolates and elucidates the transmission of emergent clones between patients. In total, 987 S. aureus isolates including both methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates were used to evaluate the novel typing approach combining high-resolution melting (HRM) analysis of multilocus sequence typing (MLST) genes (mini-MLST) and spa gene (spa-HRM). The novel approach's discriminatory ability was evaluated by whole-genome sequencing (WGS). The clonal relatedness of tested isolates was set by the BURP and BURST approach using spa and MLST data, respectively. Mini-MLST classified the S. aureus isolates into 38 clusters, followed by spa-HRM classifying the isolates into 101 clusters. The WGS proved HRM-based methods to effectively differentiate between related S. aureus isolates. Visualizing evolutionary relationships among different spa-types provided by the BURP algorithm showed comparable results to MLST/mini-MLST clonal clusters. We proved that the combination of mini-MLST and spa-HRM is rapid, reproducible, and cost-efficient. In addition to high discriminatory ability, the correlation between spa evolutionary relationships and mini-MLST clustering allows the variability in population structure to be monitored. IMPORTANCE Rapid and cost-effective molecular typing tools for Staphylococcus aureus epidemiological applications such as transmission tracking, source attribution and outbreak investigations are highly desirable. High-resolution melting based methods are effective alternative to those based on sequencing. Their good reproducibility and easy performance allow prospective typing of large set of isolates while reaching great discriminatory power. In this study, we established a new epidemiological approach to S. aureus typing. This scheme has the potential to greatly improve epidemiological investigations of S. aureus.

[Diagnosing dermatomycoses in a clinical mycology laboratory: guidelines proposed by the Czech Society for Medical Microbiology Working Group on Mycology].

This draft of guidelines for the laboratory diagnosis of dermatomycoses was developed based on discussion among members of the Czech Society for Medical Microbiology Working Group on Mycology. The document Guidelines for the Laboratory Diagnosis of Dermatomycoses was published for discussion on the Czech Society for Medical Microbiology website on 23 March 2020. Until recently, recommendations concerning this area of laboratory diagnosis in mycology were only limited to information in manuals and no comprehensive and systematic document concerning these issues was available. In an effort to fill the gap, members of the working group developed recommendations covering various laboratory aspects of mycology, from obtaining a proper history, to adequate sampling techniques, sample analyses using conventional microscopy and culture techniques, to interpretation of results. Additional information was on the diagnostic potential of novel, modern technology, in particular molecular genetic methods and mass spectrometry. The recently developed European standards for testing the susceptibility of dermatophytes to antifungals were also included in the recommendations. The document will be regularly updated based on new findings.

Rapid high-resolution melting genotyping scheme for Escherichia coli based on MLST derived single nucleotide polymorphisms.

Routinely used typing methods including MLST, rep-PCR and whole genome sequencing (WGS) are time-consuming, costly, and often low throughput. Here, we describe a novel mini-MLST scheme for Eschericha coli as an alternative method for rapid genotyping. Using the proposed mini-MLST scheme, 10,946 existing STs were converted into 1,038 Melting Types (MelTs). To validate the new mini-MLST scheme, in silico analysis was performed on 73,704 strains retrieved from EnteroBase resulting in discriminatory power D = 0.9465 (CI 95% 0.9726-0.9736) for mini-MLST and D = 0.9731 (CI 95% 0.9726-0.9736) for MLST. Moreover, validation on clinical isolates was conducted with a significant concordance between MLST, rep-PCR and WGS. To conclude, the great portability, efficient processing, cost-effectiveness, and high throughput of mini-MLST represents immense benefits, even when accompanied with a slightly lower discriminatory power than other typing methods. This study proved mini-MLST is an ideal method to screen and subgroup large sets of isolates and/or quick strain typing during outbreaks. In addition, our results clearly showed its suitability for prospective surveillance monitoring of emergent and high-risk E. coli clones'.

Trichoderma longibrachiatum and Aspergillus fischeri Infection as a Cause of Skin Graft Failure in a Patient with Critical Burns after Liver Transplantation.

Infectious complications are responsible for the majority of mortalities and morbidities of patients with critical burns. Although bacteria are the predominant etiological agents in such patients, yeasts and fungi have become relatively common causes of infections over the last decade. Here, we report a case of a young man with critical burns on 88% TBSA (total body surface area) arising as a part of polytrauma. The patient's history of orthotopic liver transplantation associated with the patient's need to use combined immunosuppressant therapy was an additional complication. Due to deep burns in the forearm region, we have (after a suitable wound bed preparation) applied a new bi-layered dermal substitute. The patient, however, developed a combined fungal infection in the region of this dermal substitute caused by Trichoderma longibrachiatum and Aspergillus fischeri (the first case ever reported). The infection caused the loss of the split-thickness skin grafts (STSGs); we had to perform repeated hydrosurgical and mechanical debridement and a systemic antifungal treatment prior to re-application of the STSGs. The subsequent skin transplant was successful.

Bronchoalveolar lavage fluid and serum 1,3-ß-D-glucan testing for invasive pulmonary aspergillosis diagnosis in hematological patients: the role of factors affecting assay performance.

Invasive fungal disease (IFD) early diagnosis improves hematological patient survival. Non-culture-based methods may reduce diagnostic time to identify IFD. As complex data on the value of 1,3-ß-D-glucan (BDG) from bronchoalveolar lavage fluid (BALF) compared to serum for the most frequent invasive pulmonary aspergillosis (IPA) diagnosis are scarce, particularly including evaluation of potential factors adversely affecting BDG assay, we provided prospective single-center analysis evaluating 172 episodes of pulmonary infiltrates with BDG detection in BALF and serum samples collected in parallel among hematological patients from 2006 to 2015. Proven and probable IPA were documented in 13.4% of the episodes. Sensitivity (SEN), specificity (SPE), positive and negative predictive value (PPV; NPV), and diagnostic odds ratio (DOR) of the BDG assay using standard (80 pg/ml) cut-off for BALF were: 56.5%; 83.2%; 34.2%; 92.5%, and 6.5, respectively, and for serum were: 56.5%; 82.6%; 33.3%; 92.5%, and 6.2, respectively. The same BDG assay parameters employing a calculated optimal cut-off for BALF (39 pg/ml) were: 78.3%; 72.5%; 30.5%; 95.6%, and 9.5, respectively; and for serum (40 pg/ml) were: 73.9%; 69.1%; 27.0%; 94.5%, and 6.3, respectively. While identifying acceptable SEN, SPE, and DOR, yet low PPV of both BALF and serum BDG assay for IPA diagnosis, neither the combination of both materials nor the new optimal BDG cut-off led to significant test quality improvement. Absolute neutrophil count and aspirated BALF volume with a significant trend affected BDG assay performance. The BDG test did not outperform galactomannan assay.

Application of mini-MLST and whole genome sequencing in low diversity hospital extended-spectrum beta-lactamase producing Klebsiella pneumoniae population.

Studying bacterial population diversity is important to understand healthcare associated infections' epidemiology and has a significant impact on dealing with multidrug resistant bacterial outbreaks. We characterised the extended-spectrum beta-lactamase producing K. pneumoniae (ESBLp KPN) population in our hospital using mini-MLST. Then we used whole genome sequencing (WGS) to compare selected isolates belonging to the most prevalent melting types (MelTs) and the colonization/infection pair isolates collected from one patient to study the ESBLp KPN population's genetic diversity. A total of 922 ESBLp KPN isolates collected between 7/2016 and 5/2018 were divided into 38 MelTs using mini-MLST with only 6 MelTs forming 82.8% of all isolates. For WGS, 14 isolates from the most prominent MelTs collected in the monitored period and 10 isolates belonging to the same MelTs collected in our hospital in 2014 were randomly selected. Resistome, virulome and ST were MelT specific and stable over time. A maximum of 23 SNV per core genome and 58 SNV per core and accessory genome were found. To determine the SNV relatedness cut-off values, 22 isolates representing colonization/infection pair samples obtained from 11 different patients were analysed by WGS with a maximum of 22 SNV in the core genome and 40 SNV in the core and accessory genome within pairs. The mini-MLST showed its potential for real-time epidemiology in clinical practice. However, for outbreak evaluation in a low diversity bacterial population, mini-MLST should be combined with more sensitive methods like WGS. Our findings showed there were only minimal differences within the core and accessory genome in the low diversity hospital population and gene based SNV analysis does not have enough discriminatory power to differentiate isolate relatedness. Thus, intergenic regions and mobile elements should be incorporated into the analysis scheme to increase discriminatory power.

Detection and identification of fungi in bronchoalveolar lavage fluid from immunocompromised patients using panfungal PCR.

Rapid diagnostics of fungal pneumonia and initiation of appropriate therapy are still challenging. In this study, we used two panfungal assays to test bronchoalveolar lavage fluid (BALF) samples to prove their ability to confirm invasive fungal disease diagnosis and identify causative agents. Two methods targeting different fungal rDNA regions were used, and the obtained PCR products were sequenced directly or after cloning. In total, 106 BALF samples from 104 patients were tested. After sequencing, we obtained 578 sequences. Four hundred thirty-seven sequences were excluded from further analysis due to duplication (n = 335) or similarity with sequences detected in the extraction control sample (n = 102); 141 unique sequences were analyzed. Altogether, 23/141 (16%) of the fungi detected belonged to pathogenic species, and 63/141 (45%) were identified as various yeasts; a variety of environmental or very rare fungal human pathogens represented 29/141 (21%) of the total and 26/141 (18%) were described as uncultured fungus. Panfungal PCR detected fungal species that would be missed by specific methods in only one case (probable cryptococcosis). Panfungal PCR followed by sequencing has limited use for testing BALF samples due to frequent commensal or environmental fungal species pickup.

The first isolation of Westerdykella dispersa in a critically burned patient.

Patients with critical thermal trauma belong to one of the most high-risk groups for development of infectious complications. Fungal infections are not among frequent complications during therapy of patients with thermal trauma, yet their incidence dramatically aggravates the prognosis for patients with this disorder. In the case report, we present the case of a young man with a critical burn, where Westerdykella dispersa was isolated. Identification of the pathogen was provided with a combination of cultivation and molecular biological confirmation. In this case, the distinction between infection and colonization was very complicated. Histopathological examination for definitive diagnosis of infection was not performed because the material from unburned soft tissue sampling could further compromise the function of the hand. Repeated cultivation and molecular identification W. dispersa before and after the necrectomy is indicative of infection rather than colonization. It is the second documented case of positive cultivation with this pathogen in humans and the first such case in a non-neutropenic host.

Investigation of next-generation sequencing data of Klebsiella pneumoniae using web-based tools.

PURPOSE: Rapid identification and characterization of multidrug-resistant Klebsiella pneumoniae strains is necessary due to the increasing frequency of severe infections in patients. The decreasing cost of next-generation sequencing enables us to obtain a comprehensive overview of genetic information in one step. The aim of this study is to demonstrate and evaluate the utility and scope of the application of web-based databases to next-generation sequenced (NGS) data. METHODOLOGY: The whole genomes of 11 clinical Klebsiella pneumoniae isolates were sequenced using Illumina MiSeq. Selected web-based tools were used to identify a variety of genetic characteristics, such as acquired antimicrobial resistance genes, multilocus sequence types, plasmid replicons, and identify virulence factors, such as virulence genes, cps clusters, urease-nickel clusters and efflux systems. RESULTS: Using web-based tools hosted by the Center for Genomic Epidemiology, we detected resistance to 8 main antimicrobial groups with at least 11 acquired resistance genes. The isolates were divided into eight sequence types (ST11, 23, 37, 323, 433, 495 and 562, and a new one, ST1646). All of the isolates carried replicons of large plasmids. Capsular types, virulence factors and genes coding AcrAB and OqxAB efflux pumps were detected using BIGSdb-Kp, whereas the selected virulence genes, identified in almost all of the isolates, were detected using CLC Genomic Workbench software. CONCLUSION: Applying appropriate web-based online tools to NGS data enables the rapid extraction of comprehensive information that can be used for more efficient diagnosis and treatment of patients, while data processing is free of charge, easy and time-efficient.

Serological Approaches.

The diagnosis of invasive fungal diseases (IFD) based on clinical, radiological, and conventional microbiological findings is not reliable and is often delayed. Non-culture-based methods with higher sensitivity and specificity may reduce diagnostic time and result in decreased IFD morbidity and mortality. These methods are now increasingly used to manage patients at risk of IFD. Among available biomarkers, fungal antigens have been investigated as an aid to early diagnosis and are predominantly used as screening tests to prompt antifungal treatment mainly in patients with hematological malignancies. The revised version of the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) consensus definitions includes some of these biological markers (galactomannan, 1,3-beta-D-glucan, cryptococcus antigen).

Validation of Minim typing for fast and accurate discrimination of extended-spectrum, beta-lactamase-producing Klebsiella pneumoniae isolates in tertiary care hospital.

Minim typing is derived from the multi-locus sequence typing (MLST). It targets the same genes, but sequencing is replaced by high resolution melt analysis. Typing can be performed by analysing six loci (6MelT), four loci (4MelT) or using data from four loci plus sequencing the tonB gene (HybridMelT). The aim of this study was to evaluate Minim typing to discriminate extended-spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-KLPN) isolates at our hospital. In total, 380 isolates were analyzed. The obtained alleles were assigned according to both the 6MelT and 4MelT typing scheme. In 97 isolates, the tonB gene was sequenced to enable HybridMelT typing. We found that the presented method is suitable to quickly monitor isolates of ESBL-KLPN; results are obtained in less than 2 hours and at a lower cost than MLST. We identified a local ESBL-KLPN outbreak and a comparison of colonizing and invasive isolates revealed a long term colonization of patients with the same strain.

Rapid detection of fungal pathogens in bronchoalveolar lavage samples using panfungal PCR combined with high resolution melting analysis.

Despite advances in the treatment of invasive fungal diseases (IFD), mortality rates remain high. Moreover, due to the expanding spectrum of causative agents, fast and accurate pathogen identification is necessary. We designed a panfungal polymerase chain reaction (PCR), which targets the highly variable ITS2 region of rDNA genes and uses high resolution melting analysis (HRM) for subsequent species identification. The sensitivity and specificity of this method was tested on a broad spectrum of the most clinically important fungal pathogens including Aspergillus spp., Candida spp. and mucormycetes. Despite the fact that fluid from bronchoalveolar lavage (BAL) is one of the most frequently tested materials there is a lack of literature sources aimed at panfungal PCR as an IFD diagnostic tool from BAL samples. The applicability of this method in routine practice was evaluated on 104 BAL samples from immunocompromised patients. Due to high ITS region variability, we obtained divergent melting peaks for different fungal species. Thirteen out of 18 patients with proven or probable IFD were positive. Therefore, the sensitivity, specificity, positive predictive value and negative predictive value of our method were 67%, 100%, 100%, and 94%, respectively. In our assay, fungal pathogens identification is based on HRM, therefore omitting the expensive and time consuming sequencing step. With the high specificity, positive and negative predictive values, short time needed to obtain a result, and low price, the presented assay is intended to be used as a quick screening method for patients at risk of IFD.

Disseminated fusariosis by Fusarium proliferatum in a patient with aplastic anaemia receiving primary posaconazole prophylaxis - case report and review of the literature.

Disseminated fusariosis is a life-threatening, invasive, opportunistic infection in immunocompromised patients, especially those with haematological malignancies. The prognosis is poor because these fungi are resistant to many of the available antifungal agents. We present a case of disseminated fusariosis caused by Fusarium proliferatum in a patient with severe aplastic anaemia complicated by a secondary infection of Aspergillus flavus, with a fatal outcome. We also review the documented Fusarium infections in immunocompromised hosts.

Rapid detection and identification of mucormycetes in bronchoalveolar lavage samples from immunocompromised patients with pulmonary infiltrates by use of high-resolution melt analysis.

Rapid differential diagnostics of pulmonary infiltrates suspected of invasive fungal disease in an immunocompromised host and early initiation of effective antifungal therapy are crucial for patient outcomes. There are no serological tests available to detect mucormycetes; therefore, PCR-based methods are highly suitable. We validated our previously published PCR followed by high-resolution melt analysis (PCR/HRMA) to detect Rhizopus spp., Rhizomucor pusillus, Lichtheimia corymbifera, and Mucor spp. in bronchoalveolar lavage (BAL) samples from immunocompromised patients who were at risk of invasive fungal disease. All PCR/HRMA-positive samples were retested using novel real-time quantitative PCR (RQ PCR) assays specific to the species identified. In total, between January 2009 and December 2012 we analyzed 99 BAL samples from 86 patients with pulmonary abnormalities using PCR/HRMA. Ninety (91%) BAL samples were negative, and 9 (9%) samples were positive. The sensitivity and specificity of PCR/HRMA were 100% and 93%, respectively. By combining the positive results of PCR/HRMA with positive RQ PCR results, the specificity was raised to 98%. PCR/HRMA, due to its high negative predictive value (99%), represents a fast and reliable tool for routine BAL sample screening for the differential diagnosis of pulmonary infiltrates in immunocompromised patients for the four most clinically important mucormycetes.

Micafungin as empirical antifungal therapy in hematological patients: a retrospective, multicenter study in the Czech and Slovak Republics.

The objective of this retrospective, multicenter study was to evaluate the efficacy and safety of micafungin as empirical antifungal therapy during febrile neutropenia (FN) in 73 hematological patients from six centers in two countries. All patients received 100 mg of micafungin/day. The overall favorable response rate (RR) was 64.8% when the resolution of fever during neutropenia was included in the response criteria and 84.5% when excluded. A significantly lower favorable RR in patients with persistent fever and non-specific pulmonary infiltrates compared to patients with persistent fever only (82.8 vs. 52.4%, respectively; p = 0.011) was not found when resolution of fever was not included in the composite endpoint criteria (93.1 vs. 78.6%, respectively; p = 0.180). Breakthrough fungal disease developed in 2.7% of patients. Treatment was discontinued in 16.4% of cases. Only one patient (1.4%) discontinued therapy due to an adverse event. Posaconazole prophylaxis improved favorable RR when defervescence was included as composite endpoint criterion (p = 0.047), but not when it was excluded (p = 0.485). However, neutrophil recovery did not influence favorable RR (p = 0.803 and p = 0.112, respectively). These data suggest that micafungin is safe and effective as an empirical therapy in patients with FN.