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Systemic infections caused by Salmonella enterica are an ongoing public health problem especially in Sub-Saharan Africa. Essentially typhoid fever is associated with high mortality particularly because of the increasing prevalence of multidrug-resistant strains. Thus, a rapid blood-culture based bacterial species diagnosis including an immediate sub-differentiation of the various serovars is mandatory. At present, MALDI-TOF based intact cell mass spectrometry (ICMS) advances to a widely used routine identification tool for bacteria and fungi. In this study, we investigated the appropriateness of ICMS to identify pathogenic bacteria derived from Sub-Saharan Africa and tested the potential of this technology to discriminate S. enterica subsp. enterica serovar Typhi (S. Typhi) from other serovars. Among blood culture isolates obtained from a study population suffering from febrile illness in Ghana, no major misidentifications were observed for the species identification process, but serovars of Salmonella enterica could not be distinguished using the commercially available Biotyper database. However, a detailed analysis of the mass spectra revealed several serovar-specific biomarker ions, allowing the discrimination of S. Typhi from others. In conclusion, ICMS is able to identify isolates from a sub-Saharan context and may facilitate the rapid discrimination of the clinically and epidemiologically important serovar S. Typhi and other non-S. Typhi serovars in future implementations.

This study was performed as a head-to-head comparison of the performance characteristics of (1) two SARS-CoV-2-specific rapid antigen assays with real-time PCR as gold standard as well as (2) a fully automated high-throughput transcription-mediated amplification (TMA) assay and real-time PCR in a latent class analysis-based test comparison without a gold standard with several hundred samples in a low prevalence “real world” setting. Recorded sensitivity and specificity of the NADAL and the LumiraDx antigen assays and the Hologic Aptima SARS-CoV-2 TMA assay were 0.1429 (0.0194, 0.5835), 0.7644 (0.7016, 0.8174), and 0.7157 (0, 1) as well as 0.4545 (0.2022, 0.7326), 0.9954 (0.9817, 0.9988), and 0.9997 (not estimable), respectively. Agreement kappa between the positive results of the two antigen-based assays was 0.060 (0.002, 0.167) and 0.659 (0.492, 0.825) for TMA and real-time PCR. Samples with low viral load as indicated by cycle threshold (Ct) values > 30 were generally missed by both antigen assays, while 1:10 pooling suggested higher sensitivity of TMA compared to real-time PCR. In conclusion, both sensitivity and specificity speak in favor of the use of the LumiraDx rather than the NADAL antigen assay, while TMA results are comparably as accurate as PCR, when applied in a low prevalence setting.

Pathogen identification is a critical step during diagnosis of infectious diseases. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF-MS) has become the gold standard for identification of microorganisms cultured on solid media in microbiology laboratories. Direct identification of microbes from liquid specimen, circumventing the need for the additional overnight cultivation step, has been successfully established for blood culture, urine and liquor. Here, we evaluate the ability of MALDI-TOF MS for direct identification of pathogens in synovial fluid after liquid enrichment in BacT/Alert blood culture bottles. Influence of synovial specimen quality on direct species identification with the MALDI BioTyper/Sepsityper was tested with samples inoculated from pretested native synovia with concomitant inoculation of blood or pus, or highly viscous fluid. Here, we achieved >90% concordance with culture on solid medium, and only mixed-species samples posed significant problems. Performance in routine diagnostics was tested prospectively on bottles inoculated by treating physicians on ward. There, we achieved >70% concordance with culture on solid media. The major contributors to test failure were the absence of a measurable mass signal and mixed-specimen samples. The Sepsityper workflow worked well on samples derived from BacT/Alert blood culture bottles inoculated with synovial fluid, giving concordant results to identification from solid media. Host remnant material in the inoculum, such as blood or pus, had no detrimental effect on identification score values of the BioTyper system after processing with the Sepsityper workflow, and neither had the initial viscosity of the synovial sample.

Background Yeasts of the CTG-clade lineage, which includes the human-infecting Candida albicans , Candida parapsilosis and Candida tropicalis species, are characterized by an altered genetic code. Instead of translating CUG codons as leucine, as happens in most eukaryotes, these yeasts, whose ancestors are thought to have lost the relevant leucine-tRNA gene, translate CUG codons as serine using a serine-tRNA with a mutated anticodon, tRNA CAG Ser . Previously reported experiments have suggested that 3–5% of the CTG-clade CUG codons are mistranslated as leucine due to mischarging of the tRNA CAG Ser . The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity. Results In this study, we reassess this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, including various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our data do not support a bias towards CUG codon mistranslation as leucine. Instead, our data suggest that (i) CUG codons are mistranslated at a frequency corresponding to the normal extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans ’ strain pathogenicity or growth form. Conclusions Our findings imply that C. albicans does not decode CUG ambiguously. This suggests that the proposed misleucylation of the tRNA CAG Ser might be as prevalent as every other misacylation or mistranslation event and, if at all, be just one of many reasons causing phenotypic diversity.

MALDI-TOF-MS of microorganisms, which identifies microbes based on masses of high abundant low molecular weight proteins, is rapidly advancing to become another standard method in clinical routine laboratory diagnostics. Allelic isoforms of these proteins result in varying masses of detectable biomarker ions. These variations give rise to a novel typing method for microorganisms named mass spectrometry-based phyloproteomics (MSPP). The base of MSPP is an amino acid sequence list of allelic isoforms caused by non-synonymous mutations in biomarker genes, which were detectable as mass shifts in an overlay of calibrated MALDI-TOF spectra. Thus, for each isolate a combination of amino acid sequences can be deduced from the scheme of recordable biomarker masses. Performing comparably to laborious multilocus and whole genome sequence typing (wgMLST)-approaches it is feasible to build phyloproteomic dendrograms using hierarchical cluster analysis. MSPP bears a high potential especially for identification of chromosomal localised virulence or antimicrobial resistance factors associated with evolutionary relatedness. In this study the principle of MSPP-typing was demonstrated on a Campylobacter jejuni ssp. jejuni isolate collection and MSPP was compared to MLST.

In SARS-CoV-2 diagnostics, cycle threshold (Ct) values from qRT-PCRs semi-quantitatively estimate a patient’s viral load. However, relevant analytical differences between qRT-PCR assays are often neglected. This study was designed (i) to identify such differences between five commonly used assays and (ii) to demonstrate a straightforward strategy to harmonize them. QRT-PCRs for SARS-CoV-2 were carried out in 85 oropharyngeal swab samples using three fully automated (Alinity m, cobas®6800 and GeneXpert) and two semi-automated (genesig® and RIDA®GENE) assays. Qualitative results (positive/negative) showed excellent comparability between the fully automated assays, but not between the Alinity m and semi-automated methods. Ct values significantly varied between all the methods, with the median values ranging from 22.76 (Alinity m) to 30.89 (RIDA®GENE) and 31.50 (genesig®), indicating the lowest sensitivity for semi-automated methods. Passing–Bablok analysis further revealed systemic biases. Assay-specific viral load concentration calculations—based on generated individual standard curves—resulted in much better comparability between the assays. Applying these calculations, significant differences were no longer detectable. This study highlights relevant analytical differences between SARS-CoV-2 qRT-PCR assays, leading to divergent decisions about the mandatory isolation of infected individuals. Secondly, we propose a strategy to harmonize qRT-PCR assays to achieve better comparability. Our findings are of particular interest for laboratories utilizing different assays.

Campylobacter jejuni, the most common bacterial pathogen causing gastroenteritis, shows a wide genetic diversity. Previously, we demonstrated by the combination of multi locus sequence typing (MLST)-based UPGMA-clustering and analysis of 16 genetic markers that twelve different C. jejuni subgroups can be distinguished. Among these are two prominent subgroups. The first subgroup contains the majority of hyperinvasive strains and is characterized by a dimeric form of the chemotaxis-receptor Tlp7(m+c). The second has an extended amino acid metabolism and is characterized by the presence of a periplasmic asparaginase (ansB) and gamma-glutamyl-transpeptidase (ggt). Phyloproteomic principal component analysis (PCA) hierarchical clustering of MALDI-TOF based intact cell mass spectrometry (ICMS) spectra was able to group particular C. jejuni subgroups of phylogenetic related isolates in distinct clusters. Especially the aforementioned Tlp7(m+c)(+) and ansB+/ ggt+ subgroups could be discriminated by PCA. Overlay of ICMS spectra of all isolates led to the identification of characteristic biomarker ions for these specific C. jejuni subgroups. Thus, mass peak shifts can be used to identify the C. jejuni subgroup with an extended amino acid metabolism. Although the PCA hierarchical clustering of ICMS-spectra groups the tested isolates into a different order as compared to MLST-based UPGMA-clustering, the isolates of the indicator-groups form predominantly coherent clusters. These clusters reflect phenotypic aspects better than phylogenetic clustering, indicating that the genes corresponding to the biomarker ions are phylogenetically coupled to the tested marker genes. Thus, PCA clustering could be an additional tool for analyzing the relatedness of bacterial isolates.

Invasive candidiasis (IC) is associated with high morbidity and mortality in hospitalized patients if not diagnosed early. Long-term use of central venous catheters is a predisposing factor for IC. Hyphal forms of Candida albicans (the major etiological agent of IC) are related to invasion of host tissues. The secreted proteins of hyphae are involved in virulence, host interaction, immune response, and immune evasion. To identify IC diagnostic biomarker candidates, we characterized the C. albicans hyphal secretome by gel-free proteomic analysis, and further assessed the antibody-reactivity patterns to this subproteome in serum pools from 12 patients with non-catheter-associated IC (ncIC), 11 patients with catheter-associated IC (cIC), and 11 non-IC patients. We identified 301 secreted hyphal proteins stratified to stem from the extracellular region, cell wall, cell surface, or intracellular compartments. ncIC and cIC patients had higher antibody levels to the hyphal secretome than non-IC patients. Seven secreted hyphal proteins were identified to be immunogenic (Bgl2, Eno1, Pgk1, Glx3, Sap5, Pra1 and Tdh3). Antibody-reactivity patterns to Bgl2, Eno1, Pgk1 and Glx3 discriminated IC patients from non-IC patients, while those to Sap5, Pra1 and Tdh3 differentiated between cIC and non-IC patients. These proteins may be useful for development of future IC diagnostic tests.

Yeasts of the Cryptococcus neoformans / gattii species complexes are human pathogens mostly in immune compromised individuals, and can cause infections from dermal lesions to fungal meningitis. Differences in virulence and antifungal drug susceptibility of species in these complexes indicate the value of full differentiation to species level in diagnostic procedures. MALDI-TOF MS has been reported to sufficiently discriminate these species. Here, we sought to re-evaluate sample pre-processing procedures and create a set of publicly available references for use with the MALDI Biotyper system. Peak content using four different pre-processing protocols was assessed, and database entries for 13 reference strains created. These were evaluated against a collection of 153 clinical isolates, typed by conventional means. The use of decapsulating protocols or mechanical disruption did not sufficiently increase the information content to justify the extra hands-on-time. Using the set of 13 reference entries created with the standard formic acid extraction, we were able to correctly classify 143/153 (93.5%) of our test isolates. The majority of the remaining ten isolates still gave correct top matches; only two isolates did not give reproducible identifications. This indicates that the log score cut-off can be lowered also in this context. Ease to identify cryptococcal isolates to the species level is improved by the workflow evaluated here. The database references are freely available from https://github.com/oliverbader/BioTyper-libraries for incorporation into local diagnostic systems.

Candida parapsilosis is a frequent cause of fungal bloodstream infections, especially in critically ill neonates or immunocompromised patients. Due to the formation of biofilms, the use of indwelling catheters and other medical devices increases the risk of infection and complicates treatment, as cells embedded in biofilms display reduced drug susceptibility. Therefore, biofilm formation may be a significant clinical parameter, guiding downstream therapeutic choices. Here, we phenotypically characterized 120 selected isolates out of a prospective collection of 215 clinical C. parapsilosis isolates, determining biofilm formation, major emerging colony morphotype, and antifungal drug susceptibility of the isolates and their biofilms. In our isolate set, increased biofilm formation capacity was independent of body site of isolation and not predictable using standard or modified European Committee on Antimicrobial Susceptibility Testing (EUCAST) drug susceptibility testing protocols. In contrast, biofilm formation was strongly correlated with the appearance of non-smooth colony morphotypes and invasiveness into agar plates. Our data suggest that the observation of non-smooth colony morphotypes in cultures of C. parapsilosis may help as an indicator to consider the initiation of anti-biofilm-active therapy, such as the switch from azole- to echinocandin- or polyene-based strategies, especially in case of infections by potent biofilm-forming strains.