Unbiased screen for pathogens in human paraffin-embedded tissue samples by whole genome sequencing and metagenomics.

Identification of bacterial pathogens in formalin fixed, paraffin embedded (FFPE) tissue samples is limited to targeted and resource-intensive methods such as sequential PCR analyses. To enable unbiased screening for pathogens in FFPE tissue samples, we established a whole genome sequencing (WGS) method that combines shotgun sequencing and metagenomics for taxonomic identification of bacterial pathogens after subtraction of human genomic reads. To validate the assay, we analyzed more than 100 samples of known composition as well as FFPE lung autopsy tissues with and without histological signs of infections. Metagenomics analysis confirmed the pathogenic species that were previously identified by species-specific PCR in 62% of samples, showing that metagenomics is less sensitive than species-specific PCR. On the other hand, metagenomics analysis identified pathogens in samples, which had been tested negative for multiple common microorganisms and showed histological signs of infection. This highlights the ability of this assay to screen for unknown pathogens and detect multi-microbial infections which is not possible by histomorphology and species-specific PCR alone.

COVID-19 Autopsies Reveal Underreporting of SARS-CoV-2 Infection and Scarcity of Co-infections.

Coronavirus disease 2019 (COVID-19) mortality can be estimated based on reliable mortality data. Variable testing procedures and heterogeneous disease course suggest that a substantial number of COVID-19 deaths is undetected. To address this question, we screened an unselected autopsy cohort for the presence of SARS-CoV-2 and a panel of common respiratory pathogens. Lung tissues from 62 consecutive autopsies, conducted during the first and second COVID-19 pandemic waves in Switzerland, were analyzed for bacterial, viral and fungal respiratory pathogens including SARS-CoV-2. SARS-CoV-2 was detected in 28 lungs of 62 deceased patients (45%), although only 18 patients (29%) were reported to have COVID-19 at the time of death. In 23 patients (37% of all), the clinical cause of death and/or autopsy findings together with the presence of SARS-CoV-2 suggested death due to COVID-19. Our autopsy results reveal a 16% higher SARS-CoV-2 infection rate and an 8% higher SARS-CoV-2 related mortality rate than reported by clinicians before death. The majority of SARS-CoV-2 infected patients (75%) did not suffer from respiratory co-infections, as long as they were treated with antibiotics. In the lungs of 5 patients (8% of all), SARS-CoV-2 was found, yet without typical clinical and/or autopsy findings. Our findings suggest that underreporting of COVID-19 contributes substantially to excess mortality. The small percentage of co-infections in SARS-CoV-2 positive patients who died with typical COVID-19 symptoms strongly suggests that the majority of SARS-CoV-2 infected patients died from and not with the virus.

High throughput sequencing reveals high specificity of TNFAIP3 mutations in ocular adnexal marginal zone B-cell lymphomas.

The majority of ocular adnexal (OA) lymphomas (OAL) are extranodal marginal zone lymphomas (MZL). First high throughput sequencing (HTS) studies on OA-MZL showed inconsistent results and the distribution of mutations in reactive lymphoid lesions of this anatomic region has not yet been sufficiently addressed. We characterized OAL and lymphoid lesions of the OA by targeted HTS. The study included 34 OA-MZL, 11 chronic conjunctivitis, five mature small cell B-cell lymphomas spreading to the OA, five diseases with increase of IgG4+ plasma cells, three Burkitt lymphomas (BL), three diffuse large B-cell lymphomas (DLBCL), three mantle cell lymphomas, three idiopathic orbital inflammations/orbital pseudo tumors (PT), and three OA lymphoid hyperplasia. All cases were negative for Chlamydia. The mutational number was highest in BL and lowest in PT. The most commonly (and exclusively) mutated gene in OA-MZL was TNFAIP3 (10 of 34 cases). Altogether, 20 out of 34 patients harbored mutually exclusive mutations of either TNFAIP3, BCL10, MYD88, ATM, BRAF, or NFKBIE, or nonexclusive mutations of IRF8, TNFRSF14, KLHL6, and TBL1XR1, all encoding for NK-κB pathway compounds or regulators. Thirteen patients (38%) had, to a great part, mutually exclusive mutations of chromatin modifier-encoding genes: KMT2D, CREBBP, BCL7A, DNMT3A, EP300, or HIST1H1E. Only four patients harbored co-occurring mutations of genes encoding for NK-κB compounds and chromatin modifiers. Finally, PTEN, KMT2D, PRDM1, and HIST1H2BK mutations were observable in reactive lymphoid lesions too, while such instances were devoid of NF-κB compound mutations and/or mutations of acetyltransferase-encoding genes. In conclusion, 80% of OA-MZL display mutations of either NK-κB compounds or chromatin modifiers. Lymphoid lesions of the OA bearing NF-κB compound mutations and/or mutations of acetyltransferase-encoding genes highly likely represent lymphomas.

Multi-fungal sepsis and mucormycosis of the central nervous system in a patient treated with ibrutinib, a case report and review of the literature.

We report the case of a 71 years old patient with chronic lymphocytic leukemia (CLL), who developed a rapidly progressing multi-fungal infection including mucormycosis of the central nervous system (CNS) during treatment with ibrutinib. On autopsy mucorales species were demonstrated intravascularly by histomorphology of several organs and lymph nodes and were characterized as Rhizomucor pusillus by polymerase-chain reaction (PCR) - analysis. In addition, invasive pulmonary Aspergillus fumigatus was found and also confirmed by PCR. To the best of our knowledge, this is the first confirmation of a multi-fungal sepsis and invasive CNS-infection with mucorales species under ibrutinib. Knowing the risk for invasive fungal disease in patients under ibrutinib, identifying the pathogen and early initiation of specific treatment is crucial for a good clinical outcome especially in mucormycosis.

Brain abscess caused by Cladophialophora bantiana: Total remission after full resection and short-course Voriconazole treatment.

We report on a brain abscess in an immunocompetent man caused by Cladophialophora bantiana, a black mold known to prefer the brain for abscess-formations with a mortality rate up to 65%. The published cases so far show different therapeutical approaches and outcomes, whereby a standardized treatment could not yet be established. We report on a case of Cladophialophora bantiana brain abscess with total remission of the abscess after full resection and a short antifungal treatment.

[Pathology of infectious diseases]. / Pathologie von Infektionskrankheiten.

Pathology of infectious diseases Abstract. The pathology of infectious diseases is an exciting interdisciplinary field, despite its niche existence that is somewhat overshadowed by tumor diagnostics. However, the strength of pathology lies in the correlation of the inflammatory patterns and pathogen detection. Moreover, corresponding tissue investigations often allow a rapid diagnosis of the disease, and additional investigations, such as immunohistochemistry or molecular pathology, enable a rapid pathogen characterization with a high sensitivity and specificity. In addition, the molecular analysis allows the detection of pathogens that are difficult, dangerous or not at all to breed. It can be assumed that complex infectious diseases will increase due to iatrogenic interventions, migration, antibiotic resistance and climate change, and that pathology, in close cooperation with its treating colleagues, will increasingly play an important role in the care of patients.

Reconstitution of mammalian cleavage factor II involved in 3' processing of mRNA precursors.

Cleavage factor II (CF II) is a poorly characterized component of the multiprotein complex catalyzing 3' cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5' kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequence-specificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.

Single-Center Experience with a Targeted Next Generation Sequencing Assay for Assessment of Relevant Somatic Alterations in Solid Tumors.

Companion diagnostics rely on genomic testing of molecular alterations to enable effective cancer treatment. Here we report the clinical application and validation of the Oncomine Focus Assay (OFA), an integrated, commercially available next-generation sequencing (NGS) assay for the rapid and simultaneous detection of single nucleotide variants, short insertions and deletions, copy number variations, and gene rearrangements in 52 cancer genes with therapeutic relevance. Two independent patient cohorts were investigated to define the workflow, turnaround times, feasibility, and reliability of OFA targeted sequencing in clinical application and using archival material. Cohort I consisted of 59 diagnostic clinical samples from the daily routine submitted for molecular testing over a 4-month time period. Cohort II consisted of 39 archival melanoma samples that were up to 15years old. Libraries were prepared from isolated nucleic acids and sequenced on the Ion Torrent PGM sequencer. Sequencing datasets were analyzed using the Ion Reporter software. Genomic alterations were identified and validated by orthogonal conventional assays including pyrosequencing and immunohistochemistry. Sequencing results of both cohorts, including archival formalin-fixed, paraffin-embedded material stored up to 15years, were consistent with published variant frequencies. A concordance of 100% between established assays and OFA targeted NGS was observed. The OFA workflow enabled a turnaround of 3½ days. Taken together, OFA was found to be a convenient tool for fast, reliable, broadly applicable and cost-effective targeted NGS of tumor samples in routine diagnostics. Thus, OFA has strong potential to become an important asset for precision oncology.

Use of argatroban: experiences in continuous renal replacement therapy in critically ill patients after cardiac surgery.

OBJECTIVES: Acute kidney injury requiring renal replacement therapy (RRT) is a common complication after cardiac surgery, complicated by suspected or proven heparin-induced thrombocytopenia (type II). The present study evaluated the use of argatroban as an anticoagulant during continuous RRT in the early period after cardiac surgery. Argatroban was compared with unfractionated heparin (UH) with respect to bleeding complications and the effectiveness of anticoagulation. METHODS: Patients requiring RRT after cardiac surgery from March 2007 to June 2009 were identified. The effectiveness of anticoagulation was measured indirectly by the duration of dialysis filter use. Bleeding was defined as clinical signs of blood loss or the need for transfusion. RESULTS: Of 94 patients, 41 received argatroban, 27 UH, and 26 required conversion from UH to argatroban. In all 3 subgroups, RRT was begun within a median postoperative period of 2.0 days. Similar levels of anticoagulation were achieved with the duration of the circuit and filter changed an average of 1.1 times daily during RRT. Liver function was comparable in all patients. Neither clinically relevant signs of bleeding nor significant differences in the hemoglobin levels or a requirement for transfusion were noted. However, the Simplified Acute Physiology Score II values during dialysis and mortality were significantly greater in the patients initially receiving argatroban compared with those who received UH alone (54 ± 2 vs 43 ± 3, P < .001; 71% vs 44%, P = .04). CONCLUSIONS: Argatroban can provide effective anticoagulation in postoperative cardiac patients receiving continuous RRT. Close monitoring and dose titration resulted in a comparable risk of bleeding for anticoagulation with both argatroban and heparin, regardless of the disease severity or impaired hepatic function.

Single FGF-23 measurement and time-averaged plasma phosphate levels in hemodialysis patients.

BACKGROUND AND OBJECTIVES: Plasma phosphate levels display considerable intraindividual variability. The phosphatonin fibroblast growth factor 23 is a central regulator of plasma phosphate levels, and it has been postulated to be a more stable marker than conventional CKD-mineral and bone disorder parameters. Thus, fibroblast growth factor 23 has been hypothesized to reflect time-averaged plasma phosphate levels in CKD patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Among 40 patients from the outpatient dialysis center, serial measurements of plasma calcium and phosphate (before every dialysis session) as well as C-terminal fibroblast growth factor 23, parathyroid hormone, and alkaline phosphatase (one time weekly) were performed over a study period of 4 weeks in November and December of 2011. Intraindividual variability of repeated plasma fibroblast growth factor 23 measurements compared with other CKD-mineral and bone disorder markers was tested, and the association of a single plasma fibroblast growth factor 23 measurement with time-averaged plasma phosphate levels was analyzed. RESULTS: Against expectations, intraindividual variability of fibroblast growth factor 23 (median coefficient of variation=27%; interquartile range=20-35) was not lower than variability of plasma phosphate (median coefficient of variation=15%; interquartile range=10-20), parathyroid hormone (median coefficient of variation=24%; interquartile range=15-39), plasma calcium (median coefficient of variation=3%; interquartile range=2-4), or alkaline phosphatase (median coefficient of variation=5%; interquartile range=3-10). Moreover, the correlation between the last fibroblast growth factor 23 measurement after 4 weeks and time-averaged plasma phosphate did not surpass the correlation between the last fibroblast growth factor 23 measurement and a single plasma phosphate value (r=0.67, P<0.001; r=0.76, P<0.001, respectively). CONCLUSIONS: Surprisingly, fibroblast growth factor 23 was not more closely associated to time-averaged plasma phosphate levels than a single plasma phosphate value, and it did not show a lower intraindividual variability than other tested markers of CKD-mineral and bone disorder. Thus, fibroblast growth factor 23 should not be used in clinical practice as a reflector of time-averaged plasma phosphate levels.

Functional group richness: implications of biodiversity for light use and lipid yield in microalgae.

Currently, very few studies address the relationship between diversity and biomass/lipid production in primary producer communities for biofuel production. Basic studies on the growth of microalgal communities, however, provide evidence of a positive relationship between diversity and biomass production. Recent studies have also shown that positive diversity-productivity relationships are related to an increase in the efficiency of light use by diverse microalgal communities. Here, we hypothesize that there is a relationship between diversity, light use, and microalgal lipid production in phytoplankton communities. Microalgae from all major freshwater algal groups were cultivated in treatments that differed in species richness and functional group richness. Polycultures with high functional group richness showed more efficient light use and higher algal lipid content with increasing species richness. There was a clear correlation between light use and lipid production in functionally diverse communities. Hence, a powerful and cost-effective way to improve biofuel production might be accomplished by incorporating diversity related, resource-use-dynamics into algal biomass production.

Total-to-ionized calcium ratio predicts mortality in continuous renal replacement therapy with citrate anticoagulation in critically ill patients.

INTRODUCTION: Regional citrate anticoagulation is safe, feasible and increasingly used in critically ill patients on continuous renal replacement therapy (CRRT). However, in patients with hepatic or multi-organ dysfunction, citrate accumulation may lead to an imbalance of calcium homeostasis. The study aimed at evaluating the incidence and prognostic relevance of an increased total to ionized calcium ratio (T/I Ca(2+) ratio) and its association to hepatic dysfunction. METHODS: We performed a prospective observational study on n = 208 critically ill patients with acute kidney injury (AKI) and necessity for CRRT with regional citrate anticoagulation (CRRT-citrate) between September 2009 and September 2011. Critical illness was estimated by Simplified Acute Physiology Score II; hepatic function was measured with indocyanine green plasma disappearance rate. After achieving a steady state of calcium homeostasis patients were classified into tertiles according to the T/I Ca(2+) ratio (<2.0 versus 2.0 - 2.39 versus ≥ 2.4). RESULTS: The T/I Ca(2+) ratio was determined as an independent predictor for 28-day mortality in critically ill patients with AKI on CRRT-citrate confirmed by receiver operating characteristics and multivariate analysis (Area under the curve 0.94 ± 0.02; p<0.001). A T/I Ca(2+) ratio ≥ 2.4 independently predicted a 33.5-fold (p<0.001) increase in 28-day mortality-rate. There was a significant correlation between the T/I Ca(2+) ratio and the hepatic clearance (p<0.001) and the severity of critical illness (p<0.001). The efficacy and safety of citrate anticoagulation, determined by blood urea nitrogen, mean filter patency and bleeding episodes, were not significantly different between the tertiles. CONCLUSIONS: In patients on CRRT-citrate T/I Ca(2+) ratio is closely related to the clinical outcome and emerged as an independent predictor of 28-day mortality. Larger studies are required to define the cut-off and predictive value for the T/I Ca(2+) ratio. This ratio is associated with hepatic and/or multi-organ dysfunction and therefore an important therapeutic target.